More importantly, both the relative XBIC and XEOL measurements appear almost exclusively below one, indicating and quantifying a local performance impairment. These techniques image competing processes, i.e., they count, respectively, generated electron‐hole pairs that diffuse to the electrodes without recombining, and photons with an energy corresponding to the bandgap emitted upon radiative recombination. XBIC appears less impaired by voids than XEOL. The larger error bars from XEOL are due to the nature of the tracked process, i.e., XEOL is intrinsically a photon‐hungry technique. Performance is locally impaired up to 8% for XBIC and 60% for XEOL. On average, the effect of voids can be quantified by the meta‐analysis (see Supporting Information for details) in a 4% loss for XBIC and 20% for XEOL (Figure ##FIG##1##2E##). XEOL, in particular, degrades more than expected from the missing absorber, which might be an indication that voids and crevices are detrimental to cell voltage. This impairment can be attributed to the amount of missing material or an enhanced recombination velocity at interfaces with voids.[\n##UREF##15##\n28\n##, ##UREF##16##\n29\n##\n] However, it is not trivial to decouple these effects, as the performance maps are effectively blurred by the diffusion length of the carriers and by the electron shower produced by the beam (see Supporting Information). Such blurring leads to an underestimation of the XBIC dips, which explains why on most voids the relative reduction of XRF exceeds its XBIC counterpart. Despite the underestimation, the measured impairment is consistent and significant, and given the high overall performance of the device, suggests that charge transport in the functioning device is mostly sustained by alternative current paths than those below the worst performing areas. The optical and electrical performance losses correlate positively with the amount of missing material (Figure ##FIG##1##2F##) (correlation coefficients ≈0.7), whereas they do not correlate, if not weakly, with the projected area of the voids.","
The local performance impairment observed through measurements of current and luminescence cannot be as clearly observed when induced by a laser beam or an electron beam. In the case of lasers, the fundamentally limited resolution does not allow us to investigate the topic, as even the largest voids are smaller than the diffraction limit of photoluminescence measurements.[\n##UREF##17##\n30\n##\n] In the case of electrons, the investigation of the effect of voids is hampered by internal scattering effects. Our complementary measurements of EBIC and CL show high‐signal spikes in the proximity of voids (see Figures ##SUPPL##0##S12## and ##SUPPL##0##S13##, Supporting Information). These spikes are not an indication of enhanced electrical or optical performance due to specific material properties, but rather a measurement artifact due to secondary electrons being reabsorbed (see Supporting Information). Whereas these scattering phenomena are negligible for X‐rays, voids can have positive effects under AM1.5G illumination due to light trapping.[\n##UREF##18##\n31\n##\n] A minor performance impairment at voids is predicted by 3D numerical simulations of simple exemplary cases, although without accounting for optical effects.[\n##REF##30479675##\n14\n##\n]\n
","Ptychographic Nanotomography","
Whereas the 2D top‐view of the sample highlights lateral differences in the operational device, it cannot provide depth information about the disclosed features. Effectively, they can be vertical projections of multiple features, in the same or different layers. In fact, it is of interest to locate their depth, as due to the Ga–In grading[\n##UREF##19##\n32\n##\n] and the vertical inhomogeneity of the CdS layer, voids can have a different impact depending on their depth in the stack. Ptychographic tomography can elucidate such features. The technique uses a set of coherent diffractive scanning projections from different angles and phase‐retrieval algorithms to map in 3D the complex refractive index of the interaction volume, whose real part δ is proportional to the electron density and whose imaginary part β is proportional to the mass absorption coefficient (see Supporting Information for details). The technique is today renowned for its astounding resolution and quantitativeness.[\n##UREF##20##\n33\n##\n] Some exemplary cuts and a volume rendering of the device under investigation are shown in Figure\n##FIG##2##\n3\n##. The δ‐tomogram shows clearly the layer stack and the voids. Spatial resolution for the δ‐tomogram was assessed in the 30–40 nm range (see Figure ##SUPPL##0##S4##, Supporting Information), with the standard deviation of measured electron densities being below 2% of the average measurement (see Figure ##SUPPL##0##S6##, Supporting Information). The edge profile across voids and interfaces (Figure ##SUPPL##0##S4##, Supporting Information) decays roughly within the same distance for δ‐ and β‐tomograms, whereas uncertainty is larger in β‐tomograms and artifacts are more severe. Quantitative values of electron density extracted from δ reveal the profile distribution of Figure ##FIG##2##3E##, which locates the voids in the upper part of the absorber, ≈500 nm below the buffer‐absorber interface. The electron‐density distribution is more uniform in the lower part of the cell, except for a slight rise in the direction of the top electrode, which is due to the Ga grading[\n##UREF##21##\n34\n##\n] and is better visible in the β‐tomogram (Figure ##SUPPL##0##S5##, Supporting Information). Moreover, very small voids are visible at the bottom of the absorber (Figure ##FIG##2##3E##, slices g‐i), which are not expected to be detrimental[\n##UREF##18##\n31\n##\n] and likely correspond to the nucleation sites of CIGS grains. Finally, lower electron‐density traits can be noticed at mid‐height (Figure ##FIG##2##3E##, slices k,l), which we may identify as grain boundaries, based on the expected grain size. It is not possible to ascertain whether the contrast for these traits is provided by a gap between grains, or by Na or Rb selenides, among which RbInSe2 is likely.[\n##UREF##22##\n35\n##, ##UREF##23##\n36\n##\n] Other secondary phases, such as Cu‐or Mo‐selenides, which would be discernible by electron‐density contrast, are not present in the device.
","
The voids were segmented in an inner 2 µm‐diameter cylinder unaffected by sample preparation artifacts with a threshold‐and‐watershed algorithm as 45 labeled regions with a spatially confined material deficit (Figure\n##FIG##3##\n4A##). The segmentation parameters were set to exclude any nonporous components of the CIGS‐CdS interface region. In particular, the threshold value was set below the expected electron density of the lightest element material (CdS), taking measurement uncertainty into account. Moreover, this segmentation excludes very small voids that are within the lower part of the CIGS and voids that are below resolution (3‐voxel size) but includes sets of voxels that are partly void and partly filled by CdS, as in spots of imperfect adherence. The voids were then fit by ellipsoids to analyze their size and orientation (Figures ##SUPPL##0##S7–S9##, Supporting Information). This analysis shows that there is no evident correlation between height and volume of the voids. Figure ##FIG##3##4B## illustrates the electron‐density distribution within the void regions, which possibly relates to the CdS filling for the largest voids, but is affected also by partially filled voxels and blurred edges in the smallest material‐deficit regions. Orthogonal views and renderings of the single voids are reported in Figure ##SUPPL##0##S10## (Supporting Information). Notable examples are voids nine and 13 which are likely occluded from the top and are not reached by the CdS chemical bath deposition (cf. Figure ##FIG##3##4B##; Figure ##SUPPL##0##S10a,b##, Supporting Information). No particular shape is detected except for a few almost spherical voids (e.g., 9,19). The general picture supported by the statistics of Figure ##SUPPL##0##S7## (Supporting Information) is that of voids with sizes between 100 and 400 nm, mostly elongated in the vertical direction, and generally with low convexity.
","
Besides the voids, we segmented every single layer of the device stack (see Supporting Information for details), which resulted in the exploded view displayed in Figure ##FIG##0##1##. This allows us to verify and quantify the assumption that a 2D map of the cell is mostly representative of the absorber. The top view projection of the full stack and groups of layers is depicted in Figure\n##FIG##4##\n5A–D##. There, we note that the other layers (including highly scattering Mo) show their own structure and features and that the dispersion in the absorber (Figure ##FIG##4##5E##) is higher than in the other layers altogether (σCIGS = 8 mrad vs σrest = 3 mrad). Consequently, the stack projection predominantly follows variations of the absorber, with the correlation coefficient between the CIGS projection and the full stack projection being R = 0.95, and the correlation coefficient between the CIGS projection and that of the stack of layers above CIGS being the second largest R = −0.67. Such a large negative value can be attributed to an overall effective filling of voids within the chemical bath deposition of CdS. Besides, the absorptance of each layer above CIGS is negligible compared to that of CIGS (Figure ##SUPPL##0##S14##, Supporting Information). Within this comparison of 2D and 3D data, we also note that regardless of a certain degree of arbitrariness involved in the choice of segmentation parameters, the void segmentation for 2D and 3D data contains notable differences. Whether segmented using the Se or the ptychography map, the voids cover an area of ≈6% of the multimodal maps vs ≈25% of the 2D projection extrapolated from the tomogram (Figure ##FIG##4##5F##). A similar disproportion is observed for the density of single voids. The reasons can be ascribed to a better intrinsic sensitivity to the void edges provided by 3D data, the inaccurate grouping of distinct overlapping voids as single, and the inability to discriminate between regions of CdS and topographical variation. The improved clarity of features obtainable in 3D is also illustrated by a minimum intensity projection (Figure ##FIG##4##5G##), which is a multiplanar image of the lowest‐density features along the projection axis. Moreover, this type of projection (cf. Figure ##SUPPL##0##S11##, Supporting Information) emphasizes the numerosity of the voids, whose area density appears significantly higher than previously reported.[\n##REF##30479675##\n14\n##\n]\n
","
Along with better sensitivity to edges, the tomogram slices show the crevices between grain boundaries in the top part of the absorber (Figure ##FIG##4##5I##), which are likely filled by CdS. These features are partly visible in 2D (Figure ##SUPPL##0##S2##, Supporting Information) and in 3D show that they form a network of voids in which most but not all are connected (Figure ##FIG##4##5H##). Whereas all voids originate from the crevices of the polycrystal, the ones that form at a lower depth are more likely to be enclosed and not be reached by the CdS. Numerical simulations show that for the same surface recombination velocity, a buried void is slightly less detrimental than an interface void.[\n##REF##30479675##\n14\n##\n] As Rb tends to segregate at grain boundaries,[\n##UREF##22##\n35\n##, ##REF##33306357##\n37\n##, ##REF##30383349##\n38\n##\n] whether CdS locally forms a p‐n junction or not, determines whether downward or upward band‐bending occurs, hence causing a detrimental or beneficial effect for charge carrier transport.[\n##REF##31484943##\n39\n##\n] In our case, only a minor downward bending and a moderate recombination velocity at the interface are expected, based on more recent measurements of PL and charge carrier lifetimes from cells of the same process flow with similar performance[\n##UREF##17##\n30\n##\n] (see Figure ##SUPPL##0##S16##, Supporting Information).
","
The network of voids is of critical importance for performance as it contains pathways for the diffusion of impurities, possibly leading to interface recombination. Recent developments for high‐energy X‐ray focusing,[\n##UREF##13##\n23\n##\n] resonant ptychographic tomography, and correlative 3D microscopy[\n##UREF##24##\n40\n##, ##REF##30406204##\n41\n##\n] can further reveal the extent to which this network is filled by CdS and impurities, and will enable the unambiguous distinction between Cd and In to determine whether p–n junctions are locally formed. Whether only the large void regions or the whole network with deep small voids are considered within the absorber, the two scenarios of the distance map in Figure ##FIG##4##5J## can be drawn (see Figure ##SUPPL##0##S6C##, Supporting Information). These maps yield an average free path well below the diffusion length,[\n##UREF##21##\n34\n##\n] 0.6 versus 3 µm in the worst case, which, along with the good cell performance, suggests that most of the voids are effectively passivated and supports the model of preferential current paths within CIGS grain.[\n##UREF##25##\n42\n##\n]\n
"],"string":"[\n \"Results and Discussion\",\n \"Multimodal 2D X‐ray Imaging\",\n \"
Some of the maps obtained through different modalities are reported in Figure\\n##FIG##1##\\n2A##. Voids are identified both through fluorescence and ptychography. Among all fluorescence signals, the most reliable and statistically relevant for this purpose is arguably the Se Kα, because of the stoichiometry of CIGS including at least twice as many atoms as the other elements and because possible secondary phases are all likely to contain Se. Nonetheless, the presence of voids can also be well noted in the Cu and Ga Kα maps (Figure ##SUPPL##0##S1##, Supporting Information). Unlike fluorescence, ptychography does not have elemental sensitivity but provides quantitative maps of the electron density. Moreover, ptychography can have a resolution that is not limited by the beam size and achieves in our case the higher resolution (30 nm estimated via Fourier ring correlation,[\\n##REF##16125414##\\n27\\n##\\n] Figure ##SUPPL##0##S2##, Supporting Information) that resolves crevices between CIGS grains. Due to these features, fluorescence and ptychography provide pictures of voids in the absorber layer, respectively, before and after the deposition of the top layers. However, these are known from previous investigations to be strongly correlated,[\\n##REF##33466442##\\n17\\n##\\n] to the extent that ultimately the ptychography map and the Se map describe essentially the same features, i.e., local material deficits and the morphology of the grains in the absorber layer. The latter is responsible for a larger length scale variation that makes it more appropriate to analyze single voids with respect to their own surroundings, i.e., the sets of nearest neighboring pixels. The average size of the segmented voids is estimated with fitting ellipses or Feret diameters as ≈300 nm but exhibits considerable variation (Figure ##FIG##1##2B##). In Figure ##FIG##1##2D## we report statistics extracted from the 26 largest voids segmented from the Se map. For each void labeled in Figure ##FIG##1##2C##, Figure ##FIG##1##2D## shows the ratio of the measurements averaged over pixels within a void and the measurements averaged over its surroundings. The XRF values fall below one by definition and the deviation from one relates to the material deficit of a void or the porosity of the filling. The ptychography values are strongly correlated to XRF values, and for this reason, statistics extracted from the segmentation of voids in XRF and ptychography do not differ substantially (cf. Figure ##SUPPL##0##S3##, Supporting Information). In fact, scanning electron microscopy (SEM) cross‐sections from previous investigations have shown that the layers deposited on the absorber mostly follow the morphology of the absorber, which leads to assuming that a top‐view image of the stack essentially depicts the absorber. Later in this work, we use tomography to validate this assumption.
\",\n \"
More importantly, both the relative XBIC and XEOL measurements appear almost exclusively below one, indicating and quantifying a local performance impairment. These techniques image competing processes, i.e., they count, respectively, generated electron‐hole pairs that diffuse to the electrodes without recombining, and photons with an energy corresponding to the bandgap emitted upon radiative recombination. XBIC appears less impaired by voids than XEOL. The larger error bars from XEOL are due to the nature of the tracked process, i.e., XEOL is intrinsically a photon‐hungry technique. Performance is locally impaired up to 8% for XBIC and 60% for XEOL. On average, the effect of voids can be quantified by the meta‐analysis (see Supporting Information for details) in a 4% loss for XBIC and 20% for XEOL (Figure ##FIG##1##2E##). XEOL, in particular, degrades more than expected from the missing absorber, which might be an indication that voids and crevices are detrimental to cell voltage. This impairment can be attributed to the amount of missing material or an enhanced recombination velocity at interfaces with voids.[\\n##UREF##15##\\n28\\n##, ##UREF##16##\\n29\\n##\\n] However, it is not trivial to decouple these effects, as the performance maps are effectively blurred by the diffusion length of the carriers and by the electron shower produced by the beam (see Supporting Information). Such blurring leads to an underestimation of the XBIC dips, which explains why on most voids the relative reduction of XRF exceeds its XBIC counterpart. Despite the underestimation, the measured impairment is consistent and significant, and given the high overall performance of the device, suggests that charge transport in the functioning device is mostly sustained by alternative current paths than those below the worst performing areas. The optical and electrical performance losses correlate positively with the amount of missing material (Figure ##FIG##1##2F##) (correlation coefficients ≈0.7), whereas they do not correlate, if not weakly, with the projected area of the voids.
\",\n \"
The local performance impairment observed through measurements of current and luminescence cannot be as clearly observed when induced by a laser beam or an electron beam. In the case of lasers, the fundamentally limited resolution does not allow us to investigate the topic, as even the largest voids are smaller than the diffraction limit of photoluminescence measurements.[\\n##UREF##17##\\n30\\n##\\n] In the case of electrons, the investigation of the effect of voids is hampered by internal scattering effects. Our complementary measurements of EBIC and CL show high‐signal spikes in the proximity of voids (see Figures ##SUPPL##0##S12## and ##SUPPL##0##S13##, Supporting Information). These spikes are not an indication of enhanced electrical or optical performance due to specific material properties, but rather a measurement artifact due to secondary electrons being reabsorbed (see Supporting Information). Whereas these scattering phenomena are negligible for X‐rays, voids can have positive effects under AM1.5G illumination due to light trapping.[\\n##UREF##18##\\n31\\n##\\n] A minor performance impairment at voids is predicted by 3D numerical simulations of simple exemplary cases, although without accounting for optical effects.[\\n##REF##30479675##\\n14\\n##\\n]\\n
\",\n \"Ptychographic Nanotomography\",\n \"
Whereas the 2D top‐view of the sample highlights lateral differences in the operational device, it cannot provide depth information about the disclosed features. Effectively, they can be vertical projections of multiple features, in the same or different layers. In fact, it is of interest to locate their depth, as due to the Ga–In grading[\\n##UREF##19##\\n32\\n##\\n] and the vertical inhomogeneity of the CdS layer, voids can have a different impact depending on their depth in the stack. Ptychographic tomography can elucidate such features. The technique uses a set of coherent diffractive scanning projections from different angles and phase‐retrieval algorithms to map in 3D the complex refractive index of the interaction volume, whose real part δ is proportional to the electron density and whose imaginary part β is proportional to the mass absorption coefficient (see Supporting Information for details). The technique is today renowned for its astounding resolution and quantitativeness.[\\n##UREF##20##\\n33\\n##\\n] Some exemplary cuts and a volume rendering of the device under investigation are shown in Figure\\n##FIG##2##\\n3\\n##. The δ‐tomogram shows clearly the layer stack and the voids. Spatial resolution for the δ‐tomogram was assessed in the 30–40 nm range (see Figure ##SUPPL##0##S4##, Supporting Information), with the standard deviation of measured electron densities being below 2% of the average measurement (see Figure ##SUPPL##0##S6##, Supporting Information). The edge profile across voids and interfaces (Figure ##SUPPL##0##S4##, Supporting Information) decays roughly within the same distance for δ‐ and β‐tomograms, whereas uncertainty is larger in β‐tomograms and artifacts are more severe. Quantitative values of electron density extracted from δ reveal the profile distribution of Figure ##FIG##2##3E##, which locates the voids in the upper part of the absorber, ≈500 nm below the buffer‐absorber interface. The electron‐density distribution is more uniform in the lower part of the cell, except for a slight rise in the direction of the top electrode, which is due to the Ga grading[\\n##UREF##21##\\n34\\n##\\n] and is better visible in the β‐tomogram (Figure ##SUPPL##0##S5##, Supporting Information). Moreover, very small voids are visible at the bottom of the absorber (Figure ##FIG##2##3E##, slices g‐i), which are not expected to be detrimental[\\n##UREF##18##\\n31\\n##\\n] and likely correspond to the nucleation sites of CIGS grains. Finally, lower electron‐density traits can be noticed at mid‐height (Figure ##FIG##2##3E##, slices k,l), which we may identify as grain boundaries, based on the expected grain size. It is not possible to ascertain whether the contrast for these traits is provided by a gap between grains, or by Na or Rb selenides, among which RbInSe2 is likely.[\\n##UREF##22##\\n35\\n##, ##UREF##23##\\n36\\n##\\n] Other secondary phases, such as Cu‐or Mo‐selenides, which would be discernible by electron‐density contrast, are not present in the device.
\",\n \"
The voids were segmented in an inner 2 µm‐diameter cylinder unaffected by sample preparation artifacts with a threshold‐and‐watershed algorithm as 45 labeled regions with a spatially confined material deficit (Figure\\n##FIG##3##\\n4A##). The segmentation parameters were set to exclude any nonporous components of the CIGS‐CdS interface region. In particular, the threshold value was set below the expected electron density of the lightest element material (CdS), taking measurement uncertainty into account. Moreover, this segmentation excludes very small voids that are within the lower part of the CIGS and voids that are below resolution (3‐voxel size) but includes sets of voxels that are partly void and partly filled by CdS, as in spots of imperfect adherence. The voids were then fit by ellipsoids to analyze their size and orientation (Figures ##SUPPL##0##S7–S9##, Supporting Information). This analysis shows that there is no evident correlation between height and volume of the voids. Figure ##FIG##3##4B## illustrates the electron‐density distribution within the void regions, which possibly relates to the CdS filling for the largest voids, but is affected also by partially filled voxels and blurred edges in the smallest material‐deficit regions. Orthogonal views and renderings of the single voids are reported in Figure ##SUPPL##0##S10## (Supporting Information). Notable examples are voids nine and 13 which are likely occluded from the top and are not reached by the CdS chemical bath deposition (cf. Figure ##FIG##3##4B##; Figure ##SUPPL##0##S10a,b##, Supporting Information). No particular shape is detected except for a few almost spherical voids (e.g., 9,19). The general picture supported by the statistics of Figure ##SUPPL##0##S7## (Supporting Information) is that of voids with sizes between 100 and 400 nm, mostly elongated in the vertical direction, and generally with low convexity.
\",\n \"
Besides the voids, we segmented every single layer of the device stack (see Supporting Information for details), which resulted in the exploded view displayed in Figure ##FIG##0##1##. This allows us to verify and quantify the assumption that a 2D map of the cell is mostly representative of the absorber. The top view projection of the full stack and groups of layers is depicted in Figure\\n##FIG##4##\\n5A–D##. There, we note that the other layers (including highly scattering Mo) show their own structure and features and that the dispersion in the absorber (Figure ##FIG##4##5E##) is higher than in the other layers altogether (σCIGS = 8 mrad vs σrest = 3 mrad). Consequently, the stack projection predominantly follows variations of the absorber, with the correlation coefficient between the CIGS projection and the full stack projection being R = 0.95, and the correlation coefficient between the CIGS projection and that of the stack of layers above CIGS being the second largest R = −0.67. Such a large negative value can be attributed to an overall effective filling of voids within the chemical bath deposition of CdS. Besides, the absorptance of each layer above CIGS is negligible compared to that of CIGS (Figure ##SUPPL##0##S14##, Supporting Information). Within this comparison of 2D and 3D data, we also note that regardless of a certain degree of arbitrariness involved in the choice of segmentation parameters, the void segmentation for 2D and 3D data contains notable differences. Whether segmented using the Se or the ptychography map, the voids cover an area of ≈6% of the multimodal maps vs ≈25% of the 2D projection extrapolated from the tomogram (Figure ##FIG##4##5F##). A similar disproportion is observed for the density of single voids. The reasons can be ascribed to a better intrinsic sensitivity to the void edges provided by 3D data, the inaccurate grouping of distinct overlapping voids as single, and the inability to discriminate between regions of CdS and topographical variation. The improved clarity of features obtainable in 3D is also illustrated by a minimum intensity projection (Figure ##FIG##4##5G##), which is a multiplanar image of the lowest‐density features along the projection axis. Moreover, this type of projection (cf. Figure ##SUPPL##0##S11##, Supporting Information) emphasizes the numerosity of the voids, whose area density appears significantly higher than previously reported.[\\n##REF##30479675##\\n14\\n##\\n]\\n
\",\n \"
Along with better sensitivity to edges, the tomogram slices show the crevices between grain boundaries in the top part of the absorber (Figure ##FIG##4##5I##), which are likely filled by CdS. These features are partly visible in 2D (Figure ##SUPPL##0##S2##, Supporting Information) and in 3D show that they form a network of voids in which most but not all are connected (Figure ##FIG##4##5H##). Whereas all voids originate from the crevices of the polycrystal, the ones that form at a lower depth are more likely to be enclosed and not be reached by the CdS. Numerical simulations show that for the same surface recombination velocity, a buried void is slightly less detrimental than an interface void.[\\n##REF##30479675##\\n14\\n##\\n] As Rb tends to segregate at grain boundaries,[\\n##UREF##22##\\n35\\n##, ##REF##33306357##\\n37\\n##, ##REF##30383349##\\n38\\n##\\n] whether CdS locally forms a p‐n junction or not, determines whether downward or upward band‐bending occurs, hence causing a detrimental or beneficial effect for charge carrier transport.[\\n##REF##31484943##\\n39\\n##\\n] In our case, only a minor downward bending and a moderate recombination velocity at the interface are expected, based on more recent measurements of PL and charge carrier lifetimes from cells of the same process flow with similar performance[\\n##UREF##17##\\n30\\n##\\n] (see Figure ##SUPPL##0##S16##, Supporting Information).
\",\n \"
The network of voids is of critical importance for performance as it contains pathways for the diffusion of impurities, possibly leading to interface recombination. Recent developments for high‐energy X‐ray focusing,[\\n##UREF##13##\\n23\\n##\\n] resonant ptychographic tomography, and correlative 3D microscopy[\\n##UREF##24##\\n40\\n##, ##REF##30406204##\\n41\\n##\\n] can further reveal the extent to which this network is filled by CdS and impurities, and will enable the unambiguous distinction between Cd and In to determine whether p–n junctions are locally formed. Whether only the large void regions or the whole network with deep small voids are considered within the absorber, the two scenarios of the distance map in Figure ##FIG##4##5J## can be drawn (see Figure ##SUPPL##0##S6C##, Supporting Information). These maps yield an average free path well below the diffusion length,[\\n##UREF##21##\\n34\\n##\\n] 0.6 versus 3 µm in the worst case, which, along with the good cell performance, suggests that most of the voids are effectively passivated and supports the model of preferential current paths within CIGS grain.[\\n##UREF##25##\\n42\\n##\\n]\\n
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","Multimodal 2D X‐ray Imaging","
Some of the maps obtained through different modalities are reported in Figure\n##FIG##1##\n2A##. Voids are identified both through fluorescence and ptychography. Among all fluorescence signals, the most reliable and statistically relevant for this purpose is arguably the Se Kα, because of the stoichiometry of CIGS including at least twice as many atoms as the other elements and because possible secondary phases are all likely to contain Se. Nonetheless, the presence of voids can also be well noted in the Cu and Ga Kα maps (Figure ##SUPPL##0##S1##, Supporting Information). Unlike fluorescence, ptychography does not have elemental sensitivity but provides quantitative maps of the electron density. Moreover, ptychography can have a resolution that is not limited by the beam size and achieves in our case the higher resolution (30 nm estimated via Fourier ring correlation,[\n##REF##16125414##\n27\n##\n] Figure ##SUPPL##0##S2##, Supporting Information) that resolves crevices between CIGS grains. Due to these features, fluorescence and ptychography provide pictures of voids in the absorber layer, respectively, before and after the deposition of the top layers. However, these are known from previous investigations to be strongly correlated,[\n##REF##33466442##\n17\n##\n] to the extent that ultimately the ptychography map and the Se map describe essentially the same features, i.e., local material deficits and the morphology of the grains in the absorber layer. The latter is responsible for a larger length scale variation that makes it more appropriate to analyze single voids with respect to their own surroundings, i.e., the sets of nearest neighboring pixels. The average size of the segmented voids is estimated with fitting ellipses or Feret diameters as ≈300 nm but exhibits considerable variation (Figure ##FIG##1##2B##). In Figure ##FIG##1##2D## we report statistics extracted from the 26 largest voids segmented from the Se map. For each void labeled in Figure ##FIG##1##2C##, Figure ##FIG##1##2D## shows the ratio of the measurements averaged over pixels within a void and the measurements averaged over its surroundings. The XRF values fall below one by definition and the deviation from one relates to the material deficit of a void or the porosity of the filling. The ptychography values are strongly correlated to XRF values, and for this reason, statistics extracted from the segmentation of voids in XRF and ptychography do not differ substantially (cf. Figure ##SUPPL##0##S3##, Supporting Information). In fact, scanning electron microscopy (SEM) cross‐sections from previous investigations have shown that the layers deposited on the absorber mostly follow the morphology of the absorber, which leads to assuming that a top‐view image of the stack essentially depicts the absorber. Later in this work, we use tomography to validate this assumption.
","
More importantly, both the relative XBIC and XEOL measurements appear almost exclusively below one, indicating and quantifying a local performance impairment. These techniques image competing processes, i.e., they count, respectively, generated electron‐hole pairs that diffuse to the electrodes without recombining, and photons with an energy corresponding to the bandgap emitted upon radiative recombination. XBIC appears less impaired by voids than XEOL. The larger error bars from XEOL are due to the nature of the tracked process, i.e., XEOL is intrinsically a photon‐hungry technique. Performance is locally impaired up to 8% for XBIC and 60% for XEOL. On average, the effect of voids can be quantified by the meta‐analysis (see Supporting Information for details) in a 4% loss for XBIC and 20% for XEOL (Figure ##FIG##1##2E##). XEOL, in particular, degrades more than expected from the missing absorber, which might be an indication that voids and crevices are detrimental to cell voltage. This impairment can be attributed to the amount of missing material or an enhanced recombination velocity at interfaces with voids.[\n##UREF##15##\n28\n##, ##UREF##16##\n29\n##\n] However, it is not trivial to decouple these effects, as the performance maps are effectively blurred by the diffusion length of the carriers and by the electron shower produced by the beam (see Supporting Information). Such blurring leads to an underestimation of the XBIC dips, which explains why on most voids the relative reduction of XRF exceeds its XBIC counterpart. Despite the underestimation, the measured impairment is consistent and significant, and given the high overall performance of the device, suggests that charge transport in the functioning device is mostly sustained by alternative current paths than those below the worst performing areas. The optical and electrical performance losses correlate positively with the amount of missing material (Figure ##FIG##1##2F##) (correlation coefficients ≈0.7), whereas they do not correlate, if not weakly, with the projected area of the voids.
","
The local performance impairment observed through measurements of current and luminescence cannot be as clearly observed when induced by a laser beam or an electron beam. In the case of lasers, the fundamentally limited resolution does not allow us to investigate the topic, as even the largest voids are smaller than the diffraction limit of photoluminescence measurements.[\n##UREF##17##\n30\n##\n] In the case of electrons, the investigation of the effect of voids is hampered by internal scattering effects. Our complementary measurements of EBIC and CL show high‐signal spikes in the proximity of voids (see Figures ##SUPPL##0##S12## and ##SUPPL##0##S13##, Supporting Information). These spikes are not an indication of enhanced electrical or optical performance due to specific material properties, but rather a measurement artifact due to secondary electrons being reabsorbed (see Supporting Information). Whereas these scattering phenomena are negligible for X‐rays, voids can have positive effects under AM1.5G illumination due to light trapping.[\n##UREF##18##\n31\n##\n] A minor performance impairment at voids is predicted by 3D numerical simulations of simple exemplary cases, although without accounting for optical effects.[\n##REF##30479675##\n14\n##\n]\n
","Ptychographic Nanotomography","
Whereas the 2D top‐view of the sample highlights lateral differences in the operational device, it cannot provide depth information about the disclosed features. Effectively, they can be vertical projections of multiple features, in the same or different layers. In fact, it is of interest to locate their depth, as due to the Ga–In grading[\n##UREF##19##\n32\n##\n] and the vertical inhomogeneity of the CdS layer, voids can have a different impact depending on their depth in the stack. Ptychographic tomography can elucidate such features. The technique uses a set of coherent diffractive scanning projections from different angles and phase‐retrieval algorithms to map in 3D the complex refractive index of the interaction volume, whose real part δ is proportional to the electron density and whose imaginary part β is proportional to the mass absorption coefficient (see Supporting Information for details). The technique is today renowned for its astounding resolution and quantitativeness.[\n##UREF##20##\n33\n##\n] Some exemplary cuts and a volume rendering of the device under investigation are shown in Figure\n##FIG##2##\n3\n##. The δ‐tomogram shows clearly the layer stack and the voids. Spatial resolution for the δ‐tomogram was assessed in the 30–40 nm range (see Figure ##SUPPL##0##S4##, Supporting Information), with the standard deviation of measured electron densities being below 2% of the average measurement (see Figure ##SUPPL##0##S6##, Supporting Information). The edge profile across voids and interfaces (Figure ##SUPPL##0##S4##, Supporting Information) decays roughly within the same distance for δ‐ and β‐tomograms, whereas uncertainty is larger in β‐tomograms and artifacts are more severe. Quantitative values of electron density extracted from δ reveal the profile distribution of Figure ##FIG##2##3E##, which locates the voids in the upper part of the absorber, ≈500 nm below the buffer‐absorber interface. The electron‐density distribution is more uniform in the lower part of the cell, except for a slight rise in the direction of the top electrode, which is due to the Ga grading[\n##UREF##21##\n34\n##\n] and is better visible in the β‐tomogram (Figure ##SUPPL##0##S5##, Supporting Information). Moreover, very small voids are visible at the bottom of the absorber (Figure ##FIG##2##3E##, slices g‐i), which are not expected to be detrimental[\n##UREF##18##\n31\n##\n] and likely correspond to the nucleation sites of CIGS grains. Finally, lower electron‐density traits can be noticed at mid‐height (Figure ##FIG##2##3E##, slices k,l), which we may identify as grain boundaries, based on the expected grain size. It is not possible to ascertain whether the contrast for these traits is provided by a gap between grains, or by Na or Rb selenides, among which RbInSe2 is likely.[\n##UREF##22##\n35\n##, ##UREF##23##\n36\n##\n] Other secondary phases, such as Cu‐or Mo‐selenides, which would be discernible by electron‐density contrast, are not present in the device.
","
The voids were segmented in an inner 2 µm‐diameter cylinder unaffected by sample preparation artifacts with a threshold‐and‐watershed algorithm as 45 labeled regions with a spatially confined material deficit (Figure\n##FIG##3##\n4A##). The segmentation parameters were set to exclude any nonporous components of the CIGS‐CdS interface region. In particular, the threshold value was set below the expected electron density of the lightest element material (CdS), taking measurement uncertainty into account. Moreover, this segmentation excludes very small voids that are within the lower part of the CIGS and voids that are below resolution (3‐voxel size) but includes sets of voxels that are partly void and partly filled by CdS, as in spots of imperfect adherence. The voids were then fit by ellipsoids to analyze their size and orientation (Figures ##SUPPL##0##S7–S9##, Supporting Information). This analysis shows that there is no evident correlation between height and volume of the voids. Figure ##FIG##3##4B## illustrates the electron‐density distribution within the void regions, which possibly relates to the CdS filling for the largest voids, but is affected also by partially filled voxels and blurred edges in the smallest material‐deficit regions. Orthogonal views and renderings of the single voids are reported in Figure ##SUPPL##0##S10## (Supporting Information). Notable examples are voids nine and 13 which are likely occluded from the top and are not reached by the CdS chemical bath deposition (cf. Figure ##FIG##3##4B##; Figure ##SUPPL##0##S10a,b##, Supporting Information). No particular shape is detected except for a few almost spherical voids (e.g., 9,19). The general picture supported by the statistics of Figure ##SUPPL##0##S7## (Supporting Information) is that of voids with sizes between 100 and 400 nm, mostly elongated in the vertical direction, and generally with low convexity.
","
Besides the voids, we segmented every single layer of the device stack (see Supporting Information for details), which resulted in the exploded view displayed in Figure ##FIG##0##1##. This allows us to verify and quantify the assumption that a 2D map of the cell is mostly representative of the absorber. The top view projection of the full stack and groups of layers is depicted in Figure\n##FIG##4##\n5A–D##. There, we note that the other layers (including highly scattering Mo) show their own structure and features and that the dispersion in the absorber (Figure ##FIG##4##5E##) is higher than in the other layers altogether (σCIGS = 8 mrad vs σrest = 3 mrad). Consequently, the stack projection predominantly follows variations of the absorber, with the correlation coefficient between the CIGS projection and the full stack projection being R = 0.95, and the correlation coefficient between the CIGS projection and that of the stack of layers above CIGS being the second largest R = −0.67. Such a large negative value can be attributed to an overall effective filling of voids within the chemical bath deposition of CdS. Besides, the absorptance of each layer above CIGS is negligible compared to that of CIGS (Figure ##SUPPL##0##S14##, Supporting Information). Within this comparison of 2D and 3D data, we also note that regardless of a certain degree of arbitrariness involved in the choice of segmentation parameters, the void segmentation for 2D and 3D data contains notable differences. Whether segmented using the Se or the ptychography map, the voids cover an area of ≈6% of the multimodal maps vs ≈25% of the 2D projection extrapolated from the tomogram (Figure ##FIG##4##5F##). A similar disproportion is observed for the density of single voids. The reasons can be ascribed to a better intrinsic sensitivity to the void edges provided by 3D data, the inaccurate grouping of distinct overlapping voids as single, and the inability to discriminate between regions of CdS and topographical variation. The improved clarity of features obtainable in 3D is also illustrated by a minimum intensity projection (Figure ##FIG##4##5G##), which is a multiplanar image of the lowest‐density features along the projection axis. Moreover, this type of projection (cf. Figure ##SUPPL##0##S11##, Supporting Information) emphasizes the numerosity of the voids, whose area density appears significantly higher than previously reported.[\n##REF##30479675##\n14\n##\n]\n
","
Along with better sensitivity to edges, the tomogram slices show the crevices between grain boundaries in the top part of the absorber (Figure ##FIG##4##5I##), which are likely filled by CdS. These features are partly visible in 2D (Figure ##SUPPL##0##S2##, Supporting Information) and in 3D show that they form a network of voids in which most but not all are connected (Figure ##FIG##4##5H##). Whereas all voids originate from the crevices of the polycrystal, the ones that form at a lower depth are more likely to be enclosed and not be reached by the CdS. Numerical simulations show that for the same surface recombination velocity, a buried void is slightly less detrimental than an interface void.[\n##REF##30479675##\n14\n##\n] As Rb tends to segregate at grain boundaries,[\n##UREF##22##\n35\n##, ##REF##33306357##\n37\n##, ##REF##30383349##\n38\n##\n] whether CdS locally forms a p‐n junction or not, determines whether downward or upward band‐bending occurs, hence causing a detrimental or beneficial effect for charge carrier transport.[\n##REF##31484943##\n39\n##\n] In our case, only a minor downward bending and a moderate recombination velocity at the interface are expected, based on more recent measurements of PL and charge carrier lifetimes from cells of the same process flow with similar performance[\n##UREF##17##\n30\n##\n] (see Figure ##SUPPL##0##S16##, Supporting Information).
","
The network of voids is of critical importance for performance as it contains pathways for the diffusion of impurities, possibly leading to interface recombination. Recent developments for high‐energy X‐ray focusing,[\n##UREF##13##\n23\n##\n] resonant ptychographic tomography, and correlative 3D microscopy[\n##UREF##24##\n40\n##, ##REF##30406204##\n41\n##\n] can further reveal the extent to which this network is filled by CdS and impurities, and will enable the unambiguous distinction between Cd and In to determine whether p–n junctions are locally formed. Whether only the large void regions or the whole network with deep small voids are considered within the absorber, the two scenarios of the distance map in Figure ##FIG##4##5J## can be drawn (see Figure ##SUPPL##0##S6C##, Supporting Information). These maps yield an average free path well below the diffusion length,[\n##UREF##21##\n34\n##\n] 0.6 versus 3 µm in the worst case, which, along with the good cell performance, suggests that most of the voids are effectively passivated and supports the model of preferential current paths within CIGS grain.[\n##UREF##25##\n42\n##\n]\n
"],"string":"[\n \"Results and Discussion\",\n \"Multimodal 2D X‐ray Imaging\",\n \"
Some of the maps obtained through different modalities are reported in Figure\\n##FIG##1##\\n2A##. Voids are identified both through fluorescence and ptychography. Among all fluorescence signals, the most reliable and statistically relevant for this purpose is arguably the Se Kα, because of the stoichiometry of CIGS including at least twice as many atoms as the other elements and because possible secondary phases are all likely to contain Se. Nonetheless, the presence of voids can also be well noted in the Cu and Ga Kα maps (Figure ##SUPPL##0##S1##, Supporting Information). Unlike fluorescence, ptychography does not have elemental sensitivity but provides quantitative maps of the electron density. Moreover, ptychography can have a resolution that is not limited by the beam size and achieves in our case the higher resolution (30 nm estimated via Fourier ring correlation,[\\n##REF##16125414##\\n27\\n##\\n] Figure ##SUPPL##0##S2##, Supporting Information) that resolves crevices between CIGS grains. Due to these features, fluorescence and ptychography provide pictures of voids in the absorber layer, respectively, before and after the deposition of the top layers. However, these are known from previous investigations to be strongly correlated,[\\n##REF##33466442##\\n17\\n##\\n] to the extent that ultimately the ptychography map and the Se map describe essentially the same features, i.e., local material deficits and the morphology of the grains in the absorber layer. The latter is responsible for a larger length scale variation that makes it more appropriate to analyze single voids with respect to their own surroundings, i.e., the sets of nearest neighboring pixels. The average size of the segmented voids is estimated with fitting ellipses or Feret diameters as ≈300 nm but exhibits considerable variation (Figure ##FIG##1##2B##). In Figure ##FIG##1##2D## we report statistics extracted from the 26 largest voids segmented from the Se map. For each void labeled in Figure ##FIG##1##2C##, Figure ##FIG##1##2D## shows the ratio of the measurements averaged over pixels within a void and the measurements averaged over its surroundings. The XRF values fall below one by definition and the deviation from one relates to the material deficit of a void or the porosity of the filling. The ptychography values are strongly correlated to XRF values, and for this reason, statistics extracted from the segmentation of voids in XRF and ptychography do not differ substantially (cf. Figure ##SUPPL##0##S3##, Supporting Information). In fact, scanning electron microscopy (SEM) cross‐sections from previous investigations have shown that the layers deposited on the absorber mostly follow the morphology of the absorber, which leads to assuming that a top‐view image of the stack essentially depicts the absorber. Later in this work, we use tomography to validate this assumption.
\",\n \"
More importantly, both the relative XBIC and XEOL measurements appear almost exclusively below one, indicating and quantifying a local performance impairment. These techniques image competing processes, i.e., they count, respectively, generated electron‐hole pairs that diffuse to the electrodes without recombining, and photons with an energy corresponding to the bandgap emitted upon radiative recombination. XBIC appears less impaired by voids than XEOL. The larger error bars from XEOL are due to the nature of the tracked process, i.e., XEOL is intrinsically a photon‐hungry technique. Performance is locally impaired up to 8% for XBIC and 60% for XEOL. On average, the effect of voids can be quantified by the meta‐analysis (see Supporting Information for details) in a 4% loss for XBIC and 20% for XEOL (Figure ##FIG##1##2E##). XEOL, in particular, degrades more than expected from the missing absorber, which might be an indication that voids and crevices are detrimental to cell voltage. This impairment can be attributed to the amount of missing material or an enhanced recombination velocity at interfaces with voids.[\\n##UREF##15##\\n28\\n##, ##UREF##16##\\n29\\n##\\n] However, it is not trivial to decouple these effects, as the performance maps are effectively blurred by the diffusion length of the carriers and by the electron shower produced by the beam (see Supporting Information). Such blurring leads to an underestimation of the XBIC dips, which explains why on most voids the relative reduction of XRF exceeds its XBIC counterpart. Despite the underestimation, the measured impairment is consistent and significant, and given the high overall performance of the device, suggests that charge transport in the functioning device is mostly sustained by alternative current paths than those below the worst performing areas. The optical and electrical performance losses correlate positively with the amount of missing material (Figure ##FIG##1##2F##) (correlation coefficients ≈0.7), whereas they do not correlate, if not weakly, with the projected area of the voids.
\",\n \"
The local performance impairment observed through measurements of current and luminescence cannot be as clearly observed when induced by a laser beam or an electron beam. In the case of lasers, the fundamentally limited resolution does not allow us to investigate the topic, as even the largest voids are smaller than the diffraction limit of photoluminescence measurements.[\\n##UREF##17##\\n30\\n##\\n] In the case of electrons, the investigation of the effect of voids is hampered by internal scattering effects. Our complementary measurements of EBIC and CL show high‐signal spikes in the proximity of voids (see Figures ##SUPPL##0##S12## and ##SUPPL##0##S13##, Supporting Information). These spikes are not an indication of enhanced electrical or optical performance due to specific material properties, but rather a measurement artifact due to secondary electrons being reabsorbed (see Supporting Information). Whereas these scattering phenomena are negligible for X‐rays, voids can have positive effects under AM1.5G illumination due to light trapping.[\\n##UREF##18##\\n31\\n##\\n] A minor performance impairment at voids is predicted by 3D numerical simulations of simple exemplary cases, although without accounting for optical effects.[\\n##REF##30479675##\\n14\\n##\\n]\\n
\",\n \"Ptychographic Nanotomography\",\n \"
Whereas the 2D top‐view of the sample highlights lateral differences in the operational device, it cannot provide depth information about the disclosed features. Effectively, they can be vertical projections of multiple features, in the same or different layers. In fact, it is of interest to locate their depth, as due to the Ga–In grading[\\n##UREF##19##\\n32\\n##\\n] and the vertical inhomogeneity of the CdS layer, voids can have a different impact depending on their depth in the stack. Ptychographic tomography can elucidate such features. The technique uses a set of coherent diffractive scanning projections from different angles and phase‐retrieval algorithms to map in 3D the complex refractive index of the interaction volume, whose real part δ is proportional to the electron density and whose imaginary part β is proportional to the mass absorption coefficient (see Supporting Information for details). The technique is today renowned for its astounding resolution and quantitativeness.[\\n##UREF##20##\\n33\\n##\\n] Some exemplary cuts and a volume rendering of the device under investigation are shown in Figure\\n##FIG##2##\\n3\\n##. The δ‐tomogram shows clearly the layer stack and the voids. Spatial resolution for the δ‐tomogram was assessed in the 30–40 nm range (see Figure ##SUPPL##0##S4##, Supporting Information), with the standard deviation of measured electron densities being below 2% of the average measurement (see Figure ##SUPPL##0##S6##, Supporting Information). The edge profile across voids and interfaces (Figure ##SUPPL##0##S4##, Supporting Information) decays roughly within the same distance for δ‐ and β‐tomograms, whereas uncertainty is larger in β‐tomograms and artifacts are more severe. Quantitative values of electron density extracted from δ reveal the profile distribution of Figure ##FIG##2##3E##, which locates the voids in the upper part of the absorber, ≈500 nm below the buffer‐absorber interface. The electron‐density distribution is more uniform in the lower part of the cell, except for a slight rise in the direction of the top electrode, which is due to the Ga grading[\\n##UREF##21##\\n34\\n##\\n] and is better visible in the β‐tomogram (Figure ##SUPPL##0##S5##, Supporting Information). Moreover, very small voids are visible at the bottom of the absorber (Figure ##FIG##2##3E##, slices g‐i), which are not expected to be detrimental[\\n##UREF##18##\\n31\\n##\\n] and likely correspond to the nucleation sites of CIGS grains. Finally, lower electron‐density traits can be noticed at mid‐height (Figure ##FIG##2##3E##, slices k,l), which we may identify as grain boundaries, based on the expected grain size. It is not possible to ascertain whether the contrast for these traits is provided by a gap between grains, or by Na or Rb selenides, among which RbInSe2 is likely.[\\n##UREF##22##\\n35\\n##, ##UREF##23##\\n36\\n##\\n] Other secondary phases, such as Cu‐or Mo‐selenides, which would be discernible by electron‐density contrast, are not present in the device.
\",\n \"
The voids were segmented in an inner 2 µm‐diameter cylinder unaffected by sample preparation artifacts with a threshold‐and‐watershed algorithm as 45 labeled regions with a spatially confined material deficit (Figure\\n##FIG##3##\\n4A##). The segmentation parameters were set to exclude any nonporous components of the CIGS‐CdS interface region. In particular, the threshold value was set below the expected electron density of the lightest element material (CdS), taking measurement uncertainty into account. Moreover, this segmentation excludes very small voids that are within the lower part of the CIGS and voids that are below resolution (3‐voxel size) but includes sets of voxels that are partly void and partly filled by CdS, as in spots of imperfect adherence. The voids were then fit by ellipsoids to analyze their size and orientation (Figures ##SUPPL##0##S7–S9##, Supporting Information). This analysis shows that there is no evident correlation between height and volume of the voids. Figure ##FIG##3##4B## illustrates the electron‐density distribution within the void regions, which possibly relates to the CdS filling for the largest voids, but is affected also by partially filled voxels and blurred edges in the smallest material‐deficit regions. Orthogonal views and renderings of the single voids are reported in Figure ##SUPPL##0##S10## (Supporting Information). Notable examples are voids nine and 13 which are likely occluded from the top and are not reached by the CdS chemical bath deposition (cf. Figure ##FIG##3##4B##; Figure ##SUPPL##0##S10a,b##, Supporting Information). No particular shape is detected except for a few almost spherical voids (e.g., 9,19). The general picture supported by the statistics of Figure ##SUPPL##0##S7## (Supporting Information) is that of voids with sizes between 100 and 400 nm, mostly elongated in the vertical direction, and generally with low convexity.
\",\n \"
Besides the voids, we segmented every single layer of the device stack (see Supporting Information for details), which resulted in the exploded view displayed in Figure ##FIG##0##1##. This allows us to verify and quantify the assumption that a 2D map of the cell is mostly representative of the absorber. The top view projection of the full stack and groups of layers is depicted in Figure\\n##FIG##4##\\n5A–D##. There, we note that the other layers (including highly scattering Mo) show their own structure and features and that the dispersion in the absorber (Figure ##FIG##4##5E##) is higher than in the other layers altogether (σCIGS = 8 mrad vs σrest = 3 mrad). Consequently, the stack projection predominantly follows variations of the absorber, with the correlation coefficient between the CIGS projection and the full stack projection being R = 0.95, and the correlation coefficient between the CIGS projection and that of the stack of layers above CIGS being the second largest R = −0.67. Such a large negative value can be attributed to an overall effective filling of voids within the chemical bath deposition of CdS. Besides, the absorptance of each layer above CIGS is negligible compared to that of CIGS (Figure ##SUPPL##0##S14##, Supporting Information). Within this comparison of 2D and 3D data, we also note that regardless of a certain degree of arbitrariness involved in the choice of segmentation parameters, the void segmentation for 2D and 3D data contains notable differences. Whether segmented using the Se or the ptychography map, the voids cover an area of ≈6% of the multimodal maps vs ≈25% of the 2D projection extrapolated from the tomogram (Figure ##FIG##4##5F##). A similar disproportion is observed for the density of single voids. The reasons can be ascribed to a better intrinsic sensitivity to the void edges provided by 3D data, the inaccurate grouping of distinct overlapping voids as single, and the inability to discriminate between regions of CdS and topographical variation. The improved clarity of features obtainable in 3D is also illustrated by a minimum intensity projection (Figure ##FIG##4##5G##), which is a multiplanar image of the lowest‐density features along the projection axis. Moreover, this type of projection (cf. Figure ##SUPPL##0##S11##, Supporting Information) emphasizes the numerosity of the voids, whose area density appears significantly higher than previously reported.[\\n##REF##30479675##\\n14\\n##\\n]\\n
\",\n \"
Along with better sensitivity to edges, the tomogram slices show the crevices between grain boundaries in the top part of the absorber (Figure ##FIG##4##5I##), which are likely filled by CdS. These features are partly visible in 2D (Figure ##SUPPL##0##S2##, Supporting Information) and in 3D show that they form a network of voids in which most but not all are connected (Figure ##FIG##4##5H##). Whereas all voids originate from the crevices of the polycrystal, the ones that form at a lower depth are more likely to be enclosed and not be reached by the CdS. Numerical simulations show that for the same surface recombination velocity, a buried void is slightly less detrimental than an interface void.[\\n##REF##30479675##\\n14\\n##\\n] As Rb tends to segregate at grain boundaries,[\\n##UREF##22##\\n35\\n##, ##REF##33306357##\\n37\\n##, ##REF##30383349##\\n38\\n##\\n] whether CdS locally forms a p‐n junction or not, determines whether downward or upward band‐bending occurs, hence causing a detrimental or beneficial effect for charge carrier transport.[\\n##REF##31484943##\\n39\\n##\\n] In our case, only a minor downward bending and a moderate recombination velocity at the interface are expected, based on more recent measurements of PL and charge carrier lifetimes from cells of the same process flow with similar performance[\\n##UREF##17##\\n30\\n##\\n] (see Figure ##SUPPL##0##S16##, Supporting Information).
\",\n \"
The network of voids is of critical importance for performance as it contains pathways for the diffusion of impurities, possibly leading to interface recombination. Recent developments for high‐energy X‐ray focusing,[\\n##UREF##13##\\n23\\n##\\n] resonant ptychographic tomography, and correlative 3D microscopy[\\n##UREF##24##\\n40\\n##, ##REF##30406204##\\n41\\n##\\n] can further reveal the extent to which this network is filled by CdS and impurities, and will enable the unambiguous distinction between Cd and In to determine whether p–n junctions are locally formed. Whether only the large void regions or the whole network with deep small voids are considered within the absorber, the two scenarios of the distance map in Figure ##FIG##4##5J## can be drawn (see Figure ##SUPPL##0##S6C##, Supporting Information). These maps yield an average free path well below the diffusion length,[\\n##UREF##21##\\n34\\n##\\n] 0.6 versus 3 µm in the worst case, which, along with the good cell performance, suggests that most of the voids are effectively passivated and supports the model of preferential current paths within CIGS grain.[\\n##UREF##25##\\n42\\n##\\n]\\n
\"\n]"},"conclusion":{"kind":"list like","value":["Conclusion and Outlook","
Altogether, we have shown in this study the 3D nature of structural defects in thin‐film CIGS solar cells and we identified local performance deficits attributable to voids. Although possibly detrimental at a local level, the high density of voids highlighted by tomography suggests that their effect cannot be dramatic at the device level, given the high efficiency of the cell. We point out that such a complex system is not easily modeled and available finite element simulation results are not directly comparable with our measurements.[\n##REF##30479675##\n14\n##\n] Our measurements with absolute electron densities quantified at the nanoscale enable the development of adequate models simulating structural and electronic defects.[\n##REF##35822496##\n19\n##\n] This investigation does not alter the void size or shape as FIB‐SEM might do, however, it does require a state‐of‐the‐art X‐ray microscopy beamline and involves a delicate sample preparation. Future experiments should aim to avoid it, probe larger areas, and explore the sample in a multi‐scale approach. Such goals may be achieved with a laminography setup.[\n##UREF##26##\n43\n##\n] Regarding the availability of beamtime, accepting a loss of resolution, similar studies can be extended to the best lab‐CT instruments. Other thin film solar cells, perovskites before all, in single‐junction or tandem configuration, demand studies of this kind to elucidate fabrication defects and improve cost‐efficiency.
","
In general, our study highlights the sensitivity at the nanoscale to a multitude of physical, chemical, and electrical properties, enabled by synchrotron imaging. These results are particularly timely in view of novel scanning X‐ray microscopes that are under development and will become operational in the coming years, in which the full set of techniques of the multimodal toolset may be performed at the same time and in 3D. The possibility of such simultaneous measurements can in turn foster future in situ and operando studies of growth, performance, and degradation,[\n##UREF##27##\n44\n##, ##REF##31911652##\n45\n##\n] which can help bridge the gap between cell and module efficiency. The enhanced brilliance of fourth‐generation sources[\n##REF##25177976##\n46\n##\n] will overcome limitations of sample size and scan duration for the 3D case.
"],"string":"[\n \"Conclusion and Outlook\",\n \"
Altogether, we have shown in this study the 3D nature of structural defects in thin‐film CIGS solar cells and we identified local performance deficits attributable to voids. Although possibly detrimental at a local level, the high density of voids highlighted by tomography suggests that their effect cannot be dramatic at the device level, given the high efficiency of the cell. We point out that such a complex system is not easily modeled and available finite element simulation results are not directly comparable with our measurements.[\\n##REF##30479675##\\n14\\n##\\n] Our measurements with absolute electron densities quantified at the nanoscale enable the development of adequate models simulating structural and electronic defects.[\\n##REF##35822496##\\n19\\n##\\n] This investigation does not alter the void size or shape as FIB‐SEM might do, however, it does require a state‐of‐the‐art X‐ray microscopy beamline and involves a delicate sample preparation. Future experiments should aim to avoid it, probe larger areas, and explore the sample in a multi‐scale approach. Such goals may be achieved with a laminography setup.[\\n##UREF##26##\\n43\\n##\\n] Regarding the availability of beamtime, accepting a loss of resolution, similar studies can be extended to the best lab‐CT instruments. Other thin film solar cells, perovskites before all, in single‐junction or tandem configuration, demand studies of this kind to elucidate fabrication defects and improve cost‐efficiency.
\",\n \"
In general, our study highlights the sensitivity at the nanoscale to a multitude of physical, chemical, and electrical properties, enabled by synchrotron imaging. These results are particularly timely in view of novel scanning X‐ray microscopes that are under development and will become operational in the coming years, in which the full set of techniques of the multimodal toolset may be performed at the same time and in 3D. The possibility of such simultaneous measurements can in turn foster future in situ and operando studies of growth, performance, and degradation,[\\n##UREF##27##\\n44\\n##, ##REF##31911652##\\n45\\n##\\n] which can help bridge the gap between cell and module efficiency. The enhanced brilliance of fourth‐generation sources[\\n##REF##25177976##\\n46\\n##\\n] will overcome limitations of sample size and scan duration for the 3D case.
Small voids in the absorber layer of thin‐film solar cells are generally suspected to impair photovoltaic performance. They have been studied on Cu(In,Ga)Se2 cells with conventional laboratory techniques, albeit limited to surface characterization and often affected by sample‐preparation artifacts. Here, synchrotron imaging is performed on a fully operational as‐deposited solar cell containing a few tens of voids. By measuring operando current and X‐ray excited optical luminescence, the local electrical and optical performance in the proximity of the voids are estimated, and via ptychographic tomography, the depth in the absorber of the voids is quantified. Besides, the complex network of material‐deficit structures between the absorber and the top electrode is highlighted. Despite certain local impairments, the massive presence of voids in the absorber suggests they only have a limited detrimental impact on performance.
","
3D X‐ray microscopy quantifies the distribution of voids in thin‐film solar cells and associated electrical performance deficits.\n\n
","
\n\n\nG.\nFevola\n, \nC.\nOssig\n, \nM.\nVerezhak\n, \nJ.\nGarrevoet\n, \nH. L.\nGuthrey\n, \nM.\nSeyrich\n, \nD.\nBrückner\n, \nJ.\nHagemann\n, \nF.\nSeiboth\n, \nA.\nSchropp\n, \nG.\nFalkenberg\n, \nP. S.\nJørgensen\n, \nA.\nSlyamov\n, \nZ. I.\nBalogh\n, \nC.\nStrelow\n, \nT.\nKipp\n, \nA.\nMews\n, \nC. G.\nSchroer\n, \nS.\nNishiwaki\n, \nR.\nCarron\n, \nJ. W.\nAndreasen\n, \nM. E.\nStuckelberger\n, 3D and Multimodal X‐Ray Microscopy Reveals the Impact of Voids in CIGS Solar Cells. Adv. Sci.\n2024, 11, 2301873. 10.1002/advs.202301873\n\n
"],"string":"[\n \"Abstract\",\n \"
Small voids in the absorber layer of thin‐film solar cells are generally suspected to impair photovoltaic performance. They have been studied on Cu(In,Ga)Se2 cells with conventional laboratory techniques, albeit limited to surface characterization and often affected by sample‐preparation artifacts. Here, synchrotron imaging is performed on a fully operational as‐deposited solar cell containing a few tens of voids. By measuring operando current and X‐ray excited optical luminescence, the local electrical and optical performance in the proximity of the voids are estimated, and via ptychographic tomography, the depth in the absorber of the voids is quantified. Besides, the complex network of material‐deficit structures between the absorber and the top electrode is highlighted. Despite certain local impairments, the massive presence of voids in the absorber suggests they only have a limited detrimental impact on performance.
\",\n \"
3D X‐ray microscopy quantifies the distribution of voids in thin‐film solar cells and associated electrical performance deficits.\\n\\n
\",\n \"
\\n\\n\\nG.\\nFevola\\n, \\nC.\\nOssig\\n, \\nM.\\nVerezhak\\n, \\nJ.\\nGarrevoet\\n, \\nH. L.\\nGuthrey\\n, \\nM.\\nSeyrich\\n, \\nD.\\nBrückner\\n, \\nJ.\\nHagemann\\n, \\nF.\\nSeiboth\\n, \\nA.\\nSchropp\\n, \\nG.\\nFalkenberg\\n, \\nP. S.\\nJørgensen\\n, \\nA.\\nSlyamov\\n, \\nZ. I.\\nBalogh\\n, \\nC.\\nStrelow\\n, \\nT.\\nKipp\\n, \\nA.\\nMews\\n, \\nC. G.\\nSchroer\\n, \\nS.\\nNishiwaki\\n, \\nR.\\nCarron\\n, \\nJ. W.\\nAndreasen\\n, \\nM. E.\\nStuckelberger\\n, 3D and Multimodal X‐Ray Microscopy Reveals the Impact of Voids in CIGS Solar Cells. Adv. Sci.\\n2024, 11, 2301873. 10.1002/advs.202301873\\n\\n
To investigate the effect of nano‐voids on performance, samples for two different synchrotron experiments were prepared. The experimental concept and setup are illustrated in Figure\n##FIG##0##\n1\n##. The layer stack of the samples under investigation comprises from top to bottom: a MgF2 antireflective coating (105 nm); Al:ZnO top contact layer (65 nm); ZnO window layer (120 nm); CdS buffer layer (20–50 nm); CIGS absorber (≈3 µm); Mo rear contact layers (500 nm); and a polyimide substrate. The samples were taken from a cell whose fabrication process resulted in a 20.2% efficiency for its best cell.[\n##UREF##4##\n7\n##\n] Details about the cell are available in the Supporting Information. In the first multimodal measurement, an area sized ca. 5 × 5 µm2 was raster‐scanned at the microprobe of PETRA‐III beamline P06 (DESY)[\n##UREF##13##\n23\n##\n] at an energy of 15.25 keV, slightly above the absorption edge of Rb. Compound refractive lenses were used with a phase plate to focus the coherent beam to a 105 nm spot size.[\n##REF##29271759##\n24\n##\n] The different techniques were performed in three successive scans and scan parameters were differently optimized for ptychography and XEOL, respectively, and registered for the analysis. For the second experiment, a pillar of 5 µm diameter covering the entire layer stack was isolated through FIB, and a PXCT scan was performed at the SLS beamline cSAXS (PSI)[\n##REF##22852697##\n25\n##\n] at an energy of 6.2 keV on the flOMNI setup.[\n##UREF##14##\n26\n##\n] Whereas the multimodal scans provide a single top view of the sample, tomography provides the full 3D image that can be represented by vertical or horizontal slices and can be further processed to independently analyze the single layers. Both for 2D and 3D images, the ultimate goal was to label pixels and voxels that present evidence of material deficit, and therefore refer to voids, i.e., volumes of thin films characterized by absence of material.[\n##REF##30479675##\n14\n##\n]\n
To investigate the effect of nano‐voids on performance, samples for two different synchrotron experiments were prepared. The experimental concept and setup are illustrated in Figure\\n##FIG##0##\\n1\\n##. The layer stack of the samples under investigation comprises from top to bottom: a MgF2 antireflective coating (105 nm); Al:ZnO top contact layer (65 nm); ZnO window layer (120 nm); CdS buffer layer (20–50 nm); CIGS absorber (≈3 µm); Mo rear contact layers (500 nm); and a polyimide substrate. The samples were taken from a cell whose fabrication process resulted in a 20.2% efficiency for its best cell.[\\n##UREF##4##\\n7\\n##\\n] Details about the cell are available in the Supporting Information. In the first multimodal measurement, an area sized ca. 5 × 5 µm2 was raster‐scanned at the microprobe of PETRA‐III beamline P06 (DESY)[\\n##UREF##13##\\n23\\n##\\n] at an energy of 15.25 keV, slightly above the absorption edge of Rb. Compound refractive lenses were used with a phase plate to focus the coherent beam to a 105 nm spot size.[\\n##REF##29271759##\\n24\\n##\\n] The different techniques were performed in three successive scans and scan parameters were differently optimized for ptychography and XEOL, respectively, and registered for the analysis. For the second experiment, a pillar of 5 µm diameter covering the entire layer stack was isolated through FIB, and a PXCT scan was performed at the SLS beamline cSAXS (PSI)[\\n##REF##22852697##\\n25\\n##\\n] at an energy of 6.2 keV on the flOMNI setup.[\\n##UREF##14##\\n26\\n##\\n] Whereas the multimodal scans provide a single top view of the sample, tomography provides the full 3D image that can be represented by vertical or horizontal slices and can be further processed to independently analyze the single layers. Both for 2D and 3D images, the ultimate goal was to label pixels and voxels that present evidence of material deficit, and therefore refer to voids, i.e., volumes of thin films characterized by absence of material.[\\n##REF##30479675##\\n14\\n##\\n]\\n
The authors acknowledge DESY, a member of the Helmholtz Association, and Paul Scherrer Institute for granting beamtime at beamlines P06 (PETRA III) and cSAXS (SLS); European Union's Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie Grant Agreements No. 701647 (M.V.) and No. 765604 (MUMMERING) (J.W.A.); Swiss Federal Office of Energy SFOE (R.C., S.N.) under the ImproCIS project (Contract no.: SI/501614‐01) (R.C., S.N.); H2020 European Research Council through the SEEWHI Consolidator grant, ERC‐2015‐CoG‐681881 (J.W.A.); U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Solar Energy Technology (H.L.G.). This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE‐AC36‐08GO28308. This research was partly supported by the Maxwell computational resources operated at DESY. Beamtime at DESY (ID: 11005475) was allocated for proposal I‐20180471. The authors acknowledge Enrico Avancini and Ayodhya Tiwari (Empa) for their contribution to the fabrication of the solar cells and Andreas Kolditz, Jan Flügge, Jan Siebels (Universität Hamburg), as well as Kathryn Spiers and the FS‐PETRA Engineering team (DESY) for experiment support.
","
Open access funding enabled and organized by Projekt DEAL.
","Data Availability Statement","
The data that support the findings of this study are available at the open‐access repository https://doi.org/10.5281/zenodo.10018968. Raw data are available from the corresponding authors upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
The authors acknowledge DESY, a member of the Helmholtz Association, and Paul Scherrer Institute for granting beamtime at beamlines P06 (PETRA III) and cSAXS (SLS); European Union's Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie Grant Agreements No. 701647 (M.V.) and No. 765604 (MUMMERING) (J.W.A.); Swiss Federal Office of Energy SFOE (R.C., S.N.) under the ImproCIS project (Contract no.: SI/501614‐01) (R.C., S.N.); H2020 European Research Council through the SEEWHI Consolidator grant, ERC‐2015‐CoG‐681881 (J.W.A.); U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Solar Energy Technology (H.L.G.). This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. Department of Energy (DOE) under Contract No. DE‐AC36‐08GO28308. This research was partly supported by the Maxwell computational resources operated at DESY. Beamtime at DESY (ID: 11005475) was allocated for proposal I‐20180471. The authors acknowledge Enrico Avancini and Ayodhya Tiwari (Empa) for their contribution to the fabrication of the solar cells and Andreas Kolditz, Jan Flügge, Jan Siebels (Universität Hamburg), as well as Kathryn Spiers and the FS‐PETRA Engineering team (DESY) for experiment support.
\",\n \"
Open access funding enabled and organized by Projekt DEAL.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available at the open‐access repository https://doi.org/10.5281/zenodo.10018968. Raw data are available from the corresponding authors upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
Experimental concept. Two samples of the same layer stack are prepared for two different experiments. In the first experiment, a sample is mounted on a printed circuit board and electrically contacted so that maps of the induced current can be measured. In the second experiment, the sample is carved out and mounted on a pin, where it is molded to a cylindrical shape. Projections from different angles are acquired to yield a 3D reconstruction that can be processed and decomposed into individual layers.
","
Multimodal 2D X‐ray imaging of CIGS solar cell. A) Stack of top‐view multimodal maps. Ptychography and fluorescence maps (top), locating voids in the absorber; XEOL and XBIC (bottom) mapping their optical and electrical performance. B) Extent of voids estimated from ellipse‐fitting or Feret diameters. Fitting dashed lines relate to the eccentricity of the voids. C) Labels of voids and surroundings segmented from the XRF map and represented in overlay transparency on the ptychography map. D) Relative measurements of XBIC, XEOL, XRF, and ptychography within labeled voids. Expressed as the ratio between average intensity within void and outside void. E) Average loss and extent of voids resulting from a meta‐analysis of measurement in (D). F) Scatter plot of XBIC and XEOL versus Se fluorescence counts. Dashed lines indicate a linear trend of increasing performance per increasing XRF counts. The size of markers in (B) and (F) is proportional to the void area.
","
Depth information from PXCT of CIGS solar cell. A) Volume rendering of cylindrical sub‐volume with electron density in greyscale. The diameter is 2 µm. B–D) Sagittal, coronal, and axial slices. Segmented voids are highlighted by colored contours E) Depth profile of electron density across the layer stack. Blue and red lines indicate the 5–95 and the 25–75 percentile variations. Most variation occurs within 500 nm below the CdS layer. Axial slices a–f) from the upper region of the CIGS layer are reported above. Slices g–l) from the lower part of the absorber, represented with truncated greyscale to enhance contrast. Scale bars are 1 µm.
","
3D segmentation and analysis of voids. A) Volume rendering of the PXCT data with segmented voids. The left inset depicts a spherical deep void, right inset depicts a group of vertically elongated voids B) Electron density within the single voids. Blue and red lines indicate the 5–95 percentile and the 25–75 percentile variations.
","
Comparison of 2D versus 3D ptychography. A–D) Vertical projections as computed from tomography of the CIGS layer, CdS‐ZnO‐MgF2, layer stack, and Mo‐polyimide (PI). E) Dispersion of values within layers illustrated in A, B, D. F) Map of overlapping voids at different heights. Areas in yellow and purple cross one and two voids respectively. G) Minimum intensity projection of the absorber. H) Volume rendering of voids as isolated (green) and connected to the buffer layer (red). I) The network of crevices connecting the voids (color highlight, slice view). J) Two hypothetical scenarios of free path distance for charge carriers across the absorber, based on measured electron density values. Void surfaces lie mostly at the large voids (left) or they lie across the whole stack (right), i.e., across unpassivated grain boundaries and minor voids.
"],"string":"[\n \"
Experimental concept. Two samples of the same layer stack are prepared for two different experiments. In the first experiment, a sample is mounted on a printed circuit board and electrically contacted so that maps of the induced current can be measured. In the second experiment, the sample is carved out and mounted on a pin, where it is molded to a cylindrical shape. Projections from different angles are acquired to yield a 3D reconstruction that can be processed and decomposed into individual layers.
\",\n \"
Multimodal 2D X‐ray imaging of CIGS solar cell. A) Stack of top‐view multimodal maps. Ptychography and fluorescence maps (top), locating voids in the absorber; XEOL and XBIC (bottom) mapping their optical and electrical performance. B) Extent of voids estimated from ellipse‐fitting or Feret diameters. Fitting dashed lines relate to the eccentricity of the voids. C) Labels of voids and surroundings segmented from the XRF map and represented in overlay transparency on the ptychography map. D) Relative measurements of XBIC, XEOL, XRF, and ptychography within labeled voids. Expressed as the ratio between average intensity within void and outside void. E) Average loss and extent of voids resulting from a meta‐analysis of measurement in (D). F) Scatter plot of XBIC and XEOL versus Se fluorescence counts. Dashed lines indicate a linear trend of increasing performance per increasing XRF counts. The size of markers in (B) and (F) is proportional to the void area.
\",\n \"
Depth information from PXCT of CIGS solar cell. A) Volume rendering of cylindrical sub‐volume with electron density in greyscale. The diameter is 2 µm. B–D) Sagittal, coronal, and axial slices. Segmented voids are highlighted by colored contours E) Depth profile of electron density across the layer stack. Blue and red lines indicate the 5–95 and the 25–75 percentile variations. Most variation occurs within 500 nm below the CdS layer. Axial slices a–f) from the upper region of the CIGS layer are reported above. Slices g–l) from the lower part of the absorber, represented with truncated greyscale to enhance contrast. Scale bars are 1 µm.
\",\n \"
3D segmentation and analysis of voids. A) Volume rendering of the PXCT data with segmented voids. The left inset depicts a spherical deep void, right inset depicts a group of vertically elongated voids B) Electron density within the single voids. Blue and red lines indicate the 5–95 percentile and the 25–75 percentile variations.
\",\n \"
Comparison of 2D versus 3D ptychography. A–D) Vertical projections as computed from tomography of the CIGS layer, CdS‐ZnO‐MgF2, layer stack, and Mo‐polyimide (PI). E) Dispersion of values within layers illustrated in A, B, D. F) Map of overlapping voids at different heights. Areas in yellow and purple cross one and two voids respectively. G) Minimum intensity projection of the absorber. H) Volume rendering of voids as isolated (green) and connected to the buffer layer (red). I) The network of crevices connecting the voids (color highlight, slice view). J) Two hypothetical scenarios of free path distance for charge carriers across the absorber, based on measured electron density values. Void surfaces lie mostly at the large voids (left) or they lie across the whole stack (right), i.e., across unpassivated grain boundaries and minor voids.
The coordination chemistry of 2‐aza‐21‐carbaporphyrin, a.k.a. N‐confused porphyrin (NCP, Figure ##FIG##0##\n1\n##) started simultaneously with the discovery of this porphyrinoid.[\n##UREF##0##\n1\n##, ##UREF##1##\n2\n##\n] For almost three decades this macrocycle has attracted the attention of the researchers involved in the coordination chemistry of macrocycles due to its unusual NNNC coordination core, several distinct coordination modes that can be adopted by this porphyrinoid, as well as stabilization of the uncommon oxidation states.[\n##UREF##2##\n3\n##, ##UREF##3##\n4\n##, ##UREF##4##\n5\n##, ##UREF##5##\n6\n##, ##UREF##6##\n7\n##, ##REF##12010019##\n8\n##, ##REF##12688742##\n9\n##, ##REF##14989654##\n10\n##, ##UREF##7##\n11\n##, ##REF##16676941##\n12\n##, ##REF##17173392##\n13\n##, ##UREF##8##\n14\n##, ##REF##18954046##\n15\n##, ##UREF##9##\n16\n##, ##REF##35230807##\n17\n##, ##REF##36852929##\n18\n##\n] The unique feature of NCP is the presence of a built‐in extra‐annular nitrogen donor that can be treated as an additional ligation site, naturally enriching the coordination chemistry of this macrocycle when compared with its isomer, i.e., regular porphyrin or various core‐modified analogues, including carbaporphyrins.[\n##REF##27657332##\n19\n##, ##REF##36771158##\n20\n##\n] Thus, for the group 12 metals, manganese(II), or iron(II) complexes, the external nitrogen N2 is coordinated to the metal center bound within the core of the adjacent NCP subunit forming a bridgeless homodimer or homotrimer.[\n##REF##12010019##\n8\n##, ##REF##14989654##\n10\n##, ##UREF##10##\n21\n##, ##UREF##11##\n22\n##, ##UREF##12##\n23\n##\n] In a quite complicated structure of mixed‐valence tetrarhodium bis(NCP) tetracarbonyl complex, two external nitrogens are coordinated to a bridging dicarbonylrhodium(0) unit.[\n##REF##17173392##\n13\n##\n] Meanwhile, in a monomeric dirhodium(I) system, one dicarbonylrhodium(I) center occupies two internal nitrogen sites and the external nitrogen is coordinated to the chlorodicarbonylrhodium(I) unit.[\n##UREF##6##\n7\n##\n] Upon coordination of two dicarbonyliridium(I) moieties, the NCP ligand undergoes inversion which results in the ligation of all four nitrogens inside the distorted macrocycle.[\n##REF##16676941##\n12\n##\n] Owing to the close location of the meso‐aryl at C20, the metal binding N2 can be a part of a six‐membered metallacycle involving C1, C20, C\nipso\n, and C\northo\n (Figure ##FIG##0##1##). Such an ortho‐metallation has been relatively rarely observed and structurally characterized for NCP derivatives. In palladium(II) and platinum(II) dimers, the NCP subunits are bridged by the metal ions,[\n##REF##11154554##\n24\n##, ##UREF##13##\n25\n##, ##REF##15018507##\n26\n##\n] while doubly‐ortho‐metallation of PtII or PtIV has been found to occur in [Pt(3,3′‐(NCP)2)] or [Pt{3,3′‐(NCP)}L1L2] comprising two directly linked NCP subunits.[\n##UREF##14##\n27\n##, ##UREF##15##\n28\n##\n] In some of these complexes, the macrocyclic core is not involved in coordination, and in all of them, the confused pyrrole is tipped from the mean plane of the regular pyrroles which may be a prerequisite for the ortho‐metallation.
","
In this paper, we report the synthesis and characterization of several late transition metal complexes comprising NCP or its derivatives with the ortho‐C20–N2 chelating motif. We focus on the structural features of the NCP complexes that can be useful for transferring chirality onto the exposed “external” metal center M1 or potential catalytic activity.
"],"string":"[\n \"Introduction\",\n \"
The coordination chemistry of 2‐aza‐21‐carbaporphyrin, a.k.a. N‐confused porphyrin (NCP, Figure ##FIG##0##\\n1\\n##) started simultaneously with the discovery of this porphyrinoid.[\\n##UREF##0##\\n1\\n##, ##UREF##1##\\n2\\n##\\n] For almost three decades this macrocycle has attracted the attention of the researchers involved in the coordination chemistry of macrocycles due to its unusual NNNC coordination core, several distinct coordination modes that can be adopted by this porphyrinoid, as well as stabilization of the uncommon oxidation states.[\\n##UREF##2##\\n3\\n##, ##UREF##3##\\n4\\n##, ##UREF##4##\\n5\\n##, ##UREF##5##\\n6\\n##, ##UREF##6##\\n7\\n##, ##REF##12010019##\\n8\\n##, ##REF##12688742##\\n9\\n##, ##REF##14989654##\\n10\\n##, ##UREF##7##\\n11\\n##, ##REF##16676941##\\n12\\n##, ##REF##17173392##\\n13\\n##, ##UREF##8##\\n14\\n##, ##REF##18954046##\\n15\\n##, ##UREF##9##\\n16\\n##, ##REF##35230807##\\n17\\n##, ##REF##36852929##\\n18\\n##\\n] The unique feature of NCP is the presence of a built‐in extra‐annular nitrogen donor that can be treated as an additional ligation site, naturally enriching the coordination chemistry of this macrocycle when compared with its isomer, i.e., regular porphyrin or various core‐modified analogues, including carbaporphyrins.[\\n##REF##27657332##\\n19\\n##, ##REF##36771158##\\n20\\n##\\n] Thus, for the group 12 metals, manganese(II), or iron(II) complexes, the external nitrogen N2 is coordinated to the metal center bound within the core of the adjacent NCP subunit forming a bridgeless homodimer or homotrimer.[\\n##REF##12010019##\\n8\\n##, ##REF##14989654##\\n10\\n##, ##UREF##10##\\n21\\n##, ##UREF##11##\\n22\\n##, ##UREF##12##\\n23\\n##\\n] In a quite complicated structure of mixed‐valence tetrarhodium bis(NCP) tetracarbonyl complex, two external nitrogens are coordinated to a bridging dicarbonylrhodium(0) unit.[\\n##REF##17173392##\\n13\\n##\\n] Meanwhile, in a monomeric dirhodium(I) system, one dicarbonylrhodium(I) center occupies two internal nitrogen sites and the external nitrogen is coordinated to the chlorodicarbonylrhodium(I) unit.[\\n##UREF##6##\\n7\\n##\\n] Upon coordination of two dicarbonyliridium(I) moieties, the NCP ligand undergoes inversion which results in the ligation of all four nitrogens inside the distorted macrocycle.[\\n##REF##16676941##\\n12\\n##\\n] Owing to the close location of the meso‐aryl at C20, the metal binding N2 can be a part of a six‐membered metallacycle involving C1, C20, C\\nipso\\n, and C\\northo\\n (Figure ##FIG##0##1##). Such an ortho‐metallation has been relatively rarely observed and structurally characterized for NCP derivatives. In palladium(II) and platinum(II) dimers, the NCP subunits are bridged by the metal ions,[\\n##REF##11154554##\\n24\\n##, ##UREF##13##\\n25\\n##, ##REF##15018507##\\n26\\n##\\n] while doubly‐ortho‐metallation of PtII or PtIV has been found to occur in [Pt(3,3′‐(NCP)2)] or [Pt{3,3′‐(NCP)}L1L2] comprising two directly linked NCP subunits.[\\n##UREF##14##\\n27\\n##, ##UREF##15##\\n28\\n##\\n] In some of these complexes, the macrocyclic core is not involved in coordination, and in all of them, the confused pyrrole is tipped from the mean plane of the regular pyrroles which may be a prerequisite for the ortho‐metallation.
\",\n \"
In this paper, we report the synthesis and characterization of several late transition metal complexes comprising NCP or its derivatives with the ortho‐C20–N2 chelating motif. We focus on the structural features of the NCP complexes that can be useful for transferring chirality onto the exposed “external” metal center M1 or potential catalytic activity.
\"\n]"},"methods":{"kind":"list like","value":["General Methods and Instrumentation","
Commercial reagents were used without further purification. Solvents were freshly distilled from the appropriate drying agents or purified under nitrogen with the mBraun MBSPS‐800 before use. Column chromatography was performed by using silica gel 60 (200–300 mesh ASTM). The NMR spectra were recorded on a Bruker Avance III spectrometer, operating at 500 MHz for 1H and 125 MHz for 13C, or a Bruker Avance III spectrometer operating at 600 MHz for 1H and 150 MHz for 13C. TMS was used as an internal reference for 1H and 13C chemical shifts and CDCl3 was used as solvent. Standard pulse programs from the Bruker library were used for homo‐ and heteronuclear 2D experiments. ESR spectra (X‐band) were recorded on a Bruker ELEXSYS E500 spectrometer. Mass spectrometry measurements were conducted by using the electrospray ionization technique on a Bruker Daltonics microTOF‐Q or using the MALDI method on a Bruker ultrafleXtreme spectrometer. Absorption UV/Vis/NIR spectra were recorded by using a Varian Cary 60 and Jasco V‐770 spectrophotometers. Circular dichroic spectra were recorded by means of Jasco 1500 spectropolarimeter equipped with a flow cell attached to the Hitachi‐Merck LaChrom HPLC system allowing detection of the chiral fraction and CD spectra measurement in a stopped‐flow technique. Enantiomer resolutions were performed using either Chirex 3010 or Chirex 3014 column (25 × 0.46 cm). The product of the catalytic reactions was analyzed utilizing an Agilent 8890 gas chromatograph equipped with an Agilent 122–5532 column (30 m × 250 µm × 0.25 µm with DB‐5 ms stationary phase) and with mass spectrometer detector Agilent 5977B. Quantitative analyses were performed on the same chromatograph with Agilent 19091S‐433UI column and FID detector. Electrochemical measurements were performed by means of Autolab (Metrohm) potentiostat/galvanostat system for dichloromethane solutions with a glassy carbon, a platinum wire, and Ag/Ag+ as the working, auxiliary, and pseudoreference electrodes, respectively. Tetrabutylammonium hexafluorophosphate was used as a supporting electrolyte. The potentials were referenced with the ferrocene/ferrocenium couple used as an internal standard.
","
The X‐ray diffraction was measured using either XtaLAB Synergy R or Xcalibur, Onyx diffractometers using a copper source of radiation (λ = 1.54184 Å), and collected with CCD camera. The standard temperature of the measurement was 100 K. The structures were solved using direct methods with SHELXT[\n##UREF##26##\n52\n##\n] and refined by the full‐matrix least‐squares method on all F\n2 data by using the SHELXL[\n##UREF##27##\n53\n##\n] incorporated in the OLEX2 program.[\n##UREF##28##\n54\n##\n] All hydrogen atoms, including those located in the difference density map, were placed in calculated positions and refined as the riding model. Crystallographic details are collected in Tables ##SUPPL##0##S2–S6## (Supporting Information). CCDC 2 284 427, 2 284 430, 2 284 431, 2 284 433, and 2 284 434 contain supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
"],"string":"[\n \"General Methods and Instrumentation\",\n \"
Commercial reagents were used without further purification. Solvents were freshly distilled from the appropriate drying agents or purified under nitrogen with the mBraun MBSPS‐800 before use. Column chromatography was performed by using silica gel 60 (200–300 mesh ASTM). The NMR spectra were recorded on a Bruker Avance III spectrometer, operating at 500 MHz for 1H and 125 MHz for 13C, or a Bruker Avance III spectrometer operating at 600 MHz for 1H and 150 MHz for 13C. TMS was used as an internal reference for 1H and 13C chemical shifts and CDCl3 was used as solvent. Standard pulse programs from the Bruker library were used for homo‐ and heteronuclear 2D experiments. ESR spectra (X‐band) were recorded on a Bruker ELEXSYS E500 spectrometer. Mass spectrometry measurements were conducted by using the electrospray ionization technique on a Bruker Daltonics microTOF‐Q or using the MALDI method on a Bruker ultrafleXtreme spectrometer. Absorption UV/Vis/NIR spectra were recorded by using a Varian Cary 60 and Jasco V‐770 spectrophotometers. Circular dichroic spectra were recorded by means of Jasco 1500 spectropolarimeter equipped with a flow cell attached to the Hitachi‐Merck LaChrom HPLC system allowing detection of the chiral fraction and CD spectra measurement in a stopped‐flow technique. Enantiomer resolutions were performed using either Chirex 3010 or Chirex 3014 column (25 × 0.46 cm). The product of the catalytic reactions was analyzed utilizing an Agilent 8890 gas chromatograph equipped with an Agilent 122–5532 column (30 m × 250 µm × 0.25 µm with DB‐5 ms stationary phase) and with mass spectrometer detector Agilent 5977B. Quantitative analyses were performed on the same chromatograph with Agilent 19091S‐433UI column and FID detector. Electrochemical measurements were performed by means of Autolab (Metrohm) potentiostat/galvanostat system for dichloromethane solutions with a glassy carbon, a platinum wire, and Ag/Ag+ as the working, auxiliary, and pseudoreference electrodes, respectively. Tetrabutylammonium hexafluorophosphate was used as a supporting electrolyte. The potentials were referenced with the ferrocene/ferrocenium couple used as an internal standard.
\",\n \"
The X‐ray diffraction was measured using either XtaLAB Synergy R or Xcalibur, Onyx diffractometers using a copper source of radiation (λ = 1.54184 Å), and collected with CCD camera. The standard temperature of the measurement was 100 K. The structures were solved using direct methods with SHELXT[\\n##UREF##26##\\n52\\n##\\n] and refined by the full‐matrix least‐squares method on all F\\n2 data by using the SHELXL[\\n##UREF##27##\\n53\\n##\\n] incorporated in the OLEX2 program.[\\n##UREF##28##\\n54\\n##\\n] All hydrogen atoms, including those located in the difference density map, were placed in calculated positions and refined as the riding model. Crystallographic details are collected in Tables ##SUPPL##0##S2–S6## (Supporting Information). CCDC 2 284 427, 2 284 430, 2 284 431, 2 284 433, and 2 284 434 contain supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
\"\n]"},"results":{"kind":"list like","value":["Results and Discussion","Syntheses and Characterizations","
As starting materials for our syntheses of bis‐metallic systems, we chose two previously reported compounds bearing a substituent at the C21 position coordinated to either nickel(II)[\n##UREF##16##\n29\n##, ##REF##11151364##\n30\n##, ##UREF##17##\n31\n##, ##REF##12467409##\n32\n##, ##REF##12950206##\n33\n##\n] or ruthenium(II).[\n##UREF##18##\n34\n##, ##REF##31944014##\n35\n##\n] The common features of these, otherwise different complexes are chirality, significant deviation from planarity of the porphyrin ring in the region of confused pyrrole, and unoccupied/non‐protonated N2. These systems were subjected to reaction with organometallic dimeric complexes of ruthenium(II), rhodium(III), or iridium(III) with two chlorides and either neutral η6‐para‐cymene (Cym) or η5‐pentamethylcyclopentadienyl anion (Cp*). Under mild conditions (reflux of DCM solution in the presence of sodium acetate as a proton scavenger) the reaction resulted in the substitution of one chloride by carbanionic ortho‐C20 and coordination of N2 atoms (Scheme ##FIG##1##\n1\n##). The ortho‐metallation efficacy varied depending on the metal ion from 35–40% reaction yield for RhIII, 54–60% for RuII, and up to 85–93% for IrIII. Slightly better results were obtained for NiMeP compared with those for RuSPy. We showed also that under the analogous condition, the reaction of [IrCl2Cp*]2 with ClNCP free base yielded ortho‐metallated derivative ClNCPIrCp* (Scheme ##FIG##1##1##) with a good outcome (83% yield).
","
The new complexes were characterized by high‐resolution mass spectrometry, 1H and 13C NMR, including homo‐ and heteronuclear 2D correlation experiments, UV–vis–NIR spectroscopy, and single crystal XRD analyses. The ESI+ HRMS indicated the presence of the incoming metal ion as well as Cym or Cp* moieties in each case, though all cations were stripped of chloride. The 1H NMR spectra revealed the preservation of most of the spectral features typical of NiMeP, RuSPy, or ClNCP in the complexes formed and the appearance of new signals related to the presence of Cp* or Cym (Figure ##FIG##2##\n2\n##). Thus, the spectra of the systems with Cp* comprise a very strong (15H) signal at about δ 0.8 ppm due to methyl protons, indicating fast rotation around the M–Cp* direction. On the other hand, the differentiation of aryl and isopropyl resonances of the RuCym moiety is in line with the slow motions of the Cym ligand at the 1H NMR time scale. Moreover, an upfield shift of all the Cym protons with respect to those observed for starting [RuCl2Cym]2, indicates the position of this moiety over the aromatic frame of the macrocyclic ligands. Significantly, this aromatic ring shielding effect is strongest for the cymene methyl Me\nc\n (Δδ 0.7–1.4 ppm) suggesting the orientation of the ligand with the isopropyl group situated rather outside, while Me\nc\n is above the macrocycle. The number of meso‐aryl distinct signals in the spectra of RuSPyIrCp* and RuSPyRuCym recorded at room temperature, reflects diastereotopic inequivalence of the macrocyclic faces and slow, at the NMR timescale, rotation of these substituents around Cipso─Cmeso bonds, resulting in differentiation of ortho‐ and meta‐H resonances of phenyls at C5, C10, and C15. Conversely, these meso‐phenyl protons in NiMePIrCp* and NiMePRuCym give rise to severely broadened signals indicating intermediate rotation rate of the substituents and diastereotopicity of the complex faces. The only sharp meso‐aryl signals are those of phenyl at the C20 position due to the complete freezing of its rotation by ortho‐metallation. The chemical shifts and coupling patterns are similar in all these ortho‐metallated complexes comprising a doublet for 20‐m at about 8.3 ppm, a doublet for 20‐o′ at about 7.7 ppm, and two triplets for 20‐m′ and 20‐p at ≈7.3 and 7.2 ppm, respectively (Figure ##FIG##2##2##). For the ortho‐metallated rhodium(III) complexes, NiMePRhCp* and RuSPyRhCp*, a close resemblance of
","
\n1H NMR spectral patterns to those of the respective IrIII systems were observed. The major difference is an additional splitting of all multiplets of the 20‐phenyl protons due to 1H‐103Rh spin–spin coupling (JRhH\n = 1.3–1.5 Hz). Significantly, the ortho‐carbon of the C20 phenyl substituent, identified on the basis of 1H,13C HSQC and 1H,13C HMBC experiments (Figure ##SUPPL##0##S7B## and ##SUPPL##0##S13B##, Supporting Information), gives rise to a doublet at δC\n 169.8 ppm with 1\nJRhC\n = 31.1 Hz for NiMePRhCp* and at δC\n 169.9 ppm with 1\nJRhC\n = 30.9 Hz for RuSPyRhCp* unequivocally proving coordination of the carbanion. The 13C–103Rh coupling could be observed also for cyclopentadiene carbon atoms in 13C NMR of both complexes giving rise to doublets at about δC\n 96 ppm with 1\nJRhC\n = 6.1 Hz. The 1H NMR characteristics of Cl\nNCPIrCp* differ from the nickel(II) and ruthenium(II) complexes with 21‐CH and 22,24‐NH resonances arising in the upfield region of the spectrum (δ −4.69 and broad signals at δ −1.05, −1.10 ppm, respectively) reflecting a free‐base character of the macrocyclic core. Interestingly, for all pentamethylcyclopentadienyl‐comprising systems, correlations of the coordinated ortho‐C with methyl protons of the η5‐Cp* ligand were observed in the 1H,13C HMBC maps (Figures ##SUPPL##0##S3B##, ##SUPPL##0##S7B##, ##SUPPL##0##S9B##, ##SUPPL##0##S13B##, and ##SUPPL##0##S15B##, Supporting Information), regardless of the metal ion. Such correlations appeared despite the fact that the coupled nuclei were separated by four bonds. The correlations may be accounted for by the anionic character of the Cp* ligand resulting in a relatively high electron density available that enhanced 1H‐13C coupling. Significantly, for the neutral η6‐Cym ligand in NiMePRuCym or RuSPyRuCym, there was no such a correlation observed, in spite of only three bonds separating aryl protons of this ligand and the RuII‐coordinating ortho‐C at 20‐Ph.
","
The electronic spectrum of ClNCPIrCp* resembles that of the starting porphyrin ClNCP with ≈65 nm and 15 nm bathochromic shifts of the lowest‐energy Q band and the Soret band, respectively observed for the complex (Figure ##SUPPL##0##S1##, Supporting Information). For the NiMeP and RuSPy, the spectral changes due to external coordination are much more profound, though similar to each other, regardless of the ortho‐metallated cation (Figure ##FIG##3##\n3\n##). These alterations involve a decrease in the relative intensity of the spectra in the Soret region near 430 nm and an increase of the absorbance in the Q band region, i.e., above 500 nm.
","Crystal Structures","
Solid state structures of the selected ortho‐metallated systems were elucidated using the single crystal X‐ray diffraction analyses (Figure ##FIG##4##\n4\n## Figures ##SUPPL##0##S39–S44##, Supporting Information). The structures determined based on diffraction data clearly showed the coordination mode of iridium(III), ruthenium(II), and rhodium(III) ions to ClNCP or metalloligands NiMeP and RuSPy. Metalloligands bind iridium(III), ruthenium(II), and rhodium(III) ions through the N2 donor atom and the ortho‐carbon of the aryl ring from the C20 meso‐position. The coordination sphere of metal ions located at the periphery of the macrocycle is supplemented by chloride and pentamethylcyclopentadienyl ligands (in ClNCPIrCp*, NiMePIrCp*, RuSPyIrCp*, and RuSPyRhCp*) or chloride and p‐cymene ligands (in NiMePRuCym). Coordinating metal ions at the edges of metalloligands retain a structural motif referred to as a “piano stool” or a half‐sandwich complex.[\n##UREF##19##\n36\n##\n] Data on bond lengths around iridium(III), ruthenium(II), and rhodium(III) ions are summarized in Table ##TAB##0##\n1\n## along with selected bond lengths for the dimeric metal sources. The average M–L distances (where L = Cp* or Cym) are significantly longer than in starting half‐sandwich organometallic chlorides, while M–Cl bond lengths are close to those observed for terminally bound chlorides in the respective dimeric precursors or only slightly longer.[\n##UREF##20##\n37\n##, ##UREF##21##\n38\n##, ##REF##30942794##\n39\n##\n] Interestingly, RuSPyIrCp* and RuSPyRhCp* are isostructural when crystallized from benzene/hexane, both forming tris(benzene) solvates.
","
The steric hindrance introduced by Cp* or Cym ligands forces them to be specifically positioned relative to methyl or mercaptopyridyl substituents at the C21 atom of metalloligand. As a consequence, they are located on opposite sides of the macrocyclic plane, and, in the case of compounds RuSPyIrCp* and RuSPyRhCp*, on the same side as the CO ligand. The specific setting of the p‐cymene relative to the macrocyclic system was established for NiMePRuCym with the isopropyl group above the metalloligand plane. Such an orientation of the Cym ligand is somewhat unexpected considering solution 1H NMR results suggesting methyl rather than the isopropyl group directed toward the macrocycle interior (vide supra). It may be accounted for by packing forces for which the ligand orientation in the crystal with a smaller substituent situated beyond the perimeter of the macrocycle is more favorable. The deviation of the porphyrin ring from planarity due to sp3 hybridization of C21, characteristic for the starting metalloligand NiMeP and RuSPy, is retained upon coordinating the metal ion to the periphery of the macrocycle. However, the external ortho‐metallation does not significantly change the bond distance between the metal ion [nickel(II) or ruthenium(II)] located in the cavity of the macrocycle and the C21 carbon atom. The Ni─C21 bond length is 2.004(4) Å[\n##UREF##16##\n29\n##\n] in NiMeP, while in NiMePIrCp* and NiMePRuCym it is 2.022(1) Å and 2.015(2) Å, respectively. For RuSPy‐containing systems, this bond length alteration is even less pronounced: from 2.118(3) Å in the metalloligand to 2.110(2) and 2.116(1) Å in RuSPyIrCp* and RuSPyRhCp*, respectively. Analyses of the out‐of‐plane porphyrin ring distortions were carried out using the PorphStruct tool[\n##REF##34061410##\n40\n##\n] which was based on the normal‐coordinate structure decomposition (NSD) approach[\n##UREF##22##\n41\n##, ##REF##9533688##\n42\n##\n] (Table ##TAB##1##\n2\n## and Figure ##FIG##5##\n5\n##). The comparative NSD analyses applied for starting systems, i.e., ClNCP, NiMeP, and RuSPy, indicated a moderate effect of the ortho‐metallation on the total out‐of‐plane distortion of the porphyrin (Doop\n). In fact, in some instances (Cl\nNCPIrCp*, RuSPyIrCp*, RuSPyRhCp*, Table ##TAB##1##2##, entries 2, 7, and 8, respectively) the chelation at the NCP perimeter led to a less pronounced displacement than that observed in the starting ligand (NCP, entry 1; RuSPy, entry 6). The most significant deviation increase due to ortho‐metallation was observed for the NiMeP metalloligand (Table ##TAB##1##2##, entry 3) upon chelation of RuCymCl moiety (entry 5). All systems indicated a significant saddling distortion which, again, increased only in NiMePRuCym. The increasing doming, ruffling, and waving distortions were observed for almost all systems upon external chelation, although these components gave rather minor contributions to Doop\n which is dominated by the saddling. Displacement of metal ions from the mean plane of the porphyrin in a metalloligand (dM‐mpln\n) slightly increased after an external metal ion was introduced, though an opposite effect was observed for the displacement from an MCNNN mean plane (dM‐ccpln\n). A common structural feature for the complexes under study as well as for NCP free base, is a pronounced deviation of the confused pyrrole plane from that of the porphyrin ring. Such a deviation can be parametrized by a dihedral angle (DH, Table ##TAB##1##2##) between the confused pyrrole mean plane and the mean plane defined by all non‐hydrogen atoms of the macrocycle, except N2, C3, and C21. Apparently, a significant increase of this angle upon external chelation was observed only for the NiMeP‐containing complexes (Table ##TAB##1##2##, entries 4 and 5). The individual atom displacements from the mean plane are typical for the saddle‐distorted porphyrins with alternate directions of the pyrrole deviation (Figure ##FIG##5##5##). Significantly, in the bimetallic systems NiMePIrCp* and NiMePRuCym, the displacement of the meso‐carbons is significantly more pronounced than in the starting NiMeP, where those atoms are located almost in the mean plane. It is particularly evident for C20 and is related to the coordination of N2 and the aryl at C20. The external chelation slightly increases the displacement of N2 and C3 in these two systems with respect to the metalloligand, in line with the increasing DH. Generally, the atoms are more displaced in NiMeP and its ortho‐metallated derivatives than in RuSPy and its complexes.
","
All ortho‐metallated complexes are chiral, as are their precursors in the solid state. However, these systems crystallized in centrosymmetric space groups as racemates and in Figure ##FIG##4##4## only one enantiomer for each of the complexes has been shown.
","Chirality","
Our attempt to separate enantiomers of the externally metallated NCP derivatives involved HPLC methods with a chiral stationary phase. The enantiomers of several of these systems are presented in Figure ##FIG##6##\n6\n## as they appear in the solid‐state structures, along with definitions of their absolute configurations. For these definitions, we took an external metallacycle which is common to all these ortho‐metallated systems, i.e., M─N2─C1─C20─C\nipso\n─C\northo\n as a chirality plane.
","
Although metalloligands NiMeP and RuSPy are intrinsically chiral due to the presence of a chirality center at the coordinated C21,[\n##REF##21226119##\n43\n##\n] the ortho‐metallation introduces its own chirality related to a differentiation of the porphyrin faces. Apparently, these two chirality sources are not independent, that is, upon external chelation of the racemic mixture of NiMeP or RuSPy only a pair of enantiomers is formed and no other NMR‐distinguishable stereoisomers can be observed. Hence, the external metallation is stereoselective although two diastereomers can be potentially formed, differing in orientation of chloride and the organometallic ligands (Cp* or Cym) at the externally chelated metal with respect to the macrocycle. The crystal structures reveal roughly the same orientation of the chloride ligand at the external metal center and the substituent at C21. Thus, the chirality of the bimetallic monomers in the solid state and solution is predefined by the starting metalloligands[\n##REF##21226119##\n43\n##, ##UREF##23##\n44\n##\n] with absolute configurations S or R at C21 in the metalloligands invariantly giving rise to the configurations P or M, respectively in the ortho‐metallated complexes. The separation of the bimetallic enantiomers by the HPLC method was expected to be effective through two approaches (Scheme ##FIG##7##\n2\n##). The first method involved a separation of enantiomers prior to the external chelation (Figure ##FIG##8##\n7A,B,C,E##), while in the second approach, separation proceeded ortho‐metallation (Figure ##FIG##8##7D,F##; Figures ##SUPPL##0##S29## and ##SUPPL##0##S30##, Supporting Information). The first method allows high enantiopurity of the bimetallic systems and, in principle, can be applied for many other metal ions resulting in chirality transfer from the metalloligand toward the external metal center which may be a site of catalytic reaction. Importantly, the absolute configurations of such complexes can be deduced directly from the absolute configuration of the metalloligand. The second method is more useful for the systems for which the external chelation is less effective, such as NiMePRhCp* or RuSPyRhCp*.
","
The asymmetry of these configurationally stable systems arises from the presence of the chiral center at the C21 atom. Conversely, the NCP free base is chiral in the solid state owing to its non‐planarity but in solution, the molecule is configurationally unstable and no enantiomer separation is possible. This is due to a flipping of the confused pyrrole allowing fast interconversion of the enantiomers, unlike in several 21‐substituted NCP derivatives.[\n##REF##22924766##\n45\n##, ##REF##24601636##\n46\n##\n] Thus, chelation of metal ion by N2 and the ortho carbon atom of the adjacent meso‐aryl such as in ClNCPIrCp* or NCPPtPPh3\n[\n##UREF##13##\n25\n##\n] as well as double chelation in directly bound 3,3′‐(NCP)2Pt[\n##UREF##15##\n28\n##\n] is sufficient to stabilize the configuration. The external ortho‐metallation provides a lock preventing fast interconversion of enantiomers and participates in the differentiation of the macrocycle faces. Thus, for ClNCPIrCp*, we were able to separate enantiomers and record their circular dichroic spectra (Figures ##SUPPL##0##S27## and ##SUPPL##0##S28##, Supporting Information) indicating the chirality of this system and its configurational stability. The absolute configurations of the separated enantiomers were assigned based on TD–DFT simulations of the CD spectra (Figures ##SUPPL##0##S23–S27##, Tables ##SUPPL##0##S7##–##SUPPL##0##S12##, Supporting Information).
","Redox Properties","
The electrochemical properties of the ortho‐metallated complexes were studied by means of cyclic and differential pulse voltammetry (Figure ##FIG##9##\n8\n##; Figure ##SUPPL##0##S35##, Supporting Information). The electrode potentials were collected in Table ##TAB##2##\n3\n##. For all but one complex system the first oxidations were reversible, and for a majority, the second oxidations appeared to be reversible as well. Conversely, for almost all complexes there was no reversible reduction. Oxidation potentials were relatively low and the external coordination did not significantly affect the first oxidation potentials compared to the metalloligands NiMeP and RuSPy (Table ##TAB##2##3##, entries 8, 9) or ligand ClNCP (Table ##TAB##2##3##, entry 10). It was not the case for RuSPyIrCp* and RuSPyRuCym (Table ##TAB##2##3##, entries 5 and 7) for which 90 and 190 mV cathodic shifts were observed, respectively.
","
The redox properties of the ortho‐metallated species and their precursors can be analyzed theoretically through comparison of the frontier orbitals energies (Figure ##FIG##10##\n9\n##). Apparently, the calculated HOMO energies are very similar for all these systems with only a small rise of potential on going from RuSPy to the ortho‐metallated derivatives which can also be noticed experimentally as an increase of oxidation potentials. The DFT‐calculated HOMO energies are in good agreement with those estimated based on the first oxidation potentials [calculated as −e(E\nOx1 + 4.8 V)], but LUMO energies are systematically higher than those derived from the first reduction potentials E\nRed1 [calculated as −e(E\nRed1 + 4.8 V)]. Consequently, the electrochemical HOMO–LUMO gaps [calculated as e(E\nOx1 − E\nRed1)] are considerably smaller (by 0.4–0.6 eV) in comparison with those based on DFT calculations (cHLG in Table ##TAB##2##3##). On the other hand, the optical HOMO–LUMO energy gaps (oHLG in Table ##TAB##2##3##.), obtained from the experimental UV–vis spectra are even lower (by 0.1–0.2 eV) than the values derived from the electrochemical potentials.
","
Spectrophotometric titration of both NiMeP and RuSPy\northo‐metallated derivatives with tris(4‐bromophenyl)ammoniumyl hexachloroantimonate (BAHA, Magic blue), a one‐electron oxidant of reduction potential 0.70 V[\n##REF##11848774##\n47\n##\n] allowed monitoring of the spectral changes upon oxidation (Figure ##FIG##11##\n10\n##). According to our electrochemical data, this oxidant was sufficiently strong for the first and the second oxidation to be achieved for all systems, except RuSPy for which E\nOx2 is too high (Table ##TAB##2##3##, entry 9). The observed changes in the spectral region between 400 and 800 nm upon the addition of one equivalent of BAHA, suggested metal‐centered oxidation in all the bimetallic systems (Figure ##FIG##11##10A,D##; Figure ##SUPPL##0##S31##–##SUPPL##0##S34##, Supporting Information). For the RuSPyMCp* complexes (M = IrIII, RhIII), the first oxidation resulted in an increase of the Soret‐like band intensity with a small (Δλ = 15 nm) bathochromic shift and a decrease of the Q‐type band (Figure ##FIG##11##10A##; Figure ##SUPPL##0##S34##, Supporting Information). Such changes suggest that the conjugation of the π‐electrons of the aromatic macrocycle remains intact after one electron is removed. Further addition of BAHA resulted in a gradual increase of the absorbance in the NIR region at ≈1500 nm which is indicative of a radical species, thus suggesting a ligand‐centered second oxidation process. These spectral changes are in sharp contrast to those observed for the RuSPy metalloligand for which a pronounced decrease of the Soret‐like and an increase of the Q‐like bands were observed and no further spectral alteration occurred after passing 1 equiv of BAHA (Figure ##FIG##11##10B##). Such a pattern of spectral changes may suggest a porphyrin‐centered oxidation to the cation metalloradical rather than Ru‐centered oxidation. On the other hand, titration of ClNCPIrCp* with BAHA indicated an initial decrease of the Soret‐like band at 448 nm and its bathochromic shift up to 463 nm upon the addition of 1 equiv of the oxidant. The changes were followed by an increase in intensity of this band with a further redshift to 469 nm which was accompanied by the absorbance increase at 816 nm and the formation of the broadband at 1500 nm when approaching 2 equiv of BAHA (Figure ##SUPPL##0##S31##, Supporting Information). Thus, apparently despite oxidation of the “empty” macrocycle in ClNCPIrCp* the aromaticity of the system is retained and typical spectral features of the porphyrinoids, i.e., the strong Soret‐like band and weaker Q‐type bands are observed even for the two‐electron oxidized species. The first oxidations of the systems comprising NiMeP are nickel‐centered. The highly anisotropic orthorhombic frozen dichloromethane ESR spectra obtained by the BAHA addition (Figure ##FIG##10##9C##) closely resemble those of various 21‐alkylated nickel(III) NCP species.[\n##REF##12467409##\n32\n##, ##REF##12950206##\n33\n##, ##UREF##24##\n48\n##, ##REF##17269762##\n49\n##\n] For NiMePIrCp*, the addition of more than 1.5 equiv of BAHA resulted in a gradual decrease of the nickel(III) signal intensity, moderate changes of the Zeeman tensor components, and in the appearance of a radical signal at g = 2.0029. Also, spectrophotometric titration with BAHA revealed fine changes in the Soret and Q regions up to 1.2 equiv, followed by a gradual increase of the band at 435 nm and the final appearance of the NIR band upon the addition of more than 2 equiv of the oxidant (Figure ##FIG##10##9D##).[\n##UREF##25##\n50\n##\n] Interestingly, for the very similar complex, i.e., NiMePRhCp*, the changes of the ESR spectra upon the addition of 1.5 or more equivalents of BAHA are different, involving slight alteration of g\n2 and g\n3 components, not the appearance of the radical signal (Figure ##SUPPL##0##S35##, Supporting Information). Similarly, the addition of BAHA to the solution of NiMePRuCym gave rise to an orthorhombic spectrum in frozen DCM (Figure ##SUPPL##0##S36##, Supporting Information) but no strong radical signal was observed upon the addition of more than 2 equiv of BAHA. For reference purposes, we performed also the ESR‐monitored oxidation for the starting metalloligand NiMeP indicating a decrease of the Zeeman tensor anisotropy upon the addition of an excess of BAHA with changes of g\n1 from 2.382 to 2.285, g\n2 from 2.168 to 2.2019, and g\n3 from 2.086 to 2.111 for the spectra recorded in the presence of 1.2 and 3 equiv, respectively (Figure ##SUPPL##0##S37##, Supporting Information). Again, no accompanying radical formation was observed. The differences among the systems comprising NiMeP in the second oxidation potentials and ESR behavior are surely related to the external coordination, although no clear tendencies can be derived from such a limited data set. It can be also rationally expected that for both groups of the ortho‐metallated complexes, oxidation of the metalloligand strongly affects electron density in the environment of the externally coordinated metal ion.
","Catalysis","
For preliminary studies of the catalytic function of the externally ortho‐metallated systems, we chose N‐heterocyclization reaction of benzylamine with 1,6‐hexanediol which has been shown to be effectively catalyzed by iridium(III) complex [IrCl2Cp*]2 in toluene under basic conditions (Table ##TAB##3##\n4\n##).[\n##REF##15387539##\n51\n##\n] Small‐scale reactions (0.5 mmol) were carried out in the presence of all ortho‐metallated iridium(III) complexes described in this paper as well as for the original catalyst [IrCl2Cp*]2, ClNCP ligand, and both metalloligands, for the reference. The samples were prepared in the inert and dry atmosphere of a glove box to avoid any interference of oxygen and moisture with the reagents, and the reactions were carried out in sealed vials. The reaction results were analyzed qualitatively using GC/MS and quantitatively using the GC/FID technique, and for the selected systems, 1H NMR quantitative analysis was applied with 2,4,6‐collidine as the internal standard. As expected, only iridium(III)‐comprising systems appeared to be catalytically active in the heterocyclizaction reaction, while neither of the ligands supported the 7‐membered ring formation. The best catalyst, i.e., ClNCPIrCp* (Table ##TAB##3##4##, entry 6) gave rise to the yield exceeding that of the original catalyst [IrCl2Cp*]2 with higher glycol conversion and better chemoselectivity. Also NiMePIrCp* seemed to be a more selective catalyst than [IrCl2Cp*]2 with a similar yield of the main reaction product (Table ##TAB##3##4##, entries 2 and 1). Surprisingly, no heterocyclization was observed for RuSPyIrCp* used as a catalyst, despite several attempts. It may be due to a less labile character of the Ir─Cl bond in this complex than in other systems. According to the proposed reaction mechanism,[\n##REF##15387539##\n51\n##\n] in the early stage of the catalytic process, coordination of the glycolic anion to the iridium center is required which implies chloride substitution (originally present in the ortho‐metallated systems). A significantly longer C─Ir bond in RuSPyIrCp* (2.083(5) Å) than those in NiMePIrCp* (2.041(3) Å) or ClNCPIrCp* (2.051(6) Å) may be responsible for a weaker trans effect of the meso‐aryl carbanion coordinated to the iridium(III) center on the opposite side to the chloride, making its exchange less effective. Of course, some other structural features, such as the presence and type of the metal ion within the macrocyclic cavity, flexibility of the molecular skeleton and its deformations, etc., may be decisive for the catalytic activity of the iridium complexes. Although at the present stage, we cannot offer any more conclusive accounting for the observed differences, it is clear that distinctions in the reaction yields among the systems used in this study indicate an influence of the porphyrin‐chelating ligand on the catalytic activity of the externally coordinated metal center.
"],"string":"[\n \"Results and Discussion\",\n \"Syntheses and Characterizations\",\n \"
As starting materials for our syntheses of bis‐metallic systems, we chose two previously reported compounds bearing a substituent at the C21 position coordinated to either nickel(II)[\\n##UREF##16##\\n29\\n##, ##REF##11151364##\\n30\\n##, ##UREF##17##\\n31\\n##, ##REF##12467409##\\n32\\n##, ##REF##12950206##\\n33\\n##\\n] or ruthenium(II).[\\n##UREF##18##\\n34\\n##, ##REF##31944014##\\n35\\n##\\n] The common features of these, otherwise different complexes are chirality, significant deviation from planarity of the porphyrin ring in the region of confused pyrrole, and unoccupied/non‐protonated N2. These systems were subjected to reaction with organometallic dimeric complexes of ruthenium(II), rhodium(III), or iridium(III) with two chlorides and either neutral η6‐para‐cymene (Cym) or η5‐pentamethylcyclopentadienyl anion (Cp*). Under mild conditions (reflux of DCM solution in the presence of sodium acetate as a proton scavenger) the reaction resulted in the substitution of one chloride by carbanionic ortho‐C20 and coordination of N2 atoms (Scheme ##FIG##1##\\n1\\n##). The ortho‐metallation efficacy varied depending on the metal ion from 35–40% reaction yield for RhIII, 54–60% for RuII, and up to 85–93% for IrIII. Slightly better results were obtained for NiMeP compared with those for RuSPy. We showed also that under the analogous condition, the reaction of [IrCl2Cp*]2 with ClNCP free base yielded ortho‐metallated derivative ClNCPIrCp* (Scheme ##FIG##1##1##) with a good outcome (83% yield).
\",\n \"
The new complexes were characterized by high‐resolution mass spectrometry, 1H and 13C NMR, including homo‐ and heteronuclear 2D correlation experiments, UV–vis–NIR spectroscopy, and single crystal XRD analyses. The ESI+ HRMS indicated the presence of the incoming metal ion as well as Cym or Cp* moieties in each case, though all cations were stripped of chloride. The 1H NMR spectra revealed the preservation of most of the spectral features typical of NiMeP, RuSPy, or ClNCP in the complexes formed and the appearance of new signals related to the presence of Cp* or Cym (Figure ##FIG##2##\\n2\\n##). Thus, the spectra of the systems with Cp* comprise a very strong (15H) signal at about δ 0.8 ppm due to methyl protons, indicating fast rotation around the M–Cp* direction. On the other hand, the differentiation of aryl and isopropyl resonances of the RuCym moiety is in line with the slow motions of the Cym ligand at the 1H NMR time scale. Moreover, an upfield shift of all the Cym protons with respect to those observed for starting [RuCl2Cym]2, indicates the position of this moiety over the aromatic frame of the macrocyclic ligands. Significantly, this aromatic ring shielding effect is strongest for the cymene methyl Me\\nc\\n (Δδ 0.7–1.4 ppm) suggesting the orientation of the ligand with the isopropyl group situated rather outside, while Me\\nc\\n is above the macrocycle. The number of meso‐aryl distinct signals in the spectra of RuSPyIrCp* and RuSPyRuCym recorded at room temperature, reflects diastereotopic inequivalence of the macrocyclic faces and slow, at the NMR timescale, rotation of these substituents around Cipso─Cmeso bonds, resulting in differentiation of ortho‐ and meta‐H resonances of phenyls at C5, C10, and C15. Conversely, these meso‐phenyl protons in NiMePIrCp* and NiMePRuCym give rise to severely broadened signals indicating intermediate rotation rate of the substituents and diastereotopicity of the complex faces. The only sharp meso‐aryl signals are those of phenyl at the C20 position due to the complete freezing of its rotation by ortho‐metallation. The chemical shifts and coupling patterns are similar in all these ortho‐metallated complexes comprising a doublet for 20‐m at about 8.3 ppm, a doublet for 20‐o′ at about 7.7 ppm, and two triplets for 20‐m′ and 20‐p at ≈7.3 and 7.2 ppm, respectively (Figure ##FIG##2##2##). For the ortho‐metallated rhodium(III) complexes, NiMePRhCp* and RuSPyRhCp*, a close resemblance of
\",\n \"
\\n1H NMR spectral patterns to those of the respective IrIII systems were observed. The major difference is an additional splitting of all multiplets of the 20‐phenyl protons due to 1H‐103Rh spin–spin coupling (JRhH\\n = 1.3–1.5 Hz). Significantly, the ortho‐carbon of the C20 phenyl substituent, identified on the basis of 1H,13C HSQC and 1H,13C HMBC experiments (Figure ##SUPPL##0##S7B## and ##SUPPL##0##S13B##, Supporting Information), gives rise to a doublet at δC\\n 169.8 ppm with 1\\nJRhC\\n = 31.1 Hz for NiMePRhCp* and at δC\\n 169.9 ppm with 1\\nJRhC\\n = 30.9 Hz for RuSPyRhCp* unequivocally proving coordination of the carbanion. The 13C–103Rh coupling could be observed also for cyclopentadiene carbon atoms in 13C NMR of both complexes giving rise to doublets at about δC\\n 96 ppm with 1\\nJRhC\\n = 6.1 Hz. The 1H NMR characteristics of Cl\\nNCPIrCp* differ from the nickel(II) and ruthenium(II) complexes with 21‐CH and 22,24‐NH resonances arising in the upfield region of the spectrum (δ −4.69 and broad signals at δ −1.05, −1.10 ppm, respectively) reflecting a free‐base character of the macrocyclic core. Interestingly, for all pentamethylcyclopentadienyl‐comprising systems, correlations of the coordinated ortho‐C with methyl protons of the η5‐Cp* ligand were observed in the 1H,13C HMBC maps (Figures ##SUPPL##0##S3B##, ##SUPPL##0##S7B##, ##SUPPL##0##S9B##, ##SUPPL##0##S13B##, and ##SUPPL##0##S15B##, Supporting Information), regardless of the metal ion. Such correlations appeared despite the fact that the coupled nuclei were separated by four bonds. The correlations may be accounted for by the anionic character of the Cp* ligand resulting in a relatively high electron density available that enhanced 1H‐13C coupling. Significantly, for the neutral η6‐Cym ligand in NiMePRuCym or RuSPyRuCym, there was no such a correlation observed, in spite of only three bonds separating aryl protons of this ligand and the RuII‐coordinating ortho‐C at 20‐Ph.
\",\n \"
The electronic spectrum of ClNCPIrCp* resembles that of the starting porphyrin ClNCP with ≈65 nm and 15 nm bathochromic shifts of the lowest‐energy Q band and the Soret band, respectively observed for the complex (Figure ##SUPPL##0##S1##, Supporting Information). For the NiMeP and RuSPy, the spectral changes due to external coordination are much more profound, though similar to each other, regardless of the ortho‐metallated cation (Figure ##FIG##3##\\n3\\n##). These alterations involve a decrease in the relative intensity of the spectra in the Soret region near 430 nm and an increase of the absorbance in the Q band region, i.e., above 500 nm.
\",\n \"Crystal Structures\",\n \"
Solid state structures of the selected ortho‐metallated systems were elucidated using the single crystal X‐ray diffraction analyses (Figure ##FIG##4##\\n4\\n## Figures ##SUPPL##0##S39–S44##, Supporting Information). The structures determined based on diffraction data clearly showed the coordination mode of iridium(III), ruthenium(II), and rhodium(III) ions to ClNCP or metalloligands NiMeP and RuSPy. Metalloligands bind iridium(III), ruthenium(II), and rhodium(III) ions through the N2 donor atom and the ortho‐carbon of the aryl ring from the C20 meso‐position. The coordination sphere of metal ions located at the periphery of the macrocycle is supplemented by chloride and pentamethylcyclopentadienyl ligands (in ClNCPIrCp*, NiMePIrCp*, RuSPyIrCp*, and RuSPyRhCp*) or chloride and p‐cymene ligands (in NiMePRuCym). Coordinating metal ions at the edges of metalloligands retain a structural motif referred to as a “piano stool” or a half‐sandwich complex.[\\n##UREF##19##\\n36\\n##\\n] Data on bond lengths around iridium(III), ruthenium(II), and rhodium(III) ions are summarized in Table ##TAB##0##\\n1\\n## along with selected bond lengths for the dimeric metal sources. The average M–L distances (where L = Cp* or Cym) are significantly longer than in starting half‐sandwich organometallic chlorides, while M–Cl bond lengths are close to those observed for terminally bound chlorides in the respective dimeric precursors or only slightly longer.[\\n##UREF##20##\\n37\\n##, ##UREF##21##\\n38\\n##, ##REF##30942794##\\n39\\n##\\n] Interestingly, RuSPyIrCp* and RuSPyRhCp* are isostructural when crystallized from benzene/hexane, both forming tris(benzene) solvates.
\",\n \"
The steric hindrance introduced by Cp* or Cym ligands forces them to be specifically positioned relative to methyl or mercaptopyridyl substituents at the C21 atom of metalloligand. As a consequence, they are located on opposite sides of the macrocyclic plane, and, in the case of compounds RuSPyIrCp* and RuSPyRhCp*, on the same side as the CO ligand. The specific setting of the p‐cymene relative to the macrocyclic system was established for NiMePRuCym with the isopropyl group above the metalloligand plane. Such an orientation of the Cym ligand is somewhat unexpected considering solution 1H NMR results suggesting methyl rather than the isopropyl group directed toward the macrocycle interior (vide supra). It may be accounted for by packing forces for which the ligand orientation in the crystal with a smaller substituent situated beyond the perimeter of the macrocycle is more favorable. The deviation of the porphyrin ring from planarity due to sp3 hybridization of C21, characteristic for the starting metalloligand NiMeP and RuSPy, is retained upon coordinating the metal ion to the periphery of the macrocycle. However, the external ortho‐metallation does not significantly change the bond distance between the metal ion [nickel(II) or ruthenium(II)] located in the cavity of the macrocycle and the C21 carbon atom. The Ni─C21 bond length is 2.004(4) Å[\\n##UREF##16##\\n29\\n##\\n] in NiMeP, while in NiMePIrCp* and NiMePRuCym it is 2.022(1) Å and 2.015(2) Å, respectively. For RuSPy‐containing systems, this bond length alteration is even less pronounced: from 2.118(3) Å in the metalloligand to 2.110(2) and 2.116(1) Å in RuSPyIrCp* and RuSPyRhCp*, respectively. Analyses of the out‐of‐plane porphyrin ring distortions were carried out using the PorphStruct tool[\\n##REF##34061410##\\n40\\n##\\n] which was based on the normal‐coordinate structure decomposition (NSD) approach[\\n##UREF##22##\\n41\\n##, ##REF##9533688##\\n42\\n##\\n] (Table ##TAB##1##\\n2\\n## and Figure ##FIG##5##\\n5\\n##). The comparative NSD analyses applied for starting systems, i.e., ClNCP, NiMeP, and RuSPy, indicated a moderate effect of the ortho‐metallation on the total out‐of‐plane distortion of the porphyrin (Doop\\n). In fact, in some instances (Cl\\nNCPIrCp*, RuSPyIrCp*, RuSPyRhCp*, Table ##TAB##1##2##, entries 2, 7, and 8, respectively) the chelation at the NCP perimeter led to a less pronounced displacement than that observed in the starting ligand (NCP, entry 1; RuSPy, entry 6). The most significant deviation increase due to ortho‐metallation was observed for the NiMeP metalloligand (Table ##TAB##1##2##, entry 3) upon chelation of RuCymCl moiety (entry 5). All systems indicated a significant saddling distortion which, again, increased only in NiMePRuCym. The increasing doming, ruffling, and waving distortions were observed for almost all systems upon external chelation, although these components gave rather minor contributions to Doop\\n which is dominated by the saddling. Displacement of metal ions from the mean plane of the porphyrin in a metalloligand (dM‐mpln\\n) slightly increased after an external metal ion was introduced, though an opposite effect was observed for the displacement from an MCNNN mean plane (dM‐ccpln\\n). A common structural feature for the complexes under study as well as for NCP free base, is a pronounced deviation of the confused pyrrole plane from that of the porphyrin ring. Such a deviation can be parametrized by a dihedral angle (DH, Table ##TAB##1##2##) between the confused pyrrole mean plane and the mean plane defined by all non‐hydrogen atoms of the macrocycle, except N2, C3, and C21. Apparently, a significant increase of this angle upon external chelation was observed only for the NiMeP‐containing complexes (Table ##TAB##1##2##, entries 4 and 5). The individual atom displacements from the mean plane are typical for the saddle‐distorted porphyrins with alternate directions of the pyrrole deviation (Figure ##FIG##5##5##). Significantly, in the bimetallic systems NiMePIrCp* and NiMePRuCym, the displacement of the meso‐carbons is significantly more pronounced than in the starting NiMeP, where those atoms are located almost in the mean plane. It is particularly evident for C20 and is related to the coordination of N2 and the aryl at C20. The external chelation slightly increases the displacement of N2 and C3 in these two systems with respect to the metalloligand, in line with the increasing DH. Generally, the atoms are more displaced in NiMeP and its ortho‐metallated derivatives than in RuSPy and its complexes.
\",\n \"
All ortho‐metallated complexes are chiral, as are their precursors in the solid state. However, these systems crystallized in centrosymmetric space groups as racemates and in Figure ##FIG##4##4## only one enantiomer for each of the complexes has been shown.
\",\n \"Chirality\",\n \"
Our attempt to separate enantiomers of the externally metallated NCP derivatives involved HPLC methods with a chiral stationary phase. The enantiomers of several of these systems are presented in Figure ##FIG##6##\\n6\\n## as they appear in the solid‐state structures, along with definitions of their absolute configurations. For these definitions, we took an external metallacycle which is common to all these ortho‐metallated systems, i.e., M─N2─C1─C20─C\\nipso\\n─C\\northo\\n as a chirality plane.
\",\n \"
Although metalloligands NiMeP and RuSPy are intrinsically chiral due to the presence of a chirality center at the coordinated C21,[\\n##REF##21226119##\\n43\\n##\\n] the ortho‐metallation introduces its own chirality related to a differentiation of the porphyrin faces. Apparently, these two chirality sources are not independent, that is, upon external chelation of the racemic mixture of NiMeP or RuSPy only a pair of enantiomers is formed and no other NMR‐distinguishable stereoisomers can be observed. Hence, the external metallation is stereoselective although two diastereomers can be potentially formed, differing in orientation of chloride and the organometallic ligands (Cp* or Cym) at the externally chelated metal with respect to the macrocycle. The crystal structures reveal roughly the same orientation of the chloride ligand at the external metal center and the substituent at C21. Thus, the chirality of the bimetallic monomers in the solid state and solution is predefined by the starting metalloligands[\\n##REF##21226119##\\n43\\n##, ##UREF##23##\\n44\\n##\\n] with absolute configurations S or R at C21 in the metalloligands invariantly giving rise to the configurations P or M, respectively in the ortho‐metallated complexes. The separation of the bimetallic enantiomers by the HPLC method was expected to be effective through two approaches (Scheme ##FIG##7##\\n2\\n##). The first method involved a separation of enantiomers prior to the external chelation (Figure ##FIG##8##\\n7A,B,C,E##), while in the second approach, separation proceeded ortho‐metallation (Figure ##FIG##8##7D,F##; Figures ##SUPPL##0##S29## and ##SUPPL##0##S30##, Supporting Information). The first method allows high enantiopurity of the bimetallic systems and, in principle, can be applied for many other metal ions resulting in chirality transfer from the metalloligand toward the external metal center which may be a site of catalytic reaction. Importantly, the absolute configurations of such complexes can be deduced directly from the absolute configuration of the metalloligand. The second method is more useful for the systems for which the external chelation is less effective, such as NiMePRhCp* or RuSPyRhCp*.
\",\n \"
The asymmetry of these configurationally stable systems arises from the presence of the chiral center at the C21 atom. Conversely, the NCP free base is chiral in the solid state owing to its non‐planarity but in solution, the molecule is configurationally unstable and no enantiomer separation is possible. This is due to a flipping of the confused pyrrole allowing fast interconversion of the enantiomers, unlike in several 21‐substituted NCP derivatives.[\\n##REF##22924766##\\n45\\n##, ##REF##24601636##\\n46\\n##\\n] Thus, chelation of metal ion by N2 and the ortho carbon atom of the adjacent meso‐aryl such as in ClNCPIrCp* or NCPPtPPh3\\n[\\n##UREF##13##\\n25\\n##\\n] as well as double chelation in directly bound 3,3′‐(NCP)2Pt[\\n##UREF##15##\\n28\\n##\\n] is sufficient to stabilize the configuration. The external ortho‐metallation provides a lock preventing fast interconversion of enantiomers and participates in the differentiation of the macrocycle faces. Thus, for ClNCPIrCp*, we were able to separate enantiomers and record their circular dichroic spectra (Figures ##SUPPL##0##S27## and ##SUPPL##0##S28##, Supporting Information) indicating the chirality of this system and its configurational stability. The absolute configurations of the separated enantiomers were assigned based on TD–DFT simulations of the CD spectra (Figures ##SUPPL##0##S23–S27##, Tables ##SUPPL##0##S7##–##SUPPL##0##S12##, Supporting Information).
\",\n \"Redox Properties\",\n \"
The electrochemical properties of the ortho‐metallated complexes were studied by means of cyclic and differential pulse voltammetry (Figure ##FIG##9##\\n8\\n##; Figure ##SUPPL##0##S35##, Supporting Information). The electrode potentials were collected in Table ##TAB##2##\\n3\\n##. For all but one complex system the first oxidations were reversible, and for a majority, the second oxidations appeared to be reversible as well. Conversely, for almost all complexes there was no reversible reduction. Oxidation potentials were relatively low and the external coordination did not significantly affect the first oxidation potentials compared to the metalloligands NiMeP and RuSPy (Table ##TAB##2##3##, entries 8, 9) or ligand ClNCP (Table ##TAB##2##3##, entry 10). It was not the case for RuSPyIrCp* and RuSPyRuCym (Table ##TAB##2##3##, entries 5 and 7) for which 90 and 190 mV cathodic shifts were observed, respectively.
\",\n \"
The redox properties of the ortho‐metallated species and their precursors can be analyzed theoretically through comparison of the frontier orbitals energies (Figure ##FIG##10##\\n9\\n##). Apparently, the calculated HOMO energies are very similar for all these systems with only a small rise of potential on going from RuSPy to the ortho‐metallated derivatives which can also be noticed experimentally as an increase of oxidation potentials. The DFT‐calculated HOMO energies are in good agreement with those estimated based on the first oxidation potentials [calculated as −e(E\\nOx1 + 4.8 V)], but LUMO energies are systematically higher than those derived from the first reduction potentials E\\nRed1 [calculated as −e(E\\nRed1 + 4.8 V)]. Consequently, the electrochemical HOMO–LUMO gaps [calculated as e(E\\nOx1 − E\\nRed1)] are considerably smaller (by 0.4–0.6 eV) in comparison with those based on DFT calculations (cHLG in Table ##TAB##2##3##). On the other hand, the optical HOMO–LUMO energy gaps (oHLG in Table ##TAB##2##3##.), obtained from the experimental UV–vis spectra are even lower (by 0.1–0.2 eV) than the values derived from the electrochemical potentials.
\",\n \"
Spectrophotometric titration of both NiMeP and RuSPy\\northo‐metallated derivatives with tris(4‐bromophenyl)ammoniumyl hexachloroantimonate (BAHA, Magic blue), a one‐electron oxidant of reduction potential 0.70 V[\\n##REF##11848774##\\n47\\n##\\n] allowed monitoring of the spectral changes upon oxidation (Figure ##FIG##11##\\n10\\n##). According to our electrochemical data, this oxidant was sufficiently strong for the first and the second oxidation to be achieved for all systems, except RuSPy for which E\\nOx2 is too high (Table ##TAB##2##3##, entry 9). The observed changes in the spectral region between 400 and 800 nm upon the addition of one equivalent of BAHA, suggested metal‐centered oxidation in all the bimetallic systems (Figure ##FIG##11##10A,D##; Figure ##SUPPL##0##S31##–##SUPPL##0##S34##, Supporting Information). For the RuSPyMCp* complexes (M = IrIII, RhIII), the first oxidation resulted in an increase of the Soret‐like band intensity with a small (Δλ = 15 nm) bathochromic shift and a decrease of the Q‐type band (Figure ##FIG##11##10A##; Figure ##SUPPL##0##S34##, Supporting Information). Such changes suggest that the conjugation of the π‐electrons of the aromatic macrocycle remains intact after one electron is removed. Further addition of BAHA resulted in a gradual increase of the absorbance in the NIR region at ≈1500 nm which is indicative of a radical species, thus suggesting a ligand‐centered second oxidation process. These spectral changes are in sharp contrast to those observed for the RuSPy metalloligand for which a pronounced decrease of the Soret‐like and an increase of the Q‐like bands were observed and no further spectral alteration occurred after passing 1 equiv of BAHA (Figure ##FIG##11##10B##). Such a pattern of spectral changes may suggest a porphyrin‐centered oxidation to the cation metalloradical rather than Ru‐centered oxidation. On the other hand, titration of ClNCPIrCp* with BAHA indicated an initial decrease of the Soret‐like band at 448 nm and its bathochromic shift up to 463 nm upon the addition of 1 equiv of the oxidant. The changes were followed by an increase in intensity of this band with a further redshift to 469 nm which was accompanied by the absorbance increase at 816 nm and the formation of the broadband at 1500 nm when approaching 2 equiv of BAHA (Figure ##SUPPL##0##S31##, Supporting Information). Thus, apparently despite oxidation of the “empty” macrocycle in ClNCPIrCp* the aromaticity of the system is retained and typical spectral features of the porphyrinoids, i.e., the strong Soret‐like band and weaker Q‐type bands are observed even for the two‐electron oxidized species. The first oxidations of the systems comprising NiMeP are nickel‐centered. The highly anisotropic orthorhombic frozen dichloromethane ESR spectra obtained by the BAHA addition (Figure ##FIG##10##9C##) closely resemble those of various 21‐alkylated nickel(III) NCP species.[\\n##REF##12467409##\\n32\\n##, ##REF##12950206##\\n33\\n##, ##UREF##24##\\n48\\n##, ##REF##17269762##\\n49\\n##\\n] For NiMePIrCp*, the addition of more than 1.5 equiv of BAHA resulted in a gradual decrease of the nickel(III) signal intensity, moderate changes of the Zeeman tensor components, and in the appearance of a radical signal at g = 2.0029. Also, spectrophotometric titration with BAHA revealed fine changes in the Soret and Q regions up to 1.2 equiv, followed by a gradual increase of the band at 435 nm and the final appearance of the NIR band upon the addition of more than 2 equiv of the oxidant (Figure ##FIG##10##9D##).[\\n##UREF##25##\\n50\\n##\\n] Interestingly, for the very similar complex, i.e., NiMePRhCp*, the changes of the ESR spectra upon the addition of 1.5 or more equivalents of BAHA are different, involving slight alteration of g\\n2 and g\\n3 components, not the appearance of the radical signal (Figure ##SUPPL##0##S35##, Supporting Information). Similarly, the addition of BAHA to the solution of NiMePRuCym gave rise to an orthorhombic spectrum in frozen DCM (Figure ##SUPPL##0##S36##, Supporting Information) but no strong radical signal was observed upon the addition of more than 2 equiv of BAHA. For reference purposes, we performed also the ESR‐monitored oxidation for the starting metalloligand NiMeP indicating a decrease of the Zeeman tensor anisotropy upon the addition of an excess of BAHA with changes of g\\n1 from 2.382 to 2.285, g\\n2 from 2.168 to 2.2019, and g\\n3 from 2.086 to 2.111 for the spectra recorded in the presence of 1.2 and 3 equiv, respectively (Figure ##SUPPL##0##S37##, Supporting Information). Again, no accompanying radical formation was observed. The differences among the systems comprising NiMeP in the second oxidation potentials and ESR behavior are surely related to the external coordination, although no clear tendencies can be derived from such a limited data set. It can be also rationally expected that for both groups of the ortho‐metallated complexes, oxidation of the metalloligand strongly affects electron density in the environment of the externally coordinated metal ion.
\",\n \"Catalysis\",\n \"
For preliminary studies of the catalytic function of the externally ortho‐metallated systems, we chose N‐heterocyclization reaction of benzylamine with 1,6‐hexanediol which has been shown to be effectively catalyzed by iridium(III) complex [IrCl2Cp*]2 in toluene under basic conditions (Table ##TAB##3##\\n4\\n##).[\\n##REF##15387539##\\n51\\n##\\n] Small‐scale reactions (0.5 mmol) were carried out in the presence of all ortho‐metallated iridium(III) complexes described in this paper as well as for the original catalyst [IrCl2Cp*]2, ClNCP ligand, and both metalloligands, for the reference. The samples were prepared in the inert and dry atmosphere of a glove box to avoid any interference of oxygen and moisture with the reagents, and the reactions were carried out in sealed vials. The reaction results were analyzed qualitatively using GC/MS and quantitatively using the GC/FID technique, and for the selected systems, 1H NMR quantitative analysis was applied with 2,4,6‐collidine as the internal standard. As expected, only iridium(III)‐comprising systems appeared to be catalytically active in the heterocyclizaction reaction, while neither of the ligands supported the 7‐membered ring formation. The best catalyst, i.e., ClNCPIrCp* (Table ##TAB##3##4##, entry 6) gave rise to the yield exceeding that of the original catalyst [IrCl2Cp*]2 with higher glycol conversion and better chemoselectivity. Also NiMePIrCp* seemed to be a more selective catalyst than [IrCl2Cp*]2 with a similar yield of the main reaction product (Table ##TAB##3##4##, entries 2 and 1). Surprisingly, no heterocyclization was observed for RuSPyIrCp* used as a catalyst, despite several attempts. It may be due to a less labile character of the Ir─Cl bond in this complex than in other systems. According to the proposed reaction mechanism,[\\n##REF##15387539##\\n51\\n##\\n] in the early stage of the catalytic process, coordination of the glycolic anion to the iridium center is required which implies chloride substitution (originally present in the ortho‐metallated systems). A significantly longer C─Ir bond in RuSPyIrCp* (2.083(5) Å) than those in NiMePIrCp* (2.041(3) Å) or ClNCPIrCp* (2.051(6) Å) may be responsible for a weaker trans effect of the meso‐aryl carbanion coordinated to the iridium(III) center on the opposite side to the chloride, making its exchange less effective. Of course, some other structural features, such as the presence and type of the metal ion within the macrocyclic cavity, flexibility of the molecular skeleton and its deformations, etc., may be decisive for the catalytic activity of the iridium complexes. Although at the present stage, we cannot offer any more conclusive accounting for the observed differences, it is clear that distinctions in the reaction yields among the systems used in this study indicate an influence of the porphyrin‐chelating ligand on the catalytic activity of the externally coordinated metal center.
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","Syntheses and Characterizations","
As starting materials for our syntheses of bis‐metallic systems, we chose two previously reported compounds bearing a substituent at the C21 position coordinated to either nickel(II)[\n##UREF##16##\n29\n##, ##REF##11151364##\n30\n##, ##UREF##17##\n31\n##, ##REF##12467409##\n32\n##, ##REF##12950206##\n33\n##\n] or ruthenium(II).[\n##UREF##18##\n34\n##, ##REF##31944014##\n35\n##\n] The common features of these, otherwise different complexes are chirality, significant deviation from planarity of the porphyrin ring in the region of confused pyrrole, and unoccupied/non‐protonated N2. These systems were subjected to reaction with organometallic dimeric complexes of ruthenium(II), rhodium(III), or iridium(III) with two chlorides and either neutral η6‐para‐cymene (Cym) or η5‐pentamethylcyclopentadienyl anion (Cp*). Under mild conditions (reflux of DCM solution in the presence of sodium acetate as a proton scavenger) the reaction resulted in the substitution of one chloride by carbanionic ortho‐C20 and coordination of N2 atoms (Scheme ##FIG##1##\n1\n##). The ortho‐metallation efficacy varied depending on the metal ion from 35–40% reaction yield for RhIII, 54–60% for RuII, and up to 85–93% for IrIII. Slightly better results were obtained for NiMeP compared with those for RuSPy. We showed also that under the analogous condition, the reaction of [IrCl2Cp*]2 with ClNCP free base yielded ortho‐metallated derivative ClNCPIrCp* (Scheme ##FIG##1##1##) with a good outcome (83% yield).
","
The new complexes were characterized by high‐resolution mass spectrometry, 1H and 13C NMR, including homo‐ and heteronuclear 2D correlation experiments, UV–vis–NIR spectroscopy, and single crystal XRD analyses. The ESI+ HRMS indicated the presence of the incoming metal ion as well as Cym or Cp* moieties in each case, though all cations were stripped of chloride. The 1H NMR spectra revealed the preservation of most of the spectral features typical of NiMeP, RuSPy, or ClNCP in the complexes formed and the appearance of new signals related to the presence of Cp* or Cym (Figure ##FIG##2##\n2\n##). Thus, the spectra of the systems with Cp* comprise a very strong (15H) signal at about δ 0.8 ppm due to methyl protons, indicating fast rotation around the M–Cp* direction. On the other hand, the differentiation of aryl and isopropyl resonances of the RuCym moiety is in line with the slow motions of the Cym ligand at the 1H NMR time scale. Moreover, an upfield shift of all the Cym protons with respect to those observed for starting [RuCl2Cym]2, indicates the position of this moiety over the aromatic frame of the macrocyclic ligands. Significantly, this aromatic ring shielding effect is strongest for the cymene methyl Me\nc\n (Δδ 0.7–1.4 ppm) suggesting the orientation of the ligand with the isopropyl group situated rather outside, while Me\nc\n is above the macrocycle. The number of meso‐aryl distinct signals in the spectra of RuSPyIrCp* and RuSPyRuCym recorded at room temperature, reflects diastereotopic inequivalence of the macrocyclic faces and slow, at the NMR timescale, rotation of these substituents around Cipso─Cmeso bonds, resulting in differentiation of ortho‐ and meta‐H resonances of phenyls at C5, C10, and C15. Conversely, these meso‐phenyl protons in NiMePIrCp* and NiMePRuCym give rise to severely broadened signals indicating intermediate rotation rate of the substituents and diastereotopicity of the complex faces. The only sharp meso‐aryl signals are those of phenyl at the C20 position due to the complete freezing of its rotation by ortho‐metallation. The chemical shifts and coupling patterns are similar in all these ortho‐metallated complexes comprising a doublet for 20‐m at about 8.3 ppm, a doublet for 20‐o′ at about 7.7 ppm, and two triplets for 20‐m′ and 20‐p at ≈7.3 and 7.2 ppm, respectively (Figure ##FIG##2##2##). For the ortho‐metallated rhodium(III) complexes, NiMePRhCp* and RuSPyRhCp*, a close resemblance of
","
\n1H NMR spectral patterns to those of the respective IrIII systems were observed. The major difference is an additional splitting of all multiplets of the 20‐phenyl protons due to 1H‐103Rh spin–spin coupling (JRhH\n = 1.3–1.5 Hz). Significantly, the ortho‐carbon of the C20 phenyl substituent, identified on the basis of 1H,13C HSQC and 1H,13C HMBC experiments (Figure ##SUPPL##0##S7B## and ##SUPPL##0##S13B##, Supporting Information), gives rise to a doublet at δC\n 169.8 ppm with 1\nJRhC\n = 31.1 Hz for NiMePRhCp* and at δC\n 169.9 ppm with 1\nJRhC\n = 30.9 Hz for RuSPyRhCp* unequivocally proving coordination of the carbanion. The 13C–103Rh coupling could be observed also for cyclopentadiene carbon atoms in 13C NMR of both complexes giving rise to doublets at about δC\n 96 ppm with 1\nJRhC\n = 6.1 Hz. The 1H NMR characteristics of Cl\nNCPIrCp* differ from the nickel(II) and ruthenium(II) complexes with 21‐CH and 22,24‐NH resonances arising in the upfield region of the spectrum (δ −4.69 and broad signals at δ −1.05, −1.10 ppm, respectively) reflecting a free‐base character of the macrocyclic core. Interestingly, for all pentamethylcyclopentadienyl‐comprising systems, correlations of the coordinated ortho‐C with methyl protons of the η5‐Cp* ligand were observed in the 1H,13C HMBC maps (Figures ##SUPPL##0##S3B##, ##SUPPL##0##S7B##, ##SUPPL##0##S9B##, ##SUPPL##0##S13B##, and ##SUPPL##0##S15B##, Supporting Information), regardless of the metal ion. Such correlations appeared despite the fact that the coupled nuclei were separated by four bonds. The correlations may be accounted for by the anionic character of the Cp* ligand resulting in a relatively high electron density available that enhanced 1H‐13C coupling. Significantly, for the neutral η6‐Cym ligand in NiMePRuCym or RuSPyRuCym, there was no such a correlation observed, in spite of only three bonds separating aryl protons of this ligand and the RuII‐coordinating ortho‐C at 20‐Ph.
","
The electronic spectrum of ClNCPIrCp* resembles that of the starting porphyrin ClNCP with ≈65 nm and 15 nm bathochromic shifts of the lowest‐energy Q band and the Soret band, respectively observed for the complex (Figure ##SUPPL##0##S1##, Supporting Information). For the NiMeP and RuSPy, the spectral changes due to external coordination are much more profound, though similar to each other, regardless of the ortho‐metallated cation (Figure ##FIG##3##\n3\n##). These alterations involve a decrease in the relative intensity of the spectra in the Soret region near 430 nm and an increase of the absorbance in the Q band region, i.e., above 500 nm.
","Crystal Structures","
Solid state structures of the selected ortho‐metallated systems were elucidated using the single crystal X‐ray diffraction analyses (Figure ##FIG##4##\n4\n## Figures ##SUPPL##0##S39–S44##, Supporting Information). The structures determined based on diffraction data clearly showed the coordination mode of iridium(III), ruthenium(II), and rhodium(III) ions to ClNCP or metalloligands NiMeP and RuSPy. Metalloligands bind iridium(III), ruthenium(II), and rhodium(III) ions through the N2 donor atom and the ortho‐carbon of the aryl ring from the C20 meso‐position. The coordination sphere of metal ions located at the periphery of the macrocycle is supplemented by chloride and pentamethylcyclopentadienyl ligands (in ClNCPIrCp*, NiMePIrCp*, RuSPyIrCp*, and RuSPyRhCp*) or chloride and p‐cymene ligands (in NiMePRuCym). Coordinating metal ions at the edges of metalloligands retain a structural motif referred to as a “piano stool” or a half‐sandwich complex.[\n##UREF##19##\n36\n##\n] Data on bond lengths around iridium(III), ruthenium(II), and rhodium(III) ions are summarized in Table ##TAB##0##\n1\n## along with selected bond lengths for the dimeric metal sources. The average M–L distances (where L = Cp* or Cym) are significantly longer than in starting half‐sandwich organometallic chlorides, while M–Cl bond lengths are close to those observed for terminally bound chlorides in the respective dimeric precursors or only slightly longer.[\n##UREF##20##\n37\n##, ##UREF##21##\n38\n##, ##REF##30942794##\n39\n##\n] Interestingly, RuSPyIrCp* and RuSPyRhCp* are isostructural when crystallized from benzene/hexane, both forming tris(benzene) solvates.
","
The steric hindrance introduced by Cp* or Cym ligands forces them to be specifically positioned relative to methyl or mercaptopyridyl substituents at the C21 atom of metalloligand. As a consequence, they are located on opposite sides of the macrocyclic plane, and, in the case of compounds RuSPyIrCp* and RuSPyRhCp*, on the same side as the CO ligand. The specific setting of the p‐cymene relative to the macrocyclic system was established for NiMePRuCym with the isopropyl group above the metalloligand plane. Such an orientation of the Cym ligand is somewhat unexpected considering solution 1H NMR results suggesting methyl rather than the isopropyl group directed toward the macrocycle interior (vide supra). It may be accounted for by packing forces for which the ligand orientation in the crystal with a smaller substituent situated beyond the perimeter of the macrocycle is more favorable. The deviation of the porphyrin ring from planarity due to sp3 hybridization of C21, characteristic for the starting metalloligand NiMeP and RuSPy, is retained upon coordinating the metal ion to the periphery of the macrocycle. However, the external ortho‐metallation does not significantly change the bond distance between the metal ion [nickel(II) or ruthenium(II)] located in the cavity of the macrocycle and the C21 carbon atom. The Ni─C21 bond length is 2.004(4) Å[\n##UREF##16##\n29\n##\n] in NiMeP, while in NiMePIrCp* and NiMePRuCym it is 2.022(1) Å and 2.015(2) Å, respectively. For RuSPy‐containing systems, this bond length alteration is even less pronounced: from 2.118(3) Å in the metalloligand to 2.110(2) and 2.116(1) Å in RuSPyIrCp* and RuSPyRhCp*, respectively. Analyses of the out‐of‐plane porphyrin ring distortions were carried out using the PorphStruct tool[\n##REF##34061410##\n40\n##\n] which was based on the normal‐coordinate structure decomposition (NSD) approach[\n##UREF##22##\n41\n##, ##REF##9533688##\n42\n##\n] (Table ##TAB##1##\n2\n## and Figure ##FIG##5##\n5\n##). The comparative NSD analyses applied for starting systems, i.e., ClNCP, NiMeP, and RuSPy, indicated a moderate effect of the ortho‐metallation on the total out‐of‐plane distortion of the porphyrin (Doop\n). In fact, in some instances (Cl\nNCPIrCp*, RuSPyIrCp*, RuSPyRhCp*, Table ##TAB##1##2##, entries 2, 7, and 8, respectively) the chelation at the NCP perimeter led to a less pronounced displacement than that observed in the starting ligand (NCP, entry 1; RuSPy, entry 6). The most significant deviation increase due to ortho‐metallation was observed for the NiMeP metalloligand (Table ##TAB##1##2##, entry 3) upon chelation of RuCymCl moiety (entry 5). All systems indicated a significant saddling distortion which, again, increased only in NiMePRuCym. The increasing doming, ruffling, and waving distortions were observed for almost all systems upon external chelation, although these components gave rather minor contributions to Doop\n which is dominated by the saddling. Displacement of metal ions from the mean plane of the porphyrin in a metalloligand (dM‐mpln\n) slightly increased after an external metal ion was introduced, though an opposite effect was observed for the displacement from an MCNNN mean plane (dM‐ccpln\n). A common structural feature for the complexes under study as well as for NCP free base, is a pronounced deviation of the confused pyrrole plane from that of the porphyrin ring. Such a deviation can be parametrized by a dihedral angle (DH, Table ##TAB##1##2##) between the confused pyrrole mean plane and the mean plane defined by all non‐hydrogen atoms of the macrocycle, except N2, C3, and C21. Apparently, a significant increase of this angle upon external chelation was observed only for the NiMeP‐containing complexes (Table ##TAB##1##2##, entries 4 and 5). The individual atom displacements from the mean plane are typical for the saddle‐distorted porphyrins with alternate directions of the pyrrole deviation (Figure ##FIG##5##5##). Significantly, in the bimetallic systems NiMePIrCp* and NiMePRuCym, the displacement of the meso‐carbons is significantly more pronounced than in the starting NiMeP, where those atoms are located almost in the mean plane. It is particularly evident for C20 and is related to the coordination of N2 and the aryl at C20. The external chelation slightly increases the displacement of N2 and C3 in these two systems with respect to the metalloligand, in line with the increasing DH. Generally, the atoms are more displaced in NiMeP and its ortho‐metallated derivatives than in RuSPy and its complexes.
","
All ortho‐metallated complexes are chiral, as are their precursors in the solid state. However, these systems crystallized in centrosymmetric space groups as racemates and in Figure ##FIG##4##4## only one enantiomer for each of the complexes has been shown.
","Chirality","
Our attempt to separate enantiomers of the externally metallated NCP derivatives involved HPLC methods with a chiral stationary phase. The enantiomers of several of these systems are presented in Figure ##FIG##6##\n6\n## as they appear in the solid‐state structures, along with definitions of their absolute configurations. For these definitions, we took an external metallacycle which is common to all these ortho‐metallated systems, i.e., M─N2─C1─C20─C\nipso\n─C\northo\n as a chirality plane.
","
Although metalloligands NiMeP and RuSPy are intrinsically chiral due to the presence of a chirality center at the coordinated C21,[\n##REF##21226119##\n43\n##\n] the ortho‐metallation introduces its own chirality related to a differentiation of the porphyrin faces. Apparently, these two chirality sources are not independent, that is, upon external chelation of the racemic mixture of NiMeP or RuSPy only a pair of enantiomers is formed and no other NMR‐distinguishable stereoisomers can be observed. Hence, the external metallation is stereoselective although two diastereomers can be potentially formed, differing in orientation of chloride and the organometallic ligands (Cp* or Cym) at the externally chelated metal with respect to the macrocycle. The crystal structures reveal roughly the same orientation of the chloride ligand at the external metal center and the substituent at C21. Thus, the chirality of the bimetallic monomers in the solid state and solution is predefined by the starting metalloligands[\n##REF##21226119##\n43\n##, ##UREF##23##\n44\n##\n] with absolute configurations S or R at C21 in the metalloligands invariantly giving rise to the configurations P or M, respectively in the ortho‐metallated complexes. The separation of the bimetallic enantiomers by the HPLC method was expected to be effective through two approaches (Scheme ##FIG##7##\n2\n##). The first method involved a separation of enantiomers prior to the external chelation (Figure ##FIG##8##\n7A,B,C,E##), while in the second approach, separation proceeded ortho‐metallation (Figure ##FIG##8##7D,F##; Figures ##SUPPL##0##S29## and ##SUPPL##0##S30##, Supporting Information). The first method allows high enantiopurity of the bimetallic systems and, in principle, can be applied for many other metal ions resulting in chirality transfer from the metalloligand toward the external metal center which may be a site of catalytic reaction. Importantly, the absolute configurations of such complexes can be deduced directly from the absolute configuration of the metalloligand. The second method is more useful for the systems for which the external chelation is less effective, such as NiMePRhCp* or RuSPyRhCp*.
","
The asymmetry of these configurationally stable systems arises from the presence of the chiral center at the C21 atom. Conversely, the NCP free base is chiral in the solid state owing to its non‐planarity but in solution, the molecule is configurationally unstable and no enantiomer separation is possible. This is due to a flipping of the confused pyrrole allowing fast interconversion of the enantiomers, unlike in several 21‐substituted NCP derivatives.[\n##REF##22924766##\n45\n##, ##REF##24601636##\n46\n##\n] Thus, chelation of metal ion by N2 and the ortho carbon atom of the adjacent meso‐aryl such as in ClNCPIrCp* or NCPPtPPh3\n[\n##UREF##13##\n25\n##\n] as well as double chelation in directly bound 3,3′‐(NCP)2Pt[\n##UREF##15##\n28\n##\n] is sufficient to stabilize the configuration. The external ortho‐metallation provides a lock preventing fast interconversion of enantiomers and participates in the differentiation of the macrocycle faces. Thus, for ClNCPIrCp*, we were able to separate enantiomers and record their circular dichroic spectra (Figures ##SUPPL##0##S27## and ##SUPPL##0##S28##, Supporting Information) indicating the chirality of this system and its configurational stability. The absolute configurations of the separated enantiomers were assigned based on TD–DFT simulations of the CD spectra (Figures ##SUPPL##0##S23–S27##, Tables ##SUPPL##0##S7##–##SUPPL##0##S12##, Supporting Information).
","Redox Properties","
The electrochemical properties of the ortho‐metallated complexes were studied by means of cyclic and differential pulse voltammetry (Figure ##FIG##9##\n8\n##; Figure ##SUPPL##0##S35##, Supporting Information). The electrode potentials were collected in Table ##TAB##2##\n3\n##. For all but one complex system the first oxidations were reversible, and for a majority, the second oxidations appeared to be reversible as well. Conversely, for almost all complexes there was no reversible reduction. Oxidation potentials were relatively low and the external coordination did not significantly affect the first oxidation potentials compared to the metalloligands NiMeP and RuSPy (Table ##TAB##2##3##, entries 8, 9) or ligand ClNCP (Table ##TAB##2##3##, entry 10). It was not the case for RuSPyIrCp* and RuSPyRuCym (Table ##TAB##2##3##, entries 5 and 7) for which 90 and 190 mV cathodic shifts were observed, respectively.
","
The redox properties of the ortho‐metallated species and their precursors can be analyzed theoretically through comparison of the frontier orbitals energies (Figure ##FIG##10##\n9\n##). Apparently, the calculated HOMO energies are very similar for all these systems with only a small rise of potential on going from RuSPy to the ortho‐metallated derivatives which can also be noticed experimentally as an increase of oxidation potentials. The DFT‐calculated HOMO energies are in good agreement with those estimated based on the first oxidation potentials [calculated as −e(E\nOx1 + 4.8 V)], but LUMO energies are systematically higher than those derived from the first reduction potentials E\nRed1 [calculated as −e(E\nRed1 + 4.8 V)]. Consequently, the electrochemical HOMO–LUMO gaps [calculated as e(E\nOx1 − E\nRed1)] are considerably smaller (by 0.4–0.6 eV) in comparison with those based on DFT calculations (cHLG in Table ##TAB##2##3##). On the other hand, the optical HOMO–LUMO energy gaps (oHLG in Table ##TAB##2##3##.), obtained from the experimental UV–vis spectra are even lower (by 0.1–0.2 eV) than the values derived from the electrochemical potentials.
","
Spectrophotometric titration of both NiMeP and RuSPy\northo‐metallated derivatives with tris(4‐bromophenyl)ammoniumyl hexachloroantimonate (BAHA, Magic blue), a one‐electron oxidant of reduction potential 0.70 V[\n##REF##11848774##\n47\n##\n] allowed monitoring of the spectral changes upon oxidation (Figure ##FIG##11##\n10\n##). According to our electrochemical data, this oxidant was sufficiently strong for the first and the second oxidation to be achieved for all systems, except RuSPy for which E\nOx2 is too high (Table ##TAB##2##3##, entry 9). The observed changes in the spectral region between 400 and 800 nm upon the addition of one equivalent of BAHA, suggested metal‐centered oxidation in all the bimetallic systems (Figure ##FIG##11##10A,D##; Figure ##SUPPL##0##S31##–##SUPPL##0##S34##, Supporting Information). For the RuSPyMCp* complexes (M = IrIII, RhIII), the first oxidation resulted in an increase of the Soret‐like band intensity with a small (Δλ = 15 nm) bathochromic shift and a decrease of the Q‐type band (Figure ##FIG##11##10A##; Figure ##SUPPL##0##S34##, Supporting Information). Such changes suggest that the conjugation of the π‐electrons of the aromatic macrocycle remains intact after one electron is removed. Further addition of BAHA resulted in a gradual increase of the absorbance in the NIR region at ≈1500 nm which is indicative of a radical species, thus suggesting a ligand‐centered second oxidation process. These spectral changes are in sharp contrast to those observed for the RuSPy metalloligand for which a pronounced decrease of the Soret‐like and an increase of the Q‐like bands were observed and no further spectral alteration occurred after passing 1 equiv of BAHA (Figure ##FIG##11##10B##). Such a pattern of spectral changes may suggest a porphyrin‐centered oxidation to the cation metalloradical rather than Ru‐centered oxidation. On the other hand, titration of ClNCPIrCp* with BAHA indicated an initial decrease of the Soret‐like band at 448 nm and its bathochromic shift up to 463 nm upon the addition of 1 equiv of the oxidant. The changes were followed by an increase in intensity of this band with a further redshift to 469 nm which was accompanied by the absorbance increase at 816 nm and the formation of the broadband at 1500 nm when approaching 2 equiv of BAHA (Figure ##SUPPL##0##S31##, Supporting Information). Thus, apparently despite oxidation of the “empty” macrocycle in ClNCPIrCp* the aromaticity of the system is retained and typical spectral features of the porphyrinoids, i.e., the strong Soret‐like band and weaker Q‐type bands are observed even for the two‐electron oxidized species. The first oxidations of the systems comprising NiMeP are nickel‐centered. The highly anisotropic orthorhombic frozen dichloromethane ESR spectra obtained by the BAHA addition (Figure ##FIG##10##9C##) closely resemble those of various 21‐alkylated nickel(III) NCP species.[\n##REF##12467409##\n32\n##, ##REF##12950206##\n33\n##, ##UREF##24##\n48\n##, ##REF##17269762##\n49\n##\n] For NiMePIrCp*, the addition of more than 1.5 equiv of BAHA resulted in a gradual decrease of the nickel(III) signal intensity, moderate changes of the Zeeman tensor components, and in the appearance of a radical signal at g = 2.0029. Also, spectrophotometric titration with BAHA revealed fine changes in the Soret and Q regions up to 1.2 equiv, followed by a gradual increase of the band at 435 nm and the final appearance of the NIR band upon the addition of more than 2 equiv of the oxidant (Figure ##FIG##10##9D##).[\n##UREF##25##\n50\n##\n] Interestingly, for the very similar complex, i.e., NiMePRhCp*, the changes of the ESR spectra upon the addition of 1.5 or more equivalents of BAHA are different, involving slight alteration of g\n2 and g\n3 components, not the appearance of the radical signal (Figure ##SUPPL##0##S35##, Supporting Information). Similarly, the addition of BAHA to the solution of NiMePRuCym gave rise to an orthorhombic spectrum in frozen DCM (Figure ##SUPPL##0##S36##, Supporting Information) but no strong radical signal was observed upon the addition of more than 2 equiv of BAHA. For reference purposes, we performed also the ESR‐monitored oxidation for the starting metalloligand NiMeP indicating a decrease of the Zeeman tensor anisotropy upon the addition of an excess of BAHA with changes of g\n1 from 2.382 to 2.285, g\n2 from 2.168 to 2.2019, and g\n3 from 2.086 to 2.111 for the spectra recorded in the presence of 1.2 and 3 equiv, respectively (Figure ##SUPPL##0##S37##, Supporting Information). Again, no accompanying radical formation was observed. The differences among the systems comprising NiMeP in the second oxidation potentials and ESR behavior are surely related to the external coordination, although no clear tendencies can be derived from such a limited data set. It can be also rationally expected that for both groups of the ortho‐metallated complexes, oxidation of the metalloligand strongly affects electron density in the environment of the externally coordinated metal ion.
","Catalysis","
For preliminary studies of the catalytic function of the externally ortho‐metallated systems, we chose N‐heterocyclization reaction of benzylamine with 1,6‐hexanediol which has been shown to be effectively catalyzed by iridium(III) complex [IrCl2Cp*]2 in toluene under basic conditions (Table ##TAB##3##\n4\n##).[\n##REF##15387539##\n51\n##\n] Small‐scale reactions (0.5 mmol) were carried out in the presence of all ortho‐metallated iridium(III) complexes described in this paper as well as for the original catalyst [IrCl2Cp*]2, ClNCP ligand, and both metalloligands, for the reference. The samples were prepared in the inert and dry atmosphere of a glove box to avoid any interference of oxygen and moisture with the reagents, and the reactions were carried out in sealed vials. The reaction results were analyzed qualitatively using GC/MS and quantitatively using the GC/FID technique, and for the selected systems, 1H NMR quantitative analysis was applied with 2,4,6‐collidine as the internal standard. As expected, only iridium(III)‐comprising systems appeared to be catalytically active in the heterocyclizaction reaction, while neither of the ligands supported the 7‐membered ring formation. The best catalyst, i.e., ClNCPIrCp* (Table ##TAB##3##4##, entry 6) gave rise to the yield exceeding that of the original catalyst [IrCl2Cp*]2 with higher glycol conversion and better chemoselectivity. Also NiMePIrCp* seemed to be a more selective catalyst than [IrCl2Cp*]2 with a similar yield of the main reaction product (Table ##TAB##3##4##, entries 2 and 1). Surprisingly, no heterocyclization was observed for RuSPyIrCp* used as a catalyst, despite several attempts. It may be due to a less labile character of the Ir─Cl bond in this complex than in other systems. According to the proposed reaction mechanism,[\n##REF##15387539##\n51\n##\n] in the early stage of the catalytic process, coordination of the glycolic anion to the iridium center is required which implies chloride substitution (originally present in the ortho‐metallated systems). A significantly longer C─Ir bond in RuSPyIrCp* (2.083(5) Å) than those in NiMePIrCp* (2.041(3) Å) or ClNCPIrCp* (2.051(6) Å) may be responsible for a weaker trans effect of the meso‐aryl carbanion coordinated to the iridium(III) center on the opposite side to the chloride, making its exchange less effective. Of course, some other structural features, such as the presence and type of the metal ion within the macrocyclic cavity, flexibility of the molecular skeleton and its deformations, etc., may be decisive for the catalytic activity of the iridium complexes. Although at the present stage, we cannot offer any more conclusive accounting for the observed differences, it is clear that distinctions in the reaction yields among the systems used in this study indicate an influence of the porphyrin‐chelating ligand on the catalytic activity of the externally coordinated metal center.
"],"string":"[\n \"Results and Discussion\",\n \"Syntheses and Characterizations\",\n \"
As starting materials for our syntheses of bis‐metallic systems, we chose two previously reported compounds bearing a substituent at the C21 position coordinated to either nickel(II)[\\n##UREF##16##\\n29\\n##, ##REF##11151364##\\n30\\n##, ##UREF##17##\\n31\\n##, ##REF##12467409##\\n32\\n##, ##REF##12950206##\\n33\\n##\\n] or ruthenium(II).[\\n##UREF##18##\\n34\\n##, ##REF##31944014##\\n35\\n##\\n] The common features of these, otherwise different complexes are chirality, significant deviation from planarity of the porphyrin ring in the region of confused pyrrole, and unoccupied/non‐protonated N2. These systems were subjected to reaction with organometallic dimeric complexes of ruthenium(II), rhodium(III), or iridium(III) with two chlorides and either neutral η6‐para‐cymene (Cym) or η5‐pentamethylcyclopentadienyl anion (Cp*). Under mild conditions (reflux of DCM solution in the presence of sodium acetate as a proton scavenger) the reaction resulted in the substitution of one chloride by carbanionic ortho‐C20 and coordination of N2 atoms (Scheme ##FIG##1##\\n1\\n##). The ortho‐metallation efficacy varied depending on the metal ion from 35–40% reaction yield for RhIII, 54–60% for RuII, and up to 85–93% for IrIII. Slightly better results were obtained for NiMeP compared with those for RuSPy. We showed also that under the analogous condition, the reaction of [IrCl2Cp*]2 with ClNCP free base yielded ortho‐metallated derivative ClNCPIrCp* (Scheme ##FIG##1##1##) with a good outcome (83% yield).
\",\n \"
The new complexes were characterized by high‐resolution mass spectrometry, 1H and 13C NMR, including homo‐ and heteronuclear 2D correlation experiments, UV–vis–NIR spectroscopy, and single crystal XRD analyses. The ESI+ HRMS indicated the presence of the incoming metal ion as well as Cym or Cp* moieties in each case, though all cations were stripped of chloride. The 1H NMR spectra revealed the preservation of most of the spectral features typical of NiMeP, RuSPy, or ClNCP in the complexes formed and the appearance of new signals related to the presence of Cp* or Cym (Figure ##FIG##2##\\n2\\n##). Thus, the spectra of the systems with Cp* comprise a very strong (15H) signal at about δ 0.8 ppm due to methyl protons, indicating fast rotation around the M–Cp* direction. On the other hand, the differentiation of aryl and isopropyl resonances of the RuCym moiety is in line with the slow motions of the Cym ligand at the 1H NMR time scale. Moreover, an upfield shift of all the Cym protons with respect to those observed for starting [RuCl2Cym]2, indicates the position of this moiety over the aromatic frame of the macrocyclic ligands. Significantly, this aromatic ring shielding effect is strongest for the cymene methyl Me\\nc\\n (Δδ 0.7–1.4 ppm) suggesting the orientation of the ligand with the isopropyl group situated rather outside, while Me\\nc\\n is above the macrocycle. The number of meso‐aryl distinct signals in the spectra of RuSPyIrCp* and RuSPyRuCym recorded at room temperature, reflects diastereotopic inequivalence of the macrocyclic faces and slow, at the NMR timescale, rotation of these substituents around Cipso─Cmeso bonds, resulting in differentiation of ortho‐ and meta‐H resonances of phenyls at C5, C10, and C15. Conversely, these meso‐phenyl protons in NiMePIrCp* and NiMePRuCym give rise to severely broadened signals indicating intermediate rotation rate of the substituents and diastereotopicity of the complex faces. The only sharp meso‐aryl signals are those of phenyl at the C20 position due to the complete freezing of its rotation by ortho‐metallation. The chemical shifts and coupling patterns are similar in all these ortho‐metallated complexes comprising a doublet for 20‐m at about 8.3 ppm, a doublet for 20‐o′ at about 7.7 ppm, and two triplets for 20‐m′ and 20‐p at ≈7.3 and 7.2 ppm, respectively (Figure ##FIG##2##2##). For the ortho‐metallated rhodium(III) complexes, NiMePRhCp* and RuSPyRhCp*, a close resemblance of
\",\n \"
\\n1H NMR spectral patterns to those of the respective IrIII systems were observed. The major difference is an additional splitting of all multiplets of the 20‐phenyl protons due to 1H‐103Rh spin–spin coupling (JRhH\\n = 1.3–1.5 Hz). Significantly, the ortho‐carbon of the C20 phenyl substituent, identified on the basis of 1H,13C HSQC and 1H,13C HMBC experiments (Figure ##SUPPL##0##S7B## and ##SUPPL##0##S13B##, Supporting Information), gives rise to a doublet at δC\\n 169.8 ppm with 1\\nJRhC\\n = 31.1 Hz for NiMePRhCp* and at δC\\n 169.9 ppm with 1\\nJRhC\\n = 30.9 Hz for RuSPyRhCp* unequivocally proving coordination of the carbanion. The 13C–103Rh coupling could be observed also for cyclopentadiene carbon atoms in 13C NMR of both complexes giving rise to doublets at about δC\\n 96 ppm with 1\\nJRhC\\n = 6.1 Hz. The 1H NMR characteristics of Cl\\nNCPIrCp* differ from the nickel(II) and ruthenium(II) complexes with 21‐CH and 22,24‐NH resonances arising in the upfield region of the spectrum (δ −4.69 and broad signals at δ −1.05, −1.10 ppm, respectively) reflecting a free‐base character of the macrocyclic core. Interestingly, for all pentamethylcyclopentadienyl‐comprising systems, correlations of the coordinated ortho‐C with methyl protons of the η5‐Cp* ligand were observed in the 1H,13C HMBC maps (Figures ##SUPPL##0##S3B##, ##SUPPL##0##S7B##, ##SUPPL##0##S9B##, ##SUPPL##0##S13B##, and ##SUPPL##0##S15B##, Supporting Information), regardless of the metal ion. Such correlations appeared despite the fact that the coupled nuclei were separated by four bonds. The correlations may be accounted for by the anionic character of the Cp* ligand resulting in a relatively high electron density available that enhanced 1H‐13C coupling. Significantly, for the neutral η6‐Cym ligand in NiMePRuCym or RuSPyRuCym, there was no such a correlation observed, in spite of only three bonds separating aryl protons of this ligand and the RuII‐coordinating ortho‐C at 20‐Ph.
\",\n \"
The electronic spectrum of ClNCPIrCp* resembles that of the starting porphyrin ClNCP with ≈65 nm and 15 nm bathochromic shifts of the lowest‐energy Q band and the Soret band, respectively observed for the complex (Figure ##SUPPL##0##S1##, Supporting Information). For the NiMeP and RuSPy, the spectral changes due to external coordination are much more profound, though similar to each other, regardless of the ortho‐metallated cation (Figure ##FIG##3##\\n3\\n##). These alterations involve a decrease in the relative intensity of the spectra in the Soret region near 430 nm and an increase of the absorbance in the Q band region, i.e., above 500 nm.
\",\n \"Crystal Structures\",\n \"
Solid state structures of the selected ortho‐metallated systems were elucidated using the single crystal X‐ray diffraction analyses (Figure ##FIG##4##\\n4\\n## Figures ##SUPPL##0##S39–S44##, Supporting Information). The structures determined based on diffraction data clearly showed the coordination mode of iridium(III), ruthenium(II), and rhodium(III) ions to ClNCP or metalloligands NiMeP and RuSPy. Metalloligands bind iridium(III), ruthenium(II), and rhodium(III) ions through the N2 donor atom and the ortho‐carbon of the aryl ring from the C20 meso‐position. The coordination sphere of metal ions located at the periphery of the macrocycle is supplemented by chloride and pentamethylcyclopentadienyl ligands (in ClNCPIrCp*, NiMePIrCp*, RuSPyIrCp*, and RuSPyRhCp*) or chloride and p‐cymene ligands (in NiMePRuCym). Coordinating metal ions at the edges of metalloligands retain a structural motif referred to as a “piano stool” or a half‐sandwich complex.[\\n##UREF##19##\\n36\\n##\\n] Data on bond lengths around iridium(III), ruthenium(II), and rhodium(III) ions are summarized in Table ##TAB##0##\\n1\\n## along with selected bond lengths for the dimeric metal sources. The average M–L distances (where L = Cp* or Cym) are significantly longer than in starting half‐sandwich organometallic chlorides, while M–Cl bond lengths are close to those observed for terminally bound chlorides in the respective dimeric precursors or only slightly longer.[\\n##UREF##20##\\n37\\n##, ##UREF##21##\\n38\\n##, ##REF##30942794##\\n39\\n##\\n] Interestingly, RuSPyIrCp* and RuSPyRhCp* are isostructural when crystallized from benzene/hexane, both forming tris(benzene) solvates.
\",\n \"
The steric hindrance introduced by Cp* or Cym ligands forces them to be specifically positioned relative to methyl or mercaptopyridyl substituents at the C21 atom of metalloligand. As a consequence, they are located on opposite sides of the macrocyclic plane, and, in the case of compounds RuSPyIrCp* and RuSPyRhCp*, on the same side as the CO ligand. The specific setting of the p‐cymene relative to the macrocyclic system was established for NiMePRuCym with the isopropyl group above the metalloligand plane. Such an orientation of the Cym ligand is somewhat unexpected considering solution 1H NMR results suggesting methyl rather than the isopropyl group directed toward the macrocycle interior (vide supra). It may be accounted for by packing forces for which the ligand orientation in the crystal with a smaller substituent situated beyond the perimeter of the macrocycle is more favorable. The deviation of the porphyrin ring from planarity due to sp3 hybridization of C21, characteristic for the starting metalloligand NiMeP and RuSPy, is retained upon coordinating the metal ion to the periphery of the macrocycle. However, the external ortho‐metallation does not significantly change the bond distance between the metal ion [nickel(II) or ruthenium(II)] located in the cavity of the macrocycle and the C21 carbon atom. The Ni─C21 bond length is 2.004(4) Å[\\n##UREF##16##\\n29\\n##\\n] in NiMeP, while in NiMePIrCp* and NiMePRuCym it is 2.022(1) Å and 2.015(2) Å, respectively. For RuSPy‐containing systems, this bond length alteration is even less pronounced: from 2.118(3) Å in the metalloligand to 2.110(2) and 2.116(1) Å in RuSPyIrCp* and RuSPyRhCp*, respectively. Analyses of the out‐of‐plane porphyrin ring distortions were carried out using the PorphStruct tool[\\n##REF##34061410##\\n40\\n##\\n] which was based on the normal‐coordinate structure decomposition (NSD) approach[\\n##UREF##22##\\n41\\n##, ##REF##9533688##\\n42\\n##\\n] (Table ##TAB##1##\\n2\\n## and Figure ##FIG##5##\\n5\\n##). The comparative NSD analyses applied for starting systems, i.e., ClNCP, NiMeP, and RuSPy, indicated a moderate effect of the ortho‐metallation on the total out‐of‐plane distortion of the porphyrin (Doop\\n). In fact, in some instances (Cl\\nNCPIrCp*, RuSPyIrCp*, RuSPyRhCp*, Table ##TAB##1##2##, entries 2, 7, and 8, respectively) the chelation at the NCP perimeter led to a less pronounced displacement than that observed in the starting ligand (NCP, entry 1; RuSPy, entry 6). The most significant deviation increase due to ortho‐metallation was observed for the NiMeP metalloligand (Table ##TAB##1##2##, entry 3) upon chelation of RuCymCl moiety (entry 5). All systems indicated a significant saddling distortion which, again, increased only in NiMePRuCym. The increasing doming, ruffling, and waving distortions were observed for almost all systems upon external chelation, although these components gave rather minor contributions to Doop\\n which is dominated by the saddling. Displacement of metal ions from the mean plane of the porphyrin in a metalloligand (dM‐mpln\\n) slightly increased after an external metal ion was introduced, though an opposite effect was observed for the displacement from an MCNNN mean plane (dM‐ccpln\\n). A common structural feature for the complexes under study as well as for NCP free base, is a pronounced deviation of the confused pyrrole plane from that of the porphyrin ring. Such a deviation can be parametrized by a dihedral angle (DH, Table ##TAB##1##2##) between the confused pyrrole mean plane and the mean plane defined by all non‐hydrogen atoms of the macrocycle, except N2, C3, and C21. Apparently, a significant increase of this angle upon external chelation was observed only for the NiMeP‐containing complexes (Table ##TAB##1##2##, entries 4 and 5). The individual atom displacements from the mean plane are typical for the saddle‐distorted porphyrins with alternate directions of the pyrrole deviation (Figure ##FIG##5##5##). Significantly, in the bimetallic systems NiMePIrCp* and NiMePRuCym, the displacement of the meso‐carbons is significantly more pronounced than in the starting NiMeP, where those atoms are located almost in the mean plane. It is particularly evident for C20 and is related to the coordination of N2 and the aryl at C20. The external chelation slightly increases the displacement of N2 and C3 in these two systems with respect to the metalloligand, in line with the increasing DH. Generally, the atoms are more displaced in NiMeP and its ortho‐metallated derivatives than in RuSPy and its complexes.
\",\n \"
All ortho‐metallated complexes are chiral, as are their precursors in the solid state. However, these systems crystallized in centrosymmetric space groups as racemates and in Figure ##FIG##4##4## only one enantiomer for each of the complexes has been shown.
\",\n \"Chirality\",\n \"
Our attempt to separate enantiomers of the externally metallated NCP derivatives involved HPLC methods with a chiral stationary phase. The enantiomers of several of these systems are presented in Figure ##FIG##6##\\n6\\n## as they appear in the solid‐state structures, along with definitions of their absolute configurations. For these definitions, we took an external metallacycle which is common to all these ortho‐metallated systems, i.e., M─N2─C1─C20─C\\nipso\\n─C\\northo\\n as a chirality plane.
\",\n \"
Although metalloligands NiMeP and RuSPy are intrinsically chiral due to the presence of a chirality center at the coordinated C21,[\\n##REF##21226119##\\n43\\n##\\n] the ortho‐metallation introduces its own chirality related to a differentiation of the porphyrin faces. Apparently, these two chirality sources are not independent, that is, upon external chelation of the racemic mixture of NiMeP or RuSPy only a pair of enantiomers is formed and no other NMR‐distinguishable stereoisomers can be observed. Hence, the external metallation is stereoselective although two diastereomers can be potentially formed, differing in orientation of chloride and the organometallic ligands (Cp* or Cym) at the externally chelated metal with respect to the macrocycle. The crystal structures reveal roughly the same orientation of the chloride ligand at the external metal center and the substituent at C21. Thus, the chirality of the bimetallic monomers in the solid state and solution is predefined by the starting metalloligands[\\n##REF##21226119##\\n43\\n##, ##UREF##23##\\n44\\n##\\n] with absolute configurations S or R at C21 in the metalloligands invariantly giving rise to the configurations P or M, respectively in the ortho‐metallated complexes. The separation of the bimetallic enantiomers by the HPLC method was expected to be effective through two approaches (Scheme ##FIG##7##\\n2\\n##). The first method involved a separation of enantiomers prior to the external chelation (Figure ##FIG##8##\\n7A,B,C,E##), while in the second approach, separation proceeded ortho‐metallation (Figure ##FIG##8##7D,F##; Figures ##SUPPL##0##S29## and ##SUPPL##0##S30##, Supporting Information). The first method allows high enantiopurity of the bimetallic systems and, in principle, can be applied for many other metal ions resulting in chirality transfer from the metalloligand toward the external metal center which may be a site of catalytic reaction. Importantly, the absolute configurations of such complexes can be deduced directly from the absolute configuration of the metalloligand. The second method is more useful for the systems for which the external chelation is less effective, such as NiMePRhCp* or RuSPyRhCp*.
\",\n \"
The asymmetry of these configurationally stable systems arises from the presence of the chiral center at the C21 atom. Conversely, the NCP free base is chiral in the solid state owing to its non‐planarity but in solution, the molecule is configurationally unstable and no enantiomer separation is possible. This is due to a flipping of the confused pyrrole allowing fast interconversion of the enantiomers, unlike in several 21‐substituted NCP derivatives.[\\n##REF##22924766##\\n45\\n##, ##REF##24601636##\\n46\\n##\\n] Thus, chelation of metal ion by N2 and the ortho carbon atom of the adjacent meso‐aryl such as in ClNCPIrCp* or NCPPtPPh3\\n[\\n##UREF##13##\\n25\\n##\\n] as well as double chelation in directly bound 3,3′‐(NCP)2Pt[\\n##UREF##15##\\n28\\n##\\n] is sufficient to stabilize the configuration. The external ortho‐metallation provides a lock preventing fast interconversion of enantiomers and participates in the differentiation of the macrocycle faces. Thus, for ClNCPIrCp*, we were able to separate enantiomers and record their circular dichroic spectra (Figures ##SUPPL##0##S27## and ##SUPPL##0##S28##, Supporting Information) indicating the chirality of this system and its configurational stability. The absolute configurations of the separated enantiomers were assigned based on TD–DFT simulations of the CD spectra (Figures ##SUPPL##0##S23–S27##, Tables ##SUPPL##0##S7##–##SUPPL##0##S12##, Supporting Information).
\",\n \"Redox Properties\",\n \"
The electrochemical properties of the ortho‐metallated complexes were studied by means of cyclic and differential pulse voltammetry (Figure ##FIG##9##\\n8\\n##; Figure ##SUPPL##0##S35##, Supporting Information). The electrode potentials were collected in Table ##TAB##2##\\n3\\n##. For all but one complex system the first oxidations were reversible, and for a majority, the second oxidations appeared to be reversible as well. Conversely, for almost all complexes there was no reversible reduction. Oxidation potentials were relatively low and the external coordination did not significantly affect the first oxidation potentials compared to the metalloligands NiMeP and RuSPy (Table ##TAB##2##3##, entries 8, 9) or ligand ClNCP (Table ##TAB##2##3##, entry 10). It was not the case for RuSPyIrCp* and RuSPyRuCym (Table ##TAB##2##3##, entries 5 and 7) for which 90 and 190 mV cathodic shifts were observed, respectively.
\",\n \"
The redox properties of the ortho‐metallated species and their precursors can be analyzed theoretically through comparison of the frontier orbitals energies (Figure ##FIG##10##\\n9\\n##). Apparently, the calculated HOMO energies are very similar for all these systems with only a small rise of potential on going from RuSPy to the ortho‐metallated derivatives which can also be noticed experimentally as an increase of oxidation potentials. The DFT‐calculated HOMO energies are in good agreement with those estimated based on the first oxidation potentials [calculated as −e(E\\nOx1 + 4.8 V)], but LUMO energies are systematically higher than those derived from the first reduction potentials E\\nRed1 [calculated as −e(E\\nRed1 + 4.8 V)]. Consequently, the electrochemical HOMO–LUMO gaps [calculated as e(E\\nOx1 − E\\nRed1)] are considerably smaller (by 0.4–0.6 eV) in comparison with those based on DFT calculations (cHLG in Table ##TAB##2##3##). On the other hand, the optical HOMO–LUMO energy gaps (oHLG in Table ##TAB##2##3##.), obtained from the experimental UV–vis spectra are even lower (by 0.1–0.2 eV) than the values derived from the electrochemical potentials.
\",\n \"
Spectrophotometric titration of both NiMeP and RuSPy\\northo‐metallated derivatives with tris(4‐bromophenyl)ammoniumyl hexachloroantimonate (BAHA, Magic blue), a one‐electron oxidant of reduction potential 0.70 V[\\n##REF##11848774##\\n47\\n##\\n] allowed monitoring of the spectral changes upon oxidation (Figure ##FIG##11##\\n10\\n##). According to our electrochemical data, this oxidant was sufficiently strong for the first and the second oxidation to be achieved for all systems, except RuSPy for which E\\nOx2 is too high (Table ##TAB##2##3##, entry 9). The observed changes in the spectral region between 400 and 800 nm upon the addition of one equivalent of BAHA, suggested metal‐centered oxidation in all the bimetallic systems (Figure ##FIG##11##10A,D##; Figure ##SUPPL##0##S31##–##SUPPL##0##S34##, Supporting Information). For the RuSPyMCp* complexes (M = IrIII, RhIII), the first oxidation resulted in an increase of the Soret‐like band intensity with a small (Δλ = 15 nm) bathochromic shift and a decrease of the Q‐type band (Figure ##FIG##11##10A##; Figure ##SUPPL##0##S34##, Supporting Information). Such changes suggest that the conjugation of the π‐electrons of the aromatic macrocycle remains intact after one electron is removed. Further addition of BAHA resulted in a gradual increase of the absorbance in the NIR region at ≈1500 nm which is indicative of a radical species, thus suggesting a ligand‐centered second oxidation process. These spectral changes are in sharp contrast to those observed for the RuSPy metalloligand for which a pronounced decrease of the Soret‐like and an increase of the Q‐like bands were observed and no further spectral alteration occurred after passing 1 equiv of BAHA (Figure ##FIG##11##10B##). Such a pattern of spectral changes may suggest a porphyrin‐centered oxidation to the cation metalloradical rather than Ru‐centered oxidation. On the other hand, titration of ClNCPIrCp* with BAHA indicated an initial decrease of the Soret‐like band at 448 nm and its bathochromic shift up to 463 nm upon the addition of 1 equiv of the oxidant. The changes were followed by an increase in intensity of this band with a further redshift to 469 nm which was accompanied by the absorbance increase at 816 nm and the formation of the broadband at 1500 nm when approaching 2 equiv of BAHA (Figure ##SUPPL##0##S31##, Supporting Information). Thus, apparently despite oxidation of the “empty” macrocycle in ClNCPIrCp* the aromaticity of the system is retained and typical spectral features of the porphyrinoids, i.e., the strong Soret‐like band and weaker Q‐type bands are observed even for the two‐electron oxidized species. The first oxidations of the systems comprising NiMeP are nickel‐centered. The highly anisotropic orthorhombic frozen dichloromethane ESR spectra obtained by the BAHA addition (Figure ##FIG##10##9C##) closely resemble those of various 21‐alkylated nickel(III) NCP species.[\\n##REF##12467409##\\n32\\n##, ##REF##12950206##\\n33\\n##, ##UREF##24##\\n48\\n##, ##REF##17269762##\\n49\\n##\\n] For NiMePIrCp*, the addition of more than 1.5 equiv of BAHA resulted in a gradual decrease of the nickel(III) signal intensity, moderate changes of the Zeeman tensor components, and in the appearance of a radical signal at g = 2.0029. Also, spectrophotometric titration with BAHA revealed fine changes in the Soret and Q regions up to 1.2 equiv, followed by a gradual increase of the band at 435 nm and the final appearance of the NIR band upon the addition of more than 2 equiv of the oxidant (Figure ##FIG##10##9D##).[\\n##UREF##25##\\n50\\n##\\n] Interestingly, for the very similar complex, i.e., NiMePRhCp*, the changes of the ESR spectra upon the addition of 1.5 or more equivalents of BAHA are different, involving slight alteration of g\\n2 and g\\n3 components, not the appearance of the radical signal (Figure ##SUPPL##0##S35##, Supporting Information). Similarly, the addition of BAHA to the solution of NiMePRuCym gave rise to an orthorhombic spectrum in frozen DCM (Figure ##SUPPL##0##S36##, Supporting Information) but no strong radical signal was observed upon the addition of more than 2 equiv of BAHA. For reference purposes, we performed also the ESR‐monitored oxidation for the starting metalloligand NiMeP indicating a decrease of the Zeeman tensor anisotropy upon the addition of an excess of BAHA with changes of g\\n1 from 2.382 to 2.285, g\\n2 from 2.168 to 2.2019, and g\\n3 from 2.086 to 2.111 for the spectra recorded in the presence of 1.2 and 3 equiv, respectively (Figure ##SUPPL##0##S37##, Supporting Information). Again, no accompanying radical formation was observed. The differences among the systems comprising NiMeP in the second oxidation potentials and ESR behavior are surely related to the external coordination, although no clear tendencies can be derived from such a limited data set. It can be also rationally expected that for both groups of the ortho‐metallated complexes, oxidation of the metalloligand strongly affects electron density in the environment of the externally coordinated metal ion.
\",\n \"Catalysis\",\n \"
For preliminary studies of the catalytic function of the externally ortho‐metallated systems, we chose N‐heterocyclization reaction of benzylamine with 1,6‐hexanediol which has been shown to be effectively catalyzed by iridium(III) complex [IrCl2Cp*]2 in toluene under basic conditions (Table ##TAB##3##\\n4\\n##).[\\n##REF##15387539##\\n51\\n##\\n] Small‐scale reactions (0.5 mmol) were carried out in the presence of all ortho‐metallated iridium(III) complexes described in this paper as well as for the original catalyst [IrCl2Cp*]2, ClNCP ligand, and both metalloligands, for the reference. The samples were prepared in the inert and dry atmosphere of a glove box to avoid any interference of oxygen and moisture with the reagents, and the reactions were carried out in sealed vials. The reaction results were analyzed qualitatively using GC/MS and quantitatively using the GC/FID technique, and for the selected systems, 1H NMR quantitative analysis was applied with 2,4,6‐collidine as the internal standard. As expected, only iridium(III)‐comprising systems appeared to be catalytically active in the heterocyclizaction reaction, while neither of the ligands supported the 7‐membered ring formation. The best catalyst, i.e., ClNCPIrCp* (Table ##TAB##3##4##, entry 6) gave rise to the yield exceeding that of the original catalyst [IrCl2Cp*]2 with higher glycol conversion and better chemoselectivity. Also NiMePIrCp* seemed to be a more selective catalyst than [IrCl2Cp*]2 with a similar yield of the main reaction product (Table ##TAB##3##4##, entries 2 and 1). Surprisingly, no heterocyclization was observed for RuSPyIrCp* used as a catalyst, despite several attempts. It may be due to a less labile character of the Ir─Cl bond in this complex than in other systems. According to the proposed reaction mechanism,[\\n##REF##15387539##\\n51\\n##\\n] in the early stage of the catalytic process, coordination of the glycolic anion to the iridium center is required which implies chloride substitution (originally present in the ortho‐metallated systems). A significantly longer C─Ir bond in RuSPyIrCp* (2.083(5) Å) than those in NiMePIrCp* (2.041(3) Å) or ClNCPIrCp* (2.051(6) Å) may be responsible for a weaker trans effect of the meso‐aryl carbanion coordinated to the iridium(III) center on the opposite side to the chloride, making its exchange less effective. Of course, some other structural features, such as the presence and type of the metal ion within the macrocyclic cavity, flexibility of the molecular skeleton and its deformations, etc., may be decisive for the catalytic activity of the iridium complexes. Although at the present stage, we cannot offer any more conclusive accounting for the observed differences, it is clear that distinctions in the reaction yields among the systems used in this study indicate an influence of the porphyrin‐chelating ligand on the catalytic activity of the externally coordinated metal center.
We have shown here that the ortho‐metallation of the NCP by representative late metals of the second and third rows of transition metals appears to be facilitated by a deviation of the confused pyrrole from the macrocycle mean plane. Such a deviation can be easily achieved when the macrocyclic crevice is empty, and thus, when the ligand is sufficiently flexible to conform the external donor set, i.e., the external N2 atom and a C\northo\n atom of the aryl at the meso‐C20 position to afford chelation. This strong out‐of‐plane deflection also allows the coordination of other ligands supplementing the coordination sphere of the metal. Importantly, in the already known ortho‐metallated NCP complexes, the metals (Pd2+, Pt2+) coordinated to the ligand exterior adopt invariantly a square‐planar geometry. In the present report, we show that a piano‐stool environment is also a suitable geometry for this type of complex, despite the presence of relatively voluminous ligands (Cym or Cp*). Although not as flexible as a free base, the C21‐substituted NCP derivatives coordinating in the macrocyclic interior to Ni2+ in the square planar or Ru2+ in the octahedral environments, comprise the confused pyrrole unit that is permanently deviated from the mean plane of the porphyrinoid. This arrangement makes complexes such as NiMeP or RuSPy effective metalloligands suitable for ortho‐metallation. The ortho‐metallated systems are chiral and can be obtained in a non‐racemic form by either separation of the final bimetallic complexes or by the external metalation of the separated enantiomers of metalloligands, as no racemization is possible upon N2–C\northo\n chelation. Favorably, the external chelation occurs stereoselectively with only one arrangement of the metal environment and the macrocycle. Thus, the chiral information can be transferred from the metalloligand to the externally situated metal center of the asymmetric environment. The redox properties of the bimetallic complexes are not profoundly altered in comparison to those of the appropriate metalloligands as has been shown here by electrochemical studies as well as by spectrophotometric and ESR‐monitored oxidation. Preliminary recognition of an influence of the ortho‐metallated ligand on the catalytic activity of the externally coordinated iridium(III) centers indicated that the heterocyclization reaction is totally absent for the system comprising RuSPy metalloligand (i.e., RuSPyIrCp*), despite the same donor set as in NiMePIrCp* or ClNCPIrCp* that effectively catalyzed this reaction. The study on these and other analogous systems directed toward recognition of their catalytic potential and redox properties will be continued in our laboratory.
"],"string":"[\n \"Conclusion\",\n \"
We have shown here that the ortho‐metallation of the NCP by representative late metals of the second and third rows of transition metals appears to be facilitated by a deviation of the confused pyrrole from the macrocycle mean plane. Such a deviation can be easily achieved when the macrocyclic crevice is empty, and thus, when the ligand is sufficiently flexible to conform the external donor set, i.e., the external N2 atom and a C\\northo\\n atom of the aryl at the meso‐C20 position to afford chelation. This strong out‐of‐plane deflection also allows the coordination of other ligands supplementing the coordination sphere of the metal. Importantly, in the already known ortho‐metallated NCP complexes, the metals (Pd2+, Pt2+) coordinated to the ligand exterior adopt invariantly a square‐planar geometry. In the present report, we show that a piano‐stool environment is also a suitable geometry for this type of complex, despite the presence of relatively voluminous ligands (Cym or Cp*). Although not as flexible as a free base, the C21‐substituted NCP derivatives coordinating in the macrocyclic interior to Ni2+ in the square planar or Ru2+ in the octahedral environments, comprise the confused pyrrole unit that is permanently deviated from the mean plane of the porphyrinoid. This arrangement makes complexes such as NiMeP or RuSPy effective metalloligands suitable for ortho‐metallation. The ortho‐metallated systems are chiral and can be obtained in a non‐racemic form by either separation of the final bimetallic complexes or by the external metalation of the separated enantiomers of metalloligands, as no racemization is possible upon N2–C\\northo\\n chelation. Favorably, the external chelation occurs stereoselectively with only one arrangement of the metal environment and the macrocycle. Thus, the chiral information can be transferred from the metalloligand to the externally situated metal center of the asymmetric environment. The redox properties of the bimetallic complexes are not profoundly altered in comparison to those of the appropriate metalloligands as has been shown here by electrochemical studies as well as by spectrophotometric and ESR‐monitored oxidation. Preliminary recognition of an influence of the ortho‐metallated ligand on the catalytic activity of the externally coordinated iridium(III) centers indicated that the heterocyclization reaction is totally absent for the system comprising RuSPy metalloligand (i.e., RuSPyIrCp*), despite the same donor set as in NiMePIrCp* or ClNCPIrCp* that effectively catalyzed this reaction. The study on these and other analogous systems directed toward recognition of their catalytic potential and redox properties will be continued in our laboratory.
A family of transition metal complexes of meso‐aryl‐2‐aza‐21‐carbaporphyrin (N‐confused porphyrin, NCP) derivatives acting as ortho‐metallating ligands for ruthenium(II), rhodium(III), and iridium(III) is synthesized and characterized by XRD, spectroscopic, and electrochemical methods. The chirality of these systems is shown by the separation of the enantiomers and analyzed by circular dichroism and DFT. A preliminary catalytic study indicates the activity of the iridium(III) ortho‐metallated complexes in the N‐heterocyclization of primary amines with diols.
","
N‐confused porphyrin and its 21‐substituted nickel(II) and ruthenium(II) complexes are used as chelating ligands for a series of late transition metals by exploiting external nitrogen of the confused pyrrole and an ortho‐carbon of the adjacent meso‐aryl. A new chirality center appears on the externally coordinated metal. Two higher oxidation states are available for the ortho‐metallated systems.\n\n
","
\n\n\nS.\nKoniarz\n, \nK.\nSzydełko\n, \nM. J.\nBiałek\n, \nK.\nHurej\n, \nP. J.\nChmielewski\n, Complexes of N‐Confused Porphyrin Derivatives as Ortho‐Metallating Ligands. Synthesis, Structure, Redox Properties, and Chirality. Adv. Sci.\n2024, 11, 2306696. 10.1002/advs.202306696\n\n
"],"string":"[\n \"Abstract\",\n \"
A family of transition metal complexes of meso‐aryl‐2‐aza‐21‐carbaporphyrin (N‐confused porphyrin, NCP) derivatives acting as ortho‐metallating ligands for ruthenium(II), rhodium(III), and iridium(III) is synthesized and characterized by XRD, spectroscopic, and electrochemical methods. The chirality of these systems is shown by the separation of the enantiomers and analyzed by circular dichroism and DFT. A preliminary catalytic study indicates the activity of the iridium(III) ortho‐metallated complexes in the N‐heterocyclization of primary amines with diols.
\",\n \"
N‐confused porphyrin and its 21‐substituted nickel(II) and ruthenium(II) complexes are used as chelating ligands for a series of late transition metals by exploiting external nitrogen of the confused pyrrole and an ortho‐carbon of the adjacent meso‐aryl. A new chirality center appears on the externally coordinated metal. Two higher oxidation states are available for the ortho‐metallated systems.\\n\\n
\",\n \"
\\n\\n\\nS.\\nKoniarz\\n, \\nK.\\nSzydełko\\n, \\nM. J.\\nBiałek\\n, \\nK.\\nHurej\\n, \\nP. J.\\nChmielewski\\n, Complexes of N‐Confused Porphyrin Derivatives as Ortho‐Metallating Ligands. Synthesis, Structure, Redox Properties, and Chirality. Adv. Sci.\\n2024, 11, 2306696. 10.1002/advs.202306696\\n\\n
\"\n]"},"body":{"kind":"list like","value":["Experimental Section","Syntheses of Precursors","
\nNCP ligands[\n##REF##10825994##\n55\n##\n] and metalloligands NiMeP\n[\n##UREF##16##\n29\n##, ##REF##11151364##\n30\n##, ##REF##12950206##\n33\n##\n] and RuSPy\n[\n##UREF##18##\n34\n##\n] were obtained by the literature methods.
","Synthesis of ClNCPIrCp*","
A sample of 0.040 g (0.054 mmol) ClNCP, 0.044 g (0.054 mmol) dichloro(pentamethylcyclopentadienyl)iridium(III) dimer, 0.044 g (0.540 mmol) anhydrous sodium acetate and 20 mL dichloromethane were placed in a two‐necked flask. The reaction mixture was purged for 20 min with N2 and then refluxed for 12 h. After this time, the reaction mixture was filtered, and the solution was concentrated and separated on a silica‐gel column. The compound was eluted using 0.5% MeOH in dichloromethane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane system. Yield 0.049 g (83%).
A sample of 0.025 g (0.036 mmol) of NiMeP, along with 0.016 g (0.020 mmol) of dichloro(pentamethylcyclopentadienyl)iridium(III) dimer, 0.016 g (0.2 mmol) anhydrous sodium acetate and 10 mL of dichloromethane were placed in a two‐neck round bottom flask. The mixture was purged for 20 min with N2 and then refluxed for 18 h. In the next stage, The reaction mixture was then filtered, concentrated, and passed down a silica gel column. The compound was eluted with 30% ethyl acetate in n‐hexane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane. Yield 0.035 g (93%).
A sample of 0.020 g (0.030 mmol) of NiMeP, 0.009 g (0.015 mmol) dichloro(p‐cymene)ruthenium(II) dimer, 0.012 g (0.150 mmol) anhydrous sodium acetate were placed in a two‐neck flask and 10 mL of dichloromethane were added. The mixture was purged for 20 min. with N2, and then refluxed for 48 h. After that, the mixture was filtered, concentrated, and subjected to silica‐gel column chromatography. The compound was eluted with 30% ethyl acetate in n‐hexane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane. Yield 0.017 g (60%).
The compound was synthesized and purified in the same way as NiMePRuCym, except that dichloro(p‐cymene)ruthenium(II) was replaced by dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (0.009 g, (0.015 mmol)). Yield 0.011 g (40%).
The compound was synthesized and purified in the same way as NiMePIrCp*. A sample of 0.020 g (0.024 mmol) of compound RuSPy, 0.010 g (0.012 mmol) dichloro(pentamethylcyclopentadienyl)iridium(III) dimer and 0.010 g (0.120 mmol) anhydrous sodium acetate were used. Yield 0.025 g (85%).
The compound was synthesized and purified in the same way as NiMePRuCym. A sample of 0.020 g (0.024 mmol) of compound RuSPy, 0.007 g (0.012 mmol) dichloro(p‐cymene) ruthenium(II) dimer and 0.010 g (0.120 mmol) of anhydrous sodium acetate were used. Yield 0.014 g (54%).
A sample of 0.020 g (0.024 mmol) of compound RuSPy, 0.007 g (0.012 mmol) dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer, 0.010 g (0.120 mmol) of anhydrous sodium acetate and 10 mL of chloroform was placed in a two‐neck round‐bottom flask. The mixture was purged with N2 gas for 20 min and then refluxed for 24 h. After this time, another portion of 0.007 g (0.012 mmol) of dichloro(cyclopentadienyl) rhodium(III) dimer and 0.010 g (0.120 mmol) of anhydrous sodium acetate was added and heated for another 24 h under reflux. In the next stage, the mixture was filtered, concentrated and separated by a silica gel chromatographic column. The compound was eluted with 30–35% ethyl acetate in n‐hexane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane. Yield 0.009 g (35%).
","Supporting information"],"string":"[\n \"Experimental Section\",\n \"Syntheses of Precursors\",\n \"
\\nNCP ligands[\\n##REF##10825994##\\n55\\n##\\n] and metalloligands NiMeP\\n[\\n##UREF##16##\\n29\\n##, ##REF##11151364##\\n30\\n##, ##REF##12950206##\\n33\\n##\\n] and RuSPy\\n[\\n##UREF##18##\\n34\\n##\\n] were obtained by the literature methods.
\",\n \"Synthesis of ClNCPIrCp*\",\n \"
A sample of 0.040 g (0.054 mmol) ClNCP, 0.044 g (0.054 mmol) dichloro(pentamethylcyclopentadienyl)iridium(III) dimer, 0.044 g (0.540 mmol) anhydrous sodium acetate and 20 mL dichloromethane were placed in a two‐necked flask. The reaction mixture was purged for 20 min with N2 and then refluxed for 12 h. After this time, the reaction mixture was filtered, and the solution was concentrated and separated on a silica‐gel column. The compound was eluted using 0.5% MeOH in dichloromethane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane system. Yield 0.049 g (83%).
\",\n \"Selected Data for <bold>ClNCP</bold>IrCp*\",\n \"
A sample of 0.025 g (0.036 mmol) of NiMeP, along with 0.016 g (0.020 mmol) of dichloro(pentamethylcyclopentadienyl)iridium(III) dimer, 0.016 g (0.2 mmol) anhydrous sodium acetate and 10 mL of dichloromethane were placed in a two‐neck round bottom flask. The mixture was purged for 20 min with N2 and then refluxed for 18 h. In the next stage, The reaction mixture was then filtered, concentrated, and passed down a silica gel column. The compound was eluted with 30% ethyl acetate in n‐hexane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane. Yield 0.035 g (93%).
A sample of 0.020 g (0.030 mmol) of NiMeP, 0.009 g (0.015 mmol) dichloro(p‐cymene)ruthenium(II) dimer, 0.012 g (0.150 mmol) anhydrous sodium acetate were placed in a two‐neck flask and 10 mL of dichloromethane were added. The mixture was purged for 20 min. with N2, and then refluxed for 48 h. After that, the mixture was filtered, concentrated, and subjected to silica‐gel column chromatography. The compound was eluted with 30% ethyl acetate in n‐hexane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane. Yield 0.017 g (60%).
The compound was synthesized and purified in the same way as NiMePRuCym, except that dichloro(p‐cymene)ruthenium(II) was replaced by dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer (0.009 g, (0.015 mmol)). Yield 0.011 g (40%).
The compound was synthesized and purified in the same way as NiMePIrCp*. A sample of 0.020 g (0.024 mmol) of compound RuSPy, 0.010 g (0.012 mmol) dichloro(pentamethylcyclopentadienyl)iridium(III) dimer and 0.010 g (0.120 mmol) anhydrous sodium acetate were used. Yield 0.025 g (85%).
The compound was synthesized and purified in the same way as NiMePRuCym. A sample of 0.020 g (0.024 mmol) of compound RuSPy, 0.007 g (0.012 mmol) dichloro(p‐cymene) ruthenium(II) dimer and 0.010 g (0.120 mmol) of anhydrous sodium acetate were used. Yield 0.014 g (54%).
A sample of 0.020 g (0.024 mmol) of compound RuSPy, 0.007 g (0.012 mmol) dichloro(pentamethylcyclopentadienyl)rhodium(III) dimer, 0.010 g (0.120 mmol) of anhydrous sodium acetate and 10 mL of chloroform was placed in a two‐neck round‐bottom flask. The mixture was purged with N2 gas for 20 min and then refluxed for 24 h. After this time, another portion of 0.007 g (0.012 mmol) of dichloro(cyclopentadienyl) rhodium(III) dimer and 0.010 g (0.120 mmol) of anhydrous sodium acetate was added and heated for another 24 h under reflux. In the next stage, the mixture was filtered, concentrated and separated by a silica gel chromatographic column. The compound was eluted with 30–35% ethyl acetate in n‐hexane. The collected fraction was evaporated to dryness and recrystallized from the dichloromethane/n‐hexane. Yield 0.009 g (35%).
This work was supported by the Polish National Science Center (2022/45/B/ST4/01229). DFT calculations were carried out using resources provided by the Wrocław Center for Networking and Supercomputing, grant 329.
","Data Availability Statement","
The data that support the findings of this study are available in the supplementary material of this article.
"],"string":"[\n \"Acknowledgements\",\n \"
This work was supported by the Polish National Science Center (2022/45/B/ST4/01229). DFT calculations were carried out using resources provided by the Wrocław Center for Networking and Supercomputing, grant 329.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available in the supplementary material of this article.
\"\n]"},"figure":{"kind":"list like","value":["
Schematic structures of NCP and its ortho‐metallated complexes.
","
Syntheses of the ortho‐metallated NCP complexes.
","
A) Selected regions of 1H NMR spectra (500 MHz, CDCl3, 300 K) of NiMePIrCp* (top) and NiMePRuCym (bottom) along with a partial signal assignment. B) Selected regions of 1H NMR spectra (500 MHz, CDCl3, 300 K) of RuSPyIrCp* (top) and RuSPyRuCym (bottom) along with a partial signal assignment. s, residual CHCl3 signal; w, dissolved water signal. The signal of impurities is marked with asterisks.
","
Optical spectra (DCM, 298 K): A) NiMeP and its ortho‐metallated derivatives and B) RuSPy and its ortho‐metallated derivatives.
","
Perspective views (50% displacement ellipsoid plots and stick diagrams) of molecular structures of A) ClNCPIrCp*, B) NiMePRuCym, C) NiMePIrCp*, and D) RuSPyIrCp*. All solvent molecules are omitted. In the stick representations of the side views, all hydrogens and all but ortho‐metallated aryl substituents are removed for clarity.
","
Atom displacements from the porphyrin mean plane calculated from SCXRD data of NiMePRuCym (black dots), NiMePIrCp* (red dots), RuSPyIrCp* (green dots), and NiMeP (violet circles).
","
Definition of absolute configuration at metalacyclic part of the systems A) and enantiomers along with chirality definitions for the selected ortho‐metallated NCP complexes, B) ClNCPIrCp*, C) NiMePRuCym, and D) RuSPyRhCp*.
","
Two approaches applicable for the separation of enantiomers of the ortho‐metallated complexes.
","
A) HPLC profiles for RuSPy on chiral stationary phase column (Chirex 3010, 1% MeOH in CH2Cl2, 2 mL min−1); top, CD and bottom, absorbance detection at 363 nm. B) CD (top) and absorbance (bottom) spectra of the HPLC‐separated fractions of RuSPy. C) superimposed CD spectra (CH2Cl2, 298 K) of S‐\nNiMeP (orange trace) and P‐NiMePIrCP* (purple trace) obtained by metalation of the former with [IrCl2Cp*]2. D) CD spectra of enantiomers of NiMePRuCym separated by the chiral stationary phase HPLC. E) superimposed CD spectra (CH2Cl2, 298 K) of S‐\nRuSPy (blue trace) and P‐RuSPyIrCP* (red trace) obtained by metallation of the former with [IrCl2Cp*]2. F) CD spectra of enantiomers of RuSPyRuCym separated by the chiral stationary phase HPLC. The absolute configurations were assigned to the enantiomers on the basis of TD‐DFT simulations of the CD spectra (Figures ##SUPPL##0##S23–S26##, Supporting Information).
","
Cyclic (CV, black traces) and differential pulse (DP, purple traces) voltammograms of A) NiMePIrCp*, B) RuSPyIrCp*, C) ClNCPIrCp*, D) NiMeRuCym, E) RuSPyRuCym, and F) RuSPyRhCp*. The experiments were carried out in a dichloromethane solution of [Bu4N]PF6 (0.1 m) using a glassy carbon working electrode, a platinum wire as an auxiliary electrode, and Ag/AgCl as a pseudoreference electrode. The green numbers associated with DP peaks are electrode potentials in volts. The partial CV scans are given to indicate the reversibility of some of the processes.
","
DFT‐calculated frontier orbitals energies of specified systems with the experimentally estimated energies of the HOMO and LUMO derived from the electrochemical data (green sticks).
","
Spectrophotometric titration of dichloromethane solutions of A) RuSPyIrCp*, B) RuSPy, and D) NiMePIrCp* with tris(4‐bromophenyl)ammoniumyl hexachloroantimonate (BAHA) and selected ESR spectra recorded in frozen dichloromethane solutions (120 K) upon addition of specified amounts of BAHA (C). The black arrows in (A) and (B) indicate the direction of the absorbance changes before, while the red arrows—after the addition of 1 equiv of BAHA.
"],"string":"[\n \"
Schematic structures of NCP and its ortho‐metallated complexes.
\",\n \"
Syntheses of the ortho‐metallated NCP complexes.
\",\n \"
A) Selected regions of 1H NMR spectra (500 MHz, CDCl3, 300 K) of NiMePIrCp* (top) and NiMePRuCym (bottom) along with a partial signal assignment. B) Selected regions of 1H NMR spectra (500 MHz, CDCl3, 300 K) of RuSPyIrCp* (top) and RuSPyRuCym (bottom) along with a partial signal assignment. s, residual CHCl3 signal; w, dissolved water signal. The signal of impurities is marked with asterisks.
\",\n \"
Optical spectra (DCM, 298 K): A) NiMeP and its ortho‐metallated derivatives and B) RuSPy and its ortho‐metallated derivatives.
\",\n \"
Perspective views (50% displacement ellipsoid plots and stick diagrams) of molecular structures of A) ClNCPIrCp*, B) NiMePRuCym, C) NiMePIrCp*, and D) RuSPyIrCp*. All solvent molecules are omitted. In the stick representations of the side views, all hydrogens and all but ortho‐metallated aryl substituents are removed for clarity.
\",\n \"
Atom displacements from the porphyrin mean plane calculated from SCXRD data of NiMePRuCym (black dots), NiMePIrCp* (red dots), RuSPyIrCp* (green dots), and NiMeP (violet circles).
\",\n \"
Definition of absolute configuration at metalacyclic part of the systems A) and enantiomers along with chirality definitions for the selected ortho‐metallated NCP complexes, B) ClNCPIrCp*, C) NiMePRuCym, and D) RuSPyRhCp*.
\",\n \"
Two approaches applicable for the separation of enantiomers of the ortho‐metallated complexes.
\",\n \"
A) HPLC profiles for RuSPy on chiral stationary phase column (Chirex 3010, 1% MeOH in CH2Cl2, 2 mL min−1); top, CD and bottom, absorbance detection at 363 nm. B) CD (top) and absorbance (bottom) spectra of the HPLC‐separated fractions of RuSPy. C) superimposed CD spectra (CH2Cl2, 298 K) of S‐\\nNiMeP (orange trace) and P‐NiMePIrCP* (purple trace) obtained by metalation of the former with [IrCl2Cp*]2. D) CD spectra of enantiomers of NiMePRuCym separated by the chiral stationary phase HPLC. E) superimposed CD spectra (CH2Cl2, 298 K) of S‐\\nRuSPy (blue trace) and P‐RuSPyIrCP* (red trace) obtained by metallation of the former with [IrCl2Cp*]2. F) CD spectra of enantiomers of RuSPyRuCym separated by the chiral stationary phase HPLC. The absolute configurations were assigned to the enantiomers on the basis of TD‐DFT simulations of the CD spectra (Figures ##SUPPL##0##S23–S26##, Supporting Information).
\",\n \"
Cyclic (CV, black traces) and differential pulse (DP, purple traces) voltammograms of A) NiMePIrCp*, B) RuSPyIrCp*, C) ClNCPIrCp*, D) NiMeRuCym, E) RuSPyRuCym, and F) RuSPyRhCp*. The experiments were carried out in a dichloromethane solution of [Bu4N]PF6 (0.1 m) using a glassy carbon working electrode, a platinum wire as an auxiliary electrode, and Ag/AgCl as a pseudoreference electrode. The green numbers associated with DP peaks are electrode potentials in volts. The partial CV scans are given to indicate the reversibility of some of the processes.
\",\n \"
DFT‐calculated frontier orbitals energies of specified systems with the experimentally estimated energies of the HOMO and LUMO derived from the electrochemical data (green sticks).
\",\n \"
Spectrophotometric titration of dichloromethane solutions of A) RuSPyIrCp*, B) RuSPy, and D) NiMePIrCp* with tris(4‐bromophenyl)ammoniumyl hexachloroantimonate (BAHA) and selected ESR spectra recorded in frozen dichloromethane solutions (120 K) upon addition of specified amounts of BAHA (C). The black arrows in (A) and (B) indicate the direction of the absorbance changes before, while the red arrows—after the addition of 1 equiv of BAHA.
\"\n]"},"table":{"kind":"list like","value":["
Bond lengths d for the externally chelated metal ions.
Compound
\nd M–N [Å]
\nd M–C [Å]
\nd M–Cl [Å]
\nd M–(ηn─L)\na)\n [Å]
\nClNCPIrCp*
2.066(5)
2.051(6)
2.406(2)
2.187(6)
\nNiMePIrCp*
2.065(2)
2.041(3)
2.407(1)
2.193(2)
\nNiMePRuCym
2.069(3)
2.047(3)
2.408(1)
2.215(3)
\nRuSPyIrCp*
2.060(4)
2.083(5)
2.420(1)
2.198(3)
\nRuSPyRhCp*
2.072(4)
2.030(4)
2.402(1)
2.201(4)
[RuCl2Cym]2\n\nb)\n\n
–
–
\n
2.444(4)\n#\n\n
\n
2.392(4)\n&\n\n
\n
2.158
[RhCl2Cp*]2\n\nc)\n\n
–
–
\n
2.458(9)\n#\n\n
\n
2.397(1)\n&\n\n
\n
2.126
[IrCl2Cp*]2\n\nd)\n\n
–
–
\n
2.453(5)\n#\n\n
\n
2.387(4)\n&\n\n
\n
2.132
John Wiley & Sons, Ltd.","
Analyses of the out‐of‐plane displacements for the macrocyclic ring of NCP calculated by means of PorphyStruct.[\n##REF##34061410##\n40\n##\n] on the basis of SCXRD structures.
Entry
Compound
\ndoming [Å]
\nsaddling [Å]
\nruffling [Å]
\nwavingX [Å]
\nwavingY [Å]
\npropellering [Å]
\ndM‐mpln\n\n\na)\n [Å]
\ndM‐ccpln\n\n\nb)\n [Å]
\nDoop\n\n\nc)\n [Å]
\nDH\n\nd)\n [deg]
1
\nNCP\n\ne)\n\n
0.196
1.360
−0.070
0.259
−0.055
0.040
–
–
1.465
27.0
2
\nClNCPIrCp*
0.341
−1.165
0.047
−0.108
−0.354
−0.026
–
–
1.325
28.0
3
\nNiMeP\n\nf)\n\n
0.271
−2.069
−0.092
−0.011
−0.353
0.003
0.022
−0.077
2.181
38.2
4
\nNiMePIrCp*
0.428
2.020
0.729
−0.515
−0.180
0.027
0.088
0.061
2.290
43.7
5
\nNiMePRuCym
−0.437
−2.233
−0.580
0.499
0.090
−0.028
−0.083
0.067
2.445
45.5
6
\nRuSPy\n\ng)\n\n
0.415
−1.804
0.282
0.058
0.326
−0.003
0.046
−0.108
1.990
40.2
7
\nRuSPyIrCp*
0.559
1.343
0.312
−0.607
−0.246
0.018
0.086
−0.098
1.689
41.3
8
\nRuSPyRhCp*
0.554
1.315
0.291
−0.590
−0.258
0.018
0.088
−0.096
1.657
41.2
11
\nNCPPtPPh3\n\nh)\n\n
−0.278
1.290
−0.320
0.107
0.437
0.008
–
–
1.501
31.5
John Wiley & Sons, Ltd.","
Electrode potentials of the orthometallated complexes and metalloligands.
Entry
Compound
\nE\nRed3 [V]
\nE\nRed2 [V]
\nE\nRed1 [V]
\nE\nOx1 [V]
\nE\nOx2 [V]
\nE\nOx3 [V]
ΔE\n\na)\n [V]
oHLG\ne)\n [eV]
cHLG\nf)\n [eV]
1
\nClNCPIrCp*
−2.03\nb)\n\n
−1.72\nb)\n\n
−1.34
0.34
0.61\nb)\n\n
0.87\nb)\n\n
1.68
1.53
2.02
2
\nNiMePIrCp*
−2.31\nb)\n\n
−1.99\nb)\n\n
−1.48\nb)\n\n
0.23
0.42
0.92\nb)\n\n
1.71
1.60
2.29
3
\nNiMePRhCp*
−2.18\nb)\n\n
−1.96\nb)\n\n
−1.46
0.29\nb)\n\n
0.52
0.91\nb)\n\n
1.75
1.56
N.A.
4
\nNiMePRuCym
–
–
−1.46\nb)\n\n
0.21
0.30
1.04\nb)\n\n
1.67
1.57
2.27
5
\nRuSPyIrCp*
–
–
−1.47\nb)\n\n
0.25
0.63
1.06\nb)\n\n
1.72
1.61
2.15
6
\nRuSPyRhCp*
–
−2.08\nb)\n\n
−1.46\nb)\n\n
0.34
0.71\nb)\n\n
–
1.80
1.63
2.19
7
\nRuSPyRuCym
−2.28\nb)\n\n
−1.94\nb)\n\n
−1.58
0.15
0.57
1.00\nb)\n\n
1.73
1.60
N.A.
8
\nNiMeP\n\nc)\n\n
–
–
−1.37
0.22
0.50\nb)\n\n
–
1.59
1.46
N.A.
9
\nRuSPy\n\nd)\n\n
–
–
−1.39\nb)\n\n
0.34
0.79
–
1.73
1.65
2.39
10
\nClNCP\n
–
−2.09\nb)\n\n
−1.42\nb)\n\n
0.36\nb)\n\n
0.63\nb)\n\n
0.84\nb)\n\n
1.78
1.61
N.A.
John Wiley & Sons, Ltd.","
Reaction conditions and yields for benzylamine (A) reacting with 1,6‐hexanediol (B) in the presence of various catalysts.
\n\n
Entry\na)\n\n
Catalyst
%mol\nb)\n\n
µmol of Ir
Conversion of B [%]
Yield C [%]
Yield D [%]
Yield E [%]
1
[IrCl2Cp*]2\n
0.6
6
90
66\nc)\n\n
43
2
2
\nNiMePIrCp*
1.0
5
96
67
18
<1
3
\nNiMeP\n
1.0
0
< 5
n.d.
n.d.
15\nd)\n\n
4
\nRuSPy\n
1.0
0
< 5
n.d.
n.d.
11\nd)\n\n
5
\nRuSPyIrCp*
1.0
5
< 5
n.d.
n.d.
18\nd)\n\n
6
\nClNCPIrCp*
1.1
6
99
86
19
2
7
\nClNCP\n
1.0
0
< 5
n.d.
n.d.
2\nd)\n\n
John Wiley & Sons, Ltd."],"string":"[\n \"
Bond lengths d for the externally chelated metal ions.
Compound
\\nd M–N [Å]
\\nd M–C [Å]
\\nd M–Cl [Å]
\\nd M–(ηn─L)\\na)\\n [Å]
\\nClNCPIrCp*
2.066(5)
2.051(6)
2.406(2)
2.187(6)
\\nNiMePIrCp*
2.065(2)
2.041(3)
2.407(1)
2.193(2)
\\nNiMePRuCym
2.069(3)
2.047(3)
2.408(1)
2.215(3)
\\nRuSPyIrCp*
2.060(4)
2.083(5)
2.420(1)
2.198(3)
\\nRuSPyRhCp*
2.072(4)
2.030(4)
2.402(1)
2.201(4)
[RuCl2Cym]2\\n\\nb)\\n\\n
–
–
\\n
2.444(4)\\n#\\n\\n
\\n
2.392(4)\\n&\\n\\n
\\n
2.158
[RhCl2Cp*]2\\n\\nc)\\n\\n
–
–
\\n
2.458(9)\\n#\\n\\n
\\n
2.397(1)\\n&\\n\\n
\\n
2.126
[IrCl2Cp*]2\\n\\nd)\\n\\n
–
–
\\n
2.453(5)\\n#\\n\\n
\\n
2.387(4)\\n&\\n\\n
\\n
2.132
John Wiley & Sons, Ltd.\",\n \"
Analyses of the out‐of‐plane displacements for the macrocyclic ring of NCP calculated by means of PorphyStruct.[\\n##REF##34061410##\\n40\\n##\\n] on the basis of SCXRD structures.
Entry
Compound
\\ndoming [Å]
\\nsaddling [Å]
\\nruffling [Å]
\\nwavingX [Å]
\\nwavingY [Å]
\\npropellering [Å]
\\ndM‐mpln\\n\\n\\na)\\n [Å]
\\ndM‐ccpln\\n\\n\\nb)\\n [Å]
\\nDoop\\n\\n\\nc)\\n [Å]
\\nDH\\n\\nd)\\n [deg]
1
\\nNCP\\n\\ne)\\n\\n
0.196
1.360
−0.070
0.259
−0.055
0.040
–
–
1.465
27.0
2
\\nClNCPIrCp*
0.341
−1.165
0.047
−0.108
−0.354
−0.026
–
–
1.325
28.0
3
\\nNiMeP\\n\\nf)\\n\\n
0.271
−2.069
−0.092
−0.011
−0.353
0.003
0.022
−0.077
2.181
38.2
4
\\nNiMePIrCp*
0.428
2.020
0.729
−0.515
−0.180
0.027
0.088
0.061
2.290
43.7
5
\\nNiMePRuCym
−0.437
−2.233
−0.580
0.499
0.090
−0.028
−0.083
0.067
2.445
45.5
6
\\nRuSPy\\n\\ng)\\n\\n
0.415
−1.804
0.282
0.058
0.326
−0.003
0.046
−0.108
1.990
40.2
7
\\nRuSPyIrCp*
0.559
1.343
0.312
−0.607
−0.246
0.018
0.086
−0.098
1.689
41.3
8
\\nRuSPyRhCp*
0.554
1.315
0.291
−0.590
−0.258
0.018
0.088
−0.096
1.657
41.2
11
\\nNCPPtPPh3\\n\\nh)\\n\\n
−0.278
1.290
−0.320
0.107
0.437
0.008
–
–
1.501
31.5
John Wiley & Sons, Ltd.\",\n \"
Electrode potentials of the orthometallated complexes and metalloligands.
Entry
Compound
\\nE\\nRed3 [V]
\\nE\\nRed2 [V]
\\nE\\nRed1 [V]
\\nE\\nOx1 [V]
\\nE\\nOx2 [V]
\\nE\\nOx3 [V]
ΔE\\n\\na)\\n [V]
oHLG\\ne)\\n [eV]
cHLG\\nf)\\n [eV]
1
\\nClNCPIrCp*
−2.03\\nb)\\n\\n
−1.72\\nb)\\n\\n
−1.34
0.34
0.61\\nb)\\n\\n
0.87\\nb)\\n\\n
1.68
1.53
2.02
2
\\nNiMePIrCp*
−2.31\\nb)\\n\\n
−1.99\\nb)\\n\\n
−1.48\\nb)\\n\\n
0.23
0.42
0.92\\nb)\\n\\n
1.71
1.60
2.29
3
\\nNiMePRhCp*
−2.18\\nb)\\n\\n
−1.96\\nb)\\n\\n
−1.46
0.29\\nb)\\n\\n
0.52
0.91\\nb)\\n\\n
1.75
1.56
N.A.
4
\\nNiMePRuCym
–
–
−1.46\\nb)\\n\\n
0.21
0.30
1.04\\nb)\\n\\n
1.67
1.57
2.27
5
\\nRuSPyIrCp*
–
–
−1.47\\nb)\\n\\n
0.25
0.63
1.06\\nb)\\n\\n
1.72
1.61
2.15
6
\\nRuSPyRhCp*
–
−2.08\\nb)\\n\\n
−1.46\\nb)\\n\\n
0.34
0.71\\nb)\\n\\n
–
1.80
1.63
2.19
7
\\nRuSPyRuCym
−2.28\\nb)\\n\\n
−1.94\\nb)\\n\\n
−1.58
0.15
0.57
1.00\\nb)\\n\\n
1.73
1.60
N.A.
8
\\nNiMeP\\n\\nc)\\n\\n
–
–
−1.37
0.22
0.50\\nb)\\n\\n
–
1.59
1.46
N.A.
9
\\nRuSPy\\n\\nd)\\n\\n
–
–
−1.39\\nb)\\n\\n
0.34
0.79
–
1.73
1.65
2.39
10
\\nClNCP\\n
–
−2.09\\nb)\\n\\n
−1.42\\nb)\\n\\n
0.36\\nb)\\n\\n
0.63\\nb)\\n\\n
0.84\\nb)\\n\\n
1.78
1.61
N.A.
John Wiley & Sons, Ltd.\",\n \"
Reaction conditions and yields for benzylamine (A) reacting with 1,6‐hexanediol (B) in the presence of various catalysts.
\\n\\n
Entry\\na)\\n\\n
Catalyst
%mol\\nb)\\n\\n
µmol of Ir
Conversion of B [%]
Yield C [%]
Yield D [%]
Yield E [%]
1
[IrCl2Cp*]2\\n
0.6
6
90
66\\nc)\\n\\n
43
2
2
\\nNiMePIrCp*
1.0
5
96
67
18
<1
3
\\nNiMeP\\n
1.0
0
< 5
n.d.
n.d.
15\\nd)\\n\\n
4
\\nRuSPy\\n
1.0
0
< 5
n.d.
n.d.
11\\nd)\\n\\n
5
\\nRuSPyIrCp*
1.0
5
< 5
n.d.
n.d.
18\\nd)\\n\\n
6
\\nClNCPIrCp*
1.1
6
99
86
19
2
7
\\nClNCP\\n
1.0
0
< 5
n.d.
n.d.
2\\nd)\\n\\n
John Wiley & Sons, Ltd.\"\n]"},"formula":{"kind":"list like","value":[],"string":"[]"},"box":{"kind":"list like","value":[""],"string":"[\n \"\"\n]"},"code":{"kind":"list like","value":[],"string":"[]"},"quote":{"kind":"list like","value":[],"string":"[]"},"chemical":{"kind":"list like","value":[],"string":"[]"},"supplementary":{"kind":"list like","value":["
Supporting Information
"],"string":"[\n \"
Supporting Information
\"\n]"},"footnote":{"kind":"list like","value":["
Mean values of the M–C distances for η\n5‐Cp*–M or η\n6‐Cym–Ru;
Data from ref. [##REF##30942794##39##];
Data from ref. [##UREF##21##38##];
Data from ref. [##UREF##20##37##];
Bridging chloride;
Terminal chloride.
","
The metal ion (NiII or RuII) displacement out of the porphyrin mean plane;
The metal ion (NiII or RuII) displacement out of the mean plane of the coordination core (MCNNN);
total out‐of‐plane distortion;
dihedral angle between the mean plane defined by all non‐hydrogen atoms of the macrocyclic ring except C21, N2, and C3 but including core‐coordinated metal, and the mean plane of the confused pyrrole;
Optical HOMO–LUMO energy gaps derived from onsets of the lowest‐energy bands in the electronic spectra;
HOMO–LUMO energy gaps derived from DFT calculations.
","
Reactions were carried out for 0.5 mmol (59 mg) of 1,6‐hexanediol B and 0.75 mmol (80 mg) of benzylamine A in the presence of 1 mg of NaHCO3. The yields were estimated employing quantitative 1H NMR of the reaction mixtures with 2,4,6‐collidine as an internal reference unless stated otherwise;
With respect to 1,6‐hexanediol;
The reported yield was 74%[\n##REF##15387539##\n51\n##\n];
Results from GC–MS/FID analysis only.
"],"string":"[\n \"
Mean values of the M–C distances for η\\n5‐Cp*–M or η\\n6‐Cym–Ru;
Data from ref. [##REF##30942794##39##];
Data from ref. [##UREF##21##38##];
Data from ref. [##UREF##20##37##];
Bridging chloride;
Terminal chloride.
\",\n \"
The metal ion (NiII or RuII) displacement out of the porphyrin mean plane;
The metal ion (NiII or RuII) displacement out of the mean plane of the coordination core (MCNNN);
total out‐of‐plane distortion;
dihedral angle between the mean plane defined by all non‐hydrogen atoms of the macrocyclic ring except C21, N2, and C3 but including core‐coordinated metal, and the mean plane of the confused pyrrole;
Optical HOMO–LUMO energy gaps derived from onsets of the lowest‐energy bands in the electronic spectra;
HOMO–LUMO energy gaps derived from DFT calculations.
\",\n \"
Reactions were carried out for 0.5 mmol (59 mg) of 1,6‐hexanediol B and 0.75 mmol (80 mg) of benzylamine A in the presence of 1 mg of NaHCO3. The yields were estimated employing quantitative 1H NMR of the reaction mixtures with 2,4,6‐collidine as an internal reference unless stated otherwise;
With respect to 1,6‐hexanediol;
The reported yield was 74%[\\n##REF##15387539##\\n51\\n##\\n];
Gastric cancer (GC) is one of the most prevalent malignant tumors globally. Owing to its indistinct early‐stage symptoms, most patients have already progressed into advanced stages at the time of initial diagnosis, resulting in poor overall prognosis.[\n##REF##33538338##\n1\n##\n] Chemotherapy is still the mainstream workforce for advanced GC.[\n##REF##29488121##\n2\n##\n] Paclitaxel (PTX), one of chemotherapeutic drugs in the first‐line regimen for advanced GC, is a cell cycle‐specific antitumor drug that inhibits mitosis and proliferation of tumor cells.[\n##REF##25240821##\n3\n##\n] Despite its excellent antitumor effect against GC, PTX shares the similar issues as other chemotherapeutic agents, such as poor water solubility, lack of targeting specificity, a short retention time, and severe toxic side effects, which have hampered its widespread use in clinical practice.[\n##REF##37655323##\n4\n##\n]\n
","
The combination of chemotherapy and molecular targeted therapy could synergistically exert antitumor effects, holding an immense prospect of its application in treating GC.[\n##REF##33592120##\n5\n##\n] Currently, the majority of targeted drugs for GC are anti‐human epidermal growth factor receptor 2 or anti‐epidermal growth factor receptor gene therapy drugs, and immunotherapy drugs.[\n##REF##24626858##\n6\n##\n] However, positive response rates of these targeted agents for GC are relatively low, and only a very few patients can benefit from targeted therapy. Significant efforts have been dedicated to the identification of novel targeted drug candidates. The phosphatidylinositol‐3‐kinase (PI3K)/Protein kinase B (AKT) signaling pathway has a crucial regulatory function in cellular processes including cell growth, proliferation, differentiation, apoptosis and glucose transport.[\n##REF##12094235##\n7\n##\n] Activation of this signaling pathway is commonly observed in different types of solid tumors and its activation may be associated with poor prognosis.[\n##REF##25037117##\n8\n##\n] A retrospective study revealed that positive expression of AKT was detected in 81.54% of GC tissues, notably higher than that in 20.8% of normal tissues.[\n##REF##29143985##\n9\n##\n] Abnormal activation of the PI3K/AKT signaling pathway enhances the proliferation, metastasis, and drug resistance of GC cells, while suppressing their apoptosis. PI3K and AKT inhibitors have been demonstrated with promising antitumor effects for GC, thus they have great potential as therapeutic targeted agents.[\n##REF##18841391##\n10\n##\n]\n
","
It was reported that the combination of AKT inhibitors and chemotherapeutic drugs could synergistically triggers apoptosis and impedes tumor growth.[\n##REF##22294718##\n11\n##\n] Capivasertib (CAP), a highly potent pan‐AKT kinase inhibitor, demonstrates a comparable inhibitory efficacy against three different subtypes of AKT. CAP inhibits the phosphorylation of AKT substrates, thereby blocking the PI3K/AKT signaling pathway. As a result, the growth of cells is inhibited, and apoptosis is promoted.[\n##REF##23394218##\n12\n##\n] Nonetheless, several limitations, such as poor water solubility and low bioavailability, have restricted its widespread application. Encouragingly, combined treatment with PTX and CAP has been shown with an enhancement in the level of PTX‐induced tumor cell apoptosis,[\n##REF##24088382##\n13\n##\n] suggesting that their combination could provide a promising new approach for the treatment of GC. It has been reported that patients could tolerate the combination of AKT inhibitors and chemotherapeutic agents, however, they suffer from serious toxic side effects such as diarrhea, infection, neutropenia, rash, and fatigue, which might be attributed to their suboptimal pharmacokinetics and poor biodistribution in the body.[\n##REF##33326257##\n14\n##\n] Hence, effective means of delivering both AKT inhibitors and chemotherapeutic agents to achieve potent combinatorial therapeutic effects and reduce their side effects have been pursued.
","
One of the most effective means for potent and low‐toxicity combine therapy is achieved via a nano‐scale drug delivery system by incorporating chemotherapeutic and targeted drugs through physical encapsulation or covalent bonding.[\n##REF##33393582##\n15\n##\n] The nano‐scale delivery system offers active or passive targeting of tumors and enables controlled release of drugs from the system within a tumor microenvironment (TME). Therefore, they can enhance drug distribution and reduce their toxic effects. Polymer‐based nano‐drug delivery systems have been showcased as the most promising one, and some of them have already progressed to clinical trials or clinical practice.[\n##UREF##0##\n16\n##\n] Sugar‐derived polymers stand out from other polymers due to their hydrophilicity, cellular affinity, and degradability. In addition, multiple hydroxyl and aldehyde/ketone groups in sugar‐based polymers could form intermolecular hydrogen bonds, contributing to stable nano‐structures.[\n##UREF##1##\n17\n##\n]\n
","
The efficacy of drugs in the polymer‐based drug delivery system largely depends on the molecular structure, composition, and molecular weight of the polymer carrier.[\n##REF##36875053##\n18\n##\n] Advances in controlled polymerization and click chemistry have expanded the portfolio of polymer carriers, paving the way for novel sugar‐based carriers with specific chemical compositions and molecular structures.[\n##UREF##2##\n19\n##\n] Our preliminary study suggests that polymers with branched structures offer better modifiability for both structure and function than linear polymers, thus they are promising templates for nano‐delivery systems.[\n##UREF##1##\n17\n##, ##REF##37908735##\n20\n##\n] Additionally, leveraging unique characteristic factors in the TME, including overexpressed enzymes, a low pH, and redox conditions, intelligent drug delivery systems, can achieve controlled drug release at tumor sites.[\n##REF##33426371##\n21\n##\n] Enzymes have advantages of their substrate specificity and mild reaction conditions. Response of nano‐delivery systems to overexpressed enzymes in the TME could minimize drug leakage and degradation during in vivo circulation, thus improving the drug delivery efficiency into tumor sites.
","
In this study, an innovative approach was proposed by utilizing 2‐lactobionamidoethylmethacrylamide (LAEMA) as a key building unit for branched glycopolymers to design and prepare an enzyme‐responsive nano‐drug delivery system for PTX and CAP. This system was engineered for precise targeting of tumor cells and controlled release of PTX and CAP. As illustrated in the Scheme ##FIG##0##\n1\n##, LAEMA‐based branched polymer frameworks as a PTX prodrug were prepared through RAFT polymerization and thiol‐ene click reaction. These frameworks displayed self‐assembly characteristics, and they were used to encapsulate CAP during the self‐assembly process. Upon encountering tumor cells, the nano‐drug system underwent rapid disintegration due to the presence of overexpressed cathepsin B in the lysosomes to release PTX and CAP. This dual‐agent strategy exhibited notable synergy for GC: PTX hindered tumor cell proliferation by inhibiting mitosis and inducing apoptosis, and CAP restrained tumor cell growth and promoted apoptosis by suppressing the PI3K/AKT pathway. The encouraging therapeutic enhancement achieved through the nano‐delivery system underlines the potential of this dual‐drug co‐delivery approach as a promising therapeutic avenue for advancing GC treatment by combining chemotherapy and AKT inhibition.
"],"string":"[\n \"Introduction\",\n \"
Gastric cancer (GC) is one of the most prevalent malignant tumors globally. Owing to its indistinct early‐stage symptoms, most patients have already progressed into advanced stages at the time of initial diagnosis, resulting in poor overall prognosis.[\\n##REF##33538338##\\n1\\n##\\n] Chemotherapy is still the mainstream workforce for advanced GC.[\\n##REF##29488121##\\n2\\n##\\n] Paclitaxel (PTX), one of chemotherapeutic drugs in the first‐line regimen for advanced GC, is a cell cycle‐specific antitumor drug that inhibits mitosis and proliferation of tumor cells.[\\n##REF##25240821##\\n3\\n##\\n] Despite its excellent antitumor effect against GC, PTX shares the similar issues as other chemotherapeutic agents, such as poor water solubility, lack of targeting specificity, a short retention time, and severe toxic side effects, which have hampered its widespread use in clinical practice.[\\n##REF##37655323##\\n4\\n##\\n]\\n
\",\n \"
The combination of chemotherapy and molecular targeted therapy could synergistically exert antitumor effects, holding an immense prospect of its application in treating GC.[\\n##REF##33592120##\\n5\\n##\\n] Currently, the majority of targeted drugs for GC are anti‐human epidermal growth factor receptor 2 or anti‐epidermal growth factor receptor gene therapy drugs, and immunotherapy drugs.[\\n##REF##24626858##\\n6\\n##\\n] However, positive response rates of these targeted agents for GC are relatively low, and only a very few patients can benefit from targeted therapy. Significant efforts have been dedicated to the identification of novel targeted drug candidates. The phosphatidylinositol‐3‐kinase (PI3K)/Protein kinase B (AKT) signaling pathway has a crucial regulatory function in cellular processes including cell growth, proliferation, differentiation, apoptosis and glucose transport.[\\n##REF##12094235##\\n7\\n##\\n] Activation of this signaling pathway is commonly observed in different types of solid tumors and its activation may be associated with poor prognosis.[\\n##REF##25037117##\\n8\\n##\\n] A retrospective study revealed that positive expression of AKT was detected in 81.54% of GC tissues, notably higher than that in 20.8% of normal tissues.[\\n##REF##29143985##\\n9\\n##\\n] Abnormal activation of the PI3K/AKT signaling pathway enhances the proliferation, metastasis, and drug resistance of GC cells, while suppressing their apoptosis. PI3K and AKT inhibitors have been demonstrated with promising antitumor effects for GC, thus they have great potential as therapeutic targeted agents.[\\n##REF##18841391##\\n10\\n##\\n]\\n
\",\n \"
It was reported that the combination of AKT inhibitors and chemotherapeutic drugs could synergistically triggers apoptosis and impedes tumor growth.[\\n##REF##22294718##\\n11\\n##\\n] Capivasertib (CAP), a highly potent pan‐AKT kinase inhibitor, demonstrates a comparable inhibitory efficacy against three different subtypes of AKT. CAP inhibits the phosphorylation of AKT substrates, thereby blocking the PI3K/AKT signaling pathway. As a result, the growth of cells is inhibited, and apoptosis is promoted.[\\n##REF##23394218##\\n12\\n##\\n] Nonetheless, several limitations, such as poor water solubility and low bioavailability, have restricted its widespread application. Encouragingly, combined treatment with PTX and CAP has been shown with an enhancement in the level of PTX‐induced tumor cell apoptosis,[\\n##REF##24088382##\\n13\\n##\\n] suggesting that their combination could provide a promising new approach for the treatment of GC. It has been reported that patients could tolerate the combination of AKT inhibitors and chemotherapeutic agents, however, they suffer from serious toxic side effects such as diarrhea, infection, neutropenia, rash, and fatigue, which might be attributed to their suboptimal pharmacokinetics and poor biodistribution in the body.[\\n##REF##33326257##\\n14\\n##\\n] Hence, effective means of delivering both AKT inhibitors and chemotherapeutic agents to achieve potent combinatorial therapeutic effects and reduce their side effects have been pursued.
\",\n \"
One of the most effective means for potent and low‐toxicity combine therapy is achieved via a nano‐scale drug delivery system by incorporating chemotherapeutic and targeted drugs through physical encapsulation or covalent bonding.[\\n##REF##33393582##\\n15\\n##\\n] The nano‐scale delivery system offers active or passive targeting of tumors and enables controlled release of drugs from the system within a tumor microenvironment (TME). Therefore, they can enhance drug distribution and reduce their toxic effects. Polymer‐based nano‐drug delivery systems have been showcased as the most promising one, and some of them have already progressed to clinical trials or clinical practice.[\\n##UREF##0##\\n16\\n##\\n] Sugar‐derived polymers stand out from other polymers due to their hydrophilicity, cellular affinity, and degradability. In addition, multiple hydroxyl and aldehyde/ketone groups in sugar‐based polymers could form intermolecular hydrogen bonds, contributing to stable nano‐structures.[\\n##UREF##1##\\n17\\n##\\n]\\n
\",\n \"
The efficacy of drugs in the polymer‐based drug delivery system largely depends on the molecular structure, composition, and molecular weight of the polymer carrier.[\\n##REF##36875053##\\n18\\n##\\n] Advances in controlled polymerization and click chemistry have expanded the portfolio of polymer carriers, paving the way for novel sugar‐based carriers with specific chemical compositions and molecular structures.[\\n##UREF##2##\\n19\\n##\\n] Our preliminary study suggests that polymers with branched structures offer better modifiability for both structure and function than linear polymers, thus they are promising templates for nano‐delivery systems.[\\n##UREF##1##\\n17\\n##, ##REF##37908735##\\n20\\n##\\n] Additionally, leveraging unique characteristic factors in the TME, including overexpressed enzymes, a low pH, and redox conditions, intelligent drug delivery systems, can achieve controlled drug release at tumor sites.[\\n##REF##33426371##\\n21\\n##\\n] Enzymes have advantages of their substrate specificity and mild reaction conditions. Response of nano‐delivery systems to overexpressed enzymes in the TME could minimize drug leakage and degradation during in vivo circulation, thus improving the drug delivery efficiency into tumor sites.
\",\n \"
In this study, an innovative approach was proposed by utilizing 2‐lactobionamidoethylmethacrylamide (LAEMA) as a key building unit for branched glycopolymers to design and prepare an enzyme‐responsive nano‐drug delivery system for PTX and CAP. This system was engineered for precise targeting of tumor cells and controlled release of PTX and CAP. As illustrated in the Scheme ##FIG##0##\\n1\\n##, LAEMA‐based branched polymer frameworks as a PTX prodrug were prepared through RAFT polymerization and thiol‐ene click reaction. These frameworks displayed self‐assembly characteristics, and they were used to encapsulate CAP during the self‐assembly process. Upon encountering tumor cells, the nano‐drug system underwent rapid disintegration due to the presence of overexpressed cathepsin B in the lysosomes to release PTX and CAP. This dual‐agent strategy exhibited notable synergy for GC: PTX hindered tumor cell proliferation by inhibiting mitosis and inducing apoptosis, and CAP restrained tumor cell growth and promoted apoptosis by suppressing the PI3K/AKT pathway. The encouraging therapeutic enhancement achieved through the nano‐delivery system underlines the potential of this dual‐drug co‐delivery approach as a promising therapeutic avenue for advancing GC treatment by combining chemotherapy and AKT inhibition.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results and Discussion","Expression of AKT in GC Tissues and its Clinical Significance","
Previous studies have validated abnormal activation of the PI3K/AKT signaling pathway in different types of tumors including GC could lead to unfavorable prognoses.[\n##REF##32333246##\n22\n##\n] The amplification of AKT mutations is believed to contribute to the activation of the PI3K/AKT signaling pathway, serving as one of the important factors responsible for this process.[\n##REF##24748656##\n23\n##\n] To reveal clinical significance of AKT expression in GC, analysis of AKT expression in GC tissues was performed and its relationship with prognosis using public databases assessed. The outcomes indicate a significantly elevated level of AKT expression in GC tissues in comparison to normal tissues (Figure\n##FIG##1##\n1A##). Furthermore, GC patients with a high level of AKT expression often have a significantly lower overall survival rate compared to those with a low level of AKT expression (Figure ##FIG##1##1B##). To validate this discovery, immunohistochemistry (IHC) analysis was conducted on clinical tissue samples obtained from the West China Hospital (Figure ##SUPPL##0##S1##, Supporting Information). The analysis results (Figure ##FIG##1##1C,D##) are well aligned with the data in Figure ##FIG##1##1A,B##, corroborating the findings reported by Gu et al.[\n##REF##24726064##\n24\n##\n] Furthermore, previous research findings have demonstrated a strong correlation between the activation of the PI3K/AKT signaling pathway and drug resistance in various tumors.[\n##REF##32973135##\n25\n##\n]\n
","
In this study, out of 100 GC patients, 25 received neoadjuvant chemotherapy, while 75 did not. IHC analysis supports a higher level of AKT expression in GC samples of patients who received neoadjuvant chemotherapy (p < 0.001) (Figure ##FIG##1##1E##). In addition, among 25 GC patients who underwent neoadjuvant chemotherapy, those with a lower level of AKT expression exhibit more pronounced tumor regression (Table ##SUPPL##0##S1##, Supporting Information). Western blot results confirm increased expression of AKT in MFC cells after PTX treatment for 24 h (Figure ##FIG##1##1F##). These results suggest chemotherapy could boost the expression of AKT in MFC cells, and the expression of AKT may be related to the inefficacy of chemotherapy. It has been reported that an AKT inhibitor, CAP, can suppress the expression of AKT, leading to an enhanced sensitivity of tumor cells to chemotherapeutic drugs such as PTX.[\n##REF##32070411##\n26\n##\n] To demonstrate the impact of CAP on the cytotoxic effect of PTX, CAP combined with PTX was applied to treatmouse forestomach carcinoma (MFC) cells and western blot results confirm that AKT expression is inhibited (Figure ##FIG##1##1F,G##). The Cell Counting Kit‐8 (CCK‐8) assay was conducted to assess the cytotoxic effect of CAP combined with free PTX on MFC cells. The results demonstrate that the combination of CAP and PTX exhibits a significantly higher level of cytotoxicity compared to free PTX (Figure ##FIG##1##1H##), which indicates that the inhibition of AKT may be an effective approach to enhancing the efficacy of chemotherapy against GC.
","
The synergistic anti‐tumor effect of free CAP and PTX was then comprehensively evaluated. The cytotoxic effects of the combination of free CAP and PTX on MFC cells were assessed at various CAP/PTX weight ratios (CAP: PTX = 4:1, 1:1, and 1:4). The results reveal that almost all synergy indexes for the combination of CAP and PTX at different weight ratios are less than 1, suggesting a synergistic effect of CAP and PTX in killing MFC cells (Figure ##FIG##1##1I##). This demonstrates tremendous potential of the combine therapy of PTX and CAP in treating GC.
","Fabrication and Characterizations of BPGP@CAP","
In this study, we utilized controlled RAFT polymerization and efficient thiol‐ene reaction for designing and synthesizing the cathepsin B‐sensitive branched glycopolymer‐PTX prodrug. As shown in Scheme ##SUPPL##0##S1## (Supporting Information), a crosslinking agent, MA‐GFLG‐MA, and a small molecular prodrug, maleimide‐GFLG‐PTX, were first synthesized. Structural confirmation and purity assessment were performed using 1H NMR and liquid chromatography‐mass spectrometry (LC‐MS), and the experimental results are presented in Figures ##SUPPL##0##S2–S7## (Supporting Information). After successful preparation of polymerizable monomers and functionalized small molecules, RAFT polymerization was conducted to produce a high‐molecular‐weight chain transfer agent, poly(LAEMA)‐CTA, illustrated in Scheme ##SUPPL##0##S2A## (Supporting Information). An approximate value of 28 kDa is obtained from molecular weight calculation via 1H NMR and the repeating LAEMA units are ≈61 (Figure ##SUPPL##0##S8##, Supporting Information). Gel permeation chromatography (GPC) analysis reveals a PDI of ≈1.06 for the chain transfer agent (Figure ##SUPPL##0##S13##, Supporting Information), indicative of a narrow molecular weight distribution.
","
Subsequently, co‐polymerization of poly(LAEMA)‐CTA, MA‐GFLG‐MA, MA‐PySS, and LAEMA yields an intermediate polymer with a branched architecture (branched poly(LAEMA)‐GFLG‐PySS). The 1H NMR spectrum of the intermediate polymer displays significant characteristic peaks of pyridine at 7.22 ppm, 7.74 ppm, and 8.31 ppm, indicating successful introduction of MA‐PySS into the polymer structure (Figure ##SUPPL##0##S9##, Supporting Information). After deprotection treatment, the characteristic peak of pyridine in the polymer disappears from the 1H NMR spectrum, while a characteristic peak of the benzene ring in GFLG is observed at 7.29 ppm (Figure ##SUPPL##0##S10##, Supporting Information). The as‐prepared intermediate polymer was sequentially conjugated with maleimide‐Cy5 and maleimide GFLG PTX, resulting in a product of a branched polymer prodrug, poly(LAEMACy5)‐GFLG‐PTX (termed as BPGP). Structural confirmation of the product is attained through its 1H NMR (Figure\n##FIG##2##\n2A##). Notably, distinct peaks corresponding to PTX and GFLG are not observed in the 1H NMR spectrum of BPGP in D2O, which is distinctly different from the spectrum of BPGP in DMSO‐d6. This disparity suggests BPGP may experience self‐assembly in an aqueous solution (Figure ##SUPPL##0##S11## and ##SUPPL##0##S12##, Supporting Information). As shown in Figure ##FIG##2##2B##, compared with unlabeled branched polymer prodrugs, the characteristic peak of Cy5 can be observed in both ultraviolet visible (UV‐vis) and fluorescence spectra, and there is no significant change in the wavelength of the characteristic peak compared to free Cy5, indicating that the optical properties of Cy5 remain after it was covalently coupled to the polymer.
","
Due to the presence of hydrophilic segments and hydrophobic drugs in the branched polymer prodrug, the prodrug could self‐assemble into nanoparticles through hydrophilic hydrophobic interactions. We used pyrene as a fluorescence probe to detect its critical micelle concentration (CAC), and its CAC value is found to be ≈2.64 µg mL−1, which indicates that the polymer prodrug possesses a remarkable self‐assembly ability (Figure ##FIG##2##2C##). Subsequently, we assessed the potential of the PTX prodrug as a polymeric nanocarrier system for drug delivery. To realize the synergy of chemotherapy and molecular targeted therapy, we utilized a solvent evaporation technique to encapsulate CAP, an AKT inhibitor, into the glycopolymer‐PTX prodrug, resulting in a nanoassembly, BPGP@CAP. Initially, the nanoassembly was fabricated at different PTX‐to‐CAP feed ratios to assess the encapsulation capability of the polymer‐PTX prodrug for CAP. As shown in Figure ##FIG##2##2D##, with an increase in the CAP feed weight, the drug loading of CAP within the nanoassembly progressively augments, signifying a great encapsulation capacity of the nanoassembly. However, it's worth noting that as the drug loading increases, the encapsulation efficiency of the nanoassembly exhibits a gradual decline. Notably, when the weight ratio of PTX to CAP drops below 1:1.5, the encapsulation efficiency of the nanoassembly experiences a substantial reduction. Consequently, the nanoassembly synthesized at a PTX‐to‐CAP weight ratio of 1:1.5 is chosen for subsequent experimental uses. The results of HPLC analysis reveal that 7.5 wt.% of PTX and 8.0 wt.% of CAP are in the nanoassembly of BPGP@CAP. As shown in Figure ##FIG##2##2E,F##, DLS measurements confirm that the hydrodynamic diameter of BPGP is ≈78.83 ± 0.35 nm, and upon encapsulation of CAP, a slight increase in the hydrodynamic size is observed (90.12 ± 0.36 nm). TEM images exhibit a relatively uniform size distribution for both BPGP and BPGP@CAP. Although encapsulation of CAP leads to a marginal size augmentation, the morphology of the nanoassembly remains unchanged. Leveraging the presence of GFLG in the polymer structure, the drug release behavior from the polymer prodrug was probed in a simulated tumor intracellular microenvironment. Since papain and cathepsin B share the same catalytic site, papain was selected to mimic cathepsin B in the tumor microenvironment. As depicted in the Figure ##FIG##2##2G##, under conditions without papain catalysis, the polymer prodrug retains stability with negligible drug release. Conversely, under enzymatic catalysis, the polymer prodrug effectively releases the loaded drug. These findings suggest the polymer prodrug could be used for stable in vivo drug delivery and controlled drug release at tumor sites.
","Cellular Uptake of BPGP@CAP by MFC Cells","
Cellular uptake of polymeric drug delivery systems by tumor cells is a crucial step to exert a drug antitumor effect. The uptake of Cy5‐labeled BPGP@CAP into MFC cells was evaluated using CLSM and flow cytometry techniques. By observing the fluorescence signal of Cy5 in MFC cells after incubation with Cy5‐labeled BPGP@CAP at different durations through CLSM, it is evident that with an increase in the incubation time, the red fluorescence signal from Cy5 gradually accumulates and becomes intensified in the cytoplasm of MFC cells (Figure\n##FIG##3##\n3A##; Figure ##SUPPL##0##S14##, Supporting Information). Flow cytometry data for uptake of Cy5‐labeled BPGP@CAP by MFC cells is in agreement with the CLSM images (Figure ##FIG##3##3C##; Figure ##SUPPL##0##S15##, Supporting Information). These results indicate that BPGP@CAP could be efficiently ingested by MFC cells, and this uptake process is time‐dependent.
","
To investigate the endocytic pathways of BPGP@CAP into MFC cells, flow cytometry was employed to detect the uptake of BPGP@CAP by MFC cells at a low temperature. The results show that the uptake of BPGP@CAP by MFC cells is significantly inhibited at an inhibition rate of 80.5%, indicating that the internalization of BPGP@CAP is an energy‐dependent process. Furthermore, upon incubating inhibitors with MFC cells, flow cytometry data demonstrate that chlorpromazine, genistein, and amiloride inhibit cellular uptake of BPGP@CAP by 70.2%, 65.4%, and 44.5% respectively. This suggests that clathrin‐mediated, caveolin‐mediated, and icropinocytosis‐mediated endocytic pathways may be involved in the uptake of BPGP@CAP (Figure ##FIG##3##3D##; Figure ##SUPPL##0##S16##, Supporting Information). Moreover, since the lysosomal cathepsin B can cleave the GFLG linker to release PTX,[\n##UREF##3##\n27\n##\n] we investigated whether BPGP@CAP would be transported to the lysosomes for degradation after cellular uptake. Through lysosomal colocalization experiments (Figure ##FIG##3##3B##), we observe a high overlapping level of the fluorescence signal for BPGP@CAP and lysosomes, suggesting BPGP@CAP may land at lysosomes before it is disintegrated by lysosomal cathepsin B. Combined with the findings from in vitro drug release experiments, it is confirmed that BPGP@CAP can be internalized by MFC cells and release drugs stimulated by lysosomal cathepsin B.
","The Toxicity of BPGP@CAP on MFC Cells","
To assess the toxicity of BPGP@CAP, we conducted CCK‐8 assays to measure the viability of MFC cells subjected to various treatment groups. The results indicate that after the treatment of BPGP@CAP, MFC cells viability significantly decreases. BPGP@CAP has an IC50 of 0.253 µg mL−1 in comparison to free PTX with an IC50 of 2.51 µg mL−1 and CAP with an IC50 of 4.74 µg mL−1. Furthermore, the cell viability in the group with the treatment of BPGP@CAP is similar to that in the group treated with free PTX+CAP with an IC50 of 0.43 µg mL−1, whereas the cell viability in the group treated with BPGP is comparable to that in the group exposed to free PTX, and the IC50 value for BPGP and free PTX is 2.52 µg mL−1 and 2.51 µg mL−1, respectively (Figure ##FIG##3##3E##). These findings suggest that BPGP@CAP could effectively release PTX and CAP after disintegration of the nanoassembly structure in the lysosomes, thus exerting an equivalent antitumor effect to a mixture of free PTX and CAP. Additionally, both the groups treated with BPGP@CAP and PTX + CAP exhibit a significantly higher cytotoxic effect on MFC cells than the groups exposed to BPGP and free PTX, indicating that the combination of CAP and PTX displays a synergistic action on MFC cells.
","
Microtubules, which are essential cytoskeletal filaments within cells, have a critical function in mitosis and intracellular vesicle transport.[\n##REF##30536951##\n28\n##\n] It is well‐established that PTX can enhance the polymerization of microtubules, hinder depolymerization, and suppress mitosis of tumor cells.[\n##REF##24435445##\n29\n##\n] In order to assess the impact of PTX derived from BPGP@CAP on microtubules, we used CLSM to observe morphological changes of microtubules in the MFC cells after various treatments. Microtubules are evenly distributed in the cytoplasm of MFC cells in the control and CAP‐treated groups. However, pronounced aggregation of microtubules is found around the cell nucleus in the groups exposed to PTX and PTX+CAP. Similar morphological changes of microtubules are observed in the groups treated with BPGP and BPGP@CAP (Figure ##FIG##3##3F##). Furthermore, in the control and CAP‐treated groups, the nuclei of MFC cells appear intact, whereas an abnormal nuclear morphology and a multinucleated structure are found in the groups treated with PTX, PTX + CAP, BPGP and BPGP@CAP, indicating that PTX released from BPGP@CAP exerts a similar effect on microtubules as free PTX.
","
Furthermore, microfilaments, which are composed of actin proteins, are integral constituents of the cellular cytoskeleton. Through CLSM, the influence of BPGP@CAP on microfilaments of MFC cells was examined. Our results indicate that actin aggregates into clusters or dots near the cell membrane in the BPGP@CAP‐treated group (Figure ##SUPPL##0##S17##, Supporting Information), leading to impaired microfilaments and reduced cell viability.
","
Moreover, we utilized flow cytometry to evaluate the impact of BPGP@CAP on the apoptosis of MFC cells. The findings indicate that the percentage of apoptotic cells in the groups exposed to BPGP@CAP (23.8%), BPGP (15.7%), PTX (17.3%), and PTX+CAP (22.3%) is significantly higher than that in the control group (4.7%) (Figure\n##FIG##4##\n4A,C##). Additionally, no significant difference is found in the percentage of apoptotic cells between the groups exposed to BPGP@CAP and PTX + CAP. Furthermore, MFC cells have a significantly higher apoptotic percentage after treatment with BPGP@CAP and PTX + CAP than those treated with PTX and CAP, further supporting their synergistic action of inducting apoptosis of MFC cells.
","
It is widely recognized that PTX exerts its effects on microtubules, causing inhibition of cell mitosis and resulting in G2/M cell cycle arrest.[\n##UREF##4##\n30\n##\n] We assessed the influence of PTX released from BPGP@CAP on the cell cycle distribution of MFC cells. As presented in Figure ##FIG##4##4B,D##, the percentages of cells in the G2/M phase in the groups treated with PTX (25.1%), BPGP (20.2%), PTX + CAP (27.8%), and BPGP@CAP (23.6%) are significantly higher than that in the control group (11.0%). Conversely, the proportions cells in the G0/G1 phase in the groups treated with PTX (43.9%), BPGP (52.2%), PTX + CAP (39.0%), and BPGP@CAP (39.6%) are significantly lower than that in the control group (76.6%). These findings support that PTX released from BPGP and BPGP@CAP can lead to cell cycle arrest in the G2/M phase. These results further confirm that BPGP@CAP can effectively releases PTX in the tumor cells, thereby displaying a similar antitumor mechanism of action as free PTX.
","Mechanism Study on the Synergistic Effect of PTX and CAP","
Our in vitro experimental results support the existence of a synergistic effect between PTX and CAP to eliminate MFC cells. To explore the underlying mechanisms behind this synergy, we conducted transcriptome sequencing on MFC cells exposed to BPGP@CAP and compared them with the control group. We detected 2799 differentially expressed genes (DEGs) between BPGP@CAP and control groups. Out of these DEGs, 1678 are upregulated and 1121 downregulated in the BPGP@CAP‐treated group (Figure\n##FIG##5##\n5A##; Figure ##SUPPL##0##S18##, Supporting Information). Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was employed to uncover the biological pathways impacted by these DEGs. The results suggest that the pathways for apoptosis and PI3K/AKT signaling are significantly upregulated, which indicates that the mechanism underlying the synergistic antitumor effects of PTX and CAP in BPGP@CAP may be related to the PI3K/AKT signaling pathway and apoptosis (Figure ##FIG##5##5B##). It has been evidenced that the PI3K/AKT signaling pathway can regulate apoptosis by modulating the phosphorylation of Bax and Bad and the regulation of their expression levels, the release of cytochrome C, and the activation of caspase ‐ 3 and 9, thus affecting cell survival.[\n##REF##23881702##\n31\n##\n] Western blot results confirm that CAP inhibits the expression of the PI3K/AKT pathway‐related proteins such as AKT, p‐mTOR, p‐S6, and p‐4EBP1 (Figure ##FIG##5##5C##), indicating that CAP can suppress the PI3K/AKT signaling pathway.
","
Furthermore, western blotting was employed to assess the expression of apoptosis‐related proteins, such as cleaved PARP, cleaved caspase‐3, Bax, and Bcl‐2, in various treatment groups. As showed in Figure ##FIG##5##5E,F##, the expression of Bax, cleaved caspase‐3, and cleaved PARP is upregulated in MFC cells after treatment with CAP, which might be credited to apoptosis after the inhibition of AKT. Furthermore, it is known that PTX can induce tumor cell apoptosis by downregulation of Bcl‐2 and upregulation of Bax and cleaved caspase‐3.[\n##REF##23735541##\n32\n##\n] Our results confirm that the expression levels of Bax, cleaved caspase‐3, and cleaved PARP were elevate, while Bcl‐2 is downregulated in the MFC cells with treatment of PTX and BPGP. In addition, when MFC was treated with the combination of free PTX and CAP or BPGP@CAP, more pronounced changes are observed in the expression of proteins including Bax, cleaved caspase −3, and cleaved PARP. Interestingly, western blot results reveal that the effect of CAP on downregulating the BCL‐2 protein expression is not pronounced as PTX, suggesting that the downregulation of Bcl‐2 may be predominantly induced by PTX, while no apparent correlation is seen between the downregulation of Bcl‐2 and the inhibition of AKT expression. However, the combination of PTX and CAP can induce more significant downregulation of Bcl‐2 compared to their individual effects, which is in agreement with a previous study.[\n##REF##32070411##\n26\n##\n] It may be explained by that CAP could significantly sensitize MFC cells to PTX‐induced apoptosis, resulting in more prominent downregulation of Bcl‐2.
","
The combination of PTX with CAP exerts synergistic effects on MFC cells via the modulation of pro and anti‐apoptotic genes, such as downregulation of Bcl‐2 and upregulation of Bax, cleaved caspase‐3, and cleaved PARP. This could be the underlying mechanism for synergistic promotion of apoptosis by PTX and CAP (Figure ##FIG##5##5G##).
","Antitumor Effect of BPGP@CAP In Vivo","\n<italic toggle=\"yes\">Distribution of BPGP@CAP</italic> In Vivo","
The accumulation of BPGP@CAP at the site of transplanted tumor plays a critical role in its antitumor efficacy in vivo. Mounting evidence has suggested that nanoparticles exhibit the enhanced permeability and retention (EPR) effect, which can extend the residence time and increase the concentration of drugs in tumors, thus achieving passive drug targeting of tumor cells.[\n##UREF##5##\n33\n##\n] The characterization results of BPGP@CAP indicate that it possesses a nanoscale size for the EPR effect. To confirm the accumulation of BPGP@CAP in tumor tissues via the EPR effect, we utilized a fluorescence imaging technique to evaluate the distribution of BPGP@CAP in the mice with MFC tumors. By administering Cy5‐labeled BPGP@CAP and free Cy5 intravenously to the tumor‐bearing nude mice, we assessed the distribution of BPGP@CAP at various time intervals using IVIS spectroscopy. The findings reveal that the fluorescence intensity of the tumor in the BPGP@CAP‐treated group is stronger than that in the free Cy5‐treated group as time elapses (Figure\n##FIG##6##\n6A##; Figure ##SUPPL##0##S19##, Supporting Information). Specifically, after 24 h, the fluorescence intensity of the tumor in the BPGP@CAP‐treated group still remains strong, while faint fluorescence signal is detected in the tumor of the free Cy5‐treated group. This indicates that BPGP@CAP can accumulate and retain in the tumor site for an extended period. Furthermore, tumor tissues were collected at 24 h post‐injection and visualized using CLSM. The images demonstrate that tumor tissues of the mice after the treatment of free Cy5 show undetectable fluorescence signal, whereas tumor tissues of the mice after the treatment of BPGP@CAP exhibit significantly strong fluorescence intensity (Figure ##FIG##6##6B##). The in vivo imaging results confirm the enrichment of BPGP@CAP in tumor tissues.
","
Furthermore, pharmacokinetic analysis in vivo reveals a slower decrease in the Cy5 concentration in the blood of the mice exposed to BPGP@CAP compared to that in the group exposed to free Cy5, and the half‐life of BPGP@CAP and free Cy5 was 488.49 and 160.85 min, respectively (Figure ##FIG##6##6C##; Table ##SUPPL##0##S2##, Supporting Information). The results might be ascribed to a high molecular weight of BPGP@CAP and a prolonged circulation time which may help enhance passive accumulation of BPGP@CAP at the tumor site, thereby improving its therapeutic efficacy against tumors.
","Antitumor Effect of BPGP@CAP","
The mice, which were subcutaneously injected with MFC cells, were divided randomly into six groups, each consisting of 5 mice. The control group were given intravenous injections of saline, while the other groups were intravenously injected with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. The concentrations of PTX remain consistent at 8 mg kg−1 for each mouse. The treatment schedule is illustrated in Figure ##FIG##6##6D##. The volume of the transplanted tumor and the body weight of each treated mouse were regularly monitored and recorded. The results reveal that the control group has the fastest tumor growth rate, while the BPGP@CAP‐treated group displays the slowest tumor growth (Figure ##FIG##6##6E##). After the treatment, the tumor volume of the control group is 1.45, 1.67, 2.04, 2.48, and 4.13 times that of the group treated with PTX, CAP, PTX+CAP, BPGP, and BPGP@CAP, respectively. The tumor inhibition rates, based on tumor volume measurements, are found to be 31.0%, 40.1%, 51.0%, 59.7%, and 75.8% after treatment with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. In addition, the tumor volumes in the groups with the treatment of free PTX and PTX + CAP are 2.85 times and 2.02 times that of the BPGP@CAP‐treated group, respectively (Figure ##FIG##6##6F##). The tumor volumes of the transplanted tumors harvested from each group are well aligned with those observed in vivo (Figure ##SUPPL##0##S20A##, Supporting Information). Comparison of the transplant tumor masses in the treatment groups reveals that the tumor mass harvested from the BPGP@CAP‐treated mice (0.28 g) is significantly lighter than that of the free control group (1.20 g), the PTX‐treated group (0.84 g), and the PTX + ACP‐treated group (0.57 g) (Figure ##FIG##6##6G##). The tumor inhibition rates, based on tumor mass measurements, are estimated to be 30.2%, 39.1%, 49.6%, 58.6%, and 75.7% for PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively (Figure ##SUPPL##0##S20B##, Supporting Information). The tumor inhibition rates based on tumor mass measurements are similar to those based on tumor volume measurements in vivo for CAP and/or PTX‐derived formulations. The difference in the tumor inhibition rates among BPGP@CAP and other groups may result from a synergistic effect of PTX and CAP, enrichment of BPGP@CAP in tumor tissues and prolonged circulation in the blood through a nanoscale drug delivery system.
","
The antitumor effect of BPGP@CAP was further verified by IHC analysis of tumor tissues. TUNEL assays of the harvested tumors indicate TUNEL‐positive tumor cells in the BPGP@CAP‐treated group are the most populous, confirming that the treatment of BPGP@CAP results in more apoptotic cells compared with other groups (Figure ##FIG##6##6I,J##). Furthermore, the mice treated with BPGP@CAP show a significant reduction in the population of Ki67‐positive tumor cells with comparison to the other groups. This observation suggests that the administration of BPGP@CAP leads to a remarkable suppression of tumor cell proliferation (Figure ##FIG##6##6I,J##), which confirms synergistic inhibition of tumor growth by both agents in vivo. These findings support the excellent anti‐tumor effect of BPGP@CAP.
","
Additionally, while the administration of free PTX and PTX + CAP effectively suppresses the growth of transplanted tumors in the mice, it is accompanied by a decrease in the body weight ranging from 9.1% to 6.3% after each administration (Figure ##FIG##6##6H##). This indicates the presence of systemic toxicity associated with free PTX. In contrast, no significant changes in the body weight are found in the BPGP and BPGP@CAP‐treated groups, suggesting that the toxicity of PTX could be remarkably reduced after PTX is delivered via a polymer prodrug. In addition, we conducted H&E staining on the major organs of the mice and analyzed the change of hematological parameters including white blood cells (WBC), red blood cells (RBC), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) to evaluate the toxicity of BPGP@CAP. The H&E staining images indicate no significant alterations in the major organs, including the heart, liver, spleen, lung, and kidney, across all treatment groups (Figure ##SUPPL##0##S21##, Supporting Information). However, compared to the control group with a WBC count of 8.20×109/L, the WBC count of the mice is found to be 3.19 × 109 per L, 3.28 × 109 per L, 5.76 × 109 per L and 5.69 × 109 per L in the group treated free PTX, PTX + CAP, BPGP, and BPGP@CAP, respectively. The most pronounced decreases in the WBC number are seen in the groups treated with PTX and PTX + CAP. Furthermore, when compared to the control group where the ALT level is 21.0 U L−1, there is no significant change in the ALT level in the BPGP+CAP‐treated group (22.0 U L−1), while a distinctive increase is seen in the PTX group (71.7 U L−1) and the PTX+CAP group (63.3 U L−1) (Figure ##SUPPL##0##S22##, Supporting Information). These results indicate that systemic administration of free PTX or PTX + CAP can cause hematopoietic system toxicity and liver toxicity, while the use of BPGP@CAP to deliver PTX and CAP can significantly reduce their toxicity.
"],"string":"[\n \"Results and Discussion\",\n \"Expression of AKT in GC Tissues and its Clinical Significance\",\n \"
Previous studies have validated abnormal activation of the PI3K/AKT signaling pathway in different types of tumors including GC could lead to unfavorable prognoses.[\\n##REF##32333246##\\n22\\n##\\n] The amplification of AKT mutations is believed to contribute to the activation of the PI3K/AKT signaling pathway, serving as one of the important factors responsible for this process.[\\n##REF##24748656##\\n23\\n##\\n] To reveal clinical significance of AKT expression in GC, analysis of AKT expression in GC tissues was performed and its relationship with prognosis using public databases assessed. The outcomes indicate a significantly elevated level of AKT expression in GC tissues in comparison to normal tissues (Figure\\n##FIG##1##\\n1A##). Furthermore, GC patients with a high level of AKT expression often have a significantly lower overall survival rate compared to those with a low level of AKT expression (Figure ##FIG##1##1B##). To validate this discovery, immunohistochemistry (IHC) analysis was conducted on clinical tissue samples obtained from the West China Hospital (Figure ##SUPPL##0##S1##, Supporting Information). The analysis results (Figure ##FIG##1##1C,D##) are well aligned with the data in Figure ##FIG##1##1A,B##, corroborating the findings reported by Gu et al.[\\n##REF##24726064##\\n24\\n##\\n] Furthermore, previous research findings have demonstrated a strong correlation between the activation of the PI3K/AKT signaling pathway and drug resistance in various tumors.[\\n##REF##32973135##\\n25\\n##\\n]\\n
\",\n \"
In this study, out of 100 GC patients, 25 received neoadjuvant chemotherapy, while 75 did not. IHC analysis supports a higher level of AKT expression in GC samples of patients who received neoadjuvant chemotherapy (p < 0.001) (Figure ##FIG##1##1E##). In addition, among 25 GC patients who underwent neoadjuvant chemotherapy, those with a lower level of AKT expression exhibit more pronounced tumor regression (Table ##SUPPL##0##S1##, Supporting Information). Western blot results confirm increased expression of AKT in MFC cells after PTX treatment for 24 h (Figure ##FIG##1##1F##). These results suggest chemotherapy could boost the expression of AKT in MFC cells, and the expression of AKT may be related to the inefficacy of chemotherapy. It has been reported that an AKT inhibitor, CAP, can suppress the expression of AKT, leading to an enhanced sensitivity of tumor cells to chemotherapeutic drugs such as PTX.[\\n##REF##32070411##\\n26\\n##\\n] To demonstrate the impact of CAP on the cytotoxic effect of PTX, CAP combined with PTX was applied to treatmouse forestomach carcinoma (MFC) cells and western blot results confirm that AKT expression is inhibited (Figure ##FIG##1##1F,G##). The Cell Counting Kit‐8 (CCK‐8) assay was conducted to assess the cytotoxic effect of CAP combined with free PTX on MFC cells. The results demonstrate that the combination of CAP and PTX exhibits a significantly higher level of cytotoxicity compared to free PTX (Figure ##FIG##1##1H##), which indicates that the inhibition of AKT may be an effective approach to enhancing the efficacy of chemotherapy against GC.
\",\n \"
The synergistic anti‐tumor effect of free CAP and PTX was then comprehensively evaluated. The cytotoxic effects of the combination of free CAP and PTX on MFC cells were assessed at various CAP/PTX weight ratios (CAP: PTX = 4:1, 1:1, and 1:4). The results reveal that almost all synergy indexes for the combination of CAP and PTX at different weight ratios are less than 1, suggesting a synergistic effect of CAP and PTX in killing MFC cells (Figure ##FIG##1##1I##). This demonstrates tremendous potential of the combine therapy of PTX and CAP in treating GC.
\",\n \"Fabrication and Characterizations of BPGP@CAP\",\n \"
In this study, we utilized controlled RAFT polymerization and efficient thiol‐ene reaction for designing and synthesizing the cathepsin B‐sensitive branched glycopolymer‐PTX prodrug. As shown in Scheme ##SUPPL##0##S1## (Supporting Information), a crosslinking agent, MA‐GFLG‐MA, and a small molecular prodrug, maleimide‐GFLG‐PTX, were first synthesized. Structural confirmation and purity assessment were performed using 1H NMR and liquid chromatography‐mass spectrometry (LC‐MS), and the experimental results are presented in Figures ##SUPPL##0##S2–S7## (Supporting Information). After successful preparation of polymerizable monomers and functionalized small molecules, RAFT polymerization was conducted to produce a high‐molecular‐weight chain transfer agent, poly(LAEMA)‐CTA, illustrated in Scheme ##SUPPL##0##S2A## (Supporting Information). An approximate value of 28 kDa is obtained from molecular weight calculation via 1H NMR and the repeating LAEMA units are ≈61 (Figure ##SUPPL##0##S8##, Supporting Information). Gel permeation chromatography (GPC) analysis reveals a PDI of ≈1.06 for the chain transfer agent (Figure ##SUPPL##0##S13##, Supporting Information), indicative of a narrow molecular weight distribution.
\",\n \"
Subsequently, co‐polymerization of poly(LAEMA)‐CTA, MA‐GFLG‐MA, MA‐PySS, and LAEMA yields an intermediate polymer with a branched architecture (branched poly(LAEMA)‐GFLG‐PySS). The 1H NMR spectrum of the intermediate polymer displays significant characteristic peaks of pyridine at 7.22 ppm, 7.74 ppm, and 8.31 ppm, indicating successful introduction of MA‐PySS into the polymer structure (Figure ##SUPPL##0##S9##, Supporting Information). After deprotection treatment, the characteristic peak of pyridine in the polymer disappears from the 1H NMR spectrum, while a characteristic peak of the benzene ring in GFLG is observed at 7.29 ppm (Figure ##SUPPL##0##S10##, Supporting Information). The as‐prepared intermediate polymer was sequentially conjugated with maleimide‐Cy5 and maleimide GFLG PTX, resulting in a product of a branched polymer prodrug, poly(LAEMACy5)‐GFLG‐PTX (termed as BPGP). Structural confirmation of the product is attained through its 1H NMR (Figure\\n##FIG##2##\\n2A##). Notably, distinct peaks corresponding to PTX and GFLG are not observed in the 1H NMR spectrum of BPGP in D2O, which is distinctly different from the spectrum of BPGP in DMSO‐d6. This disparity suggests BPGP may experience self‐assembly in an aqueous solution (Figure ##SUPPL##0##S11## and ##SUPPL##0##S12##, Supporting Information). As shown in Figure ##FIG##2##2B##, compared with unlabeled branched polymer prodrugs, the characteristic peak of Cy5 can be observed in both ultraviolet visible (UV‐vis) and fluorescence spectra, and there is no significant change in the wavelength of the characteristic peak compared to free Cy5, indicating that the optical properties of Cy5 remain after it was covalently coupled to the polymer.
\",\n \"
Due to the presence of hydrophilic segments and hydrophobic drugs in the branched polymer prodrug, the prodrug could self‐assemble into nanoparticles through hydrophilic hydrophobic interactions. We used pyrene as a fluorescence probe to detect its critical micelle concentration (CAC), and its CAC value is found to be ≈2.64 µg mL−1, which indicates that the polymer prodrug possesses a remarkable self‐assembly ability (Figure ##FIG##2##2C##). Subsequently, we assessed the potential of the PTX prodrug as a polymeric nanocarrier system for drug delivery. To realize the synergy of chemotherapy and molecular targeted therapy, we utilized a solvent evaporation technique to encapsulate CAP, an AKT inhibitor, into the glycopolymer‐PTX prodrug, resulting in a nanoassembly, BPGP@CAP. Initially, the nanoassembly was fabricated at different PTX‐to‐CAP feed ratios to assess the encapsulation capability of the polymer‐PTX prodrug for CAP. As shown in Figure ##FIG##2##2D##, with an increase in the CAP feed weight, the drug loading of CAP within the nanoassembly progressively augments, signifying a great encapsulation capacity of the nanoassembly. However, it's worth noting that as the drug loading increases, the encapsulation efficiency of the nanoassembly exhibits a gradual decline. Notably, when the weight ratio of PTX to CAP drops below 1:1.5, the encapsulation efficiency of the nanoassembly experiences a substantial reduction. Consequently, the nanoassembly synthesized at a PTX‐to‐CAP weight ratio of 1:1.5 is chosen for subsequent experimental uses. The results of HPLC analysis reveal that 7.5 wt.% of PTX and 8.0 wt.% of CAP are in the nanoassembly of BPGP@CAP. As shown in Figure ##FIG##2##2E,F##, DLS measurements confirm that the hydrodynamic diameter of BPGP is ≈78.83 ± 0.35 nm, and upon encapsulation of CAP, a slight increase in the hydrodynamic size is observed (90.12 ± 0.36 nm). TEM images exhibit a relatively uniform size distribution for both BPGP and BPGP@CAP. Although encapsulation of CAP leads to a marginal size augmentation, the morphology of the nanoassembly remains unchanged. Leveraging the presence of GFLG in the polymer structure, the drug release behavior from the polymer prodrug was probed in a simulated tumor intracellular microenvironment. Since papain and cathepsin B share the same catalytic site, papain was selected to mimic cathepsin B in the tumor microenvironment. As depicted in the Figure ##FIG##2##2G##, under conditions without papain catalysis, the polymer prodrug retains stability with negligible drug release. Conversely, under enzymatic catalysis, the polymer prodrug effectively releases the loaded drug. These findings suggest the polymer prodrug could be used for stable in vivo drug delivery and controlled drug release at tumor sites.
\",\n \"Cellular Uptake of BPGP@CAP by MFC Cells\",\n \"
Cellular uptake of polymeric drug delivery systems by tumor cells is a crucial step to exert a drug antitumor effect. The uptake of Cy5‐labeled BPGP@CAP into MFC cells was evaluated using CLSM and flow cytometry techniques. By observing the fluorescence signal of Cy5 in MFC cells after incubation with Cy5‐labeled BPGP@CAP at different durations through CLSM, it is evident that with an increase in the incubation time, the red fluorescence signal from Cy5 gradually accumulates and becomes intensified in the cytoplasm of MFC cells (Figure\\n##FIG##3##\\n3A##; Figure ##SUPPL##0##S14##, Supporting Information). Flow cytometry data for uptake of Cy5‐labeled BPGP@CAP by MFC cells is in agreement with the CLSM images (Figure ##FIG##3##3C##; Figure ##SUPPL##0##S15##, Supporting Information). These results indicate that BPGP@CAP could be efficiently ingested by MFC cells, and this uptake process is time‐dependent.
\",\n \"
To investigate the endocytic pathways of BPGP@CAP into MFC cells, flow cytometry was employed to detect the uptake of BPGP@CAP by MFC cells at a low temperature. The results show that the uptake of BPGP@CAP by MFC cells is significantly inhibited at an inhibition rate of 80.5%, indicating that the internalization of BPGP@CAP is an energy‐dependent process. Furthermore, upon incubating inhibitors with MFC cells, flow cytometry data demonstrate that chlorpromazine, genistein, and amiloride inhibit cellular uptake of BPGP@CAP by 70.2%, 65.4%, and 44.5% respectively. This suggests that clathrin‐mediated, caveolin‐mediated, and icropinocytosis‐mediated endocytic pathways may be involved in the uptake of BPGP@CAP (Figure ##FIG##3##3D##; Figure ##SUPPL##0##S16##, Supporting Information). Moreover, since the lysosomal cathepsin B can cleave the GFLG linker to release PTX,[\\n##UREF##3##\\n27\\n##\\n] we investigated whether BPGP@CAP would be transported to the lysosomes for degradation after cellular uptake. Through lysosomal colocalization experiments (Figure ##FIG##3##3B##), we observe a high overlapping level of the fluorescence signal for BPGP@CAP and lysosomes, suggesting BPGP@CAP may land at lysosomes before it is disintegrated by lysosomal cathepsin B. Combined with the findings from in vitro drug release experiments, it is confirmed that BPGP@CAP can be internalized by MFC cells and release drugs stimulated by lysosomal cathepsin B.
\",\n \"The Toxicity of BPGP@CAP on MFC Cells\",\n \"
To assess the toxicity of BPGP@CAP, we conducted CCK‐8 assays to measure the viability of MFC cells subjected to various treatment groups. The results indicate that after the treatment of BPGP@CAP, MFC cells viability significantly decreases. BPGP@CAP has an IC50 of 0.253 µg mL−1 in comparison to free PTX with an IC50 of 2.51 µg mL−1 and CAP with an IC50 of 4.74 µg mL−1. Furthermore, the cell viability in the group with the treatment of BPGP@CAP is similar to that in the group treated with free PTX+CAP with an IC50 of 0.43 µg mL−1, whereas the cell viability in the group treated with BPGP is comparable to that in the group exposed to free PTX, and the IC50 value for BPGP and free PTX is 2.52 µg mL−1 and 2.51 µg mL−1, respectively (Figure ##FIG##3##3E##). These findings suggest that BPGP@CAP could effectively release PTX and CAP after disintegration of the nanoassembly structure in the lysosomes, thus exerting an equivalent antitumor effect to a mixture of free PTX and CAP. Additionally, both the groups treated with BPGP@CAP and PTX + CAP exhibit a significantly higher cytotoxic effect on MFC cells than the groups exposed to BPGP and free PTX, indicating that the combination of CAP and PTX displays a synergistic action on MFC cells.
\",\n \"
Microtubules, which are essential cytoskeletal filaments within cells, have a critical function in mitosis and intracellular vesicle transport.[\\n##REF##30536951##\\n28\\n##\\n] It is well‐established that PTX can enhance the polymerization of microtubules, hinder depolymerization, and suppress mitosis of tumor cells.[\\n##REF##24435445##\\n29\\n##\\n] In order to assess the impact of PTX derived from BPGP@CAP on microtubules, we used CLSM to observe morphological changes of microtubules in the MFC cells after various treatments. Microtubules are evenly distributed in the cytoplasm of MFC cells in the control and CAP‐treated groups. However, pronounced aggregation of microtubules is found around the cell nucleus in the groups exposed to PTX and PTX+CAP. Similar morphological changes of microtubules are observed in the groups treated with BPGP and BPGP@CAP (Figure ##FIG##3##3F##). Furthermore, in the control and CAP‐treated groups, the nuclei of MFC cells appear intact, whereas an abnormal nuclear morphology and a multinucleated structure are found in the groups treated with PTX, PTX + CAP, BPGP and BPGP@CAP, indicating that PTX released from BPGP@CAP exerts a similar effect on microtubules as free PTX.
\",\n \"
Furthermore, microfilaments, which are composed of actin proteins, are integral constituents of the cellular cytoskeleton. Through CLSM, the influence of BPGP@CAP on microfilaments of MFC cells was examined. Our results indicate that actin aggregates into clusters or dots near the cell membrane in the BPGP@CAP‐treated group (Figure ##SUPPL##0##S17##, Supporting Information), leading to impaired microfilaments and reduced cell viability.
\",\n \"
Moreover, we utilized flow cytometry to evaluate the impact of BPGP@CAP on the apoptosis of MFC cells. The findings indicate that the percentage of apoptotic cells in the groups exposed to BPGP@CAP (23.8%), BPGP (15.7%), PTX (17.3%), and PTX+CAP (22.3%) is significantly higher than that in the control group (4.7%) (Figure\\n##FIG##4##\\n4A,C##). Additionally, no significant difference is found in the percentage of apoptotic cells between the groups exposed to BPGP@CAP and PTX + CAP. Furthermore, MFC cells have a significantly higher apoptotic percentage after treatment with BPGP@CAP and PTX + CAP than those treated with PTX and CAP, further supporting their synergistic action of inducting apoptosis of MFC cells.
\",\n \"
It is widely recognized that PTX exerts its effects on microtubules, causing inhibition of cell mitosis and resulting in G2/M cell cycle arrest.[\\n##UREF##4##\\n30\\n##\\n] We assessed the influence of PTX released from BPGP@CAP on the cell cycle distribution of MFC cells. As presented in Figure ##FIG##4##4B,D##, the percentages of cells in the G2/M phase in the groups treated with PTX (25.1%), BPGP (20.2%), PTX + CAP (27.8%), and BPGP@CAP (23.6%) are significantly higher than that in the control group (11.0%). Conversely, the proportions cells in the G0/G1 phase in the groups treated with PTX (43.9%), BPGP (52.2%), PTX + CAP (39.0%), and BPGP@CAP (39.6%) are significantly lower than that in the control group (76.6%). These findings support that PTX released from BPGP and BPGP@CAP can lead to cell cycle arrest in the G2/M phase. These results further confirm that BPGP@CAP can effectively releases PTX in the tumor cells, thereby displaying a similar antitumor mechanism of action as free PTX.
\",\n \"Mechanism Study on the Synergistic Effect of PTX and CAP\",\n \"
Our in vitro experimental results support the existence of a synergistic effect between PTX and CAP to eliminate MFC cells. To explore the underlying mechanisms behind this synergy, we conducted transcriptome sequencing on MFC cells exposed to BPGP@CAP and compared them with the control group. We detected 2799 differentially expressed genes (DEGs) between BPGP@CAP and control groups. Out of these DEGs, 1678 are upregulated and 1121 downregulated in the BPGP@CAP‐treated group (Figure\\n##FIG##5##\\n5A##; Figure ##SUPPL##0##S18##, Supporting Information). Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was employed to uncover the biological pathways impacted by these DEGs. The results suggest that the pathways for apoptosis and PI3K/AKT signaling are significantly upregulated, which indicates that the mechanism underlying the synergistic antitumor effects of PTX and CAP in BPGP@CAP may be related to the PI3K/AKT signaling pathway and apoptosis (Figure ##FIG##5##5B##). It has been evidenced that the PI3K/AKT signaling pathway can regulate apoptosis by modulating the phosphorylation of Bax and Bad and the regulation of their expression levels, the release of cytochrome C, and the activation of caspase ‐ 3 and 9, thus affecting cell survival.[\\n##REF##23881702##\\n31\\n##\\n] Western blot results confirm that CAP inhibits the expression of the PI3K/AKT pathway‐related proteins such as AKT, p‐mTOR, p‐S6, and p‐4EBP1 (Figure ##FIG##5##5C##), indicating that CAP can suppress the PI3K/AKT signaling pathway.
\",\n \"
Furthermore, western blotting was employed to assess the expression of apoptosis‐related proteins, such as cleaved PARP, cleaved caspase‐3, Bax, and Bcl‐2, in various treatment groups. As showed in Figure ##FIG##5##5E,F##, the expression of Bax, cleaved caspase‐3, and cleaved PARP is upregulated in MFC cells after treatment with CAP, which might be credited to apoptosis after the inhibition of AKT. Furthermore, it is known that PTX can induce tumor cell apoptosis by downregulation of Bcl‐2 and upregulation of Bax and cleaved caspase‐3.[\\n##REF##23735541##\\n32\\n##\\n] Our results confirm that the expression levels of Bax, cleaved caspase‐3, and cleaved PARP were elevate, while Bcl‐2 is downregulated in the MFC cells with treatment of PTX and BPGP. In addition, when MFC was treated with the combination of free PTX and CAP or BPGP@CAP, more pronounced changes are observed in the expression of proteins including Bax, cleaved caspase −3, and cleaved PARP. Interestingly, western blot results reveal that the effect of CAP on downregulating the BCL‐2 protein expression is not pronounced as PTX, suggesting that the downregulation of Bcl‐2 may be predominantly induced by PTX, while no apparent correlation is seen between the downregulation of Bcl‐2 and the inhibition of AKT expression. However, the combination of PTX and CAP can induce more significant downregulation of Bcl‐2 compared to their individual effects, which is in agreement with a previous study.[\\n##REF##32070411##\\n26\\n##\\n] It may be explained by that CAP could significantly sensitize MFC cells to PTX‐induced apoptosis, resulting in more prominent downregulation of Bcl‐2.
\",\n \"
The combination of PTX with CAP exerts synergistic effects on MFC cells via the modulation of pro and anti‐apoptotic genes, such as downregulation of Bcl‐2 and upregulation of Bax, cleaved caspase‐3, and cleaved PARP. This could be the underlying mechanism for synergistic promotion of apoptosis by PTX and CAP (Figure ##FIG##5##5G##).
\",\n \"Antitumor Effect of BPGP@CAP In Vivo\",\n \"\\n<italic toggle=\\\"yes\\\">Distribution of BPGP@CAP</italic> In Vivo\",\n \"
The accumulation of BPGP@CAP at the site of transplanted tumor plays a critical role in its antitumor efficacy in vivo. Mounting evidence has suggested that nanoparticles exhibit the enhanced permeability and retention (EPR) effect, which can extend the residence time and increase the concentration of drugs in tumors, thus achieving passive drug targeting of tumor cells.[\\n##UREF##5##\\n33\\n##\\n] The characterization results of BPGP@CAP indicate that it possesses a nanoscale size for the EPR effect. To confirm the accumulation of BPGP@CAP in tumor tissues via the EPR effect, we utilized a fluorescence imaging technique to evaluate the distribution of BPGP@CAP in the mice with MFC tumors. By administering Cy5‐labeled BPGP@CAP and free Cy5 intravenously to the tumor‐bearing nude mice, we assessed the distribution of BPGP@CAP at various time intervals using IVIS spectroscopy. The findings reveal that the fluorescence intensity of the tumor in the BPGP@CAP‐treated group is stronger than that in the free Cy5‐treated group as time elapses (Figure\\n##FIG##6##\\n6A##; Figure ##SUPPL##0##S19##, Supporting Information). Specifically, after 24 h, the fluorescence intensity of the tumor in the BPGP@CAP‐treated group still remains strong, while faint fluorescence signal is detected in the tumor of the free Cy5‐treated group. This indicates that BPGP@CAP can accumulate and retain in the tumor site for an extended period. Furthermore, tumor tissues were collected at 24 h post‐injection and visualized using CLSM. The images demonstrate that tumor tissues of the mice after the treatment of free Cy5 show undetectable fluorescence signal, whereas tumor tissues of the mice after the treatment of BPGP@CAP exhibit significantly strong fluorescence intensity (Figure ##FIG##6##6B##). The in vivo imaging results confirm the enrichment of BPGP@CAP in tumor tissues.
\",\n \"
Furthermore, pharmacokinetic analysis in vivo reveals a slower decrease in the Cy5 concentration in the blood of the mice exposed to BPGP@CAP compared to that in the group exposed to free Cy5, and the half‐life of BPGP@CAP and free Cy5 was 488.49 and 160.85 min, respectively (Figure ##FIG##6##6C##; Table ##SUPPL##0##S2##, Supporting Information). The results might be ascribed to a high molecular weight of BPGP@CAP and a prolonged circulation time which may help enhance passive accumulation of BPGP@CAP at the tumor site, thereby improving its therapeutic efficacy against tumors.
\",\n \"Antitumor Effect of BPGP@CAP\",\n \"
The mice, which were subcutaneously injected with MFC cells, were divided randomly into six groups, each consisting of 5 mice. The control group were given intravenous injections of saline, while the other groups were intravenously injected with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. The concentrations of PTX remain consistent at 8 mg kg−1 for each mouse. The treatment schedule is illustrated in Figure ##FIG##6##6D##. The volume of the transplanted tumor and the body weight of each treated mouse were regularly monitored and recorded. The results reveal that the control group has the fastest tumor growth rate, while the BPGP@CAP‐treated group displays the slowest tumor growth (Figure ##FIG##6##6E##). After the treatment, the tumor volume of the control group is 1.45, 1.67, 2.04, 2.48, and 4.13 times that of the group treated with PTX, CAP, PTX+CAP, BPGP, and BPGP@CAP, respectively. The tumor inhibition rates, based on tumor volume measurements, are found to be 31.0%, 40.1%, 51.0%, 59.7%, and 75.8% after treatment with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. In addition, the tumor volumes in the groups with the treatment of free PTX and PTX + CAP are 2.85 times and 2.02 times that of the BPGP@CAP‐treated group, respectively (Figure ##FIG##6##6F##). The tumor volumes of the transplanted tumors harvested from each group are well aligned with those observed in vivo (Figure ##SUPPL##0##S20A##, Supporting Information). Comparison of the transplant tumor masses in the treatment groups reveals that the tumor mass harvested from the BPGP@CAP‐treated mice (0.28 g) is significantly lighter than that of the free control group (1.20 g), the PTX‐treated group (0.84 g), and the PTX + ACP‐treated group (0.57 g) (Figure ##FIG##6##6G##). The tumor inhibition rates, based on tumor mass measurements, are estimated to be 30.2%, 39.1%, 49.6%, 58.6%, and 75.7% for PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively (Figure ##SUPPL##0##S20B##, Supporting Information). The tumor inhibition rates based on tumor mass measurements are similar to those based on tumor volume measurements in vivo for CAP and/or PTX‐derived formulations. The difference in the tumor inhibition rates among BPGP@CAP and other groups may result from a synergistic effect of PTX and CAP, enrichment of BPGP@CAP in tumor tissues and prolonged circulation in the blood through a nanoscale drug delivery system.
\",\n \"
The antitumor effect of BPGP@CAP was further verified by IHC analysis of tumor tissues. TUNEL assays of the harvested tumors indicate TUNEL‐positive tumor cells in the BPGP@CAP‐treated group are the most populous, confirming that the treatment of BPGP@CAP results in more apoptotic cells compared with other groups (Figure ##FIG##6##6I,J##). Furthermore, the mice treated with BPGP@CAP show a significant reduction in the population of Ki67‐positive tumor cells with comparison to the other groups. This observation suggests that the administration of BPGP@CAP leads to a remarkable suppression of tumor cell proliferation (Figure ##FIG##6##6I,J##), which confirms synergistic inhibition of tumor growth by both agents in vivo. These findings support the excellent anti‐tumor effect of BPGP@CAP.
\",\n \"
Additionally, while the administration of free PTX and PTX + CAP effectively suppresses the growth of transplanted tumors in the mice, it is accompanied by a decrease in the body weight ranging from 9.1% to 6.3% after each administration (Figure ##FIG##6##6H##). This indicates the presence of systemic toxicity associated with free PTX. In contrast, no significant changes in the body weight are found in the BPGP and BPGP@CAP‐treated groups, suggesting that the toxicity of PTX could be remarkably reduced after PTX is delivered via a polymer prodrug. In addition, we conducted H&E staining on the major organs of the mice and analyzed the change of hematological parameters including white blood cells (WBC), red blood cells (RBC), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) to evaluate the toxicity of BPGP@CAP. The H&E staining images indicate no significant alterations in the major organs, including the heart, liver, spleen, lung, and kidney, across all treatment groups (Figure ##SUPPL##0##S21##, Supporting Information). However, compared to the control group with a WBC count of 8.20×109/L, the WBC count of the mice is found to be 3.19 × 109 per L, 3.28 × 109 per L, 5.76 × 109 per L and 5.69 × 109 per L in the group treated free PTX, PTX + CAP, BPGP, and BPGP@CAP, respectively. The most pronounced decreases in the WBC number are seen in the groups treated with PTX and PTX + CAP. Furthermore, when compared to the control group where the ALT level is 21.0 U L−1, there is no significant change in the ALT level in the BPGP+CAP‐treated group (22.0 U L−1), while a distinctive increase is seen in the PTX group (71.7 U L−1) and the PTX+CAP group (63.3 U L−1) (Figure ##SUPPL##0##S22##, Supporting Information). These results indicate that systemic administration of free PTX or PTX + CAP can cause hematopoietic system toxicity and liver toxicity, while the use of BPGP@CAP to deliver PTX and CAP can significantly reduce their toxicity.
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","Expression of AKT in GC Tissues and its Clinical Significance","
Previous studies have validated abnormal activation of the PI3K/AKT signaling pathway in different types of tumors including GC could lead to unfavorable prognoses.[\n##REF##32333246##\n22\n##\n] The amplification of AKT mutations is believed to contribute to the activation of the PI3K/AKT signaling pathway, serving as one of the important factors responsible for this process.[\n##REF##24748656##\n23\n##\n] To reveal clinical significance of AKT expression in GC, analysis of AKT expression in GC tissues was performed and its relationship with prognosis using public databases assessed. The outcomes indicate a significantly elevated level of AKT expression in GC tissues in comparison to normal tissues (Figure\n##FIG##1##\n1A##). Furthermore, GC patients with a high level of AKT expression often have a significantly lower overall survival rate compared to those with a low level of AKT expression (Figure ##FIG##1##1B##). To validate this discovery, immunohistochemistry (IHC) analysis was conducted on clinical tissue samples obtained from the West China Hospital (Figure ##SUPPL##0##S1##, Supporting Information). The analysis results (Figure ##FIG##1##1C,D##) are well aligned with the data in Figure ##FIG##1##1A,B##, corroborating the findings reported by Gu et al.[\n##REF##24726064##\n24\n##\n] Furthermore, previous research findings have demonstrated a strong correlation between the activation of the PI3K/AKT signaling pathway and drug resistance in various tumors.[\n##REF##32973135##\n25\n##\n]\n
","
In this study, out of 100 GC patients, 25 received neoadjuvant chemotherapy, while 75 did not. IHC analysis supports a higher level of AKT expression in GC samples of patients who received neoadjuvant chemotherapy (p < 0.001) (Figure ##FIG##1##1E##). In addition, among 25 GC patients who underwent neoadjuvant chemotherapy, those with a lower level of AKT expression exhibit more pronounced tumor regression (Table ##SUPPL##0##S1##, Supporting Information). Western blot results confirm increased expression of AKT in MFC cells after PTX treatment for 24 h (Figure ##FIG##1##1F##). These results suggest chemotherapy could boost the expression of AKT in MFC cells, and the expression of AKT may be related to the inefficacy of chemotherapy. It has been reported that an AKT inhibitor, CAP, can suppress the expression of AKT, leading to an enhanced sensitivity of tumor cells to chemotherapeutic drugs such as PTX.[\n##REF##32070411##\n26\n##\n] To demonstrate the impact of CAP on the cytotoxic effect of PTX, CAP combined with PTX was applied to treatmouse forestomach carcinoma (MFC) cells and western blot results confirm that AKT expression is inhibited (Figure ##FIG##1##1F,G##). The Cell Counting Kit‐8 (CCK‐8) assay was conducted to assess the cytotoxic effect of CAP combined with free PTX on MFC cells. The results demonstrate that the combination of CAP and PTX exhibits a significantly higher level of cytotoxicity compared to free PTX (Figure ##FIG##1##1H##), which indicates that the inhibition of AKT may be an effective approach to enhancing the efficacy of chemotherapy against GC.
","
The synergistic anti‐tumor effect of free CAP and PTX was then comprehensively evaluated. The cytotoxic effects of the combination of free CAP and PTX on MFC cells were assessed at various CAP/PTX weight ratios (CAP: PTX = 4:1, 1:1, and 1:4). The results reveal that almost all synergy indexes for the combination of CAP and PTX at different weight ratios are less than 1, suggesting a synergistic effect of CAP and PTX in killing MFC cells (Figure ##FIG##1##1I##). This demonstrates tremendous potential of the combine therapy of PTX and CAP in treating GC.
","Fabrication and Characterizations of BPGP@CAP","
In this study, we utilized controlled RAFT polymerization and efficient thiol‐ene reaction for designing and synthesizing the cathepsin B‐sensitive branched glycopolymer‐PTX prodrug. As shown in Scheme ##SUPPL##0##S1## (Supporting Information), a crosslinking agent, MA‐GFLG‐MA, and a small molecular prodrug, maleimide‐GFLG‐PTX, were first synthesized. Structural confirmation and purity assessment were performed using 1H NMR and liquid chromatography‐mass spectrometry (LC‐MS), and the experimental results are presented in Figures ##SUPPL##0##S2–S7## (Supporting Information). After successful preparation of polymerizable monomers and functionalized small molecules, RAFT polymerization was conducted to produce a high‐molecular‐weight chain transfer agent, poly(LAEMA)‐CTA, illustrated in Scheme ##SUPPL##0##S2A## (Supporting Information). An approximate value of 28 kDa is obtained from molecular weight calculation via 1H NMR and the repeating LAEMA units are ≈61 (Figure ##SUPPL##0##S8##, Supporting Information). Gel permeation chromatography (GPC) analysis reveals a PDI of ≈1.06 for the chain transfer agent (Figure ##SUPPL##0##S13##, Supporting Information), indicative of a narrow molecular weight distribution.
","
Subsequently, co‐polymerization of poly(LAEMA)‐CTA, MA‐GFLG‐MA, MA‐PySS, and LAEMA yields an intermediate polymer with a branched architecture (branched poly(LAEMA)‐GFLG‐PySS). The 1H NMR spectrum of the intermediate polymer displays significant characteristic peaks of pyridine at 7.22 ppm, 7.74 ppm, and 8.31 ppm, indicating successful introduction of MA‐PySS into the polymer structure (Figure ##SUPPL##0##S9##, Supporting Information). After deprotection treatment, the characteristic peak of pyridine in the polymer disappears from the 1H NMR spectrum, while a characteristic peak of the benzene ring in GFLG is observed at 7.29 ppm (Figure ##SUPPL##0##S10##, Supporting Information). The as‐prepared intermediate polymer was sequentially conjugated with maleimide‐Cy5 and maleimide GFLG PTX, resulting in a product of a branched polymer prodrug, poly(LAEMACy5)‐GFLG‐PTX (termed as BPGP). Structural confirmation of the product is attained through its 1H NMR (Figure\n##FIG##2##\n2A##). Notably, distinct peaks corresponding to PTX and GFLG are not observed in the 1H NMR spectrum of BPGP in D2O, which is distinctly different from the spectrum of BPGP in DMSO‐d6. This disparity suggests BPGP may experience self‐assembly in an aqueous solution (Figure ##SUPPL##0##S11## and ##SUPPL##0##S12##, Supporting Information). As shown in Figure ##FIG##2##2B##, compared with unlabeled branched polymer prodrugs, the characteristic peak of Cy5 can be observed in both ultraviolet visible (UV‐vis) and fluorescence spectra, and there is no significant change in the wavelength of the characteristic peak compared to free Cy5, indicating that the optical properties of Cy5 remain after it was covalently coupled to the polymer.
","
Due to the presence of hydrophilic segments and hydrophobic drugs in the branched polymer prodrug, the prodrug could self‐assemble into nanoparticles through hydrophilic hydrophobic interactions. We used pyrene as a fluorescence probe to detect its critical micelle concentration (CAC), and its CAC value is found to be ≈2.64 µg mL−1, which indicates that the polymer prodrug possesses a remarkable self‐assembly ability (Figure ##FIG##2##2C##). Subsequently, we assessed the potential of the PTX prodrug as a polymeric nanocarrier system for drug delivery. To realize the synergy of chemotherapy and molecular targeted therapy, we utilized a solvent evaporation technique to encapsulate CAP, an AKT inhibitor, into the glycopolymer‐PTX prodrug, resulting in a nanoassembly, BPGP@CAP. Initially, the nanoassembly was fabricated at different PTX‐to‐CAP feed ratios to assess the encapsulation capability of the polymer‐PTX prodrug for CAP. As shown in Figure ##FIG##2##2D##, with an increase in the CAP feed weight, the drug loading of CAP within the nanoassembly progressively augments, signifying a great encapsulation capacity of the nanoassembly. However, it's worth noting that as the drug loading increases, the encapsulation efficiency of the nanoassembly exhibits a gradual decline. Notably, when the weight ratio of PTX to CAP drops below 1:1.5, the encapsulation efficiency of the nanoassembly experiences a substantial reduction. Consequently, the nanoassembly synthesized at a PTX‐to‐CAP weight ratio of 1:1.5 is chosen for subsequent experimental uses. The results of HPLC analysis reveal that 7.5 wt.% of PTX and 8.0 wt.% of CAP are in the nanoassembly of BPGP@CAP. As shown in Figure ##FIG##2##2E,F##, DLS measurements confirm that the hydrodynamic diameter of BPGP is ≈78.83 ± 0.35 nm, and upon encapsulation of CAP, a slight increase in the hydrodynamic size is observed (90.12 ± 0.36 nm). TEM images exhibit a relatively uniform size distribution for both BPGP and BPGP@CAP. Although encapsulation of CAP leads to a marginal size augmentation, the morphology of the nanoassembly remains unchanged. Leveraging the presence of GFLG in the polymer structure, the drug release behavior from the polymer prodrug was probed in a simulated tumor intracellular microenvironment. Since papain and cathepsin B share the same catalytic site, papain was selected to mimic cathepsin B in the tumor microenvironment. As depicted in the Figure ##FIG##2##2G##, under conditions without papain catalysis, the polymer prodrug retains stability with negligible drug release. Conversely, under enzymatic catalysis, the polymer prodrug effectively releases the loaded drug. These findings suggest the polymer prodrug could be used for stable in vivo drug delivery and controlled drug release at tumor sites.
","Cellular Uptake of BPGP@CAP by MFC Cells","
Cellular uptake of polymeric drug delivery systems by tumor cells is a crucial step to exert a drug antitumor effect. The uptake of Cy5‐labeled BPGP@CAP into MFC cells was evaluated using CLSM and flow cytometry techniques. By observing the fluorescence signal of Cy5 in MFC cells after incubation with Cy5‐labeled BPGP@CAP at different durations through CLSM, it is evident that with an increase in the incubation time, the red fluorescence signal from Cy5 gradually accumulates and becomes intensified in the cytoplasm of MFC cells (Figure\n##FIG##3##\n3A##; Figure ##SUPPL##0##S14##, Supporting Information). Flow cytometry data for uptake of Cy5‐labeled BPGP@CAP by MFC cells is in agreement with the CLSM images (Figure ##FIG##3##3C##; Figure ##SUPPL##0##S15##, Supporting Information). These results indicate that BPGP@CAP could be efficiently ingested by MFC cells, and this uptake process is time‐dependent.
","
To investigate the endocytic pathways of BPGP@CAP into MFC cells, flow cytometry was employed to detect the uptake of BPGP@CAP by MFC cells at a low temperature. The results show that the uptake of BPGP@CAP by MFC cells is significantly inhibited at an inhibition rate of 80.5%, indicating that the internalization of BPGP@CAP is an energy‐dependent process. Furthermore, upon incubating inhibitors with MFC cells, flow cytometry data demonstrate that chlorpromazine, genistein, and amiloride inhibit cellular uptake of BPGP@CAP by 70.2%, 65.4%, and 44.5% respectively. This suggests that clathrin‐mediated, caveolin‐mediated, and icropinocytosis‐mediated endocytic pathways may be involved in the uptake of BPGP@CAP (Figure ##FIG##3##3D##; Figure ##SUPPL##0##S16##, Supporting Information). Moreover, since the lysosomal cathepsin B can cleave the GFLG linker to release PTX,[\n##UREF##3##\n27\n##\n] we investigated whether BPGP@CAP would be transported to the lysosomes for degradation after cellular uptake. Through lysosomal colocalization experiments (Figure ##FIG##3##3B##), we observe a high overlapping level of the fluorescence signal for BPGP@CAP and lysosomes, suggesting BPGP@CAP may land at lysosomes before it is disintegrated by lysosomal cathepsin B. Combined with the findings from in vitro drug release experiments, it is confirmed that BPGP@CAP can be internalized by MFC cells and release drugs stimulated by lysosomal cathepsin B.
","The Toxicity of BPGP@CAP on MFC Cells","
To assess the toxicity of BPGP@CAP, we conducted CCK‐8 assays to measure the viability of MFC cells subjected to various treatment groups. The results indicate that after the treatment of BPGP@CAP, MFC cells viability significantly decreases. BPGP@CAP has an IC50 of 0.253 µg mL−1 in comparison to free PTX with an IC50 of 2.51 µg mL−1 and CAP with an IC50 of 4.74 µg mL−1. Furthermore, the cell viability in the group with the treatment of BPGP@CAP is similar to that in the group treated with free PTX+CAP with an IC50 of 0.43 µg mL−1, whereas the cell viability in the group treated with BPGP is comparable to that in the group exposed to free PTX, and the IC50 value for BPGP and free PTX is 2.52 µg mL−1 and 2.51 µg mL−1, respectively (Figure ##FIG##3##3E##). These findings suggest that BPGP@CAP could effectively release PTX and CAP after disintegration of the nanoassembly structure in the lysosomes, thus exerting an equivalent antitumor effect to a mixture of free PTX and CAP. Additionally, both the groups treated with BPGP@CAP and PTX + CAP exhibit a significantly higher cytotoxic effect on MFC cells than the groups exposed to BPGP and free PTX, indicating that the combination of CAP and PTX displays a synergistic action on MFC cells.
","
Microtubules, which are essential cytoskeletal filaments within cells, have a critical function in mitosis and intracellular vesicle transport.[\n##REF##30536951##\n28\n##\n] It is well‐established that PTX can enhance the polymerization of microtubules, hinder depolymerization, and suppress mitosis of tumor cells.[\n##REF##24435445##\n29\n##\n] In order to assess the impact of PTX derived from BPGP@CAP on microtubules, we used CLSM to observe morphological changes of microtubules in the MFC cells after various treatments. Microtubules are evenly distributed in the cytoplasm of MFC cells in the control and CAP‐treated groups. However, pronounced aggregation of microtubules is found around the cell nucleus in the groups exposed to PTX and PTX+CAP. Similar morphological changes of microtubules are observed in the groups treated with BPGP and BPGP@CAP (Figure ##FIG##3##3F##). Furthermore, in the control and CAP‐treated groups, the nuclei of MFC cells appear intact, whereas an abnormal nuclear morphology and a multinucleated structure are found in the groups treated with PTX, PTX + CAP, BPGP and BPGP@CAP, indicating that PTX released from BPGP@CAP exerts a similar effect on microtubules as free PTX.
","
Furthermore, microfilaments, which are composed of actin proteins, are integral constituents of the cellular cytoskeleton. Through CLSM, the influence of BPGP@CAP on microfilaments of MFC cells was examined. Our results indicate that actin aggregates into clusters or dots near the cell membrane in the BPGP@CAP‐treated group (Figure ##SUPPL##0##S17##, Supporting Information), leading to impaired microfilaments and reduced cell viability.
","
Moreover, we utilized flow cytometry to evaluate the impact of BPGP@CAP on the apoptosis of MFC cells. The findings indicate that the percentage of apoptotic cells in the groups exposed to BPGP@CAP (23.8%), BPGP (15.7%), PTX (17.3%), and PTX+CAP (22.3%) is significantly higher than that in the control group (4.7%) (Figure\n##FIG##4##\n4A,C##). Additionally, no significant difference is found in the percentage of apoptotic cells between the groups exposed to BPGP@CAP and PTX + CAP. Furthermore, MFC cells have a significantly higher apoptotic percentage after treatment with BPGP@CAP and PTX + CAP than those treated with PTX and CAP, further supporting their synergistic action of inducting apoptosis of MFC cells.
","
It is widely recognized that PTX exerts its effects on microtubules, causing inhibition of cell mitosis and resulting in G2/M cell cycle arrest.[\n##UREF##4##\n30\n##\n] We assessed the influence of PTX released from BPGP@CAP on the cell cycle distribution of MFC cells. As presented in Figure ##FIG##4##4B,D##, the percentages of cells in the G2/M phase in the groups treated with PTX (25.1%), BPGP (20.2%), PTX + CAP (27.8%), and BPGP@CAP (23.6%) are significantly higher than that in the control group (11.0%). Conversely, the proportions cells in the G0/G1 phase in the groups treated with PTX (43.9%), BPGP (52.2%), PTX + CAP (39.0%), and BPGP@CAP (39.6%) are significantly lower than that in the control group (76.6%). These findings support that PTX released from BPGP and BPGP@CAP can lead to cell cycle arrest in the G2/M phase. These results further confirm that BPGP@CAP can effectively releases PTX in the tumor cells, thereby displaying a similar antitumor mechanism of action as free PTX.
","Mechanism Study on the Synergistic Effect of PTX and CAP","
Our in vitro experimental results support the existence of a synergistic effect between PTX and CAP to eliminate MFC cells. To explore the underlying mechanisms behind this synergy, we conducted transcriptome sequencing on MFC cells exposed to BPGP@CAP and compared them with the control group. We detected 2799 differentially expressed genes (DEGs) between BPGP@CAP and control groups. Out of these DEGs, 1678 are upregulated and 1121 downregulated in the BPGP@CAP‐treated group (Figure\n##FIG##5##\n5A##; Figure ##SUPPL##0##S18##, Supporting Information). Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was employed to uncover the biological pathways impacted by these DEGs. The results suggest that the pathways for apoptosis and PI3K/AKT signaling are significantly upregulated, which indicates that the mechanism underlying the synergistic antitumor effects of PTX and CAP in BPGP@CAP may be related to the PI3K/AKT signaling pathway and apoptosis (Figure ##FIG##5##5B##). It has been evidenced that the PI3K/AKT signaling pathway can regulate apoptosis by modulating the phosphorylation of Bax and Bad and the regulation of their expression levels, the release of cytochrome C, and the activation of caspase ‐ 3 and 9, thus affecting cell survival.[\n##REF##23881702##\n31\n##\n] Western blot results confirm that CAP inhibits the expression of the PI3K/AKT pathway‐related proteins such as AKT, p‐mTOR, p‐S6, and p‐4EBP1 (Figure ##FIG##5##5C##), indicating that CAP can suppress the PI3K/AKT signaling pathway.
","
Furthermore, western blotting was employed to assess the expression of apoptosis‐related proteins, such as cleaved PARP, cleaved caspase‐3, Bax, and Bcl‐2, in various treatment groups. As showed in Figure ##FIG##5##5E,F##, the expression of Bax, cleaved caspase‐3, and cleaved PARP is upregulated in MFC cells after treatment with CAP, which might be credited to apoptosis after the inhibition of AKT. Furthermore, it is known that PTX can induce tumor cell apoptosis by downregulation of Bcl‐2 and upregulation of Bax and cleaved caspase‐3.[\n##REF##23735541##\n32\n##\n] Our results confirm that the expression levels of Bax, cleaved caspase‐3, and cleaved PARP were elevate, while Bcl‐2 is downregulated in the MFC cells with treatment of PTX and BPGP. In addition, when MFC was treated with the combination of free PTX and CAP or BPGP@CAP, more pronounced changes are observed in the expression of proteins including Bax, cleaved caspase −3, and cleaved PARP. Interestingly, western blot results reveal that the effect of CAP on downregulating the BCL‐2 protein expression is not pronounced as PTX, suggesting that the downregulation of Bcl‐2 may be predominantly induced by PTX, while no apparent correlation is seen between the downregulation of Bcl‐2 and the inhibition of AKT expression. However, the combination of PTX and CAP can induce more significant downregulation of Bcl‐2 compared to their individual effects, which is in agreement with a previous study.[\n##REF##32070411##\n26\n##\n] It may be explained by that CAP could significantly sensitize MFC cells to PTX‐induced apoptosis, resulting in more prominent downregulation of Bcl‐2.
","
The combination of PTX with CAP exerts synergistic effects on MFC cells via the modulation of pro and anti‐apoptotic genes, such as downregulation of Bcl‐2 and upregulation of Bax, cleaved caspase‐3, and cleaved PARP. This could be the underlying mechanism for synergistic promotion of apoptosis by PTX and CAP (Figure ##FIG##5##5G##).
","Antitumor Effect of BPGP@CAP In Vivo","\n<italic toggle=\"yes\">Distribution of BPGP@CAP</italic> In Vivo","
The accumulation of BPGP@CAP at the site of transplanted tumor plays a critical role in its antitumor efficacy in vivo. Mounting evidence has suggested that nanoparticles exhibit the enhanced permeability and retention (EPR) effect, which can extend the residence time and increase the concentration of drugs in tumors, thus achieving passive drug targeting of tumor cells.[\n##UREF##5##\n33\n##\n] The characterization results of BPGP@CAP indicate that it possesses a nanoscale size for the EPR effect. To confirm the accumulation of BPGP@CAP in tumor tissues via the EPR effect, we utilized a fluorescence imaging technique to evaluate the distribution of BPGP@CAP in the mice with MFC tumors. By administering Cy5‐labeled BPGP@CAP and free Cy5 intravenously to the tumor‐bearing nude mice, we assessed the distribution of BPGP@CAP at various time intervals using IVIS spectroscopy. The findings reveal that the fluorescence intensity of the tumor in the BPGP@CAP‐treated group is stronger than that in the free Cy5‐treated group as time elapses (Figure\n##FIG##6##\n6A##; Figure ##SUPPL##0##S19##, Supporting Information). Specifically, after 24 h, the fluorescence intensity of the tumor in the BPGP@CAP‐treated group still remains strong, while faint fluorescence signal is detected in the tumor of the free Cy5‐treated group. This indicates that BPGP@CAP can accumulate and retain in the tumor site for an extended period. Furthermore, tumor tissues were collected at 24 h post‐injection and visualized using CLSM. The images demonstrate that tumor tissues of the mice after the treatment of free Cy5 show undetectable fluorescence signal, whereas tumor tissues of the mice after the treatment of BPGP@CAP exhibit significantly strong fluorescence intensity (Figure ##FIG##6##6B##). The in vivo imaging results confirm the enrichment of BPGP@CAP in tumor tissues.
","
Furthermore, pharmacokinetic analysis in vivo reveals a slower decrease in the Cy5 concentration in the blood of the mice exposed to BPGP@CAP compared to that in the group exposed to free Cy5, and the half‐life of BPGP@CAP and free Cy5 was 488.49 and 160.85 min, respectively (Figure ##FIG##6##6C##; Table ##SUPPL##0##S2##, Supporting Information). The results might be ascribed to a high molecular weight of BPGP@CAP and a prolonged circulation time which may help enhance passive accumulation of BPGP@CAP at the tumor site, thereby improving its therapeutic efficacy against tumors.
","Antitumor Effect of BPGP@CAP","
The mice, which were subcutaneously injected with MFC cells, were divided randomly into six groups, each consisting of 5 mice. The control group were given intravenous injections of saline, while the other groups were intravenously injected with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. The concentrations of PTX remain consistent at 8 mg kg−1 for each mouse. The treatment schedule is illustrated in Figure ##FIG##6##6D##. The volume of the transplanted tumor and the body weight of each treated mouse were regularly monitored and recorded. The results reveal that the control group has the fastest tumor growth rate, while the BPGP@CAP‐treated group displays the slowest tumor growth (Figure ##FIG##6##6E##). After the treatment, the tumor volume of the control group is 1.45, 1.67, 2.04, 2.48, and 4.13 times that of the group treated with PTX, CAP, PTX+CAP, BPGP, and BPGP@CAP, respectively. The tumor inhibition rates, based on tumor volume measurements, are found to be 31.0%, 40.1%, 51.0%, 59.7%, and 75.8% after treatment with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. In addition, the tumor volumes in the groups with the treatment of free PTX and PTX + CAP are 2.85 times and 2.02 times that of the BPGP@CAP‐treated group, respectively (Figure ##FIG##6##6F##). The tumor volumes of the transplanted tumors harvested from each group are well aligned with those observed in vivo (Figure ##SUPPL##0##S20A##, Supporting Information). Comparison of the transplant tumor masses in the treatment groups reveals that the tumor mass harvested from the BPGP@CAP‐treated mice (0.28 g) is significantly lighter than that of the free control group (1.20 g), the PTX‐treated group (0.84 g), and the PTX + ACP‐treated group (0.57 g) (Figure ##FIG##6##6G##). The tumor inhibition rates, based on tumor mass measurements, are estimated to be 30.2%, 39.1%, 49.6%, 58.6%, and 75.7% for PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively (Figure ##SUPPL##0##S20B##, Supporting Information). The tumor inhibition rates based on tumor mass measurements are similar to those based on tumor volume measurements in vivo for CAP and/or PTX‐derived formulations. The difference in the tumor inhibition rates among BPGP@CAP and other groups may result from a synergistic effect of PTX and CAP, enrichment of BPGP@CAP in tumor tissues and prolonged circulation in the blood through a nanoscale drug delivery system.
","
The antitumor effect of BPGP@CAP was further verified by IHC analysis of tumor tissues. TUNEL assays of the harvested tumors indicate TUNEL‐positive tumor cells in the BPGP@CAP‐treated group are the most populous, confirming that the treatment of BPGP@CAP results in more apoptotic cells compared with other groups (Figure ##FIG##6##6I,J##). Furthermore, the mice treated with BPGP@CAP show a significant reduction in the population of Ki67‐positive tumor cells with comparison to the other groups. This observation suggests that the administration of BPGP@CAP leads to a remarkable suppression of tumor cell proliferation (Figure ##FIG##6##6I,J##), which confirms synergistic inhibition of tumor growth by both agents in vivo. These findings support the excellent anti‐tumor effect of BPGP@CAP.
","
Additionally, while the administration of free PTX and PTX + CAP effectively suppresses the growth of transplanted tumors in the mice, it is accompanied by a decrease in the body weight ranging from 9.1% to 6.3% after each administration (Figure ##FIG##6##6H##). This indicates the presence of systemic toxicity associated with free PTX. In contrast, no significant changes in the body weight are found in the BPGP and BPGP@CAP‐treated groups, suggesting that the toxicity of PTX could be remarkably reduced after PTX is delivered via a polymer prodrug. In addition, we conducted H&E staining on the major organs of the mice and analyzed the change of hematological parameters including white blood cells (WBC), red blood cells (RBC), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) to evaluate the toxicity of BPGP@CAP. The H&E staining images indicate no significant alterations in the major organs, including the heart, liver, spleen, lung, and kidney, across all treatment groups (Figure ##SUPPL##0##S21##, Supporting Information). However, compared to the control group with a WBC count of 8.20×109/L, the WBC count of the mice is found to be 3.19 × 109 per L, 3.28 × 109 per L, 5.76 × 109 per L and 5.69 × 109 per L in the group treated free PTX, PTX + CAP, BPGP, and BPGP@CAP, respectively. The most pronounced decreases in the WBC number are seen in the groups treated with PTX and PTX + CAP. Furthermore, when compared to the control group where the ALT level is 21.0 U L−1, there is no significant change in the ALT level in the BPGP+CAP‐treated group (22.0 U L−1), while a distinctive increase is seen in the PTX group (71.7 U L−1) and the PTX+CAP group (63.3 U L−1) (Figure ##SUPPL##0##S22##, Supporting Information). These results indicate that systemic administration of free PTX or PTX + CAP can cause hematopoietic system toxicity and liver toxicity, while the use of BPGP@CAP to deliver PTX and CAP can significantly reduce their toxicity.
"],"string":"[\n \"Results and Discussion\",\n \"Expression of AKT in GC Tissues and its Clinical Significance\",\n \"
Previous studies have validated abnormal activation of the PI3K/AKT signaling pathway in different types of tumors including GC could lead to unfavorable prognoses.[\\n##REF##32333246##\\n22\\n##\\n] The amplification of AKT mutations is believed to contribute to the activation of the PI3K/AKT signaling pathway, serving as one of the important factors responsible for this process.[\\n##REF##24748656##\\n23\\n##\\n] To reveal clinical significance of AKT expression in GC, analysis of AKT expression in GC tissues was performed and its relationship with prognosis using public databases assessed. The outcomes indicate a significantly elevated level of AKT expression in GC tissues in comparison to normal tissues (Figure\\n##FIG##1##\\n1A##). Furthermore, GC patients with a high level of AKT expression often have a significantly lower overall survival rate compared to those with a low level of AKT expression (Figure ##FIG##1##1B##). To validate this discovery, immunohistochemistry (IHC) analysis was conducted on clinical tissue samples obtained from the West China Hospital (Figure ##SUPPL##0##S1##, Supporting Information). The analysis results (Figure ##FIG##1##1C,D##) are well aligned with the data in Figure ##FIG##1##1A,B##, corroborating the findings reported by Gu et al.[\\n##REF##24726064##\\n24\\n##\\n] Furthermore, previous research findings have demonstrated a strong correlation between the activation of the PI3K/AKT signaling pathway and drug resistance in various tumors.[\\n##REF##32973135##\\n25\\n##\\n]\\n
\",\n \"
In this study, out of 100 GC patients, 25 received neoadjuvant chemotherapy, while 75 did not. IHC analysis supports a higher level of AKT expression in GC samples of patients who received neoadjuvant chemotherapy (p < 0.001) (Figure ##FIG##1##1E##). In addition, among 25 GC patients who underwent neoadjuvant chemotherapy, those with a lower level of AKT expression exhibit more pronounced tumor regression (Table ##SUPPL##0##S1##, Supporting Information). Western blot results confirm increased expression of AKT in MFC cells after PTX treatment for 24 h (Figure ##FIG##1##1F##). These results suggest chemotherapy could boost the expression of AKT in MFC cells, and the expression of AKT may be related to the inefficacy of chemotherapy. It has been reported that an AKT inhibitor, CAP, can suppress the expression of AKT, leading to an enhanced sensitivity of tumor cells to chemotherapeutic drugs such as PTX.[\\n##REF##32070411##\\n26\\n##\\n] To demonstrate the impact of CAP on the cytotoxic effect of PTX, CAP combined with PTX was applied to treatmouse forestomach carcinoma (MFC) cells and western blot results confirm that AKT expression is inhibited (Figure ##FIG##1##1F,G##). The Cell Counting Kit‐8 (CCK‐8) assay was conducted to assess the cytotoxic effect of CAP combined with free PTX on MFC cells. The results demonstrate that the combination of CAP and PTX exhibits a significantly higher level of cytotoxicity compared to free PTX (Figure ##FIG##1##1H##), which indicates that the inhibition of AKT may be an effective approach to enhancing the efficacy of chemotherapy against GC.
\",\n \"
The synergistic anti‐tumor effect of free CAP and PTX was then comprehensively evaluated. The cytotoxic effects of the combination of free CAP and PTX on MFC cells were assessed at various CAP/PTX weight ratios (CAP: PTX = 4:1, 1:1, and 1:4). The results reveal that almost all synergy indexes for the combination of CAP and PTX at different weight ratios are less than 1, suggesting a synergistic effect of CAP and PTX in killing MFC cells (Figure ##FIG##1##1I##). This demonstrates tremendous potential of the combine therapy of PTX and CAP in treating GC.
\",\n \"Fabrication and Characterizations of BPGP@CAP\",\n \"
In this study, we utilized controlled RAFT polymerization and efficient thiol‐ene reaction for designing and synthesizing the cathepsin B‐sensitive branched glycopolymer‐PTX prodrug. As shown in Scheme ##SUPPL##0##S1## (Supporting Information), a crosslinking agent, MA‐GFLG‐MA, and a small molecular prodrug, maleimide‐GFLG‐PTX, were first synthesized. Structural confirmation and purity assessment were performed using 1H NMR and liquid chromatography‐mass spectrometry (LC‐MS), and the experimental results are presented in Figures ##SUPPL##0##S2–S7## (Supporting Information). After successful preparation of polymerizable monomers and functionalized small molecules, RAFT polymerization was conducted to produce a high‐molecular‐weight chain transfer agent, poly(LAEMA)‐CTA, illustrated in Scheme ##SUPPL##0##S2A## (Supporting Information). An approximate value of 28 kDa is obtained from molecular weight calculation via 1H NMR and the repeating LAEMA units are ≈61 (Figure ##SUPPL##0##S8##, Supporting Information). Gel permeation chromatography (GPC) analysis reveals a PDI of ≈1.06 for the chain transfer agent (Figure ##SUPPL##0##S13##, Supporting Information), indicative of a narrow molecular weight distribution.
\",\n \"
Subsequently, co‐polymerization of poly(LAEMA)‐CTA, MA‐GFLG‐MA, MA‐PySS, and LAEMA yields an intermediate polymer with a branched architecture (branched poly(LAEMA)‐GFLG‐PySS). The 1H NMR spectrum of the intermediate polymer displays significant characteristic peaks of pyridine at 7.22 ppm, 7.74 ppm, and 8.31 ppm, indicating successful introduction of MA‐PySS into the polymer structure (Figure ##SUPPL##0##S9##, Supporting Information). After deprotection treatment, the characteristic peak of pyridine in the polymer disappears from the 1H NMR spectrum, while a characteristic peak of the benzene ring in GFLG is observed at 7.29 ppm (Figure ##SUPPL##0##S10##, Supporting Information). The as‐prepared intermediate polymer was sequentially conjugated with maleimide‐Cy5 and maleimide GFLG PTX, resulting in a product of a branched polymer prodrug, poly(LAEMACy5)‐GFLG‐PTX (termed as BPGP). Structural confirmation of the product is attained through its 1H NMR (Figure\\n##FIG##2##\\n2A##). Notably, distinct peaks corresponding to PTX and GFLG are not observed in the 1H NMR spectrum of BPGP in D2O, which is distinctly different from the spectrum of BPGP in DMSO‐d6. This disparity suggests BPGP may experience self‐assembly in an aqueous solution (Figure ##SUPPL##0##S11## and ##SUPPL##0##S12##, Supporting Information). As shown in Figure ##FIG##2##2B##, compared with unlabeled branched polymer prodrugs, the characteristic peak of Cy5 can be observed in both ultraviolet visible (UV‐vis) and fluorescence spectra, and there is no significant change in the wavelength of the characteristic peak compared to free Cy5, indicating that the optical properties of Cy5 remain after it was covalently coupled to the polymer.
\",\n \"
Due to the presence of hydrophilic segments and hydrophobic drugs in the branched polymer prodrug, the prodrug could self‐assemble into nanoparticles through hydrophilic hydrophobic interactions. We used pyrene as a fluorescence probe to detect its critical micelle concentration (CAC), and its CAC value is found to be ≈2.64 µg mL−1, which indicates that the polymer prodrug possesses a remarkable self‐assembly ability (Figure ##FIG##2##2C##). Subsequently, we assessed the potential of the PTX prodrug as a polymeric nanocarrier system for drug delivery. To realize the synergy of chemotherapy and molecular targeted therapy, we utilized a solvent evaporation technique to encapsulate CAP, an AKT inhibitor, into the glycopolymer‐PTX prodrug, resulting in a nanoassembly, BPGP@CAP. Initially, the nanoassembly was fabricated at different PTX‐to‐CAP feed ratios to assess the encapsulation capability of the polymer‐PTX prodrug for CAP. As shown in Figure ##FIG##2##2D##, with an increase in the CAP feed weight, the drug loading of CAP within the nanoassembly progressively augments, signifying a great encapsulation capacity of the nanoassembly. However, it's worth noting that as the drug loading increases, the encapsulation efficiency of the nanoassembly exhibits a gradual decline. Notably, when the weight ratio of PTX to CAP drops below 1:1.5, the encapsulation efficiency of the nanoassembly experiences a substantial reduction. Consequently, the nanoassembly synthesized at a PTX‐to‐CAP weight ratio of 1:1.5 is chosen for subsequent experimental uses. The results of HPLC analysis reveal that 7.5 wt.% of PTX and 8.0 wt.% of CAP are in the nanoassembly of BPGP@CAP. As shown in Figure ##FIG##2##2E,F##, DLS measurements confirm that the hydrodynamic diameter of BPGP is ≈78.83 ± 0.35 nm, and upon encapsulation of CAP, a slight increase in the hydrodynamic size is observed (90.12 ± 0.36 nm). TEM images exhibit a relatively uniform size distribution for both BPGP and BPGP@CAP. Although encapsulation of CAP leads to a marginal size augmentation, the morphology of the nanoassembly remains unchanged. Leveraging the presence of GFLG in the polymer structure, the drug release behavior from the polymer prodrug was probed in a simulated tumor intracellular microenvironment. Since papain and cathepsin B share the same catalytic site, papain was selected to mimic cathepsin B in the tumor microenvironment. As depicted in the Figure ##FIG##2##2G##, under conditions without papain catalysis, the polymer prodrug retains stability with negligible drug release. Conversely, under enzymatic catalysis, the polymer prodrug effectively releases the loaded drug. These findings suggest the polymer prodrug could be used for stable in vivo drug delivery and controlled drug release at tumor sites.
\",\n \"Cellular Uptake of BPGP@CAP by MFC Cells\",\n \"
Cellular uptake of polymeric drug delivery systems by tumor cells is a crucial step to exert a drug antitumor effect. The uptake of Cy5‐labeled BPGP@CAP into MFC cells was evaluated using CLSM and flow cytometry techniques. By observing the fluorescence signal of Cy5 in MFC cells after incubation with Cy5‐labeled BPGP@CAP at different durations through CLSM, it is evident that with an increase in the incubation time, the red fluorescence signal from Cy5 gradually accumulates and becomes intensified in the cytoplasm of MFC cells (Figure\\n##FIG##3##\\n3A##; Figure ##SUPPL##0##S14##, Supporting Information). Flow cytometry data for uptake of Cy5‐labeled BPGP@CAP by MFC cells is in agreement with the CLSM images (Figure ##FIG##3##3C##; Figure ##SUPPL##0##S15##, Supporting Information). These results indicate that BPGP@CAP could be efficiently ingested by MFC cells, and this uptake process is time‐dependent.
\",\n \"
To investigate the endocytic pathways of BPGP@CAP into MFC cells, flow cytometry was employed to detect the uptake of BPGP@CAP by MFC cells at a low temperature. The results show that the uptake of BPGP@CAP by MFC cells is significantly inhibited at an inhibition rate of 80.5%, indicating that the internalization of BPGP@CAP is an energy‐dependent process. Furthermore, upon incubating inhibitors with MFC cells, flow cytometry data demonstrate that chlorpromazine, genistein, and amiloride inhibit cellular uptake of BPGP@CAP by 70.2%, 65.4%, and 44.5% respectively. This suggests that clathrin‐mediated, caveolin‐mediated, and icropinocytosis‐mediated endocytic pathways may be involved in the uptake of BPGP@CAP (Figure ##FIG##3##3D##; Figure ##SUPPL##0##S16##, Supporting Information). Moreover, since the lysosomal cathepsin B can cleave the GFLG linker to release PTX,[\\n##UREF##3##\\n27\\n##\\n] we investigated whether BPGP@CAP would be transported to the lysosomes for degradation after cellular uptake. Through lysosomal colocalization experiments (Figure ##FIG##3##3B##), we observe a high overlapping level of the fluorescence signal for BPGP@CAP and lysosomes, suggesting BPGP@CAP may land at lysosomes before it is disintegrated by lysosomal cathepsin B. Combined with the findings from in vitro drug release experiments, it is confirmed that BPGP@CAP can be internalized by MFC cells and release drugs stimulated by lysosomal cathepsin B.
\",\n \"The Toxicity of BPGP@CAP on MFC Cells\",\n \"
To assess the toxicity of BPGP@CAP, we conducted CCK‐8 assays to measure the viability of MFC cells subjected to various treatment groups. The results indicate that after the treatment of BPGP@CAP, MFC cells viability significantly decreases. BPGP@CAP has an IC50 of 0.253 µg mL−1 in comparison to free PTX with an IC50 of 2.51 µg mL−1 and CAP with an IC50 of 4.74 µg mL−1. Furthermore, the cell viability in the group with the treatment of BPGP@CAP is similar to that in the group treated with free PTX+CAP with an IC50 of 0.43 µg mL−1, whereas the cell viability in the group treated with BPGP is comparable to that in the group exposed to free PTX, and the IC50 value for BPGP and free PTX is 2.52 µg mL−1 and 2.51 µg mL−1, respectively (Figure ##FIG##3##3E##). These findings suggest that BPGP@CAP could effectively release PTX and CAP after disintegration of the nanoassembly structure in the lysosomes, thus exerting an equivalent antitumor effect to a mixture of free PTX and CAP. Additionally, both the groups treated with BPGP@CAP and PTX + CAP exhibit a significantly higher cytotoxic effect on MFC cells than the groups exposed to BPGP and free PTX, indicating that the combination of CAP and PTX displays a synergistic action on MFC cells.
\",\n \"
Microtubules, which are essential cytoskeletal filaments within cells, have a critical function in mitosis and intracellular vesicle transport.[\\n##REF##30536951##\\n28\\n##\\n] It is well‐established that PTX can enhance the polymerization of microtubules, hinder depolymerization, and suppress mitosis of tumor cells.[\\n##REF##24435445##\\n29\\n##\\n] In order to assess the impact of PTX derived from BPGP@CAP on microtubules, we used CLSM to observe morphological changes of microtubules in the MFC cells after various treatments. Microtubules are evenly distributed in the cytoplasm of MFC cells in the control and CAP‐treated groups. However, pronounced aggregation of microtubules is found around the cell nucleus in the groups exposed to PTX and PTX+CAP. Similar morphological changes of microtubules are observed in the groups treated with BPGP and BPGP@CAP (Figure ##FIG##3##3F##). Furthermore, in the control and CAP‐treated groups, the nuclei of MFC cells appear intact, whereas an abnormal nuclear morphology and a multinucleated structure are found in the groups treated with PTX, PTX + CAP, BPGP and BPGP@CAP, indicating that PTX released from BPGP@CAP exerts a similar effect on microtubules as free PTX.
\",\n \"
Furthermore, microfilaments, which are composed of actin proteins, are integral constituents of the cellular cytoskeleton. Through CLSM, the influence of BPGP@CAP on microfilaments of MFC cells was examined. Our results indicate that actin aggregates into clusters or dots near the cell membrane in the BPGP@CAP‐treated group (Figure ##SUPPL##0##S17##, Supporting Information), leading to impaired microfilaments and reduced cell viability.
\",\n \"
Moreover, we utilized flow cytometry to evaluate the impact of BPGP@CAP on the apoptosis of MFC cells. The findings indicate that the percentage of apoptotic cells in the groups exposed to BPGP@CAP (23.8%), BPGP (15.7%), PTX (17.3%), and PTX+CAP (22.3%) is significantly higher than that in the control group (4.7%) (Figure\\n##FIG##4##\\n4A,C##). Additionally, no significant difference is found in the percentage of apoptotic cells between the groups exposed to BPGP@CAP and PTX + CAP. Furthermore, MFC cells have a significantly higher apoptotic percentage after treatment with BPGP@CAP and PTX + CAP than those treated with PTX and CAP, further supporting their synergistic action of inducting apoptosis of MFC cells.
\",\n \"
It is widely recognized that PTX exerts its effects on microtubules, causing inhibition of cell mitosis and resulting in G2/M cell cycle arrest.[\\n##UREF##4##\\n30\\n##\\n] We assessed the influence of PTX released from BPGP@CAP on the cell cycle distribution of MFC cells. As presented in Figure ##FIG##4##4B,D##, the percentages of cells in the G2/M phase in the groups treated with PTX (25.1%), BPGP (20.2%), PTX + CAP (27.8%), and BPGP@CAP (23.6%) are significantly higher than that in the control group (11.0%). Conversely, the proportions cells in the G0/G1 phase in the groups treated with PTX (43.9%), BPGP (52.2%), PTX + CAP (39.0%), and BPGP@CAP (39.6%) are significantly lower than that in the control group (76.6%). These findings support that PTX released from BPGP and BPGP@CAP can lead to cell cycle arrest in the G2/M phase. These results further confirm that BPGP@CAP can effectively releases PTX in the tumor cells, thereby displaying a similar antitumor mechanism of action as free PTX.
\",\n \"Mechanism Study on the Synergistic Effect of PTX and CAP\",\n \"
Our in vitro experimental results support the existence of a synergistic effect between PTX and CAP to eliminate MFC cells. To explore the underlying mechanisms behind this synergy, we conducted transcriptome sequencing on MFC cells exposed to BPGP@CAP and compared them with the control group. We detected 2799 differentially expressed genes (DEGs) between BPGP@CAP and control groups. Out of these DEGs, 1678 are upregulated and 1121 downregulated in the BPGP@CAP‐treated group (Figure\\n##FIG##5##\\n5A##; Figure ##SUPPL##0##S18##, Supporting Information). Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was employed to uncover the biological pathways impacted by these DEGs. The results suggest that the pathways for apoptosis and PI3K/AKT signaling are significantly upregulated, which indicates that the mechanism underlying the synergistic antitumor effects of PTX and CAP in BPGP@CAP may be related to the PI3K/AKT signaling pathway and apoptosis (Figure ##FIG##5##5B##). It has been evidenced that the PI3K/AKT signaling pathway can regulate apoptosis by modulating the phosphorylation of Bax and Bad and the regulation of their expression levels, the release of cytochrome C, and the activation of caspase ‐ 3 and 9, thus affecting cell survival.[\\n##REF##23881702##\\n31\\n##\\n] Western blot results confirm that CAP inhibits the expression of the PI3K/AKT pathway‐related proteins such as AKT, p‐mTOR, p‐S6, and p‐4EBP1 (Figure ##FIG##5##5C##), indicating that CAP can suppress the PI3K/AKT signaling pathway.
\",\n \"
Furthermore, western blotting was employed to assess the expression of apoptosis‐related proteins, such as cleaved PARP, cleaved caspase‐3, Bax, and Bcl‐2, in various treatment groups. As showed in Figure ##FIG##5##5E,F##, the expression of Bax, cleaved caspase‐3, and cleaved PARP is upregulated in MFC cells after treatment with CAP, which might be credited to apoptosis after the inhibition of AKT. Furthermore, it is known that PTX can induce tumor cell apoptosis by downregulation of Bcl‐2 and upregulation of Bax and cleaved caspase‐3.[\\n##REF##23735541##\\n32\\n##\\n] Our results confirm that the expression levels of Bax, cleaved caspase‐3, and cleaved PARP were elevate, while Bcl‐2 is downregulated in the MFC cells with treatment of PTX and BPGP. In addition, when MFC was treated with the combination of free PTX and CAP or BPGP@CAP, more pronounced changes are observed in the expression of proteins including Bax, cleaved caspase −3, and cleaved PARP. Interestingly, western blot results reveal that the effect of CAP on downregulating the BCL‐2 protein expression is not pronounced as PTX, suggesting that the downregulation of Bcl‐2 may be predominantly induced by PTX, while no apparent correlation is seen between the downregulation of Bcl‐2 and the inhibition of AKT expression. However, the combination of PTX and CAP can induce more significant downregulation of Bcl‐2 compared to their individual effects, which is in agreement with a previous study.[\\n##REF##32070411##\\n26\\n##\\n] It may be explained by that CAP could significantly sensitize MFC cells to PTX‐induced apoptosis, resulting in more prominent downregulation of Bcl‐2.
\",\n \"
The combination of PTX with CAP exerts synergistic effects on MFC cells via the modulation of pro and anti‐apoptotic genes, such as downregulation of Bcl‐2 and upregulation of Bax, cleaved caspase‐3, and cleaved PARP. This could be the underlying mechanism for synergistic promotion of apoptosis by PTX and CAP (Figure ##FIG##5##5G##).
\",\n \"Antitumor Effect of BPGP@CAP In Vivo\",\n \"\\n<italic toggle=\\\"yes\\\">Distribution of BPGP@CAP</italic> In Vivo\",\n \"
The accumulation of BPGP@CAP at the site of transplanted tumor plays a critical role in its antitumor efficacy in vivo. Mounting evidence has suggested that nanoparticles exhibit the enhanced permeability and retention (EPR) effect, which can extend the residence time and increase the concentration of drugs in tumors, thus achieving passive drug targeting of tumor cells.[\\n##UREF##5##\\n33\\n##\\n] The characterization results of BPGP@CAP indicate that it possesses a nanoscale size for the EPR effect. To confirm the accumulation of BPGP@CAP in tumor tissues via the EPR effect, we utilized a fluorescence imaging technique to evaluate the distribution of BPGP@CAP in the mice with MFC tumors. By administering Cy5‐labeled BPGP@CAP and free Cy5 intravenously to the tumor‐bearing nude mice, we assessed the distribution of BPGP@CAP at various time intervals using IVIS spectroscopy. The findings reveal that the fluorescence intensity of the tumor in the BPGP@CAP‐treated group is stronger than that in the free Cy5‐treated group as time elapses (Figure\\n##FIG##6##\\n6A##; Figure ##SUPPL##0##S19##, Supporting Information). Specifically, after 24 h, the fluorescence intensity of the tumor in the BPGP@CAP‐treated group still remains strong, while faint fluorescence signal is detected in the tumor of the free Cy5‐treated group. This indicates that BPGP@CAP can accumulate and retain in the tumor site for an extended period. Furthermore, tumor tissues were collected at 24 h post‐injection and visualized using CLSM. The images demonstrate that tumor tissues of the mice after the treatment of free Cy5 show undetectable fluorescence signal, whereas tumor tissues of the mice after the treatment of BPGP@CAP exhibit significantly strong fluorescence intensity (Figure ##FIG##6##6B##). The in vivo imaging results confirm the enrichment of BPGP@CAP in tumor tissues.
\",\n \"
Furthermore, pharmacokinetic analysis in vivo reveals a slower decrease in the Cy5 concentration in the blood of the mice exposed to BPGP@CAP compared to that in the group exposed to free Cy5, and the half‐life of BPGP@CAP and free Cy5 was 488.49 and 160.85 min, respectively (Figure ##FIG##6##6C##; Table ##SUPPL##0##S2##, Supporting Information). The results might be ascribed to a high molecular weight of BPGP@CAP and a prolonged circulation time which may help enhance passive accumulation of BPGP@CAP at the tumor site, thereby improving its therapeutic efficacy against tumors.
\",\n \"Antitumor Effect of BPGP@CAP\",\n \"
The mice, which were subcutaneously injected with MFC cells, were divided randomly into six groups, each consisting of 5 mice. The control group were given intravenous injections of saline, while the other groups were intravenously injected with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. The concentrations of PTX remain consistent at 8 mg kg−1 for each mouse. The treatment schedule is illustrated in Figure ##FIG##6##6D##. The volume of the transplanted tumor and the body weight of each treated mouse were regularly monitored and recorded. The results reveal that the control group has the fastest tumor growth rate, while the BPGP@CAP‐treated group displays the slowest tumor growth (Figure ##FIG##6##6E##). After the treatment, the tumor volume of the control group is 1.45, 1.67, 2.04, 2.48, and 4.13 times that of the group treated with PTX, CAP, PTX+CAP, BPGP, and BPGP@CAP, respectively. The tumor inhibition rates, based on tumor volume measurements, are found to be 31.0%, 40.1%, 51.0%, 59.7%, and 75.8% after treatment with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively. In addition, the tumor volumes in the groups with the treatment of free PTX and PTX + CAP are 2.85 times and 2.02 times that of the BPGP@CAP‐treated group, respectively (Figure ##FIG##6##6F##). The tumor volumes of the transplanted tumors harvested from each group are well aligned with those observed in vivo (Figure ##SUPPL##0##S20A##, Supporting Information). Comparison of the transplant tumor masses in the treatment groups reveals that the tumor mass harvested from the BPGP@CAP‐treated mice (0.28 g) is significantly lighter than that of the free control group (1.20 g), the PTX‐treated group (0.84 g), and the PTX + ACP‐treated group (0.57 g) (Figure ##FIG##6##6G##). The tumor inhibition rates, based on tumor mass measurements, are estimated to be 30.2%, 39.1%, 49.6%, 58.6%, and 75.7% for PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, respectively (Figure ##SUPPL##0##S20B##, Supporting Information). The tumor inhibition rates based on tumor mass measurements are similar to those based on tumor volume measurements in vivo for CAP and/or PTX‐derived formulations. The difference in the tumor inhibition rates among BPGP@CAP and other groups may result from a synergistic effect of PTX and CAP, enrichment of BPGP@CAP in tumor tissues and prolonged circulation in the blood through a nanoscale drug delivery system.
\",\n \"
The antitumor effect of BPGP@CAP was further verified by IHC analysis of tumor tissues. TUNEL assays of the harvested tumors indicate TUNEL‐positive tumor cells in the BPGP@CAP‐treated group are the most populous, confirming that the treatment of BPGP@CAP results in more apoptotic cells compared with other groups (Figure ##FIG##6##6I,J##). Furthermore, the mice treated with BPGP@CAP show a significant reduction in the population of Ki67‐positive tumor cells with comparison to the other groups. This observation suggests that the administration of BPGP@CAP leads to a remarkable suppression of tumor cell proliferation (Figure ##FIG##6##6I,J##), which confirms synergistic inhibition of tumor growth by both agents in vivo. These findings support the excellent anti‐tumor effect of BPGP@CAP.
\",\n \"
Additionally, while the administration of free PTX and PTX + CAP effectively suppresses the growth of transplanted tumors in the mice, it is accompanied by a decrease in the body weight ranging from 9.1% to 6.3% after each administration (Figure ##FIG##6##6H##). This indicates the presence of systemic toxicity associated with free PTX. In contrast, no significant changes in the body weight are found in the BPGP and BPGP@CAP‐treated groups, suggesting that the toxicity of PTX could be remarkably reduced after PTX is delivered via a polymer prodrug. In addition, we conducted H&E staining on the major organs of the mice and analyzed the change of hematological parameters including white blood cells (WBC), red blood cells (RBC), platelets (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) to evaluate the toxicity of BPGP@CAP. The H&E staining images indicate no significant alterations in the major organs, including the heart, liver, spleen, lung, and kidney, across all treatment groups (Figure ##SUPPL##0##S21##, Supporting Information). However, compared to the control group with a WBC count of 8.20×109/L, the WBC count of the mice is found to be 3.19 × 109 per L, 3.28 × 109 per L, 5.76 × 109 per L and 5.69 × 109 per L in the group treated free PTX, PTX + CAP, BPGP, and BPGP@CAP, respectively. The most pronounced decreases in the WBC number are seen in the groups treated with PTX and PTX + CAP. Furthermore, when compared to the control group where the ALT level is 21.0 U L−1, there is no significant change in the ALT level in the BPGP+CAP‐treated group (22.0 U L−1), while a distinctive increase is seen in the PTX group (71.7 U L−1) and the PTX+CAP group (63.3 U L−1) (Figure ##SUPPL##0##S22##, Supporting Information). These results indicate that systemic administration of free PTX or PTX + CAP can cause hematopoietic system toxicity and liver toxicity, while the use of BPGP@CAP to deliver PTX and CAP can significantly reduce their toxicity.
In this study, a cathepsin B‐responsive drug delivery system has been established for co‐delivery of PTX and CAP using controlled RAFT polymerization and efficient click chemistry, aiming to achieve a synergistic therapeutic outcome from the combine therapy of chemotherapy and targeted therapy for GC. The BPGP@CAP nanoassembly enables targeted delivery of both PTX and CAP into the tumor site through the EPR effect, and precise release of PTX and CAP from BPGP@CAP after intelligently responding to overexpressed cathepsin B in the lysosomes of tumor cells. The released CAP suppresses the expression of AKT and its downstream PI3K/AKT pathway, and it synergizes with the released PTX to exert an enhanced anti‐tumor effect. Meanwhile, the toxicity of PTX and CAP is significantly reduced after their delivery in this glycopolymer‐based drug delivery system. Therefore, this enzyme‐responsive nanomedicine based on glycopolymer prodrugs could be utilized for co‐delivery of multiple therapeutic agents, providing a novel approach for GC treatment.
"],"string":"[\n \"Conclusions\",\n \"
In this study, a cathepsin B‐responsive drug delivery system has been established for co‐delivery of PTX and CAP using controlled RAFT polymerization and efficient click chemistry, aiming to achieve a synergistic therapeutic outcome from the combine therapy of chemotherapy and targeted therapy for GC. The BPGP@CAP nanoassembly enables targeted delivery of both PTX and CAP into the tumor site through the EPR effect, and precise release of PTX and CAP from BPGP@CAP after intelligently responding to overexpressed cathepsin B in the lysosomes of tumor cells. The released CAP suppresses the expression of AKT and its downstream PI3K/AKT pathway, and it synergizes with the released PTX to exert an enhanced anti‐tumor effect. Meanwhile, the toxicity of PTX and CAP is significantly reduced after their delivery in this glycopolymer‐based drug delivery system. Therefore, this enzyme‐responsive nanomedicine based on glycopolymer prodrugs could be utilized for co‐delivery of multiple therapeutic agents, providing a novel approach for GC treatment.
Combined chemotherapy and targeted therapy holds immense potential in the management of advanced gastric cancer (GC). GC tissues exhibit an elevated expression level of protein kinase B (AKT), which contributes to disease progression and poor chemotherapeutic responsiveness. Inhibition of AKT expression through an AKT inhibitor, capivasertib (CAP), to enhance cytotoxicity of paclitaxel (PTX) toward GC cells is demonstrated in this study. A cathepsin B‐responsive polymeric nanoparticle prodrug system is employed for co‐delivery of PTX and CAP, resulting in a polymeric nano‐drug BPGP@CAP. The release of PTX and CAP is triggered in an environment with overexpressed cathepsin B upon lysosomal uptake of BPGP@CAP. A synergistic therapeutic effect of PTX and CAP on killing GC cells is confirmed by in vitro and in vivo experiments. Mechanistic investigations suggested that CAP may inhibit AKT expression, leading to suppression of the phosphoinositide 3‐kinase (PI3K)/AKT signaling pathway. Encouragingly, CAP can synergize with PTX to exert potent antitumor effects against GC after they are co‐delivered via a polymeric drug delivery system, and this delivery system helped reduce their toxic side effects, which provides an effective therapeutic strategy for treating GC.
","
A cathepsin B‐responsive polymeric nanoparticle prodrug system is established for co‐delivery of paclitaxel (PTX) and capivasertib (CAP) to obtain BPGP@CAP, which selectively accumulates in the tumor tissue and releases PTX and CAP in response to overexpressed cathepsin B in lysosomes to exert a synergistic therapeutic effect to eradicate gastric cancer cells.\n\n
","
\n\n\nX.\nSong\n, \nH.\nCai\n, \nZ.\nShi\n, \nZ.\nLi\n, \nX.\nZheng\n, \nK.\nYang\n, \nQ.\nGong\n, \nZ.\nGu\n, \nJ.\nHu\n, \nK.\nLuo\n, Enzyme‐Responsive Branched Glycopolymer‐Based Nanoassembly for Co‐Delivery of Paclitaxel and Akt Inhibitor toward Synergistic Therapy of Gastric Cancer. Adv. Sci.\n2024, 11, 2306230. 10.1002/advs.202306230\n\n
"],"string":"[\n \"Abstract\",\n \"
Combined chemotherapy and targeted therapy holds immense potential in the management of advanced gastric cancer (GC). GC tissues exhibit an elevated expression level of protein kinase B (AKT), which contributes to disease progression and poor chemotherapeutic responsiveness. Inhibition of AKT expression through an AKT inhibitor, capivasertib (CAP), to enhance cytotoxicity of paclitaxel (PTX) toward GC cells is demonstrated in this study. A cathepsin B‐responsive polymeric nanoparticle prodrug system is employed for co‐delivery of PTX and CAP, resulting in a polymeric nano‐drug BPGP@CAP. The release of PTX and CAP is triggered in an environment with overexpressed cathepsin B upon lysosomal uptake of BPGP@CAP. A synergistic therapeutic effect of PTX and CAP on killing GC cells is confirmed by in vitro and in vivo experiments. Mechanistic investigations suggested that CAP may inhibit AKT expression, leading to suppression of the phosphoinositide 3‐kinase (PI3K)/AKT signaling pathway. Encouragingly, CAP can synergize with PTX to exert potent antitumor effects against GC after they are co‐delivered via a polymeric drug delivery system, and this delivery system helped reduce their toxic side effects, which provides an effective therapeutic strategy for treating GC.
\",\n \"
A cathepsin B‐responsive polymeric nanoparticle prodrug system is established for co‐delivery of paclitaxel (PTX) and capivasertib (CAP) to obtain BPGP@CAP, which selectively accumulates in the tumor tissue and releases PTX and CAP in response to overexpressed cathepsin B in lysosomes to exert a synergistic therapeutic effect to eradicate gastric cancer cells.\\n\\n
\",\n \"
\\n\\n\\nX.\\nSong\\n, \\nH.\\nCai\\n, \\nZ.\\nShi\\n, \\nZ.\\nLi\\n, \\nX.\\nZheng\\n, \\nK.\\nYang\\n, \\nQ.\\nGong\\n, \\nZ.\\nGu\\n, \\nJ.\\nHu\\n, \\nK.\\nLuo\\n, Enzyme‐Responsive Branched Glycopolymer‐Based Nanoassembly for Co‐Delivery of Paclitaxel and Akt Inhibitor toward Synergistic Therapy of Gastric Cancer. Adv. Sci.\\n2024, 11, 2306230. 10.1002/advs.202306230\\n\\n
CAP and PTX were obtained from Selleck Chemicals (Shanghai, China). Maleimide cyanine5 was acquired from Confluore Biotech CO. Ltd. (Xian, China). Chlorpromazine hydrochloride, amiloride hydrochloride, and dynasore were bought from APExBIO (Houston, USA). A terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling (TUNEL) Apoptosis Assay Kit was bought from Promega (Beijing, China). Hoechst 33342 and Cell Apoptosis Kit were purchased from Yeasen (Shanghai, China). Actin‐Tracker Green, Tubulin‐Tracker Red and Lyso‐Tracter Green were purchased from Beyotime (Shanghai, China). CCK‐8 was bought from MCE (Shanghai, China).
","Synthesis of LAEMA‐Based Branched Polymeric Prodrug","
\nSynthesis of Poly (LAEMA)‐CTA: 4‐cyano‐4‐((phenylcarbonothioyl)thio)pentanoic acid (chain transfer agent, CTA) (18.6 mg, 66.7 µmol) and LAEMA (2001.6 mg, 4.275 mmol) were mixed within a small‐necked 25 mL flask. The flask was subjected to argon purging, and the process was repeated thrice. Under an ice bath condition, an initiator solution containing VA044 (7.2 mg, 22.2 µmol) in a solvent mixture of water and methanol (H2O/CH3OH = 3:2, 9.1 mL) was introduced into the flask via syringe injection. The reaction mixture was thereafter subjected to bubbling with a continuous stream of argon for a duration of 30 min. The flask was then placed in a light‐protected environment at 45 °C and the reaction continued for 20 h. After the polymerization was completed, the reaction was quenched using liquid nitrogen. The reaction mixture underwent dialysis against deionized water (MWCO 3500 Da) for 1.5 days and was subsequently freeze‐dried to yield the product poly (LAEMA)‐CTA with a 78% yield.
","
\nSynthesis of Branched Poly (LAEMA)‐GFLG‐PySS: To synthesize the branched polymer poly(LAEMA)‐GFLG‐PySS, poly(LAEMA)‐CTA (270 mg), a monomer MA‐PySS (29 mg, 113 µmol), LAEMA (107 mg, 229 µmol), and a crosslinker MA‐GFLG‐MA (15 mg, 26 µmol) were mixed in a small‐necked 5 mL round‐bottom flask. The flask was purged with argon gas three times. Under an ice bath condition, a solution containing the initiator, VA044 (1.62 mg), in a solvent mixture of water and dimethylformamide (H2O/DMF = 2:7, 1.9 mL) was introduced into the flask via syringe injection. The reaction mixture was continuously bubbled with argon for 30 min. The flask was then placed in a light‐protected environment at 45 °C and the reaction continued for 24 h. Upon completion of the reaction, the reaction was quenched using liquid nitrogen. The reaction mixture underwent dialysis against deionized water (MWCO 3500 Da) for a duration of 1.5 days, followed by freeze‐drying to obtain the final product, branched poly(LAEMA)‐GFLG‐PySS, with a yield of 67%.
","
\nSynthesis of Branched Poly(LAEMA)‐GFLG‐PTX: In a round‐bottom flask, 200 mg of branched poly(LAEMA)‐GFLG‐PySS was accurately weighed and dissolved in 8 mL of a mixture of DMSO:H2O (4:1, v/v). After complete dissolution, 30 mg of dithiothreitol (DTT) was introduced, and the reaction mixture underwent 12 h reaction at room temperature. Subsequently, the reaction mixture was subjected to dialysis to remove organic solvents, followed by freeze‐drying to yield the product, branched poly(LAEMA)‐GFLG‐SH.
","
Branched poly (LAEMA)‐GFLG‐SH(146 mg) was carefully weighed and dissolved in 8 mL of a mixture of DMSO:H2O (4:1, v/v) in a round‐bottom flask. After complete dissolution, 1.4 mg of maleimide‐Cy5 dissolved in DMSO was added to the reaction mixture. The reaction proceeded overnight at room temperature. 48.7 mg of maleimide‐GFLG‐PTX was then introduced into the reaction mixture, and the reaction continued at room temperature for an additional 12 h. After the completion of the reaction, the mixture was sequentially dialyzed with DMF and deionized water (MWCO = 3500 Da). The resulting product was collected, filtered through a membrane, and subjected to freeze‐drying, resulting in the formation of branched poly(LAEMACy5)‐GFLG‐PTX. The drug loading of PTX in the polymer was determined through HPLC analysis.
","Physicochemical Characterizations of BPGP and BPGP@CAP","
The assessment of the CAC of BPGP was accomplished with the aid of pyrene as a fluorescence agent. Herein, 200 µL of a pyrene‐acetone solution (0.67 × 10−6m) was introduced into a 10 mL sample vial. After complete evaporation of acetone, an aliquot of the BPGP aqueous solution (ranging from 0.015 to 1000 µg mL−1) was added into the sample vial. After 2 h incubation, the fluorescence spectra were captured and recorded via a fluorescence spectrophotometer.
","
The AKT inhibitor capivasertib ‐loaded branched poly(LAEMACy5)‐GFLG‐PTX was prepared using the solvent evaporation method. In brief, a methanol/acetonitrile mixed solution (0.4 mL) containing 1.125 mg of CAP was slowly added dropwise to a 5 mL aqueous solution of BPGP (2 mg mL−1, PTX:CAP = 1 (wt):1.5 (wt)) under stirring conditions. The mixture was stirred at room temperature until the organic solvents were completely evaporated. The remaining solution was then filtered through a membrane (0.45 µm) to remove unencapsulated CAP. Finally, the obtained filtrate was freeze‐dried to yield the desired product, BPGP@CAP. BPGP@CAP with different weight ratios of PTX and CAP was prepared by adjusting the weighting ratio of PTX and CAP. The loading contents of CAP in nanoassemblies were determined by HPLC system. The drug loading content (DLC) and the drug loading efficiency (DLE) were calculated using the following equations:
","
DLC (wt.%) = (weight of drug loaded/total weight of polymer and loaded drug) × 100
","
DLE (wt.%) = (weight of drug loaded/initial weight of drug) × 100
","
The hydrodynamic diameter of the obtained nanoassemblies were measured by dynamic light scattering (DLS, Brookhaven ZetaPALS, USA). The morphology of the obtained nanoassemblies was examined under a transmission electron microscope (TEM, Tecnai GF20S‐TWIN, USA). The enzyme‐responsive drug release capacity of nanoassembly was investigated under simulated physiological conditions, with and without the presence of papain.
","Cell Line and Animals","
MFC cell, a cell line derived from GC, was acquired from the Chinese Academy of Medical Sciences (Shanghai, China), which were cultured in a temperature‐controlled incubator at 37 °C with 5% CO2 in a humidified environment. Male BALB/c nude mice (4‐6 weeks old, weighing 18–20 g) were procured from Hfk Bioscience Co., Ltd (Beijing, China). Subcutaneous GC MFC tumor models were generated through injecting 5 × 105 MFC cells into the right back of the mice. Approval for all animal experiments was granted by the Animal Ethics Committee of West China Hospital, Sichuan University (Approval No. 2018154A).
","Clinical Tissue Sampling","
The AKT expression and its relationship was examined with the prognosis of GC using the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) cohort data via Kaplan‐Meier Plotter (http://kmplot.com/analysis/index.php?p = background). Tumor and normal tissues of 100 GC patients were collected from the West China Hospital of Sichuan University. IHC was employed for assessing the expression of AKT. Based on the IHC scores, 100 GC patients were categorized into high and low AKT expression groups. The Kaplan–Meier method was used for the survival curve of both groups. This study regarding clinical tissue sampling received approval from the Ethics Committee of The West China Hospital of Sichuan University (2023 Review, No. 549) and all patients provided informed consent for participation in the study.
","Cellular Uptake, Endocytic Pathways, and Subcellular Localization","
MFC cells were inoculated in a confocal chamber (1 × 105 cells per well) and incubated overnight. They were then incubated with culture medium containing Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) for 1, 2, and 4 h, respectively. Following washing with PBS, Hoechst 33342 was added for nucleus staining. Cellular uptake of BPGP@CAP by MFC cells was determined by analyzing the Cy5 signal using laser scanning confocal microscopy (CLSM) (Nikon, Tokyo, Japan). The mean fluorescence intensity was obtained from semi‐quantitative analysis using Image J software. Additionally, flow cytometry (BD FACSCelesta, New Jersey, USA) was employed for detecting cellular uptake of BPGP@CAP by analyzing the Cy5 signal in MFC cells treated with Cy5‐labeled BPGP@CAP for various durations (1 h, 2 h, and 4 h).
","
To investigate the endocytic pathways of BPGP@CAP, MFC cells were pre‐treated with the culture medium containing 8 µg mL−1 chlorpromazine hydrochloride, 0.5 mm amiloride hydrochloride or 0.5 mm dynasore for 2 h. Subsequently, those MFC cells was incubated with medium containing Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) for an additional 2 h. Quantitative analysis of cellular uptake of BPGP@CAP was conducted using flow cytometry. Furthermore, to determine if the cellular uptake of BPGP@CAP was relied on energy, cells were exposed in the culture medium with Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) and incubated at 4 °C for a duration of 2 h and flow cytometry was employed to check the uptake level of BPGP@CAP.
","
For subcellular localization studies of BPGP@CAP, MFC cells were first exposed to Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) for 2 h. Following the staining of MFC cell lysosomes with Lyso Tracker Green and the nucleus with Hoechst 33342 for a duration of 15–20 min, observation and imaging of MFC cells were conducted using CLSM.
","Cell Cytotoxicity Assay","
After MFC cells were inoculated in 96‐well plates (3 × 103 cells per well) and grown overnight, MFC cells were exposed to various formulations (such as PTX, CAP, a mixture of PTX and CAP (PTX+CAP), BPGP, and BPGP@CAP) at different concentrations for 48 h. MFC cells treated with fresh culture medium were included as a control group. Following the treatment, those cells were exposed to a medium containing a 10% CCK‐8 solution and incubated at 37 °C for 1 h. A microplate reader (Tecan, Switzerland) was utilized to measure the OD450 value, which was then used to evaluate the relative cell viability.
","Cell Apoptosis and Cell Cycle Assay","
MFC cells were inoculated in a 6‐well plate with 2 × 105 cells per well and grown for 12 h. Subsequently, those cells were incubated with the culture medium containing PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for 48 h. MFC cells treated with fresh culture medium were included as a control group. Those cells were collected and stained with an Annexin V‐PI reagent for 30 min. Flow cytometry was employed for assessing apoptosis of MFC cells.
","
For cell cycle assays, cells that received treatment with PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for a duration of 48 h were collected and subjected to overnight fixation in 75% ethanol. Following gentle washing with PBS, they were delicately incubated with a 0.5 mL solution of the PI/RNase dye. The flow cytometry was used to discern the cellular distribution at different cell cycle phases. Each experiment had three replicates, and the acquired data was analyzed through the Flowjo software.
","Morphological Examination of Microtubules and Microfilaments","
The culture medium containing PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP was used to treat MFC cells for 24 h to examine the morphology of microtubules. Following washing with PBS, those MFC cells were fixed with a 4% paraformaldehyde solution. After washing with PBS containing 0.1% Triton X‐100, the cells were subjected to Tubulin‐tracker Red staining for 1 h. Subsequently, nucleus staining was performed with DAPI. The cells were then observed and imaged under CLSM. For morphological analysis of microfilaments, MFC cells were stained with Actin‐tracker Green and DAPI and they were then observed and imaged via CLSM.
","Transcriptome Analysis by RNA‐Seq","
MFC cells were inoculated in a 6‐well plate (2 × 105 cells per well) and grown for 12 h. Thereafter, they were exposed to BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for a duration of 48 h. MFC cells treated with fresh culture medium were included as a control group. Total RNA of MFC cells treated with BPGP@CAP in fresh culture medium was extracted. Genechem Co., Ltd (Shanghai, China) conducted the subsequent steps of quality control, library preparation, and RNA sequencing. Gene Ontology (GO) and KEGG pathway analyses were performed through the R package. Significant differences between the BPGP@CAP treatment group and the control cohort were robustly identified with a p‐adjust < 0.05 and a |log2FC| > 1.
","Western Blots","
MFC cells were treated with PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for 48 h. Untreated MFC cells were set as a control group. After collecting the cells from each experimental group, total proteins were collected from cell lysates in a lysis buffer. Subsequently, they underwent SDS‐PAGE gel electrophoresis and were then transferred to a 0.45 µm PVDF membrane (Millipore). Following a blockade using a 5% milk TBST solution, the membrane was subjected to an overnight incubation with primary antibodies in a refrigerator at 4°C. After a thorough TBST wash, HRP‐conjugated secondary antibodies were applied to the membrane and incubated at room temperature for 1 h. Detection of the proteins was achieved through the Syngene GeneGenius gel imaging system (Syngene, Cambridge, UK).
","In Vivo Pharmacokinetics Studies","
Normal male balb/c nude mice were randomly allocated into two groups (n = 5) for intravenous administration of free Cy5 and Cy5‐labelled BPGP@CAP (Cy5: 1.5 mg kg−1). At various time points (0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, and 24 h) after injection, blood samples were obtained from the orbital vein and immediately mixed with double distilled water. After the samples were then supplemented with DMSO and refrigerated overnight at 4 °C, they were centrifuged at 12 000 rpm for 15 min, and the supernatant from each sample was transferred to a 96‐well plate for fluorescence measurement using a microplate reader. The pharmacokinetic parameters were calculated using the PKsolver software.
","In Vivo Distribution","
The MFC tumor‐bearing nude mice (n = 3) received intravenous injections via the tail with free Cy5 and Cy5‐labeled BPGP@CAP (Cy5: 1.5 mg kg−1) in order to assess their in vivo biodistribution. At various time points following the injection (1 h, 2 h, 3 h, 6 h, 9 h, 12 h, and 24 h), the mice were observed and imaged using an IVIS Spectrum (Caliper Life Sciences, Cambridge, UK). Upon completion of the imaging experiment, tumor samples from the mice were obtained and subjected to frozen sectioning. After staining with DAPI to label cell nuclei, the sections were observed and imaged using CLSM.
","In Vivo Anti‐Tumor Study","
The mice with MFC tumors received different formulations via tail vein injection (n = 5): (1) Saline; (2) PTX; (3) CAP; (4) PTX + CAP; (5) BPGP; and (6) BPGP@CAP (the PTX concentration: 8.0 mg kg−1 and the corresponding concentration of CAP: 8.5 mg kg−1). The drugs were administered every other day until the completion of the treatment. The body weight of the mice, the length (L) and width (W) of the tumor were regularly recorded. The tumor volume (V) was determined using the formula: V = (L × W2)/2.
","
After the treatment, the mice were humanely euthanized by cervical dislocation, and their tumors were surgically excised and weighed. Furthermore, the tumors and vital organs underwent a process of fixation, embedding, and sectioning. The tumor sections were subjected to staining with Ki67 and TUNEL to evaluate proliferation and apoptosis of tumor cells. In addition, hematoxylin and eosin (H&E) staining was performed to assess the biocompatibility of these formulations.
","
Moreover, the biosafety of these formulations was evaluated in MFC tumor‐bearing nude mice. Hematological and biochemical analyses were conducted using collected whole blood and serum samples. Healthy balb/c nude mice were included as a control group for comparison.
","Statistical Analysis","
GraphPad Prism 9 software was employed to analyze data obtained from at least three independent measurements. The results were presented as the mean ± standard deviation (± S.D.). Statistical significance was determined by performing a two‐tailed t‐test between two groups, and a P‐value less than 0.05 was considered to be statistically significant.
CAP and PTX were obtained from Selleck Chemicals (Shanghai, China). Maleimide cyanine5 was acquired from Confluore Biotech CO. Ltd. (Xian, China). Chlorpromazine hydrochloride, amiloride hydrochloride, and dynasore were bought from APExBIO (Houston, USA). A terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling (TUNEL) Apoptosis Assay Kit was bought from Promega (Beijing, China). Hoechst 33342 and Cell Apoptosis Kit were purchased from Yeasen (Shanghai, China). Actin‐Tracker Green, Tubulin‐Tracker Red and Lyso‐Tracter Green were purchased from Beyotime (Shanghai, China). CCK‐8 was bought from MCE (Shanghai, China).
\",\n \"Synthesis of LAEMA‐Based Branched Polymeric Prodrug\",\n \"
\\nSynthesis of Poly (LAEMA)‐CTA: 4‐cyano‐4‐((phenylcarbonothioyl)thio)pentanoic acid (chain transfer agent, CTA) (18.6 mg, 66.7 µmol) and LAEMA (2001.6 mg, 4.275 mmol) were mixed within a small‐necked 25 mL flask. The flask was subjected to argon purging, and the process was repeated thrice. Under an ice bath condition, an initiator solution containing VA044 (7.2 mg, 22.2 µmol) in a solvent mixture of water and methanol (H2O/CH3OH = 3:2, 9.1 mL) was introduced into the flask via syringe injection. The reaction mixture was thereafter subjected to bubbling with a continuous stream of argon for a duration of 30 min. The flask was then placed in a light‐protected environment at 45 °C and the reaction continued for 20 h. After the polymerization was completed, the reaction was quenched using liquid nitrogen. The reaction mixture underwent dialysis against deionized water (MWCO 3500 Da) for 1.5 days and was subsequently freeze‐dried to yield the product poly (LAEMA)‐CTA with a 78% yield.
\",\n \"
\\nSynthesis of Branched Poly (LAEMA)‐GFLG‐PySS: To synthesize the branched polymer poly(LAEMA)‐GFLG‐PySS, poly(LAEMA)‐CTA (270 mg), a monomer MA‐PySS (29 mg, 113 µmol), LAEMA (107 mg, 229 µmol), and a crosslinker MA‐GFLG‐MA (15 mg, 26 µmol) were mixed in a small‐necked 5 mL round‐bottom flask. The flask was purged with argon gas three times. Under an ice bath condition, a solution containing the initiator, VA044 (1.62 mg), in a solvent mixture of water and dimethylformamide (H2O/DMF = 2:7, 1.9 mL) was introduced into the flask via syringe injection. The reaction mixture was continuously bubbled with argon for 30 min. The flask was then placed in a light‐protected environment at 45 °C and the reaction continued for 24 h. Upon completion of the reaction, the reaction was quenched using liquid nitrogen. The reaction mixture underwent dialysis against deionized water (MWCO 3500 Da) for a duration of 1.5 days, followed by freeze‐drying to obtain the final product, branched poly(LAEMA)‐GFLG‐PySS, with a yield of 67%.
\",\n \"
\\nSynthesis of Branched Poly(LAEMA)‐GFLG‐PTX: In a round‐bottom flask, 200 mg of branched poly(LAEMA)‐GFLG‐PySS was accurately weighed and dissolved in 8 mL of a mixture of DMSO:H2O (4:1, v/v). After complete dissolution, 30 mg of dithiothreitol (DTT) was introduced, and the reaction mixture underwent 12 h reaction at room temperature. Subsequently, the reaction mixture was subjected to dialysis to remove organic solvents, followed by freeze‐drying to yield the product, branched poly(LAEMA)‐GFLG‐SH.
\",\n \"
Branched poly (LAEMA)‐GFLG‐SH(146 mg) was carefully weighed and dissolved in 8 mL of a mixture of DMSO:H2O (4:1, v/v) in a round‐bottom flask. After complete dissolution, 1.4 mg of maleimide‐Cy5 dissolved in DMSO was added to the reaction mixture. The reaction proceeded overnight at room temperature. 48.7 mg of maleimide‐GFLG‐PTX was then introduced into the reaction mixture, and the reaction continued at room temperature for an additional 12 h. After the completion of the reaction, the mixture was sequentially dialyzed with DMF and deionized water (MWCO = 3500 Da). The resulting product was collected, filtered through a membrane, and subjected to freeze‐drying, resulting in the formation of branched poly(LAEMACy5)‐GFLG‐PTX. The drug loading of PTX in the polymer was determined through HPLC analysis.
\",\n \"Physicochemical Characterizations of BPGP and BPGP@CAP\",\n \"
The assessment of the CAC of BPGP was accomplished with the aid of pyrene as a fluorescence agent. Herein, 200 µL of a pyrene‐acetone solution (0.67 × 10−6m) was introduced into a 10 mL sample vial. After complete evaporation of acetone, an aliquot of the BPGP aqueous solution (ranging from 0.015 to 1000 µg mL−1) was added into the sample vial. After 2 h incubation, the fluorescence spectra were captured and recorded via a fluorescence spectrophotometer.
\",\n \"
The AKT inhibitor capivasertib ‐loaded branched poly(LAEMACy5)‐GFLG‐PTX was prepared using the solvent evaporation method. In brief, a methanol/acetonitrile mixed solution (0.4 mL) containing 1.125 mg of CAP was slowly added dropwise to a 5 mL aqueous solution of BPGP (2 mg mL−1, PTX:CAP = 1 (wt):1.5 (wt)) under stirring conditions. The mixture was stirred at room temperature until the organic solvents were completely evaporated. The remaining solution was then filtered through a membrane (0.45 µm) to remove unencapsulated CAP. Finally, the obtained filtrate was freeze‐dried to yield the desired product, BPGP@CAP. BPGP@CAP with different weight ratios of PTX and CAP was prepared by adjusting the weighting ratio of PTX and CAP. The loading contents of CAP in nanoassemblies were determined by HPLC system. The drug loading content (DLC) and the drug loading efficiency (DLE) were calculated using the following equations:
\",\n \"
DLC (wt.%) = (weight of drug loaded/total weight of polymer and loaded drug) × 100
\",\n \"
DLE (wt.%) = (weight of drug loaded/initial weight of drug) × 100
\",\n \"
The hydrodynamic diameter of the obtained nanoassemblies were measured by dynamic light scattering (DLS, Brookhaven ZetaPALS, USA). The morphology of the obtained nanoassemblies was examined under a transmission electron microscope (TEM, Tecnai GF20S‐TWIN, USA). The enzyme‐responsive drug release capacity of nanoassembly was investigated under simulated physiological conditions, with and without the presence of papain.
\",\n \"Cell Line and Animals\",\n \"
MFC cell, a cell line derived from GC, was acquired from the Chinese Academy of Medical Sciences (Shanghai, China), which were cultured in a temperature‐controlled incubator at 37 °C with 5% CO2 in a humidified environment. Male BALB/c nude mice (4‐6 weeks old, weighing 18–20 g) were procured from Hfk Bioscience Co., Ltd (Beijing, China). Subcutaneous GC MFC tumor models were generated through injecting 5 × 105 MFC cells into the right back of the mice. Approval for all animal experiments was granted by the Animal Ethics Committee of West China Hospital, Sichuan University (Approval No. 2018154A).
\",\n \"Clinical Tissue Sampling\",\n \"
The AKT expression and its relationship was examined with the prognosis of GC using the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/) cohort data via Kaplan‐Meier Plotter (http://kmplot.com/analysis/index.php?p = background). Tumor and normal tissues of 100 GC patients were collected from the West China Hospital of Sichuan University. IHC was employed for assessing the expression of AKT. Based on the IHC scores, 100 GC patients were categorized into high and low AKT expression groups. The Kaplan–Meier method was used for the survival curve of both groups. This study regarding clinical tissue sampling received approval from the Ethics Committee of The West China Hospital of Sichuan University (2023 Review, No. 549) and all patients provided informed consent for participation in the study.
\",\n \"Cellular Uptake, Endocytic Pathways, and Subcellular Localization\",\n \"
MFC cells were inoculated in a confocal chamber (1 × 105 cells per well) and incubated overnight. They were then incubated with culture medium containing Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) for 1, 2, and 4 h, respectively. Following washing with PBS, Hoechst 33342 was added for nucleus staining. Cellular uptake of BPGP@CAP by MFC cells was determined by analyzing the Cy5 signal using laser scanning confocal microscopy (CLSM) (Nikon, Tokyo, Japan). The mean fluorescence intensity was obtained from semi‐quantitative analysis using Image J software. Additionally, flow cytometry (BD FACSCelesta, New Jersey, USA) was employed for detecting cellular uptake of BPGP@CAP by analyzing the Cy5 signal in MFC cells treated with Cy5‐labeled BPGP@CAP for various durations (1 h, 2 h, and 4 h).
\",\n \"
To investigate the endocytic pathways of BPGP@CAP, MFC cells were pre‐treated with the culture medium containing 8 µg mL−1 chlorpromazine hydrochloride, 0.5 mm amiloride hydrochloride or 0.5 mm dynasore for 2 h. Subsequently, those MFC cells was incubated with medium containing Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) for an additional 2 h. Quantitative analysis of cellular uptake of BPGP@CAP was conducted using flow cytometry. Furthermore, to determine if the cellular uptake of BPGP@CAP was relied on energy, cells were exposed in the culture medium with Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) and incubated at 4 °C for a duration of 2 h and flow cytometry was employed to check the uptake level of BPGP@CAP.
\",\n \"
For subcellular localization studies of BPGP@CAP, MFC cells were first exposed to Cy5‐labeled BPGP@CAP (Cy5: 0.5 µg mL−1) for 2 h. Following the staining of MFC cell lysosomes with Lyso Tracker Green and the nucleus with Hoechst 33342 for a duration of 15–20 min, observation and imaging of MFC cells were conducted using CLSM.
\",\n \"Cell Cytotoxicity Assay\",\n \"
After MFC cells were inoculated in 96‐well plates (3 × 103 cells per well) and grown overnight, MFC cells were exposed to various formulations (such as PTX, CAP, a mixture of PTX and CAP (PTX+CAP), BPGP, and BPGP@CAP) at different concentrations for 48 h. MFC cells treated with fresh culture medium were included as a control group. Following the treatment, those cells were exposed to a medium containing a 10% CCK‐8 solution and incubated at 37 °C for 1 h. A microplate reader (Tecan, Switzerland) was utilized to measure the OD450 value, which was then used to evaluate the relative cell viability.
\",\n \"Cell Apoptosis and Cell Cycle Assay\",\n \"
MFC cells were inoculated in a 6‐well plate with 2 × 105 cells per well and grown for 12 h. Subsequently, those cells were incubated with the culture medium containing PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for 48 h. MFC cells treated with fresh culture medium were included as a control group. Those cells were collected and stained with an Annexin V‐PI reagent for 30 min. Flow cytometry was employed for assessing apoptosis of MFC cells.
\",\n \"
For cell cycle assays, cells that received treatment with PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for a duration of 48 h were collected and subjected to overnight fixation in 75% ethanol. Following gentle washing with PBS, they were delicately incubated with a 0.5 mL solution of the PI/RNase dye. The flow cytometry was used to discern the cellular distribution at different cell cycle phases. Each experiment had three replicates, and the acquired data was analyzed through the Flowjo software.
\",\n \"Morphological Examination of Microtubules and Microfilaments\",\n \"
The culture medium containing PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP was used to treat MFC cells for 24 h to examine the morphology of microtubules. Following washing with PBS, those MFC cells were fixed with a 4% paraformaldehyde solution. After washing with PBS containing 0.1% Triton X‐100, the cells were subjected to Tubulin‐tracker Red staining for 1 h. Subsequently, nucleus staining was performed with DAPI. The cells were then observed and imaged under CLSM. For morphological analysis of microfilaments, MFC cells were stained with Actin‐tracker Green and DAPI and they were then observed and imaged via CLSM.
\",\n \"Transcriptome Analysis by RNA‐Seq\",\n \"
MFC cells were inoculated in a 6‐well plate (2 × 105 cells per well) and grown for 12 h. Thereafter, they were exposed to BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for a duration of 48 h. MFC cells treated with fresh culture medium were included as a control group. Total RNA of MFC cells treated with BPGP@CAP in fresh culture medium was extracted. Genechem Co., Ltd (Shanghai, China) conducted the subsequent steps of quality control, library preparation, and RNA sequencing. Gene Ontology (GO) and KEGG pathway analyses were performed through the R package. Significant differences between the BPGP@CAP treatment group and the control cohort were robustly identified with a p‐adjust < 0.05 and a |log2FC| > 1.
\",\n \"Western Blots\",\n \"
MFC cells were treated with PTX, CAP, PTX + CAP, BPGP, or BPGP@CAP (the PTX concentration: 2.0 µg mL−1 and the corresponding concentration of CAP: 2.1 µg mL−1) for 48 h. Untreated MFC cells were set as a control group. After collecting the cells from each experimental group, total proteins were collected from cell lysates in a lysis buffer. Subsequently, they underwent SDS‐PAGE gel electrophoresis and were then transferred to a 0.45 µm PVDF membrane (Millipore). Following a blockade using a 5% milk TBST solution, the membrane was subjected to an overnight incubation with primary antibodies in a refrigerator at 4°C. After a thorough TBST wash, HRP‐conjugated secondary antibodies were applied to the membrane and incubated at room temperature for 1 h. Detection of the proteins was achieved through the Syngene GeneGenius gel imaging system (Syngene, Cambridge, UK).
\",\n \"In Vivo Pharmacokinetics Studies\",\n \"
Normal male balb/c nude mice were randomly allocated into two groups (n = 5) for intravenous administration of free Cy5 and Cy5‐labelled BPGP@CAP (Cy5: 1.5 mg kg−1). At various time points (0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, and 24 h) after injection, blood samples were obtained from the orbital vein and immediately mixed with double distilled water. After the samples were then supplemented with DMSO and refrigerated overnight at 4 °C, they were centrifuged at 12 000 rpm for 15 min, and the supernatant from each sample was transferred to a 96‐well plate for fluorescence measurement using a microplate reader. The pharmacokinetic parameters were calculated using the PKsolver software.
\",\n \"In Vivo Distribution\",\n \"
The MFC tumor‐bearing nude mice (n = 3) received intravenous injections via the tail with free Cy5 and Cy5‐labeled BPGP@CAP (Cy5: 1.5 mg kg−1) in order to assess their in vivo biodistribution. At various time points following the injection (1 h, 2 h, 3 h, 6 h, 9 h, 12 h, and 24 h), the mice were observed and imaged using an IVIS Spectrum (Caliper Life Sciences, Cambridge, UK). Upon completion of the imaging experiment, tumor samples from the mice were obtained and subjected to frozen sectioning. After staining with DAPI to label cell nuclei, the sections were observed and imaged using CLSM.
\",\n \"In Vivo Anti‐Tumor Study\",\n \"
The mice with MFC tumors received different formulations via tail vein injection (n = 5): (1) Saline; (2) PTX; (3) CAP; (4) PTX + CAP; (5) BPGP; and (6) BPGP@CAP (the PTX concentration: 8.0 mg kg−1 and the corresponding concentration of CAP: 8.5 mg kg−1). The drugs were administered every other day until the completion of the treatment. The body weight of the mice, the length (L) and width (W) of the tumor were regularly recorded. The tumor volume (V) was determined using the formula: V = (L × W2)/2.
\",\n \"
After the treatment, the mice were humanely euthanized by cervical dislocation, and their tumors were surgically excised and weighed. Furthermore, the tumors and vital organs underwent a process of fixation, embedding, and sectioning. The tumor sections were subjected to staining with Ki67 and TUNEL to evaluate proliferation and apoptosis of tumor cells. In addition, hematoxylin and eosin (H&E) staining was performed to assess the biocompatibility of these formulations.
\",\n \"
Moreover, the biosafety of these formulations was evaluated in MFC tumor‐bearing nude mice. Hematological and biochemical analyses were conducted using collected whole blood and serum samples. Healthy balb/c nude mice were included as a control group for comparison.
\",\n \"Statistical Analysis\",\n \"
GraphPad Prism 9 software was employed to analyze data obtained from at least three independent measurements. The results were presented as the mean ± standard deviation (± S.D.). Statistical significance was determined by performing a two‐tailed t‐test between two groups, and a P‐value less than 0.05 was considered to be statistically significant.
X.S. and H.C. contributed equally to this work. This work was supported by National Natural Science Foundation of China (32271445, 52073193, 82072688, 82202322), 1·3·5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC21013, ZYJC21006) and the Sichuan Science and Technology Program (2023NSFSC1592). The authors also thank Li Li, Fei Chen, and Chunjuan Bao (institute of Clinical Pathology, West China Hospital) for their help in processing histological staining. The authors would like to thank Lei Wu and Yaping Wu (Histology and Imaging Platform, Research Core Facility, West China Hospital, Sichuan University) for their help in imaging studies. The authors also thank Qianyu Zhu (Laboratory of Gastric Cancer, West China Hospital, Sichuan University) for her help in data analysis. The authors also acknowledge Qiaorong Huang, Xue Li and Wentong Meng (Laboratory of Stem Cell Biology, West China Hospital, Sichuan University) for help in flow cytometer. Scheme ##FIG##0##1##, Figures ##FIG##5##5G## and ##FIG##6##6D## of contents entry were created with BioRender.com.
","Data Availability Statement","
The data that support the findings of this study are available from the corresponding author upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
X.S. and H.C. contributed equally to this work. This work was supported by National Natural Science Foundation of China (32271445, 52073193, 82072688, 82202322), 1·3·5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (ZYJC21013, ZYJC21006) and the Sichuan Science and Technology Program (2023NSFSC1592). The authors also thank Li Li, Fei Chen, and Chunjuan Bao (institute of Clinical Pathology, West China Hospital) for their help in processing histological staining. The authors would like to thank Lei Wu and Yaping Wu (Histology and Imaging Platform, Research Core Facility, West China Hospital, Sichuan University) for their help in imaging studies. The authors also thank Qianyu Zhu (Laboratory of Gastric Cancer, West China Hospital, Sichuan University) for her help in data analysis. The authors also acknowledge Qiaorong Huang, Xue Li and Wentong Meng (Laboratory of Stem Cell Biology, West China Hospital, Sichuan University) for help in flow cytometer. Scheme ##FIG##0##1##, Figures ##FIG##5##5G## and ##FIG##6##6D## of contents entry were created with BioRender.com.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available from the corresponding author upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
Schematic illustration of enzyme‐responsive branched glycopolymer‐based nanoassembly (BPGP@CAP) for synergistic antitumor therapy. A branched poly(LAEMA)‐GFLG‐PTX prodrug self‐assembles through hydrophobic‐hydrophilic interactions and this prodrug encapsulates CAP as an AKT inhibitor to form a stable nanoassembly, BPGP@CAP. Through the enhanced permeability and retention (EPR) effect, BPGP@CAP selectively accumulates at tumor tissues. Within tumor cells, overexpressed cathepsin B triggers specific cleavage of the GFLG peptide in the polymer structure, leading to carrier degradation and concomitant drug release. The released PTX and CAP synergistically exhibit antitumor effects by inducing apoptosis.
","
Clinical significance of AKT in gastric cancer (GC). A) The RNA expression of AKT in GC samples and normal gastric tissues was analyzed by utilizing the GEO cohort data. B) Overall survival rates of GC patients with a high or low level of AKT expression were analyzed through Kaplan‐Meier analysis. C) The AKT expression detected by IHC in GC samples and normal gastric tissues obtained from West China Hospital. D) Kaplan–Meier analysis of overall survival rates of GC patients based on the AKT expression level using the West China Hospital cohort data. E) IHC analysis of AKT expression in GC samples treated with and without neoadjuvant therapy (NAC) in West China Hospital. F) Western blot results of AKT expression in MFC cells treated with PTX (1 µg mL‐1) and PTX (1 µg mL‐1) + CAP (1 µg mL‐1). G) Semiquantitative analysis of the AKT protein level in different groups in Figure ##FIG##1##1F##. H) Cytotoxicity of PTX, CAP, and PTX + CAP toward MFC cells. I) Combination index (CI) of CAP and PTX for treating MFC cells at different CAP/PTX weight ratios (n = 5). Data is displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, ***p < 0.001.
","
Characterizations of physicochemical properties of BPGP and BPGP@CAP. A) 1H NMR of the branched poly(LAEMA)‐GFLG‐PTX prodrug in DMSO‐d6. B) UV–vis and fluorescence spectra of the branched poly(LAEMA)‐GFLG‐PTX prodrug (dissolved in DMSO) with or without Cy5 labeling. The characteristic UV–vis absorption peak of Cy5 appears at 648 nm, and the fluorescence characteristic peak at 667 nm (Ex = 647 nm). C) CAC of the branched poly(LAEMA)‐GFLG‐PTX prodrug. D) The drug loading content (DLC) and the drug loading efficiency (DLE) of CAP in BPGP at various weight ratios of PTX to CAP. E,F) Hydrodynamic diameters and representative TEM images of BPGP and BPGP@CAP, respectively. Scale bars = 100 nm. G) HPLC chromatograms of poly (LAEMA)‐GFLG‐PTX with or without papain, and free PTX as a control.
","
Cellular uptake and cytotoxicity of BPGP@CAP in vitro. A) CLSM images of MFC cells exposed to Cy5‐labelled BPGP@CAP (red: Cy5; blue: nuclei). Scale bar = 20 µm. B) Colocalization of lysosomes and BPGP@CAP in MFC cells. Lyso‐Tracker was used to label and visualize the lysosomes (green), while Hoechst 33342 was employed to stain the nuclei (blue). Scale bar = 20 µm. C) Mean fluorescence intensity (MFI) of MFC cells after exposure to Cy5‐labelled BPGP@CAP for 1 h, 2 h, and 4 h from flow cytometry analysis. D) The inhibitory rate of cellular uptake of BPGP@CAP by MFC cells treated with different inhibitors or at a low temperature. E) Viabilities of MFC cells after treatment with PTX, CAP, PTX + CAP, BPGP and BPGP@CAP. F) Microtubule aggregation in MFC cells induced by various treatments for 24 h. Tubulin Tracker was utilized to stain and detect tubulin (red), while DAPI was employed for staining the nuclei (blue). Scale bar = 20 µm.
","
Analysis of apoptosis and cell cycle arrest in MFC cells after exposure to various treatments through flow cytometry. A,C) Percentages of apoptotic MFC cells after exposure to PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP for 48 h (n = 3). B,D) Cell cycle distribution of MFC cells after exposure to PTX, CAP, PTX + CAP, BPGP and BPGP@CAP for 48 h (n = 3). Data is displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, **p < 0.01, ***p < 0.001 and ns for p > 0.05.
","
Mechanisms underlying synergistic effect of PTX and CAP for anticancer treatment. A) Volcano plots of DEGs in BPGP@CAP‐treated MFC cells compared with the control group. B) Top 20 enriched KEGG pathways from DEGs in MFC cells exposed to BPGP@CAP compared to the control cells. C, D) Western blotting analysis of proteins associated with the PI3K/AKT signal pathway in MFC cells treated with CAP, including AKT, p‐mTOR, p‐4E‐BP1, and p‐S6. The proteins in the control group are significantly lower than those in the groups treated with CAP at 0.3 µg mL‐1 and 1.0 µg mL‐1, and the P‐values are less than 0.001. E,F) Western blot results of proteins associated with apoptosis in MFC cells after exposure to PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, including Bax, Bcl‐2, cleaved‐caspase‐3 and cleavedPARP. The proteins in the control group are significantly lower than those in the groups treated with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, and the P‐values are less than 0.001. G) Schematic diagram for the mechanisms for the anticancer effect by the combine therapy with PTX and CAP against GC. Data were displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, ***p < 0.001.
","
Biodistribution and tumor growth inhibition of BPGP@CAP in vivo. A) Fluorescent images of the mice with MFC tumors exposed to Cy5‐labelled BPGP@CAP and free Cy5 at different time‐points. B) Immunofluorescence staining images of tumor tissues obtained from the mice treated with free Cy5 and Cy5‐labelled BPGP@CAP for 24 h. Red for Cy5‐labeled BPGP@CAP or free Cy5, and blue for DAPI‐stained cell nuclei. Scale bar = 100 µm. C) Pharmacokinetic curves of the Cy5 concentration after intravenous injection of Cy5‐labelled BPGP@CAP and free Cy5, respectively (n = 5). D) Establishment of subcutaneous GC MFC tumor models and therapeutic treatment schedule. E) Growth curves of the tumors in individual mice across different treatment groups (n = 5). F) Average tumor growth curves in different treatments groups (n = 5). G) Tumor weights of the mice with tumors in different treatment groups (n = 5). H) Body weights of the mice with tumors after different treatments (n = 5). The statistical difference is obtained from the body weights of the BPGP@CAP‐treated group in comparison with those of the PTX‐treated group and the PTX+CAP‐treated group. I) H&E, TUNEL and Ki67 staining images for tumor tissues from the mice after different treatments (scale bar = 100 µm). J) The proportion of TUNEL and Ki67‐positive cells in the tumors after different treatments. The proportion of TUNEL and Ki67‐positive cells of the BPGP@CAP‐treated group is compared with that in the control group, the PTX‐treated group and the PTX+CAP‐treated group. Data are displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, ***p < 0.001.
"],"string":"[\n \"
Schematic illustration of enzyme‐responsive branched glycopolymer‐based nanoassembly (BPGP@CAP) for synergistic antitumor therapy. A branched poly(LAEMA)‐GFLG‐PTX prodrug self‐assembles through hydrophobic‐hydrophilic interactions and this prodrug encapsulates CAP as an AKT inhibitor to form a stable nanoassembly, BPGP@CAP. Through the enhanced permeability and retention (EPR) effect, BPGP@CAP selectively accumulates at tumor tissues. Within tumor cells, overexpressed cathepsin B triggers specific cleavage of the GFLG peptide in the polymer structure, leading to carrier degradation and concomitant drug release. The released PTX and CAP synergistically exhibit antitumor effects by inducing apoptosis.
\",\n \"
Clinical significance of AKT in gastric cancer (GC). A) The RNA expression of AKT in GC samples and normal gastric tissues was analyzed by utilizing the GEO cohort data. B) Overall survival rates of GC patients with a high or low level of AKT expression were analyzed through Kaplan‐Meier analysis. C) The AKT expression detected by IHC in GC samples and normal gastric tissues obtained from West China Hospital. D) Kaplan–Meier analysis of overall survival rates of GC patients based on the AKT expression level using the West China Hospital cohort data. E) IHC analysis of AKT expression in GC samples treated with and without neoadjuvant therapy (NAC) in West China Hospital. F) Western blot results of AKT expression in MFC cells treated with PTX (1 µg mL‐1) and PTX (1 µg mL‐1) + CAP (1 µg mL‐1). G) Semiquantitative analysis of the AKT protein level in different groups in Figure ##FIG##1##1F##. H) Cytotoxicity of PTX, CAP, and PTX + CAP toward MFC cells. I) Combination index (CI) of CAP and PTX for treating MFC cells at different CAP/PTX weight ratios (n = 5). Data is displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, ***p < 0.001.
\",\n \"
Characterizations of physicochemical properties of BPGP and BPGP@CAP. A) 1H NMR of the branched poly(LAEMA)‐GFLG‐PTX prodrug in DMSO‐d6. B) UV–vis and fluorescence spectra of the branched poly(LAEMA)‐GFLG‐PTX prodrug (dissolved in DMSO) with or without Cy5 labeling. The characteristic UV–vis absorption peak of Cy5 appears at 648 nm, and the fluorescence characteristic peak at 667 nm (Ex = 647 nm). C) CAC of the branched poly(LAEMA)‐GFLG‐PTX prodrug. D) The drug loading content (DLC) and the drug loading efficiency (DLE) of CAP in BPGP at various weight ratios of PTX to CAP. E,F) Hydrodynamic diameters and representative TEM images of BPGP and BPGP@CAP, respectively. Scale bars = 100 nm. G) HPLC chromatograms of poly (LAEMA)‐GFLG‐PTX with or without papain, and free PTX as a control.
\",\n \"
Cellular uptake and cytotoxicity of BPGP@CAP in vitro. A) CLSM images of MFC cells exposed to Cy5‐labelled BPGP@CAP (red: Cy5; blue: nuclei). Scale bar = 20 µm. B) Colocalization of lysosomes and BPGP@CAP in MFC cells. Lyso‐Tracker was used to label and visualize the lysosomes (green), while Hoechst 33342 was employed to stain the nuclei (blue). Scale bar = 20 µm. C) Mean fluorescence intensity (MFI) of MFC cells after exposure to Cy5‐labelled BPGP@CAP for 1 h, 2 h, and 4 h from flow cytometry analysis. D) The inhibitory rate of cellular uptake of BPGP@CAP by MFC cells treated with different inhibitors or at a low temperature. E) Viabilities of MFC cells after treatment with PTX, CAP, PTX + CAP, BPGP and BPGP@CAP. F) Microtubule aggregation in MFC cells induced by various treatments for 24 h. Tubulin Tracker was utilized to stain and detect tubulin (red), while DAPI was employed for staining the nuclei (blue). Scale bar = 20 µm.
\",\n \"
Analysis of apoptosis and cell cycle arrest in MFC cells after exposure to various treatments through flow cytometry. A,C) Percentages of apoptotic MFC cells after exposure to PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP for 48 h (n = 3). B,D) Cell cycle distribution of MFC cells after exposure to PTX, CAP, PTX + CAP, BPGP and BPGP@CAP for 48 h (n = 3). Data is displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, **p < 0.01, ***p < 0.001 and ns for p > 0.05.
\",\n \"
Mechanisms underlying synergistic effect of PTX and CAP for anticancer treatment. A) Volcano plots of DEGs in BPGP@CAP‐treated MFC cells compared with the control group. B) Top 20 enriched KEGG pathways from DEGs in MFC cells exposed to BPGP@CAP compared to the control cells. C, D) Western blotting analysis of proteins associated with the PI3K/AKT signal pathway in MFC cells treated with CAP, including AKT, p‐mTOR, p‐4E‐BP1, and p‐S6. The proteins in the control group are significantly lower than those in the groups treated with CAP at 0.3 µg mL‐1 and 1.0 µg mL‐1, and the P‐values are less than 0.001. E,F) Western blot results of proteins associated with apoptosis in MFC cells after exposure to PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, including Bax, Bcl‐2, cleaved‐caspase‐3 and cleavedPARP. The proteins in the control group are significantly lower than those in the groups treated with PTX, CAP, PTX + CAP, BPGP, and BPGP@CAP, and the P‐values are less than 0.001. G) Schematic diagram for the mechanisms for the anticancer effect by the combine therapy with PTX and CAP against GC. Data were displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, ***p < 0.001.
\",\n \"
Biodistribution and tumor growth inhibition of BPGP@CAP in vivo. A) Fluorescent images of the mice with MFC tumors exposed to Cy5‐labelled BPGP@CAP and free Cy5 at different time‐points. B) Immunofluorescence staining images of tumor tissues obtained from the mice treated with free Cy5 and Cy5‐labelled BPGP@CAP for 24 h. Red for Cy5‐labeled BPGP@CAP or free Cy5, and blue for DAPI‐stained cell nuclei. Scale bar = 100 µm. C) Pharmacokinetic curves of the Cy5 concentration after intravenous injection of Cy5‐labelled BPGP@CAP and free Cy5, respectively (n = 5). D) Establishment of subcutaneous GC MFC tumor models and therapeutic treatment schedule. E) Growth curves of the tumors in individual mice across different treatment groups (n = 5). F) Average tumor growth curves in different treatments groups (n = 5). G) Tumor weights of the mice with tumors in different treatment groups (n = 5). H) Body weights of the mice with tumors after different treatments (n = 5). The statistical difference is obtained from the body weights of the BPGP@CAP‐treated group in comparison with those of the PTX‐treated group and the PTX+CAP‐treated group. I) H&E, TUNEL and Ki67 staining images for tumor tissues from the mice after different treatments (scale bar = 100 µm). J) The proportion of TUNEL and Ki67‐positive cells in the tumors after different treatments. The proportion of TUNEL and Ki67‐positive cells of the BPGP@CAP‐treated group is compared with that in the control group, the PTX‐treated group and the PTX+CAP‐treated group. Data are displayed as mean ± SD. A two‐sided unpaired Student's t‐test was employed for assessing statistical significance, ***p < 0.001.
Over the past few decades, significant efforts have been devoted to developing advanced energy storage devices and new renewable energy sources to cope with the fossil energy crisis and environmental problems. Since the C║LiCoO2 battery was commercialized by Sony in 1991,[\n##REF##11713543##\n1\n##\n] lithium‐ion batteries (LIBs) have been used in a wide range of portable electronic devices, hybrid and full electric vehicles and other energy‐storing devices because of their high energy density, long cyclability, low self‐discharge and absence of memory effect.[\n##UREF##0##\n2\n##, ##REF##23584142##\n3\n##\n] Reducing greenhouse gas emission has now become global consensus in response to the greenhouse effect. For example, in October 2021 the Chinese government has proposed the goals of peak carbon dioxide emissions and carbon neutrality which will be achieved in 2030 and 2060, respectively. In this context, there is no doubt that the new energy technology is inevitable to achieve carbon peak and carbon neutrality. Thus, as a representative of clean energy storage devices, LIBs stand for an ideal candidate due to high energy density, low cost, safety, and environmental sustainability, which are of particular importance.
","
LIBs are mainly made of cathode, separator, electrolyte and anode, where the cathode limits the energy density and dominates the final cost of the battery.[\n##REF##32214093##\n4\n##, ##UREF##1##\n5\n##, ##UREF##2##\n6\n##\n] Up to now, three main types of cathode materials have been commercialized, including layered lithium transition metal oxide LiTMO2 (TM = Co, Ni, Mn), spinel LiMn2O4, and olivine‐structure lithium iron phosphate LiFePO4 (Figure\n##FIG##0##\n1a##).[\n##REF##24202440##\n7\n##, ##UREF##3##\n8\n##\n] In terms of cathode materials, metal elements play an essential role in the advancement of energy storage. As shown in Figure ##FIG##0##1b##, the crustal content of common metals is provided, including nickel (Ni), cobalt (Co), iron (Fe), aluminum (Al) and manganese (Mn), which is related to the cost of batteries. To better compare the characteristics of the state‐of‐the‐art cathodes that are being used in industry today (LiCoO2, LiNi0.33Co0.33Mn0.33O2, LiNi0.80Co0.15Al0.05O2, LiMn2O4, LiNi0.5Mn1.5O4, LiFePO4), Figure ##FIG##0##1c–i## provides the specific parameters of them.[\n##REF##34705441##\n9\n##, ##REF##29956705##\n10\n##, ##UREF##4##\n11\n##, ##UREF##5##\n12\n##, ##UREF##6##\n13\n##, ##UREF##7##\n14\n##, ##UREF##8##\n15\n##, ##UREF##9##\n16\n##\n]\n
","
LiCoO2 was successfully developed by Goodenough in the 1980s,[\n##UREF##10##\n17\n##\n] which became the typical layered cathode material and remained the dominant cathode material for the portable electronics market. However, it can be seen that the content of Co is the lowest among above mentioned metals, leading to high cost. Additionally, toxicity and its adverse effects on the environment will be a major constraint on its development.[\n##UREF##11##\n18\n##\n] Thus, Ni‐layered oxides mixed Co and Mn emerge as the time requires. LiNi0.80Co0.15Mn0.05 has an energy density of 760 kw kg−1 at 4.3 V thanks to the high nickel fraction.[\n##UREF##12##\n19\n##\n] Nevertheless, A high nickel fraction would promote the cation disorder, resulting in the collapse of the crystal structure and the release of oxygen during the process of cycling.[\n##UREF##13##\n20\n##\n] That is, although the cost of nickel is lower than that of Co, the low safety of ternary cathode materials mainly caused by the metal Ni cannot be ignored.[\n##UREF##14##\n21\n##, ##UREF##15##\n22\n##, ##UREF##16##\n23\n##\n]\n
","
Therefore, iron and manganese demonstrate great promise for cathode materials owing to their non‐toxicity, abundant resources, and low cost. And from a sustainability perspective, Mn‐based cathodes and LiFePO4 demonstrate an attractive prospect. Here it should be noted that the sustainability of materials is rated in terms of supply‐demand relations, mining technologies, environmental impacts, waste management, etc,[\n##UREF##17##\n24\n##\n] and value 5 for sustainability in Figure ##FIG##0##1c## represents the highest level. LiFePO4 is commonly used in commercial electric vehicles owing to its high level of thermal stability and safety.[\n##UREF##18##\n25\n##\n] However, the theoretical capacity of olivine LiFePO4 (170 mAh g−1) is significantly lower than that of other cathode materials (LiCoO2: 274 mAh g−1, LiNi1−\n\nx\n\n−\n\ny\nCo\nx\nMn\ny\nO2: 273—285 mAh g−1). Moreover, compared with the theoretical density of LiCoO2, LiNiO2, and LiMn2O4 (5.1, 4.8, 4.2 g cm−3, respectively), LiFePO4 shows much lower theoretical density (3.6 g cm−3), which gives a seriously adverse effect on the energy density and specific capacity.[\n##UREF##19##\n26\n##\n] Meanwhile, LiFePO4 has poor electronic conductivity and ionic conductivity, leading to initial capacity loss.[\n##UREF##20##\n27\n##\n] Recently, Co‐free high‐voltage spinel LiNi0.5Mn1.5O4 has attracted attention due to its high operating voltage of 4.7 V (vs Li/Li+). However, high voltage brings about rapid capacity degradation and the decomposition of electrolytes.[\n##UREF##21##\n28\n##, ##UREF##22##\n29\n##, ##UREF##23##\n30\n##\n] Hence, manganese‐based cathodes have lately received great attention for the development of Co‐free cathode materials.[\n##UREF##24##\n31\n##\n] Compared with LiMn2O4 (148 mAh g−1), LiMnO2 possesses a higher theoretical capacity (285 mAh g−1).
","
As early as 2001, Ammundsen and Paulsen reviewed the progress of LiMnO2.[\n##UREF##25##\n32\n##\n] In the past two decades, more researchers have extensively investigated LiMnO2 cathode, especially in terms of synthesis methods and modification manners. To get a more comprehensive understanding of LiMnO2 cathode, we deliver the review. This review is organized as follows. Firstly, in Section 2, the structural characteristics and drawbacks of LiMnO2 cathode are presented, as well as the synthesis methods discussed briefly. To improve the weaknesses of LiMnO2, modifications like element doping, surface coating, composites designing and finding compatible electrolyte are reviewed in Section 3. Finally, we describe our conclusions with an outlook for LiMnO2 cathode in Section 4.
"],"string":"[\n \"Introduction\",\n \"
Over the past few decades, significant efforts have been devoted to developing advanced energy storage devices and new renewable energy sources to cope with the fossil energy crisis and environmental problems. Since the C║LiCoO2 battery was commercialized by Sony in 1991,[\\n##REF##11713543##\\n1\\n##\\n] lithium‐ion batteries (LIBs) have been used in a wide range of portable electronic devices, hybrid and full electric vehicles and other energy‐storing devices because of their high energy density, long cyclability, low self‐discharge and absence of memory effect.[\\n##UREF##0##\\n2\\n##, ##REF##23584142##\\n3\\n##\\n] Reducing greenhouse gas emission has now become global consensus in response to the greenhouse effect. For example, in October 2021 the Chinese government has proposed the goals of peak carbon dioxide emissions and carbon neutrality which will be achieved in 2030 and 2060, respectively. In this context, there is no doubt that the new energy technology is inevitable to achieve carbon peak and carbon neutrality. Thus, as a representative of clean energy storage devices, LIBs stand for an ideal candidate due to high energy density, low cost, safety, and environmental sustainability, which are of particular importance.
\",\n \"
LIBs are mainly made of cathode, separator, electrolyte and anode, where the cathode limits the energy density and dominates the final cost of the battery.[\\n##REF##32214093##\\n4\\n##, ##UREF##1##\\n5\\n##, ##UREF##2##\\n6\\n##\\n] Up to now, three main types of cathode materials have been commercialized, including layered lithium transition metal oxide LiTMO2 (TM = Co, Ni, Mn), spinel LiMn2O4, and olivine‐structure lithium iron phosphate LiFePO4 (Figure\\n##FIG##0##\\n1a##).[\\n##REF##24202440##\\n7\\n##, ##UREF##3##\\n8\\n##\\n] In terms of cathode materials, metal elements play an essential role in the advancement of energy storage. As shown in Figure ##FIG##0##1b##, the crustal content of common metals is provided, including nickel (Ni), cobalt (Co), iron (Fe), aluminum (Al) and manganese (Mn), which is related to the cost of batteries. To better compare the characteristics of the state‐of‐the‐art cathodes that are being used in industry today (LiCoO2, LiNi0.33Co0.33Mn0.33O2, LiNi0.80Co0.15Al0.05O2, LiMn2O4, LiNi0.5Mn1.5O4, LiFePO4), Figure ##FIG##0##1c–i## provides the specific parameters of them.[\\n##REF##34705441##\\n9\\n##, ##REF##29956705##\\n10\\n##, ##UREF##4##\\n11\\n##, ##UREF##5##\\n12\\n##, ##UREF##6##\\n13\\n##, ##UREF##7##\\n14\\n##, ##UREF##8##\\n15\\n##, ##UREF##9##\\n16\\n##\\n]\\n
\",\n \"
LiCoO2 was successfully developed by Goodenough in the 1980s,[\\n##UREF##10##\\n17\\n##\\n] which became the typical layered cathode material and remained the dominant cathode material for the portable electronics market. However, it can be seen that the content of Co is the lowest among above mentioned metals, leading to high cost. Additionally, toxicity and its adverse effects on the environment will be a major constraint on its development.[\\n##UREF##11##\\n18\\n##\\n] Thus, Ni‐layered oxides mixed Co and Mn emerge as the time requires. LiNi0.80Co0.15Mn0.05 has an energy density of 760 kw kg−1 at 4.3 V thanks to the high nickel fraction.[\\n##UREF##12##\\n19\\n##\\n] Nevertheless, A high nickel fraction would promote the cation disorder, resulting in the collapse of the crystal structure and the release of oxygen during the process of cycling.[\\n##UREF##13##\\n20\\n##\\n] That is, although the cost of nickel is lower than that of Co, the low safety of ternary cathode materials mainly caused by the metal Ni cannot be ignored.[\\n##UREF##14##\\n21\\n##, ##UREF##15##\\n22\\n##, ##UREF##16##\\n23\\n##\\n]\\n
\",\n \"
Therefore, iron and manganese demonstrate great promise for cathode materials owing to their non‐toxicity, abundant resources, and low cost. And from a sustainability perspective, Mn‐based cathodes and LiFePO4 demonstrate an attractive prospect. Here it should be noted that the sustainability of materials is rated in terms of supply‐demand relations, mining technologies, environmental impacts, waste management, etc,[\\n##UREF##17##\\n24\\n##\\n] and value 5 for sustainability in Figure ##FIG##0##1c## represents the highest level. LiFePO4 is commonly used in commercial electric vehicles owing to its high level of thermal stability and safety.[\\n##UREF##18##\\n25\\n##\\n] However, the theoretical capacity of olivine LiFePO4 (170 mAh g−1) is significantly lower than that of other cathode materials (LiCoO2: 274 mAh g−1, LiNi1−\\n\\nx\\n\\n−\\n\\ny\\nCo\\nx\\nMn\\ny\\nO2: 273—285 mAh g−1). Moreover, compared with the theoretical density of LiCoO2, LiNiO2, and LiMn2O4 (5.1, 4.8, 4.2 g cm−3, respectively), LiFePO4 shows much lower theoretical density (3.6 g cm−3), which gives a seriously adverse effect on the energy density and specific capacity.[\\n##UREF##19##\\n26\\n##\\n] Meanwhile, LiFePO4 has poor electronic conductivity and ionic conductivity, leading to initial capacity loss.[\\n##UREF##20##\\n27\\n##\\n] Recently, Co‐free high‐voltage spinel LiNi0.5Mn1.5O4 has attracted attention due to its high operating voltage of 4.7 V (vs Li/Li+). However, high voltage brings about rapid capacity degradation and the decomposition of electrolytes.[\\n##UREF##21##\\n28\\n##, ##UREF##22##\\n29\\n##, ##UREF##23##\\n30\\n##\\n] Hence, manganese‐based cathodes have lately received great attention for the development of Co‐free cathode materials.[\\n##UREF##24##\\n31\\n##\\n] Compared with LiMn2O4 (148 mAh g−1), LiMnO2 possesses a higher theoretical capacity (285 mAh g−1).
\",\n \"
As early as 2001, Ammundsen and Paulsen reviewed the progress of LiMnO2.[\\n##UREF##25##\\n32\\n##\\n] In the past two decades, more researchers have extensively investigated LiMnO2 cathode, especially in terms of synthesis methods and modification manners. To get a more comprehensive understanding of LiMnO2 cathode, we deliver the review. This review is organized as follows. Firstly, in Section 2, the structural characteristics and drawbacks of LiMnO2 cathode are presented, as well as the synthesis methods discussed briefly. To improve the weaknesses of LiMnO2, modifications like element doping, surface coating, composites designing and finding compatible electrolyte are reviewed in Section 3. Finally, we describe our conclusions with an outlook for LiMnO2 cathode in Section 4.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":[],"string":"[]"},"discussion":{"kind":"list like","value":[],"string":"[]"},"conclusion":{"kind":"list like","value":["Conclusion and Outlook","
It has been 30 years since the commercialization of LIBs in 1991, and seeking for electrodes of low cost, high energy density, high safety and long life is always the primary aim to develop new‐generation LIBs in the future.
","
Facing resource depletion and the quest for non‐toxic green resources, lithium manganese oxide as a cathode material shows highly favorable advantages. Previous studies have shown that elemental doping is a good way to keep the LiMnO2 layers in place by suppressing phase transitions caused by the J–T effect due to high spin Mn3+. But it is not even close to commercialization because the charge/discharge mostly remains ≈50 cycles, especially for onefold doping. For another thing, surface coating is another effective approach to resolve the Mn dissolution and improve the cycling stability of LiMnO2, however, it also suffers from the complex procedures during coating and electrochemical‐inert coating layers that decrease the capacity of LiMnO2.
","
Thus, further efforts are still required to address the key issues and make commercialization achievable for LiMnO2. Special attention needs to be paid to the following aspects such as design of Li‐rich lithium manganese oxides, multi‐functional coating layers and new liquid electrolytes to achieve higher energy and more stable performance. Meanwhile, from the viewpoint of practical application with high sustainability, all‐solid‐state batteries systems with LiMnO2 as cathodes should be further exploited and the repairing/recycling techniques of spent LiMnO2 are necessary to carry forward.\n
In the LiTMO2 family, Li‐rich lithium manganese oxides (like Li1+\n\nx\nTM1‐\n\nx\nO2) with the disorder‐rocksalt structure are a class of cathode materials that can deliver ultrahigh capacity (> 300 mAh g−1) and energy density (>1000 Wh kg−1). It is aspired that effective methods could be developed for the controlled preparation of Li‐rich lithium manganese oxides to meet the request in the area of high‐energy applications.
It is highly desirable to construct multi‐functional coating layers with excellent mechanical stability to avoid Mn dissolution and side reactions, electrochemical activity to contribute capacity and electronic/ionic conductivity to facilitate charge transfer, which needs to design composite coating layers with elaborate element combinations other than traditional ones.
To reduce side reactions of LiMnO2 with electrolyte solution and Mn dissolution at most, the design and development of new electrolytes matching LiMnO2 cathodes is also a good solution.
All‐solid‐state batteries feature high energy density and high safety. LiMnO2‐based all‐solid‐state batteries are deemed to eliminate the problem of Mn dissolution and to deliver higher energy density. However, LiMnO2‐based all‐solid‐state batteries have been seldomly reported, which should be paid special attention to and developed as a priority for purpose of practical applications.
The resource shortage of lithium has raised the price of raw materials of cathode materials as well as the cost of batteries. Therefore, to address the concern in cost as well as the environment, the repairing and recycling techniques are urgent for spent LiMnO2 cathode materials.
\n
"],"string":"[\n \"Conclusion and Outlook\",\n \"
It has been 30 years since the commercialization of LIBs in 1991, and seeking for electrodes of low cost, high energy density, high safety and long life is always the primary aim to develop new‐generation LIBs in the future.
\",\n \"
Facing resource depletion and the quest for non‐toxic green resources, lithium manganese oxide as a cathode material shows highly favorable advantages. Previous studies have shown that elemental doping is a good way to keep the LiMnO2 layers in place by suppressing phase transitions caused by the J–T effect due to high spin Mn3+. But it is not even close to commercialization because the charge/discharge mostly remains ≈50 cycles, especially for onefold doping. For another thing, surface coating is another effective approach to resolve the Mn dissolution and improve the cycling stability of LiMnO2, however, it also suffers from the complex procedures during coating and electrochemical‐inert coating layers that decrease the capacity of LiMnO2.
\",\n \"
Thus, further efforts are still required to address the key issues and make commercialization achievable for LiMnO2. Special attention needs to be paid to the following aspects such as design of Li‐rich lithium manganese oxides, multi‐functional coating layers and new liquid electrolytes to achieve higher energy and more stable performance. Meanwhile, from the viewpoint of practical application with high sustainability, all‐solid‐state batteries systems with LiMnO2 as cathodes should be further exploited and the repairing/recycling techniques of spent LiMnO2 are necessary to carry forward.\\n
In the LiTMO2 family, Li‐rich lithium manganese oxides (like Li1+\\n\\nx\\nTM1‐\\n\\nx\\nO2) with the disorder‐rocksalt structure are a class of cathode materials that can deliver ultrahigh capacity (> 300 mAh g−1) and energy density (>1000 Wh kg−1). It is aspired that effective methods could be developed for the controlled preparation of Li‐rich lithium manganese oxides to meet the request in the area of high‐energy applications.
It is highly desirable to construct multi‐functional coating layers with excellent mechanical stability to avoid Mn dissolution and side reactions, electrochemical activity to contribute capacity and electronic/ionic conductivity to facilitate charge transfer, which needs to design composite coating layers with elaborate element combinations other than traditional ones.
To reduce side reactions of LiMnO2 with electrolyte solution and Mn dissolution at most, the design and development of new electrolytes matching LiMnO2 cathodes is also a good solution.
All‐solid‐state batteries feature high energy density and high safety. LiMnO2‐based all‐solid‐state batteries are deemed to eliminate the problem of Mn dissolution and to deliver higher energy density. However, LiMnO2‐based all‐solid‐state batteries have been seldomly reported, which should be paid special attention to and developed as a priority for purpose of practical applications.
The resource shortage of lithium has raised the price of raw materials of cathode materials as well as the cost of batteries. Therefore, to address the concern in cost as well as the environment, the repairing and recycling techniques are urgent for spent LiMnO2 cathode materials.
Lithium manganese oxides are considered as promising cathodes for lithium‐ion batteries due to their low cost and available resources. Layered LiMnO2 with orthorhombic or monoclinic structure has attracted tremendous interest thanks to its ultrahigh theoretical capacity (285 mAh g−1) that almost doubles that of commercialized spinel LiMn2O4 (148 mAh g−1). However, LiMnO2 undergoes phase transition to spinel upon cycling cause by the Jahn‐Teller effect of the high‐spin Mn3+. In addition, soluble Mn2+ generates from the disproportionation of Mn3+ and oxygen release during electrochemical processes may cause poor cycle performance. To address the critical issues, tremendous efforts have been made. This paper provides a general review of layered LiMnO2 materials including their crystal structures, synthesis methods, structural/elemental modifications, and electrochemical performance. In brief, first the crystal structures of LiMnO2 and synthetic methods have been summarized. Subsequently, modification strategies for improving electrochemical performance are comprehensively reviewed, including element doping to suppress its phase transition, surface coating to resist manganese dissolution into the electrolyte and impede surface reactions, designing LiMnO2 composites to improve electronic conductivity and Li+ diffusion, and finding compatible electrolytes to enhance safety. At last, future efforts on the research frontier and practical application of LiMnO2 have been discussed.
","
A LiMnO2 cathode with high theoretical capacity shows great commercial value due to its low price and nontoxicity. This review summarizes the overall progress and challenges of LiMnO2, focusing on crystallographic structures, synthesis and modifications like element doping, surface coating, composites designing, and electrolytes matching for electrochemical performance improvement. Future perspectives on the research frontier and practical application are also discussed.\n\n
","
\n\n\nJ.\nMa\n, \nT.\nLiu\n, \nJ.\nMa\n, \nC.\nZhang\n, \nJ.\nYang\n, Progress, Challenge, and Prospect of LiMnO2: An Adventure toward High‐Energy and Low‐Cost Li‐Ion Batteries. Adv. Sci.\n2024, 11, 2304938. 10.1002/advs.202304938\n\n
"],"string":"[\n \"Abstract\",\n \"
Lithium manganese oxides are considered as promising cathodes for lithium‐ion batteries due to their low cost and available resources. Layered LiMnO2 with orthorhombic or monoclinic structure has attracted tremendous interest thanks to its ultrahigh theoretical capacity (285 mAh g−1) that almost doubles that of commercialized spinel LiMn2O4 (148 mAh g−1). However, LiMnO2 undergoes phase transition to spinel upon cycling cause by the Jahn‐Teller effect of the high‐spin Mn3+. In addition, soluble Mn2+ generates from the disproportionation of Mn3+ and oxygen release during electrochemical processes may cause poor cycle performance. To address the critical issues, tremendous efforts have been made. This paper provides a general review of layered LiMnO2 materials including their crystal structures, synthesis methods, structural/elemental modifications, and electrochemical performance. In brief, first the crystal structures of LiMnO2 and synthetic methods have been summarized. Subsequently, modification strategies for improving electrochemical performance are comprehensively reviewed, including element doping to suppress its phase transition, surface coating to resist manganese dissolution into the electrolyte and impede surface reactions, designing LiMnO2 composites to improve electronic conductivity and Li+ diffusion, and finding compatible electrolytes to enhance safety. At last, future efforts on the research frontier and practical application of LiMnO2 have been discussed.
\",\n \"
A LiMnO2 cathode with high theoretical capacity shows great commercial value due to its low price and nontoxicity. This review summarizes the overall progress and challenges of LiMnO2, focusing on crystallographic structures, synthesis and modifications like element doping, surface coating, composites designing, and electrolytes matching for electrochemical performance improvement. Future perspectives on the research frontier and practical application are also discussed.\\n\\n
\",\n \"
\\n\\n\\nJ.\\nMa\\n, \\nT.\\nLiu\\n, \\nJ.\\nMa\\n, \\nC.\\nZhang\\n, \\nJ.\\nYang\\n, Progress, Challenge, and Prospect of LiMnO2: An Adventure toward High‐Energy and Low‐Cost Li‐Ion Batteries. Adv. Sci.\\n2024, 11, 2304938. 10.1002/advs.202304938\\n\\n
\"\n]"},"body":{"kind":"list like","value":["LiMnO<sub>2</sub>\n","Crystal Structure and Comparison","
Layered LiMnO2 and spinel LiMn2O4 are two typical manganese‐based cathodes. The spinel LiMn2O4 belongs to the space group Fd3¯m, where Li cations occupy the 8a site in 1/8 of the tetrahedron, Mn cations fill in the 16d site in 1/2 of the octahedral void, and O anions arranged by face‐centered cubes (FCC) lie in the 32e site (Figure\n##FIG##1##\n2a##).[\n##UREF##26##\n33\n##\n] In this structure, unoccupied oxygen tetrahedra and oxygen octahedra are connected in a shared plane and line, which forms 3D interconnected channels for the diffusion of Li+.[\n##UREF##27##\n34\n##\n] While layered LiMnO2 is a polymorphous compound including two quintessentially crystallographic structure (Figure ##FIG##1##2b and c##) and a cation disordered rocksalt polymorph (Figure ##FIG##1##2d##).[\n##UREF##1##\n5\n##, ##UREF##28##\n35\n##\n] The orthorhombic LiMnO2 (referred to as o‐LiMnO2 in the following) having β‐NaMnO2‐like structures belongs to Pmnm space group (a = 0.2805 nm; b = 0.5757 nm; c = 0.4572 nm; Z = 2). In the crystal with a close‐packed oxygen lattice, all MnO6 octahedra shares a common edge with each other,[\n##UREF##29##\n36\n##\n] as well as MnO6 and LiO6 are arranged in a corrugated interaction. The monoclinic LiMnO2 (herein referred to as m‐LiMnO2) is equipped with a structure of α‐NaFeO2 type, which belongs to the C2/m space group (a = 0.5439 nm; b = 0.2809 nm; c = 0.5395 nm; Z = 2), similar to LiCoO2 and LiNiO2.[\n##UREF##30##\n37\n##\n] It can also be said that Li and Mn cations alternate to form zigzag layers along the (010) in o‐LiMnO2. Different from o‐LiMnO2, the Li and Mn cations of m‐LiMnO2 occupy octahedral site layers that are parallel to the (111) plane of the cubic oxygen sublattice.[\n##UREF##31##\n38\n##\n] Consequently, Li cations fill in tetrahedral sites and Mn cations are in octahedral sites of the spinel LiMn2O4, while both Mn and Li cations occupy octahedral sites in the layered structure.[\n##REF##15669161##\n39\n##\n]\n
","
Additionally, Yabuuchi et al. designed a cation‐disordered rocksalt‐type LiMnO2 by mechanical milling o‐LiMnO2. RIETAN‐FP (a software for crystal structure refinement) was used to analyze the structure of the samples before and after milling. The results show that o‐LiMnO2 is in a zigzag‐type layered structure with a high crystallinity. Whereas the sample lacks the structural features embodied in o‐LiMnO2 after mechanical milling. Meanwhile, the broad diffraction peaks of X‐ray diffraction (XRD) patterns suggest that the zigzag‐type structure of o‐LiMnO2 transforms into the cation‐disordered rocksalt phase.[\n##UREF##1##\n5\n##\n]\n
","Challenges that LiMnO<sub>2</sub> Faced","
Unfortunately, commercial applications of layered LiMnO2 have been plagued by the following problems. The Mn cations in LiMnO2 all present as Mn3+.[\n##UREF##32##\n40\n##\n] Three electrons in the d orbitals of high‐spin Mn3+ stay in the t2g orbital with the same spin, and only one electron occupies an eg orbital. It is important to note that eg orbitals with dx2−dy2and dz2 are doubly degenerate orbitals obtained by splitting 3d orbitals (Figure\n##FIG##2##\n3a##). Consequently, this t2g\n3eg\n1 electronic configuration of Mn3+ results in the asymmetric occupation of eg orbitals. Meanwhile, the electrons in the dx2−dy2and dz2 orbitals exhibit shielding for the Mn nucleus to varying degrees in different directions, resulting in an unstable state of central Mn3+. In order to maintain the stability of Mn3+, the two longitudinal Mn‐O bonds are gradually elongated, accompanying the shrinkage of the other four horizontal Mn‐O bonds (Figure ##FIG##2##3b##). In this case, the symmetry of MnO6 octahedron changes from Oh to D4h, giving rise to the Jahn–Teller (J–T) distortion.[\n##UREF##33##\n41\n##, ##UREF##34##\n42\n##, ##UREF##35##\n43\n##\n] Simultaneously, the volume change from the cubic to tetragonal coordination results in the structural degradation of LiMnO2. Figure ##FIG##2##3c## illustrates the changes in XRD patterns of the o‐LiMnO2 electrodes after different cycles. The intensity of the o‐LiMnO2 peak gradually decreases with the progress of the cycle and some new peaks appear. After three cycles, there are no o‐LiMnO2 peaks in the XRD patterns, which provides compelling evidence that J–T distortion triggers the rapid structure transformation from layered into the spinel or the rock salt upon cycling.[\n##REF##34073268##\n44\n##, ##UREF##36##\n45\n##\n] It was confirmed by in situ X‐ray studies that o‐LiMnO2 was irreversibly converted to spinel structure.[\n##UREF##37##\n46\n##\n] In addition, high stacking faults induced by structural disorder at Li/Mn sites are more likely to lead to phase transformation to a spinel‐like structure and display capacity fading in 3 V.[\n##UREF##38##\n47\n##\n] According to the study by Lu et al.,[\n##UREF##39##\n48\n##\n] additional plateau in the discharging curves (4.0 V) indicated spinel phase Li\nx\nMn2O4 was formed from o‐LiMnO2 (Figure ##FIG##2##3d##). Based on charge/discharge curves analysis and structural studies, Molenda et al. proposed that phase transition (o‐LiMnO2↔LiMn2O4) was reversible in o‐LiMnO2.[\n##UREF##40##\n49\n##\n] However, in‐depth examinations are required to understand the complex kinetics in the process of the phase transition.
","
On the other hand, the cycling performance of LiMnO2 is also restricted by the disproportionation of Mn element (Mn3+ → Mn2+ + Mn4+) during the discharge process. Seriously, Mn2+ is freely soluble in electrolyte solution. For one thing, Mn ions migrating and depositing on the anode will have major implications for solid electrolyte interphase (SEI). For another, Mn dissolution leads to the reduction of the active material, directly leading to structural degradation and capacity loss.[\n##UREF##41##\n50\n##, ##UREF##42##\n51\n##, ##UREF##43##\n52\n##\n]\n
","
It is concluded that phase transformation and manganese dissolution are the critical challenges for the application of layered LiMnO2. In addition, the loss of oxygen and the preparation of pure‐phase LiMnO2 also need to be cracked.
","Synthesis and Electrochemical Performance","
The synthetic route has a significant effect on the phase and structure of LiMnO2 as well as electrochemical properties. Similar to other cathode materials, the main synthesis methods of layered LiMnO2 include ion‐exchange method, solid‐state process, hydrothermal method, sol‐gel method and co‐precipitation.
","
In 1996, Armstrong et al. prepared the stoichiometric m‐LiMnO2 by the ion‐exchange method.[\n##UREF##44##\n53\n##\n] First, NaMnO2 was prepared by heating Na2CO3 and Mn2O3 at high temperatures under the argon atmosphere and then refluxing the mixture of NaMnO2 and excess lithium compounds (LiCl or LiBr) in n‐hexanol. The initial charge capacity of obtained m‐LiMnO2 was up to 270 mAh g−1. In the same year, Delmas et al. also reported layered m‐LiMnO2 with metastable character by ion‐exchange process.[\n##UREF##45##\n54\n##\n] It needs to be pointed out that refluxing experiments are carried out under the argon atmosphere and with a large excess of LiCl to protect the oxidation of any material. A differential scanning calorimetry (DSC) and a high temperature X‐ray diffractometer were applied to evaluate the thermal stability of m‐LiMnO2. Both measurement results indicated it is metastable. More specifically, exothermic effects at 300 °C are observed in the DSC curve. Meanwhile, the diffraction lines of o‐LiMnO2 disappear at 300 °C. In addition, Shao‐Horn et al. obtained a LiMnO2 material accompanied by spinel phase and orthorhombic LiMnO2 by ion exchange method.[\n##UREF##46##\n55\n##\n]\n
","
Zhou and co‐workers reported the one‐step synthesis of pure‐phase m‐LiMnO2 at a lower temperature (450 °C). Electrolytic manganese dioxide (EMD) was used as a manganese precursor to realize carbothermal reduction under the Ar atmosphere.[\n##UREF##47##\n56\n##\n] An initial reversible capacity of 180 mAh g−1 is obtained in the synthesized sample. Using one‐step hydrothermal methods, the controlled preparation of LiMnO2 mixed orthorhombic and monoclinic phases was successfully achieved.[\n##UREF##48##\n57\n##\n] It presented a higher discharge capacity (112.5 mAh g−1) after 50 cycles in a voltage range of 2.0–4.5 V than that of both structures (m‐LiMnO2: 94.5 mAh g−1, o‐LiMnO2: 106.8 mAh g−1). The fabrication of pure m‐LiMnO2 is considered to be relatively difficult because of its thermodynamic instability. Therefore, o‐LiMnO2 has been extensively investigated.[\n##UREF##49##\n58\n##, ##UREF##50##\n59\n##\n]\n
","
As early as the 1990s, there were studies through high‐temperature solid‐phase approaches to prepare layered LiMnO2, which was performed by calcining the mixture of MnO2 and lithium metal salts (LiOH/Li2CO3) at high temperatures (600–1000 °C) under an argon atmosphere.[\n##UREF##51##\n60\n##, ##UREF##52##\n61\n##\n] To prevent manganese from being oxidized to the tetravalent state, a reducing agent like carbon was even required. Despite undergoing a phase transition to spinel in LiMnO2 during charging and discharging, an interesting phenomenon was found that a small amount of spinel in the original LiMnO2 cathode could improve electrochemical performance than pure LiMnO2 cathodes. In addition, its electrochemical performance was better than that of pure LiMnO2 cathodes. Lee et al. obtained the pure o‐LiMnO2 phase by a distinctive quenching process, which employed the reaction of LiOH with γ‐MnOOH at 1000 °C in an argon atmosphere.[\n##UREF##53##\n62\n##\n] It delivered a high discharge capacity of 201 mAh g−1 in the first cycle and remained 200 mAh g−1 after 50 cycles at a current density of 0.4 mA cm−2 between 4.3 and 2.0 V. The group of Wang reported o‐LiMnO2 with a discharge specific capacity of 180—190 mAh g−1 by a two‐step solid‐state reaction.[\n##UREF##54##\n63\n##\n] First, the precursors were prepared by firing mixtures of Mn2O3 and LiOH⋅H2O at 450 °C for 5 h, and then at higher 600 °C for 12 h with argon flow. Furthermore, one‐step synthesis by the solid‐state process using glucose as a reducing agent to prepare o‐LiMnO2 is also feasible.[\n##UREF##55##\n64\n##\n] MnO2, LiOH⋅H2O and C6H12O6 mixed with a Li/Mn/C molar ratio of 5/4/2 were well ground, and then annealed at 750 °C for 15 hours in a tube furnace under nitrogen flow. In this way, heating temperature and time need to be strictly controlled, otherwise impurity phases are accompanied. However, severe particle agglomeration and high energy consumption are inevitable problems in solid‐phase synthesis.
","
The hydrothermal method with low energy consumption is an ideal way to prepare o‐LiMnO2 for getting uniform and fine particles. In the processes of two‐step hydrothermal synthesis of LiMnO2, there are three precursors (γ‐MnOOH, Mn3O4, and Mn2O3) formed by the redox reaction of the manganese source. The crystalline o‐LiMnO2 can be obtained by reacting γ‐MnOOH as the precursor with LiOH solution in a Teflon‐lined autoclave at 180 °C for 24 h.[\n##UREF##56##\n65\n##\n] The γ‐MnOOH nanorods as precursors were synthesized by magnetically stirring a mixed solution of polyethylene glycol (PEG‐10000) and Mn(NO)3 at 140 °C for 24 h, namely a novel polymer‐assisted low‐temperature hydrothermal method. The attempts to synthesize o‐LiMnO2 hydrothermally with Mn3O4 as a precursor were completed by Komaba et al. in 2002.[\n##UREF##57##\n66\n##, ##UREF##58##\n67\n##\n] Dark brown Mn3O4 powders were prepared by stirring the aqueous mixed solution of Mn(CH3COO)2 and KOH at 80 °C for 24 h with bubbling O2 gas. It is important to wash the Mn3O4 powders with deionized water until the pH value of 7 for the synthesis of o‐LiMnO2. Interestingly, replacing γ‐ MnOOH and Mn2O3 with Mn3O4 as a precursor can obtain highly crystalline o‐LiMnO in hydrothermal synthesis, enabling initial reversible capacity of 210 mAh g−1.[\n##UREF##59##\n68\n##\n] Nanocrystalline Mn3O4 particles can also be prepared through the reduction of high‐valent manganese compounds (KMnO4) with methanol or ethanol in an autoclave at the temperature of 80—100 °C for 12–48 h.[\n##UREF##60##\n69\n##\n] In addition, Yang group introduced a rapid method to synthesize nanosized o‐LiMnO2, which was based on microwave‐solvothermal approach. This technique mainly uses α‐Mn2O3 as the precursor mixed with LiOH·H2O for ≈30 min at a low temperature of 160 °C.[\n##UREF##61##\n70\n##\n] The microwave hydrothermal synthesis was carried out in the microwave digestion system. Introducing hydrazine hydrate (N2H4⋅H2O) as a reducing agent in the hydrothermal route, submicron‐sized o‐LiMnO2 crystals can be successfully prepared by using LiOH and the precursors of porous Mn2O3 at the temperature of 180 °C for 16 h.[\n##UREF##62##\n71\n##\n] The Mn2O3 precursor was formed by calcining MnCO3 under the air atmosphere at 620 °C for 6 h. The submicron‐sized o‐LiMnO2 displayed a superior discharge capacity of 216 mAh g−1.
","
Nevertheless, the time‐consuming and complex procedures involved in multi‐step processes are the undeniable disadvantages of the two‐step synthesis mentioned above. Hence, the facile and simple one‐step hydrothermal approach was chosen to obtain LiMnO2 materials.[\n##UREF##63##\n72\n##, ##UREF##64##\n73\n##\n] This way is conventionally achieved by employing the reactants of Mn source (Mn2O3/MnO2) and LiOH·H2O aqueous solution in a Teflon‐lined autoclave. Unfortunately, this method may introduce small amounts of impurities (e.g., Li2MnO3) in samples.[\n##UREF##65##\n74\n##\n] Li2MnO3 is also a layered lithium‐manganese oxide with an α‐NaFeO2 type structure and the C2∖m monoclinic crystal system. In this structure, Li cations occupy the interslab octahedral position in the rock salt structure, and 1/3 of Li cations and 2/3 of Mn cations occupy slab octahedral sites of the transition metal layer,[\n##UREF##66##\n75\n##\n] so it can also be written as the conventional layered structural formula of Li[Li1/3Mn2/3]O2. There are two hypotheses regarding the explanation of the electrochemical activity of Li2MnO3. With the aid of flame emission, atomic absorption, X‐ray photoelectron spectroscopy (XPS), thermogravimetric analysis coupled with mass spectrometry (TGA/MS), Bruce et al. investigated the electrochemical activity of Li2MnO3 in organic electrolytes. They indicated that the activity of Li2MnO3 could be attributed to the exchange of Li+ by H+ in organic electrolytes rather than removal of O2−, and there was no oxidation of Mn4+ to Mn5+.[\n##UREF##67##\n76\n##\n] Another hypothesis was proposed by Dahn in 2002. They believed oxygen was irreversibly released during the first charge to 4.8 V in Li/Li[Ni\nx\nLi(1/3‐2\n\nx\n\n/3)Mn(2/3‐\n\nx\n\n/3)]O2 material.[\n##UREF##68##\n77\n##\n] In 2006, Armstrong et al. used in situ differential electrochemical mass spectrometry (DEMS) to directly detect the oxidation of O2− in Li2MnO3 structure and the accompanying Li+ extraction, which provided a reliable basis for the mechanism of oxygen loss to the electrochemical activity of Li2MnO3.[\n##REF##16802836##\n78\n##\n] Ethylenediaminetetraacetic acid disodium salt (EDTA‐2Na) acted as both a chelating reagent and reducing agent to prepare pure o‐LiMnO2 via hydrothermal synthesis. EDTA‐2Na not only prevents residual oxygen from oxidation in the reacting system effectively but also ingeniously suppresses the formation of Li2MnO3 (Figure\n##FIG##3##\n4a##).[\n##UREF##69##\n79\n##\n] Figure ##FIG##3##4b,c## show the influence of the concentration of EDTA‐2Na and the reactive temperatures on the appearance of Li2MnO3, respectively.\n
","
Particularly, the conditions for soft hydrothermal synthesis can affect the properties of the material. Thus, adjusting the synthesis conditions is an effective way to improve electrochemical performance. A clear understanding of the formation and growth mechanism of materials (including the nucleation time and growth process) is usually expected when conducting synthetic experiments, and in situ hydrothermal synthesis might be a good way to do this.[\n##REF##22747718##\n80\n##\n]\n
","
To the best of our knowledge, the electrochemical performance is heavily affected by the morphology and structure of the material. Accordingly, LiMnO2 with specific nanostructures and morphologies (e.g., nanoplates, nanoparticles, nanorods, and microcubes) have spurred an intensive search for excellent electrochemical performance (Figure\n##FIG##4##\n5\n##).[\n##UREF##70##\n81\n##, ##UREF##71##\n82\n##, ##UREF##72##\n83\n##, ##UREF##73##\n84\n##\n] In 2007, Liu et al. used needle‐like MnOOH as the precursor to prepare o‐LiMnO2 with a nanorod‐like shape. Notably, the platy shape, platy with irregular shape and rod‐like crystals corresponded to the appearance of Mn3O4 phase, cubic Li0.2Mn2O4 phase, and pure o‐LiMnO2 after 2 h, 3 and 4 h hydrothermal treatment, respectively (Figure\n##FIG##5##\n6\n##).[\n##UREF##74##\n85\n##\n] It is also a good evidence that LiOH has the two important roles in providing a source of lithium and controlling both morphology and particle size. Xiao et al. prepared orthorhombic LiMnO2 nanoparticles and LiMnO2 nanorods by different hydrothermal methods.[\n##UREF##75##\n86\n##\n] Compared with LiMnO2 nanoparticles prepared through simple one‐step routes, LiMnO2 nanorods synthesized by γ‐MnOOH precursors showed a higher discharge capacity (200 mAh g−1) as well as better cyclability (180 mAh g−1 after 30 cycles) due to favorable electronic transport of 1D electronic pathways. Zhao et al. combined Mn2O3 nanorods as a template and thermal decomposition process to prepare o‐LiMnO2, which delivered outstanding performance. As a consequence, the one‐dimensional crystalline nanostructure could effectively promote the transport of charge/electron and increase the electrode‐filled ratio.[\n##UREF##72##\n83\n##\n]\n
","
To overcome the difficulty in synthesis of layered LiMnO2, there are some novel methods developed, including the emulsion‐drying method, reverse‐microemulsion preparation, microwave irradiation, in.situ oxidation coupling with ion exchange, and in situ carbothermal reduction. It is reported that o‐LiMnO2 materials by emulsion‐drying method show only a 10% capacity loss after 300 cycles at 45 mA g−1. The emulsion‐drying method could mix cations homogeneously at the atomic level and obtain fine single‐crystal oxide cathode materials.[\n##UREF##76##\n87\n##\n] Reverse‐microemulsion preparation is another nice way to allow cations to mix and interact in an atomic scale. Obtaining a thermodynamically stable and transparent microemulsion, in which nano‐sized water is well dispersed into the oil phase, is the key to reverse microemulsion preparation.[\n##UREF##39##\n48\n##\n] One of the new technologies based on hydrothermal synthesis is microwave hydrothermal. With the advantage of microwave radiation, it significantly reduces reaction time and temperature in some processes.[\n##UREF##77##\n88\n##\n] Li et al. described the synthesis of high‐purity and highly crystallized o‐LiMnO2 via the process of in situ oxidation coupling with ion exchange.[\n##UREF##78##\n89\n##\n] During the synthesis process, MnOOH was produced by the oxidation of Mn(OH)2 with the oxidizing agent (NH4)2S2O8 in a LiOH‐existing strong basic environment. Despite 80 °C, deoxygenated distilled water and nitrogen were required to eliminate the impact of oxygen in the air. Komarnenib's group prepared nanorod‐like o‐LiMnO2 with high purity and excellent electrochemical behavior by in situ carbothermal reduction.[\n##UREF##79##\n90\n##\n] In this synthesis process, MnO2, LiOH·H2O and affordable glucose (C6H12O6) as a reducing agent were homogeneously mixed followed by high temperature treatment in an argon atmosphere for several hours. Additionally, sol‐gel and co‐precipitation methods have also been used for the synthesis of LiMnO2. As for the sol‐gel approach to get LiMnO2, citric acid is a common chelating agent.[\n##UREF##80##\n91\n##\n] Zeng group obtained spherical o‐LiMnO2 powders with a discharge capacity of 152 mAh g−1 by a carbonate coprecipitation method using NH4HCO3 and MnCl2 as raw materials.[\n##UREF##81##\n92\n##\n]\n
","
Briefly, it is relatively difficult to synthesize pure layered LiMnO2 for its thermodynamic sub‐stability, especially for monoclinic LiMnO2. Li2MnO3 and manganese oxide as impurities are frequently seen in the XRD analysis of the product. So, to avoid the various side reactions during synthesis, the use of inert gas is the recommended choice. Both solid‐state synthesis and hydrothermal synthesis should strictly control the Li/Mn ratio, reaction temperature, and reaction time. Moreover, in situ detection techniques can be used for precise material preparation.
","Modifications","
To overcome the drawbacks mentioned in Section 2, tremendous efforts such as element doping, surface coating, nanocomposite designing and compatible electrolyte have been adopted to improve the electrochemical performance of LiMnO2 cathode materials.
","Element Doping","
Element doping is one of the most extensive approaches for modifying cathode materials to improve the basic physical properties of materials. The elements doping can suppress phase transitions, and enhance capacity. For LiMnO2 cathode, the research on doping strategies mainly includes element types, element content, doping sites, synthesis conditions, morphology, and structural effects. The efficacy of element substitution by doping is mainly manifested in the following aspects: 1) inhibiting J–T distortion and making structures stable for improved cycling performance; 2) providing favorable Li+ diffusion. For substituting at the Mn site, doping ions with smaller radius (Al3+, Co3+, Cr3+) than Mn3+ result in a contracted crystal lattice and microstrains owing to the shorter and stronger transition metal‐oxygen bonds than Mn─O bonds; substitutes of larger radius (Y3+) show larger lattice parameters and lead to the free movement of Li+ in the oxide, which makes it possible to increase the rate of capability. For substituting at the Li site, the metal element with a larger radius ion can provide a pillaring effect to enhance the stability of cathode materials. For instance, Li0.53Na0.03MnO2 with a pseudo‐spinel structure displays an increasing discharge capacity, which reaches 200 mAh g−1 in the 50th cycle.[\n##UREF##82##\n93\n##\n] Up to now, various doping elements have been found to improve the electrochemical properties of layered LiMnO2, including Na, Cu, Mg, Zn, Fe, Ni, Al, Co, Cr, Y, In, S, Ti, V, Nb, Ru, B, F, BO3\n3−, PO4\n3−and SiO3\n3−.
","Cations Doping","Divalent Cations","
Transition metal elements usually occupy the Mn site of lithium manganese oxide to improve electrochemical performance. The ionic radius of the doping Cu2+ (0.072 nm) is larger than that of Mn3+, which is beneficial for faster mobility of Li+ in bulk, contributing to the decreased charge transfer impedance and Warburg impedance of LiMnO2. Such lower resistance has an important role in improving high‐rate capability by 65.5%.[\n##UREF##83##\n94\n##\n] Unlike the doping of transition metal elements, the Mg may occupy both Li and Mn sites. During the charge/discharge cycling, the presence of Mg at the Li sites is beneficial for maintaining the stability of the structure but reduces the electrochemical extraction of lithium ions.[\n##UREF##84##\n95\n##\n]\n
","
Suresh et al. investigated the doping effect of Zn and Fe elements using the ion‐exchange method.[\n##UREF##85##\n96\n##\n] It was only 5% Zn/Fe (LiMn0.95Zn0.05O2/LiMn0.95Fe0.05O2) substituted into LiMnO2 that could exhibit a better discharge capacity of 180 and 193 mAh g−1, respectively. The substitution of Zn can improve the retention of capacity. However, increasing the content of Zn leads to the capacity fading due to decreasing of electrochemically active Mn3+. The decreased intensity of Mn3+/Mn4+ redox peak in the cyclic voltammograms (CV) curves confirms it.
","Mixed Bivalent and Trivalent Cations","
Quine et al. reported that Li\nx\nMn0.95Ni0.05O2 with the O3 (α‐NaFeO2) structure formed via an ion‐exchange route. Li\nx\nMn0.95Ni0.05O2 delivered a high capacity of 220 mAh g−1 above 2.5 V.[\n##UREF##86##\n97\n##\n] Inevitably, a phase transition to spinel occurred after the cycling. In that case, Ni substituted in the layered LiMnO2 was in a state of Ni2+ thus accompanied by a reaction from Mn3+ to Mn4+. Drawing support from the x‐ray absorption spectroscopy (XAS) spectrum, Wadati et al. pointed out that Li deficiencies could not be created by the Ni2+ valence and the change from Mn3+ to Mn4+.[\n##UREF##87##\n98\n##\n] However, the Ni3+ was observed in the CV curves, corresponding to anodic peak of 3.05 V.[\n##UREF##84##\n95\n##\n] In addition, when 5% Ni is substituted, the compound provides the highest capacity of 220 mAh g−1 but is followed by a rapid capacity decay on repeated cycling. The high Ni contents can provide stable capacity, due to the decrease of Mn3+ contents, while the loss of capacity is inevitable. Nahm and co‐workers found that O2‐Li0.7[Ni1/6Mn5/6]O2 instead of Li0.7[Li1/6Mn5/6]O2 exhibited no phase change after 30 cycles.[\n##UREF##88##\n99\n##\n] In the first cycle, this sample delivered a discharge capacity of 198 mAh g−1 between the voltage of 4.6 and 2.0 V. Meanwhile, it exhibited a 96% capacity retention after 25 cycles at 1/3 C. Therefore, Ni doping can exert a positive effect on the stability of the structure and the retention of capacity.
","Trivalent Cations","
In the 1990s and early 2000s, interests in modifying layered LiMnO2 were focused more on the solid solution formed between LiMnO2 and LiMO2 analogues (LiAlO2, LiCoO2, and LiCrO2). Although pure LiAlO2 is electrochemically inert, the solid solution consisting of LiAlO2 and other lithiated transition‐metal oxides probably possesses a high intercalation potential, leading to high energy density.[\n##UREF##89##\n100\n##\n] And two effects can explain why Al3+ doping can stabilize the monoclinic phase:1) AlO6 octahedra is not distorted because of non‐J‐T Al3+; 2) the smaller size of Al3+ (0.054 nm) has advantages in building stable transition metal‐oxygen bond.[\n##UREF##90##\n101\n##, ##UREF##91##\n102\n##\n] Chiang et al. indicated that the Al‐doped LiMnO2 contained the monoclinic and orthorhombic ordered rock‐salt structures, and exhibited better cycling performance and higher discharge capacities under the test conditions of elevated temperatures.[\n##UREF##92##\n103\n##\n] But m‐Li\nx\nAl0.05Mn0.95O2 showed a higher discharge capacity of 188 mAh g−1 as well as higher energy densities of 602 Wh kg−1 than o‐Li\nx\nAl0.05Mn0.95O2 (146 mAh g−1 and 465 Wh kg−1) at 55 °C. The doping of an element not only directly improves the electrochemical performance of materials, but also affects the structure and morphology of materials. Regan et al. elucidated the impacts of Al doping on the structures and surface properties of material[\n##UREF##93##\n104\n##\n] The existence of Al favors on Mn‐Li inversion in alternating slabs of the LiAl\nx\nMn1‐\n\nx\nO2. Moreover, it is manifested by XPS that Al is uniformly incorporated in the whole grains up to the limit of its solubility. And no aggregation of Al is observed on the surface. The group of Yang described the facile hydrothermal synthesis of o‐LiMn1‐\n\nx\nAl\nx\nO2 and investigated the changes in the morphology with varying Al content in the layer.[\n##UREF##94##\n105\n##\n] The results indicate that Al3+ in a small content contributes to growth in the b‐axis direction (i.e., (010) direction) and formation of cuboid crystals, but the growth is inhibited and changes to cubic crystals with a high content of Al3+. In 1999, Robertson prepared Co‐substituted Li(Mn1‐\n\ny\nCo\ny\n)O2 in the range 0≤y≤0.5 using a solution‐based route with the assistance of ion exchange.[\n##UREF##95##\n106\n##\n] On the one hand, it pointed out that Co entered the layered LiMnO2 as Co3+ rather than Co2+. On the other hand, it indicated that higher content of Co caused somewhat lower capacities, and the best amount of doping was 2.5%.[\n##UREF##96##\n107\n##, ##UREF##97##\n108\n##\n] The LiMn1‐\n\ny\nCo\ny\nO2 from the hydrothermal synthesis gave different optimal doping content (10%).[\n##UREF##98##\n109\n##\n] Doping of Al, Co, and Cr makes sense in stabilizing LiMnO2 structure and improving cycle retention. In addition, all doped compounds show contracted crystal lattices by comparison with nonsubstituted LiMnO2 due to a relatively small radius of doping cations. Although LiMn1‐yCoyO2 and LiMn1‐yAlyO2 undergo a spinel phase transition at 4 V,[\n##UREF##99##\n110\n##, ##UREF##100##\n111\n##\n] this has almost no harmful effect on cycling performance and even increases discharge capacity.[\n##UREF##101##\n112\n##\n] But low levels of Cr3+‐substituted materials show insignificant transformation in cycling.[\n##UREF##102##\n113\n##\n] The key points of Cr doping depend on 1) forming a continuous solid solution in LiMnO2 across the whole range of composition from LiMnO2 to LiCrO2;[\n##UREF##103##\n114\n##\n] 2) more regular local coordination symmetry around Cr3+ than Mn3+; 3) higher coordination between Cr3+ and oxygen than Mn3+.[\n##UREF##104##\n115\n##\n] Furthermore, with the help of 6Li magic‐angle spinning NMR spectroscopy, it has been found that Cr3+ doping can partially disrupt the Mn‐Mn antiferromagnetic correlations, which is important for the stabilization of the layered structure over the orthorhombic structure.[\n##UREF##103##\n114\n##, ##UREF##105##\n116\n##\n] Alternatively, Pang observed that even though Cr doping into o‐LiMnO2 brought a change in the topography from orthorhombic to monoclinic geometry employing Pechini's synthesis method and XRD patterns,[\n##UREF##106##\n117\n##\n] it does not matter on improving cycling performance and reversible capacity. Xiao et al. reported the rheological phase method to prepare m‐LiMn1‐\n\nx\nCr\nx\nO2,[\n##UREF##107##\n118\n##\n] forming ultrafine spherical particles with the size of 60–200 nm. Relative to the preparation through solid‐state reaction at the higher temperature of 1000 °C, LiMn0.85Cr0.15O2 by rheological phase method calcined at 800 °C yields a much higher initial discharge capacity (180 mAh g−1) and capacity retention as well (94% after 40 cycles).
","
Y3+ as a substituent in place of Mn3+ in LiMnO2 was first explored by Zong and partners.[\n##UREF##108##\n119\n##\n] There is no 4 V plateau associated with spinel LiMn2O4 in the CV and charge/discharge curves of the Li/LiMn0.98Y0.02O2 cell, which can be attributed to the pillaring effect of Y3+. In addition, doping with a low content of Fe is beneficial for capacity and cycling stability.[\n##UREF##85##\n96\n##\n] Whereas, Myung found o‐LiMnO2 of Fe substitution by a hydrothermal reaction obtained much lower capacity than that of undoped one,[\n##UREF##109##\n120\n##\n] which is down to quasi‐reversible Fe4+/Fe3+ redox couple and gives unimpressive battery performance. The introduction of Ti3+ into o‐LiMnO2 is an effective attempt to moderate the phase evolution and stabilize the structure.[\n##UREF##110##\n121\n##\n] Unfortunately, the secondary phase m‐Li2MnO3 in LiMnO2 has a low conductivity. By doping Ti3+ into LiMnO2, c‐LiTiO2 with better electrical conductivity (ca. 10−6 S cm−1) can replace above mentioned secondary phase. And Ti─O bonds are stronger than Mn‐O bonds, which militates in favor of improved structural stability of o‐LiMn1‐\n\nx\nTi\nx\nO2. In addition, B‐doped m‐LiMnO2 was prepared by carbothermal reduction using LiOH and MnO2 as reactants under the argon atmosphere.[\n##UREF##47##\n56\n##\n] Even though the transformation from layer to spinel cannot be suppressed by B‐doping, the cycle performance and Coulombic efficiency are improved. 5% B‐doping (m‐LiB0.05Mn0.95O2) gives the best electrochemical performance among B‐doped samples with different ratios, particularly at elevated temperatures (60 °C). The average Coulombic efficiency increases from 69.7% to 99.1% at 60 °C.
","Anions Doping","
Aside from cations doping, doping anions is another feasible approach to modify the LiMnO2 cathode. The S‐doped LiMnO2 can deliver a discharge capacity of 220 mAh g−1 after 50 cycles with a voltage of 2.0–4.6 V.[\n##UREF##111##\n122\n##\n] The F substitution can stabilize the host structure due to high electronegativity, showing better cycle performance compared with F‐free LiMnO2.[\n##UREF##112##\n123\n##\n] Because the S2− (0.184 nm) and F− (0.136 nm) are greater than the O2− (0.132 nm), the lattice constant of LiMnO2 doped with S2 and F− increases. However, above mentioned S and F‐doping cannot ultimately suppress the transformation from layer to spinel.
","
Larger polyanions (BO3\n3−, PO4\n3−and SiO3\n3−) in place of O2− ions in nonstoichiometric Li\nx\nMnO2 can generate an open 3D framework, which enhances the mobility of Li+ ions. Decreased Warburg impedance is also observed, which is reflected in the improved electrochemical properties of the high‐rate charge/discharge capability. Unfortunately, polyanion doping presents increased charge‐transfer resistance due to the poor electrochemical activity of polyanions relative to cations. Surprisingly, although the discharge capacity of Li\nx\nMnO1.99X0.01 (X = BO3\n3−, PO4\n3−and SiO3\n3−) has decreased, the cycling ability has been improved.[\n##UREF##83##\n94\n##\n]\n
","Co‐Doping","
Furthermore, doping multiple elements into LiMnO2 endows electrodes with a synergistic effect. Suresh doped 5% Ni and 5% Fe into LiMnO2,[\n##UREF##113##\n124\n##\n] forming a pure and homogeneous phase with a layered structure. The low content of Ni and F exhibited a discharge capacity of 250 mAh g−1 at the rate of 0.1 C, and improved the cycling stability. The co‐doping of cations and anions, such as Li+ and F− ions, into o‐LiMnO2 has been realized through a solid‐state reaction.[\n##UREF##114##\n125\n##\n] Scanning electron microscopy characterization indicates o‐Li1.07Mn0.93O1.92F0.08 presents a smooth surface even after cycling. Co‐doping of Li+ and F− ions reduces the contact area with the electrolyte as well as suppresses the reaction of the Mn dissolution. Eventually, the products improved cycling stability and rate capability at a higher temperature (55 °C). However, doped LiMnO2 with multiple elements still shows some reserved disadvantages, such as low specific capacity.
","
Su investigated In3+ and S2− co‐doped o‐LiMnO2 synthesized via the hydrothermal method.[\n##UREF##115##\n126\n##\n] Compared to pristine and single doping (In or S), dual doping reduced the crystallinity of LiMnO2. Meanwhile, In and S dual doping exhibited the partial structural transformation from orthorhombic to spinel after 10 cycles at 50 mA g−1, but excellent cycle performance was obtained at various discharge current densities. In brief, In and S dual doping provides a great method to improve the rate performance of LiMnO2 cathode.
","Theoretical Calculations on Doping Effect","
As a quantum mechanical method for the study of the electronic structure of multi‐electron systems, density functional theory (DFT) is very effective in offering a deeper elaboration of elemental doping. An ab initio study pointed out that 10% Co‐substitution makes the antiferromagnetic spin orthorhombic structure more stable than the monoclinic structure, and 60% substitution helps stabilize the disordered local moment (DLM) rhombohedral layered structure over the ferromagnetic orthorhombic phase and offers easy migration of lithium during intercalation.[\n##UREF##116##\n127\n##\n]\n
","
Prasad et al. reported their first‐principles calculations of doped rhombohedral LiMnO2 against J–T distortion.[\n##UREF##117##\n128\n##\n] All calculations indicate that, for dopants, their oxidation state is the first significant factor for effectively stabilizing the rhombohedral structure, and then for a certain oxidation state, the t2g or eg subshell filling is the second. In addition, results show that divalent dopants (such as Mg, Zn) are more effective in the suppression of the J–T distortion than trivalent dopants because each divalent dopant can remove two Mn3+ ions from the sublattice.[\n##UREF##118##\n129\n##\n] Based on generalized gradient approximation, it is found that divalent dopants (Mg and Co) destabilized the monoclinic structure than the trivalent dopant (Fe).[\n##UREF##119##\n130\n##\n] Using the hybrid eigenvector‐following and DFT approach, Grey groups studied the effect of trivalent dopants on the Mn migration leading to spinel transformation, and found that dopants with a small ionic radius (Al3+ and Cr3+) can raise the migration barrier of Mn, but only Cr3+ does not move to tetrahedral sites within the Li layer.[\n##UREF##120##\n131\n##\n] Kong et al. investigated the effects of ten cationic (Mg, Ti, V, Nb, Fe, Ru, Co, Ni, Cu, and Al) and two anionic (N and F) dopants of LiMnO2 using ab initio DFT simulations.[\n##UREF##121##\n132\n##\n] The findings indicate that Mg, Ti, V, Nb, Ru as well as F can effectively reduce the redox potential, Ti, V, Nb, and Ru can increase the hole conductivity to inhibit the formation of the hole polaron. However, only Ni can decrease the diffusion batteries of Li+ by 0.23 V and only the N‐doped phase is thermodynamically unstable. When doping at the Li site, it shows unfavorable thermodynamics than Mn‐site doped configurations.[\n##UREF##122##\n133\n##\n] Similarly, Khang Hoang indicates that substitutes (Al, Fe) are more favorable at the Mn site.[\n##UREF##123##\n134\n##\n]\n
","
To summarize, a systematical comparison of ionic radius and electrochemical performance of different elements doped into LiMnO2 is presented in Table\n##TAB##0##\n1\n##. Element doping is effective in stabilizing structures and improving the electrochemical performance of LiMnO2. To one degree or another, it ameliorates the adverse effects of transformation from a layer to a spinel crystal structure. For Co and Cr doping, it shows good capacity retention upon cycling. However, element doping in LiMnO2 still suffers from some limitations. For instance, it is inevitable for Al‐doped LiMnO2 to convert into a spinel phase at 4 V. Keeping the high capacity retention for long cycles is still the biggest problem. Decreasing the amount of Li also needs to be solved during the process of electrochemical extraction, when a dopant occurs at the Li site
","Surface Coating","
Element doping is particularly effective in stabilizing the bulk structure, while coating makes great sense to protect the surface of electrode materials for resisting manganese dissolution into the electrolyte and impeding surface reactions.
","
Al2O3, an ionic and electronic insulation, is often used as a coating for the cathode to prevent active electrode materials from being corroded by electrolytes. Cho et al. confirmed that the capacity loss of the Al2O3‐coated o‐LiMnO2 (only 2% after 50 cycles) was significantly lower than that of the bare one (35% loss).[\n##UREF##124##\n135\n##\n] Most significantly, there are no Li2Mn2O4 phases in the Al2O3‐coated electrode during the process of cycling compared with the uncoated one, implying that the protective coating layer can keep the lattice stable and suppress the J–T distortion. However, the LiMn1‐\n\nx\nAl\nx\nO2 solid solution or LiAlO2 may be formed during the process of coating Al2O3 with sol‐gel and heating treatment.[\n##UREF##125##\n136\n##\n] For the materials prepared at high temperatures (600 °C and 700 °C), the uniform formation of the LiMn1‐\n\nx\nAl\nx\nO2 solid solution across the particle instead of staying on the surface may bring the rapid capacity decrease. The LiAlO2 peak can be observed from the XRD pattern, indicating that the Al2O3 gel solution would react with the o‐LiMnO2 on the surface of materials. Likewise, similar effects can be observed in CoO‐coated o‐LiMnO2.[\n##UREF##126##\n137\n##\n] Even if CoO‐coated LiMnO2 exhibits a much lower initial discharge capacity (127 mAh g−1) compared to that of the bare one (162 mAh g−1), the former presents rapidly increasing capacity (185 mAh g−1 after 15–20 cycles) and just 12% capacity decay after 50 cycles. A significant amount of Co atoms appears in the range of 2‐µm range near the surface than in the bulk, which is a sign of solid solution formation on the surface. Thanks to the presence of solid solutions, the electrochemical stability of CoO‐coated LiMnO2 has been improved at 55 °C. In addition, the disappearance of the Li2MnO3 impurity phase after Al2O3/CoO coating implies that the coating layer lies on the surface of LiMnO2.\n[\n##UREF##127##\n138\n##\n] It may be a reference for the removal of Li2MnO3 impurity that occurred in the synthesis of related lithium‐manganese oxygen cathodes. There are many similar effects in Al2O3 and CoO‐coated o‐LiMnO2. However, only CoO‐coated LiMnO2 displays an additional voltage plateau at 2 V probably causing capacity decay. This phenomenon illustrates the structural disorder of Al2O3/CoO coating is different.
","
Lithium boron oxide (LBO) with good ionic conductivity has been successfully used to coat spinel and binary/ternary layered cathode materials for improving cycling stability. There is no doubt that above mentioned cycling stability is also observed in the LBO‐modified o‐LiMnO2. Nagasubramanian et al. studied the cycling performance of the LBO‐coated o‐LiMnO2 and its potential explanation.[\n##UREF##128##\n139\n##\n] During the first charge‐discharge, the intercalation/deintercalation behavior of Li+ is considered to be irreversible, which can be attributed to the structure transformation from the layered to the spinel. But as the phase transition is completed during cycles, the maximum capacity is raised to 189 mAh g−1 and the electrode retains 91% of the maximum capacities after 70 cycles, while only 77% of capacity can be obtained for uncoated one. By observing the contribution of discharge capacity from different platforms (3 V and 4 V) in the charge/discharge curves before and after coating, it can be seen that the major capacity loss is occurring from the 3 V region. Coupled with significantly reduced charge‐transfer resistance in the corresponding voltage range, it is obvious that LBO is beneficial for Li+ inserting into the octahedral voids in the LiMn2O4 structure, resulting in less capacity loss.
","
In recent years, electrode materials with core‐shell structures have been aggressively developed to resolve the problem of low capacity and poor cyclic instability.[\n##REF##21879116##\n140\n##\n] Generally speaking, the shell can be formed by coating and acts as a protection layer to make better overall performance of the materials. Guo et al. confirm that designing an o‐LiMnO2@Li2CO3 nanosheet array cathode with a core‐shell structure presents better electrochemical behavior.[\n##REF##27270124##\n141\n##\n] Intriguingly, the o‐LiMnO2@Li2CO3 electrode shows 80% and 79% initial capacity after 400 cycles at 2C at 20 °C and 60 °C, respectively (Figure\n##FIG##6##\n7a## and ##FIG##5##6b##), yet the control electrode without Li2CO3 coating at 0.5 C only exhibits capacity retention of 18% at 20 °C and less than 1% at 60 °C after 400 cycles (Figure ##FIG##6##7c##). The reasons for the excellent cycling performance of o‐LiMnO2@Li2CO3 nanosheet array electrode can be explained in Figure ##FIG##6##7d–f##. First, the outer layer of Li2CO3 on the o‐LiMnO2 surface can be confirmed by high resolution transmission electron microscope (HRTEM) (Figure ##FIG##6##7d##). The outer layer can significantly minimize or avoid adverse reactions (o‐LMO dissolution and oxygen release) (Figure ##FIG##6##7e##). The Li2CO3 is one of the parts of the SEI layer, the Li2CO3 layer is beneficial to electrochemical stability at elevated temperatures. Second, the nanosheet array structure is not only equipped with a 1D pathway for fast electron transport but also shortens the diffusion length of lithium, which ensures sufficient contact of active material with electrolyte (Figure ##FIG##6##7f##). Huang et al. induced two different core‐shell microstructures of Li‐Mn‐O materials by heating treatment (Figure\n##FIG##7##\n8a##).[\n##UREF##129##\n142\n##\n] The phase transformation is summarized in detail in Figure ##FIG##7##8b##, there are three stages at 300 °C and 700 °C, corresponding to two transformations. TGA analysis was provided in Figure ##FIG##7##8c##. When annealed to 350°C, the oxygen on the surface of Li1.11Mn0.76O2 (LMO) is released and the lithium diffuses to form Li2O. Thus, the lithium vacancies are occupied by Mn to form spinel, resulting in a layered core and spinel shell. As the annealing proceeds to higher temperatures, the internal Li+ diffuses to the external spinel, forming a LiMnO2 shell. That is, a LiMnO2 shell wrapped around a spinel core is formed at 750 °C. The cycling performance and rate capability of these two core‐shell structural materials are shown in Figure ##FIG##7##8d,e##. Notably, the annealing‐induced synthesis method ensures the homogeneity of the core‐shell structure. Compared with conventional coating methods, this approach presents atomic‐level contacts between the cores and shells.
","
In brief, modification by coating has resulted in a relatively feasible solution of optimizing the electrode surface to improve electrochemical performance. For instance, the coating layer reduces phase transformation (from the layer to spinel), decreases capacity loss, facilitates Li+ transfer, and increases capacities. Particularly, the coating delivers high capacity retention even at elevated temperatures, which may be attributed to impeding the active materials touch with the electrolyte solution, thus the side effects of LiMnO2 are minimized during cycling, especially manganese dissolution. Maybe coating is just like eggshells, keeping the active material safe in its role. But how to get the depth of the coating to hit the spot in the process of material synthesis still needs further study. Additionally, research on LiMnO2 coating appears to be slightly less than other cathodes (such as LiCoO2 and LiMn2O4). Co‐coating can be considered as a new modification method to improve the electrochemical performance of LiMnO2, due to its synergistic effect.
","Composites Designing","
To break through the bottleneck of imperfect LiMnO2 cathode, introducing other favorable materials to fabricate composites has been investigated in recent years. These materials are conducive to the electrochemical performance of electrodes, especially for carbon materials. LiMnO2‐carbon composites are usually prepared by mixing carbon materials (carbon nanotubes (CNTs), reduced graphene oxide (rGO) and graphene nanoplatelet (GNP)) with prepared LiMnO2 or with raw material for the preparation of LiMnO2. The carbon materials in LiMnO2‐carbon composites can offer high electronic conductivity and fast Li+ diffusion, which contributes to enhanced specific capacity, improved capacity retention and reduced electrochemical impedance relative to pristine LiMnO2.[\n##UREF##130##\n143\n##, ##UREF##131##\n144\n##\n]\n
","
\nFigure\n##FIG##8##\n9\n## is the scheme and scanning electron microscope (SEM) images of orthorhombic LiMnO2/CNTs nanocomposites.[\n##UREF##132##\n145\n##, ##UREF##133##\n146\n##\n] As schematically illustrated in Figure ##FIG##8##9a##, o‐LiMnO2/CNTs composites were prepared by a one‐step dynamic hydrothermal way. And the corresponding SEM images show that LiMnO2 particles are “confined” among CNTs (Figure ##FIG##8##9b,c##).
","
Figure ##FIG##8##9a## is the prepared scheme of o‐LiMnO2/CNTs composites through a one‐step dynamic hydrothermal way, and the corresponding SEM images show that LiMnO2 particles are “confined” among CNTs (Figure ##FIG##8##9b,c##). The composite with 5 wt% CNTs brings about the optimum specific capacity of 204.9 mAh g−1. The o‐LiMnO2‐MWCNTs nanocomposites, where CNTs instead of carbon black worked as an additive material, display the intricate network in Figure ##FIG##8##9d##. The addition of CNTs reduces the charge transfer resistance from 190 to 105 kΩ. The 3D network provided by CNTs increases electronic conductivity of the samples and facilitates the Li+ diffusion across the interface of electrolyte. Tian et al. synthesized LiMnO2@rGO composites via a one‐pot hydrothermal route at 200 °C for 12 h,[\n##REF##33043900##\n147\n##\n] in which LiMnO2 nanoparticles were uniformly anchored onto rGO nanosheets. It is rewarding to note that the rGO with superior conductivity provides a stable conductive net, thus the first discharge capacity of LiMnO2@rGO could be improved to 175 mAh g−1, as well as capacity retention increased from 53.53% to 80.97% after 100 cycles.
","
Although manganese‐based cathode materials are of great significance for the sustainability of LIBs, the J–T distortion of Mn3+ has become a bottleneck to improving their structural stability. Zhu et al first proposed a nice idea to suppress the J–T distortion of Mn3+ using interfacial orbital ordering.[\n##UREF##134##\n148\n##\n] As shown in Figure\n##FIG##9##\n10a,b##, the in situ electrochemical conversion from Mn3O4 was used to prepare heterostructured spinel‐layered LiMnO2 (SPL‐LMO). Layered domains within the spinel matrix and the orientation of orbitals at the spinel‐layered interface can be seen in Figure ##FIG##9##10c–e##, where MnO6 of the layered and spinel phases is linked with an included angle of 83.8°. Significantly, the SPL‐LMO materials exhibit superior rate performance (Figure ##FIG##9##10f##) as well as cycling stability of the full cell at a current density of 1.0 A g−1 for 1000 cycles (Figure ##FIG##9##10g##), which is superior to layered or spinel LiMnO2 cathodes reported previously.
","Compatible Electrolyte","
The electrolyte is one of the most important components of the battery, as it endows with the ability to establish ionic conductive channels and block the electronic conductivity between the cathode and anode. The development of electrolytes for LiMnO2 has been underway for several years.
","
In 1997, the group of Davidson researched the electrochemistry of LiMnO2 over the range of 1.0‐4.6 V with a variety of electrolytes.[\n##UREF##135##\n149\n##\n] Li/LiMnO2 cells exhibit a higher discharge capacity in 1 M LiClO4 ethylene carbonate (EC)/dimethyl carbonate (DMC) electrolyte (257 mAh g−1) than that of 1 M LiPF6 EC/DMC electrolyte (220 mAh g−1) between 1.0 and 4.4 V. In addition, the voltage range also has a significant effect on the charge and discharge capacity. For example, with 1 M LiClO4 in propylene carbonate (PC)/EC, charge and discharge capacities are 253 and 250 mAh g−1 over the range of 1.0–4.4 V, respectively. But when charging to 4.2 V, it displays only the charge capacity of 228 mAh g−1. However, they give no further explanation. Thus, substantial differences occur when varying the electrolytes, which also needs to adjust the voltage range for better compatibility.
","
Based on 1, 3‐dioxolane‐LiAsF6 solutions, Tadiran developed the LiMnO2 rechargeable battery with excellent electrochemical performance.[\n##UREF##136##\n150\n##\n] In 1, 3‐dioxolane‐LiAsF6 solutions, the surface film consisting of Li alkoxides, Li formate, LiF and Li\nx\nAsF\ny\n is formed on the Li anode, which induces uniform and smooth Li deposition. Thus, the smooth morphology of Li deposition provides a high cycle life.
","
The layered lithium transition metal oxides tend to release oxygen in a highly delithiated state, possibly accompanied by an exothermic reaction with the electrolyte. These could be the most serious threat to the safety of batteries. Reassuringly, when o‐LiMnO2 cathode contacts with 1 M LiPF6 solution in 1:1:1 wt.% EC/diethyl carbonate (DEC)/DMC solvent, DSC measurements show a much lower heat effect during the first process compared with other cathode materials.[\n##UREF##137##\n151\n##\n]\n
","
Solid polymer electrolyte (SPE) has the advantage of excellent processability and flexible tunability. All of these advantages make it particularly valuable in designing and developing high‐energy, long‐life solid state lithium batteries. Lithium polymer batteries (LPBs) involved LiMnO2 cathode have been investigated in recent years, which relates to SPE such as polyethylene oxide (PEO), polyvinylidene fluoride‐hexafluoropropylene (PVDF‐HFP), and polyvinylidene fluoride (PVDF). Xia et al. evaluated the thermal stability of LiMn2O4 and LiMnO2 in PEO polymer electrolyte. DSC curves show that delithiated LiMn2O4 decomposes at 230 °C, while the decomposition of LiMnO2 with partial charge occurs at 350 °C. It is worth noting that the LiMnO2 cathode displays much higher thermal stability than that of LiMn2O4 when the PEO polymer is used as an electrolyte.[\n##UREF##138##\n152\n##\n] Gu's group obtained PVDF‐HFP‐PC10EC10LiClO4 as SPE to fabricate LiMnO2/SPE/Li battery. First, they prepared a mixed solution consisting of LiClO4, EC, PC, and 25 wt.% PVDF, and then casting and quickly heating it at 110 °C for 10 min.[\n##UREF##139##\n153\n##\n] Compared with LiMnO2/Li battery with the 1 M LiPF6 in EC/DMC (EC:DMC = 1:1) liquid electrolyte, LiMnO2/SPE/Li battery shows a greater discharge capacity of 124 mAh g−1.[\n##UREF##140##\n154\n##\n] In addition, LiMnO2‐PAn‐DMcT composite cathode was prepared by mixing LiMnO2 powder with polyaniline (PAn), 2,5‐dimercapto‐1,3,4‐thiadiazole (DMcT) and carbon in N‐mehtylpyrrolidinone (NMP).[\n##UREF##141##\n155\n##\n] LiMnO2‐PAn‐DMcT composite cathode can increase the capacity of lithium polymer battery. In addition, PVDF‐based SPE has been intensively studied due to its high mechanical strength and good thermal stability. In 2021, Fouladvand et al. prepared a PVDF/SGO polymer electrolyte by introducing sulfonated graphene oxide (SGO) as an effective nanofiller. LiMnO2 cathode matched with PVDF/SGO polymer electrolyte obtains a high discharge capacity of 204 mAh g−1 at 0.1 C.[\n##UREF##142##\n156\n##\n]\n
","
All solid‐state batteries (ASSBs) are the strong contender for the new energy industry due to their longer cycle life, higher energy density and safety compared to conventional liquid batteries. And further research into competitive high energy‐density ASSB is focused on small‐size cathode composite material consisting of active material, solid state electrolyte (SSE) and conductive additive. In order to enhance the charge transfer of Li+, active material must bond strongly with SSE by high temperature annealing. Rumpel et al. investigated the thermal stabilities of LiMnO2 and LiMnPO4 (LMP) in ASSB, which was assembled with the ceramic electrolyte Li1.3Al0.3Ti1.7(PO4)3 (LATP).[\n##UREF##143##\n157\n##\n] Unfortunately, LiMnO2 decomposes into Mn3O4 and LiMn2O4 at <500 °C because of the disproportionation of Mn3+. In contrast, LATP remains thermally stable even under 800 °C in an argon atmosphere.
","
In conclusion, LiMnO2 as a cathode can be used in various batteries, including non‐aqueous batteries, LPBs, and ASSBs. To facilitate the commercialization of LiMnO2 cathode, the design of electrolytes is of great significance. Thus, the application of LiMnO2 cathode can be researched in more fields.
","Conflict of Interest","
The authors declare no conflict of interest.
"],"string":"[\n \"LiMnO<sub>2</sub>\\n\",\n \"Crystal Structure and Comparison\",\n \"
Layered LiMnO2 and spinel LiMn2O4 are two typical manganese‐based cathodes. The spinel LiMn2O4 belongs to the space group Fd3¯m, where Li cations occupy the 8a site in 1/8 of the tetrahedron, Mn cations fill in the 16d site in 1/2 of the octahedral void, and O anions arranged by face‐centered cubes (FCC) lie in the 32e site (Figure\\n##FIG##1##\\n2a##).[\\n##UREF##26##\\n33\\n##\\n] In this structure, unoccupied oxygen tetrahedra and oxygen octahedra are connected in a shared plane and line, which forms 3D interconnected channels for the diffusion of Li+.[\\n##UREF##27##\\n34\\n##\\n] While layered LiMnO2 is a polymorphous compound including two quintessentially crystallographic structure (Figure ##FIG##1##2b and c##) and a cation disordered rocksalt polymorph (Figure ##FIG##1##2d##).[\\n##UREF##1##\\n5\\n##, ##UREF##28##\\n35\\n##\\n] The orthorhombic LiMnO2 (referred to as o‐LiMnO2 in the following) having β‐NaMnO2‐like structures belongs to Pmnm space group (a = 0.2805 nm; b = 0.5757 nm; c = 0.4572 nm; Z = 2). In the crystal with a close‐packed oxygen lattice, all MnO6 octahedra shares a common edge with each other,[\\n##UREF##29##\\n36\\n##\\n] as well as MnO6 and LiO6 are arranged in a corrugated interaction. The monoclinic LiMnO2 (herein referred to as m‐LiMnO2) is equipped with a structure of α‐NaFeO2 type, which belongs to the C2/m space group (a = 0.5439 nm; b = 0.2809 nm; c = 0.5395 nm; Z = 2), similar to LiCoO2 and LiNiO2.[\\n##UREF##30##\\n37\\n##\\n] It can also be said that Li and Mn cations alternate to form zigzag layers along the (010) in o‐LiMnO2. Different from o‐LiMnO2, the Li and Mn cations of m‐LiMnO2 occupy octahedral site layers that are parallel to the (111) plane of the cubic oxygen sublattice.[\\n##UREF##31##\\n38\\n##\\n] Consequently, Li cations fill in tetrahedral sites and Mn cations are in octahedral sites of the spinel LiMn2O4, while both Mn and Li cations occupy octahedral sites in the layered structure.[\\n##REF##15669161##\\n39\\n##\\n]\\n
\",\n \"
Additionally, Yabuuchi et al. designed a cation‐disordered rocksalt‐type LiMnO2 by mechanical milling o‐LiMnO2. RIETAN‐FP (a software for crystal structure refinement) was used to analyze the structure of the samples before and after milling. The results show that o‐LiMnO2 is in a zigzag‐type layered structure with a high crystallinity. Whereas the sample lacks the structural features embodied in o‐LiMnO2 after mechanical milling. Meanwhile, the broad diffraction peaks of X‐ray diffraction (XRD) patterns suggest that the zigzag‐type structure of o‐LiMnO2 transforms into the cation‐disordered rocksalt phase.[\\n##UREF##1##\\n5\\n##\\n]\\n
\",\n \"Challenges that LiMnO<sub>2</sub> Faced\",\n \"
Unfortunately, commercial applications of layered LiMnO2 have been plagued by the following problems. The Mn cations in LiMnO2 all present as Mn3+.[\\n##UREF##32##\\n40\\n##\\n] Three electrons in the d orbitals of high‐spin Mn3+ stay in the t2g orbital with the same spin, and only one electron occupies an eg orbital. It is important to note that eg orbitals with dx2−dy2and dz2 are doubly degenerate orbitals obtained by splitting 3d orbitals (Figure\\n##FIG##2##\\n3a##). Consequently, this t2g\\n3eg\\n1 electronic configuration of Mn3+ results in the asymmetric occupation of eg orbitals. Meanwhile, the electrons in the dx2−dy2and dz2 orbitals exhibit shielding for the Mn nucleus to varying degrees in different directions, resulting in an unstable state of central Mn3+. In order to maintain the stability of Mn3+, the two longitudinal Mn‐O bonds are gradually elongated, accompanying the shrinkage of the other four horizontal Mn‐O bonds (Figure ##FIG##2##3b##). In this case, the symmetry of MnO6 octahedron changes from Oh to D4h, giving rise to the Jahn–Teller (J–T) distortion.[\\n##UREF##33##\\n41\\n##, ##UREF##34##\\n42\\n##, ##UREF##35##\\n43\\n##\\n] Simultaneously, the volume change from the cubic to tetragonal coordination results in the structural degradation of LiMnO2. Figure ##FIG##2##3c## illustrates the changes in XRD patterns of the o‐LiMnO2 electrodes after different cycles. The intensity of the o‐LiMnO2 peak gradually decreases with the progress of the cycle and some new peaks appear. After three cycles, there are no o‐LiMnO2 peaks in the XRD patterns, which provides compelling evidence that J–T distortion triggers the rapid structure transformation from layered into the spinel or the rock salt upon cycling.[\\n##REF##34073268##\\n44\\n##, ##UREF##36##\\n45\\n##\\n] It was confirmed by in situ X‐ray studies that o‐LiMnO2 was irreversibly converted to spinel structure.[\\n##UREF##37##\\n46\\n##\\n] In addition, high stacking faults induced by structural disorder at Li/Mn sites are more likely to lead to phase transformation to a spinel‐like structure and display capacity fading in 3 V.[\\n##UREF##38##\\n47\\n##\\n] According to the study by Lu et al.,[\\n##UREF##39##\\n48\\n##\\n] additional plateau in the discharging curves (4.0 V) indicated spinel phase Li\\nx\\nMn2O4 was formed from o‐LiMnO2 (Figure ##FIG##2##3d##). Based on charge/discharge curves analysis and structural studies, Molenda et al. proposed that phase transition (o‐LiMnO2↔LiMn2O4) was reversible in o‐LiMnO2.[\\n##UREF##40##\\n49\\n##\\n] However, in‐depth examinations are required to understand the complex kinetics in the process of the phase transition.
\",\n \"
On the other hand, the cycling performance of LiMnO2 is also restricted by the disproportionation of Mn element (Mn3+ → Mn2+ + Mn4+) during the discharge process. Seriously, Mn2+ is freely soluble in electrolyte solution. For one thing, Mn ions migrating and depositing on the anode will have major implications for solid electrolyte interphase (SEI). For another, Mn dissolution leads to the reduction of the active material, directly leading to structural degradation and capacity loss.[\\n##UREF##41##\\n50\\n##, ##UREF##42##\\n51\\n##, ##UREF##43##\\n52\\n##\\n]\\n
\",\n \"
It is concluded that phase transformation and manganese dissolution are the critical challenges for the application of layered LiMnO2. In addition, the loss of oxygen and the preparation of pure‐phase LiMnO2 also need to be cracked.
\",\n \"Synthesis and Electrochemical Performance\",\n \"
The synthetic route has a significant effect on the phase and structure of LiMnO2 as well as electrochemical properties. Similar to other cathode materials, the main synthesis methods of layered LiMnO2 include ion‐exchange method, solid‐state process, hydrothermal method, sol‐gel method and co‐precipitation.
\",\n \"
In 1996, Armstrong et al. prepared the stoichiometric m‐LiMnO2 by the ion‐exchange method.[\\n##UREF##44##\\n53\\n##\\n] First, NaMnO2 was prepared by heating Na2CO3 and Mn2O3 at high temperatures under the argon atmosphere and then refluxing the mixture of NaMnO2 and excess lithium compounds (LiCl or LiBr) in n‐hexanol. The initial charge capacity of obtained m‐LiMnO2 was up to 270 mAh g−1. In the same year, Delmas et al. also reported layered m‐LiMnO2 with metastable character by ion‐exchange process.[\\n##UREF##45##\\n54\\n##\\n] It needs to be pointed out that refluxing experiments are carried out under the argon atmosphere and with a large excess of LiCl to protect the oxidation of any material. A differential scanning calorimetry (DSC) and a high temperature X‐ray diffractometer were applied to evaluate the thermal stability of m‐LiMnO2. Both measurement results indicated it is metastable. More specifically, exothermic effects at 300 °C are observed in the DSC curve. Meanwhile, the diffraction lines of o‐LiMnO2 disappear at 300 °C. In addition, Shao‐Horn et al. obtained a LiMnO2 material accompanied by spinel phase and orthorhombic LiMnO2 by ion exchange method.[\\n##UREF##46##\\n55\\n##\\n]\\n
\",\n \"
Zhou and co‐workers reported the one‐step synthesis of pure‐phase m‐LiMnO2 at a lower temperature (450 °C). Electrolytic manganese dioxide (EMD) was used as a manganese precursor to realize carbothermal reduction under the Ar atmosphere.[\\n##UREF##47##\\n56\\n##\\n] An initial reversible capacity of 180 mAh g−1 is obtained in the synthesized sample. Using one‐step hydrothermal methods, the controlled preparation of LiMnO2 mixed orthorhombic and monoclinic phases was successfully achieved.[\\n##UREF##48##\\n57\\n##\\n] It presented a higher discharge capacity (112.5 mAh g−1) after 50 cycles in a voltage range of 2.0–4.5 V than that of both structures (m‐LiMnO2: 94.5 mAh g−1, o‐LiMnO2: 106.8 mAh g−1). The fabrication of pure m‐LiMnO2 is considered to be relatively difficult because of its thermodynamic instability. Therefore, o‐LiMnO2 has been extensively investigated.[\\n##UREF##49##\\n58\\n##, ##UREF##50##\\n59\\n##\\n]\\n
\",\n \"
As early as the 1990s, there were studies through high‐temperature solid‐phase approaches to prepare layered LiMnO2, which was performed by calcining the mixture of MnO2 and lithium metal salts (LiOH/Li2CO3) at high temperatures (600–1000 °C) under an argon atmosphere.[\\n##UREF##51##\\n60\\n##, ##UREF##52##\\n61\\n##\\n] To prevent manganese from being oxidized to the tetravalent state, a reducing agent like carbon was even required. Despite undergoing a phase transition to spinel in LiMnO2 during charging and discharging, an interesting phenomenon was found that a small amount of spinel in the original LiMnO2 cathode could improve electrochemical performance than pure LiMnO2 cathodes. In addition, its electrochemical performance was better than that of pure LiMnO2 cathodes. Lee et al. obtained the pure o‐LiMnO2 phase by a distinctive quenching process, which employed the reaction of LiOH with γ‐MnOOH at 1000 °C in an argon atmosphere.[\\n##UREF##53##\\n62\\n##\\n] It delivered a high discharge capacity of 201 mAh g−1 in the first cycle and remained 200 mAh g−1 after 50 cycles at a current density of 0.4 mA cm−2 between 4.3 and 2.0 V. The group of Wang reported o‐LiMnO2 with a discharge specific capacity of 180—190 mAh g−1 by a two‐step solid‐state reaction.[\\n##UREF##54##\\n63\\n##\\n] First, the precursors were prepared by firing mixtures of Mn2O3 and LiOH⋅H2O at 450 °C for 5 h, and then at higher 600 °C for 12 h with argon flow. Furthermore, one‐step synthesis by the solid‐state process using glucose as a reducing agent to prepare o‐LiMnO2 is also feasible.[\\n##UREF##55##\\n64\\n##\\n] MnO2, LiOH⋅H2O and C6H12O6 mixed with a Li/Mn/C molar ratio of 5/4/2 were well ground, and then annealed at 750 °C for 15 hours in a tube furnace under nitrogen flow. In this way, heating temperature and time need to be strictly controlled, otherwise impurity phases are accompanied. However, severe particle agglomeration and high energy consumption are inevitable problems in solid‐phase synthesis.
\",\n \"
The hydrothermal method with low energy consumption is an ideal way to prepare o‐LiMnO2 for getting uniform and fine particles. In the processes of two‐step hydrothermal synthesis of LiMnO2, there are three precursors (γ‐MnOOH, Mn3O4, and Mn2O3) formed by the redox reaction of the manganese source. The crystalline o‐LiMnO2 can be obtained by reacting γ‐MnOOH as the precursor with LiOH solution in a Teflon‐lined autoclave at 180 °C for 24 h.[\\n##UREF##56##\\n65\\n##\\n] The γ‐MnOOH nanorods as precursors were synthesized by magnetically stirring a mixed solution of polyethylene glycol (PEG‐10000) and Mn(NO)3 at 140 °C for 24 h, namely a novel polymer‐assisted low‐temperature hydrothermal method. The attempts to synthesize o‐LiMnO2 hydrothermally with Mn3O4 as a precursor were completed by Komaba et al. in 2002.[\\n##UREF##57##\\n66\\n##, ##UREF##58##\\n67\\n##\\n] Dark brown Mn3O4 powders were prepared by stirring the aqueous mixed solution of Mn(CH3COO)2 and KOH at 80 °C for 24 h with bubbling O2 gas. It is important to wash the Mn3O4 powders with deionized water until the pH value of 7 for the synthesis of o‐LiMnO2. Interestingly, replacing γ‐ MnOOH and Mn2O3 with Mn3O4 as a precursor can obtain highly crystalline o‐LiMnO in hydrothermal synthesis, enabling initial reversible capacity of 210 mAh g−1.[\\n##UREF##59##\\n68\\n##\\n] Nanocrystalline Mn3O4 particles can also be prepared through the reduction of high‐valent manganese compounds (KMnO4) with methanol or ethanol in an autoclave at the temperature of 80—100 °C for 12–48 h.[\\n##UREF##60##\\n69\\n##\\n] In addition, Yang group introduced a rapid method to synthesize nanosized o‐LiMnO2, which was based on microwave‐solvothermal approach. This technique mainly uses α‐Mn2O3 as the precursor mixed with LiOH·H2O for ≈30 min at a low temperature of 160 °C.[\\n##UREF##61##\\n70\\n##\\n] The microwave hydrothermal synthesis was carried out in the microwave digestion system. Introducing hydrazine hydrate (N2H4⋅H2O) as a reducing agent in the hydrothermal route, submicron‐sized o‐LiMnO2 crystals can be successfully prepared by using LiOH and the precursors of porous Mn2O3 at the temperature of 180 °C for 16 h.[\\n##UREF##62##\\n71\\n##\\n] The Mn2O3 precursor was formed by calcining MnCO3 under the air atmosphere at 620 °C for 6 h. The submicron‐sized o‐LiMnO2 displayed a superior discharge capacity of 216 mAh g−1.
\",\n \"
Nevertheless, the time‐consuming and complex procedures involved in multi‐step processes are the undeniable disadvantages of the two‐step synthesis mentioned above. Hence, the facile and simple one‐step hydrothermal approach was chosen to obtain LiMnO2 materials.[\\n##UREF##63##\\n72\\n##, ##UREF##64##\\n73\\n##\\n] This way is conventionally achieved by employing the reactants of Mn source (Mn2O3/MnO2) and LiOH·H2O aqueous solution in a Teflon‐lined autoclave. Unfortunately, this method may introduce small amounts of impurities (e.g., Li2MnO3) in samples.[\\n##UREF##65##\\n74\\n##\\n] Li2MnO3 is also a layered lithium‐manganese oxide with an α‐NaFeO2 type structure and the C2∖m monoclinic crystal system. In this structure, Li cations occupy the interslab octahedral position in the rock salt structure, and 1/3 of Li cations and 2/3 of Mn cations occupy slab octahedral sites of the transition metal layer,[\\n##UREF##66##\\n75\\n##\\n] so it can also be written as the conventional layered structural formula of Li[Li1/3Mn2/3]O2. There are two hypotheses regarding the explanation of the electrochemical activity of Li2MnO3. With the aid of flame emission, atomic absorption, X‐ray photoelectron spectroscopy (XPS), thermogravimetric analysis coupled with mass spectrometry (TGA/MS), Bruce et al. investigated the electrochemical activity of Li2MnO3 in organic electrolytes. They indicated that the activity of Li2MnO3 could be attributed to the exchange of Li+ by H+ in organic electrolytes rather than removal of O2−, and there was no oxidation of Mn4+ to Mn5+.[\\n##UREF##67##\\n76\\n##\\n] Another hypothesis was proposed by Dahn in 2002. They believed oxygen was irreversibly released during the first charge to 4.8 V in Li/Li[Ni\\nx\\nLi(1/3‐2\\n\\nx\\n\\n/3)Mn(2/3‐\\n\\nx\\n\\n/3)]O2 material.[\\n##UREF##68##\\n77\\n##\\n] In 2006, Armstrong et al. used in situ differential electrochemical mass spectrometry (DEMS) to directly detect the oxidation of O2− in Li2MnO3 structure and the accompanying Li+ extraction, which provided a reliable basis for the mechanism of oxygen loss to the electrochemical activity of Li2MnO3.[\\n##REF##16802836##\\n78\\n##\\n] Ethylenediaminetetraacetic acid disodium salt (EDTA‐2Na) acted as both a chelating reagent and reducing agent to prepare pure o‐LiMnO2 via hydrothermal synthesis. EDTA‐2Na not only prevents residual oxygen from oxidation in the reacting system effectively but also ingeniously suppresses the formation of Li2MnO3 (Figure\\n##FIG##3##\\n4a##).[\\n##UREF##69##\\n79\\n##\\n] Figure ##FIG##3##4b,c## show the influence of the concentration of EDTA‐2Na and the reactive temperatures on the appearance of Li2MnO3, respectively.\\n
\",\n \"
Particularly, the conditions for soft hydrothermal synthesis can affect the properties of the material. Thus, adjusting the synthesis conditions is an effective way to improve electrochemical performance. A clear understanding of the formation and growth mechanism of materials (including the nucleation time and growth process) is usually expected when conducting synthetic experiments, and in situ hydrothermal synthesis might be a good way to do this.[\\n##REF##22747718##\\n80\\n##\\n]\\n
\",\n \"
To the best of our knowledge, the electrochemical performance is heavily affected by the morphology and structure of the material. Accordingly, LiMnO2 with specific nanostructures and morphologies (e.g., nanoplates, nanoparticles, nanorods, and microcubes) have spurred an intensive search for excellent electrochemical performance (Figure\\n##FIG##4##\\n5\\n##).[\\n##UREF##70##\\n81\\n##, ##UREF##71##\\n82\\n##, ##UREF##72##\\n83\\n##, ##UREF##73##\\n84\\n##\\n] In 2007, Liu et al. used needle‐like MnOOH as the precursor to prepare o‐LiMnO2 with a nanorod‐like shape. Notably, the platy shape, platy with irregular shape and rod‐like crystals corresponded to the appearance of Mn3O4 phase, cubic Li0.2Mn2O4 phase, and pure o‐LiMnO2 after 2 h, 3 and 4 h hydrothermal treatment, respectively (Figure\\n##FIG##5##\\n6\\n##).[\\n##UREF##74##\\n85\\n##\\n] It is also a good evidence that LiOH has the two important roles in providing a source of lithium and controlling both morphology and particle size. Xiao et al. prepared orthorhombic LiMnO2 nanoparticles and LiMnO2 nanorods by different hydrothermal methods.[\\n##UREF##75##\\n86\\n##\\n] Compared with LiMnO2 nanoparticles prepared through simple one‐step routes, LiMnO2 nanorods synthesized by γ‐MnOOH precursors showed a higher discharge capacity (200 mAh g−1) as well as better cyclability (180 mAh g−1 after 30 cycles) due to favorable electronic transport of 1D electronic pathways. Zhao et al. combined Mn2O3 nanorods as a template and thermal decomposition process to prepare o‐LiMnO2, which delivered outstanding performance. As a consequence, the one‐dimensional crystalline nanostructure could effectively promote the transport of charge/electron and increase the electrode‐filled ratio.[\\n##UREF##72##\\n83\\n##\\n]\\n
\",\n \"
To overcome the difficulty in synthesis of layered LiMnO2, there are some novel methods developed, including the emulsion‐drying method, reverse‐microemulsion preparation, microwave irradiation, in.situ oxidation coupling with ion exchange, and in situ carbothermal reduction. It is reported that o‐LiMnO2 materials by emulsion‐drying method show only a 10% capacity loss after 300 cycles at 45 mA g−1. The emulsion‐drying method could mix cations homogeneously at the atomic level and obtain fine single‐crystal oxide cathode materials.[\\n##UREF##76##\\n87\\n##\\n] Reverse‐microemulsion preparation is another nice way to allow cations to mix and interact in an atomic scale. Obtaining a thermodynamically stable and transparent microemulsion, in which nano‐sized water is well dispersed into the oil phase, is the key to reverse microemulsion preparation.[\\n##UREF##39##\\n48\\n##\\n] One of the new technologies based on hydrothermal synthesis is microwave hydrothermal. With the advantage of microwave radiation, it significantly reduces reaction time and temperature in some processes.[\\n##UREF##77##\\n88\\n##\\n] Li et al. described the synthesis of high‐purity and highly crystallized o‐LiMnO2 via the process of in situ oxidation coupling with ion exchange.[\\n##UREF##78##\\n89\\n##\\n] During the synthesis process, MnOOH was produced by the oxidation of Mn(OH)2 with the oxidizing agent (NH4)2S2O8 in a LiOH‐existing strong basic environment. Despite 80 °C, deoxygenated distilled water and nitrogen were required to eliminate the impact of oxygen in the air. Komarnenib's group prepared nanorod‐like o‐LiMnO2 with high purity and excellent electrochemical behavior by in situ carbothermal reduction.[\\n##UREF##79##\\n90\\n##\\n] In this synthesis process, MnO2, LiOH·H2O and affordable glucose (C6H12O6) as a reducing agent were homogeneously mixed followed by high temperature treatment in an argon atmosphere for several hours. Additionally, sol‐gel and co‐precipitation methods have also been used for the synthesis of LiMnO2. As for the sol‐gel approach to get LiMnO2, citric acid is a common chelating agent.[\\n##UREF##80##\\n91\\n##\\n] Zeng group obtained spherical o‐LiMnO2 powders with a discharge capacity of 152 mAh g−1 by a carbonate coprecipitation method using NH4HCO3 and MnCl2 as raw materials.[\\n##UREF##81##\\n92\\n##\\n]\\n
\",\n \"
Briefly, it is relatively difficult to synthesize pure layered LiMnO2 for its thermodynamic sub‐stability, especially for monoclinic LiMnO2. Li2MnO3 and manganese oxide as impurities are frequently seen in the XRD analysis of the product. So, to avoid the various side reactions during synthesis, the use of inert gas is the recommended choice. Both solid‐state synthesis and hydrothermal synthesis should strictly control the Li/Mn ratio, reaction temperature, and reaction time. Moreover, in situ detection techniques can be used for precise material preparation.
\",\n \"Modifications\",\n \"
To overcome the drawbacks mentioned in Section 2, tremendous efforts such as element doping, surface coating, nanocomposite designing and compatible electrolyte have been adopted to improve the electrochemical performance of LiMnO2 cathode materials.
\",\n \"Element Doping\",\n \"
Element doping is one of the most extensive approaches for modifying cathode materials to improve the basic physical properties of materials. The elements doping can suppress phase transitions, and enhance capacity. For LiMnO2 cathode, the research on doping strategies mainly includes element types, element content, doping sites, synthesis conditions, morphology, and structural effects. The efficacy of element substitution by doping is mainly manifested in the following aspects: 1) inhibiting J–T distortion and making structures stable for improved cycling performance; 2) providing favorable Li+ diffusion. For substituting at the Mn site, doping ions with smaller radius (Al3+, Co3+, Cr3+) than Mn3+ result in a contracted crystal lattice and microstrains owing to the shorter and stronger transition metal‐oxygen bonds than Mn─O bonds; substitutes of larger radius (Y3+) show larger lattice parameters and lead to the free movement of Li+ in the oxide, which makes it possible to increase the rate of capability. For substituting at the Li site, the metal element with a larger radius ion can provide a pillaring effect to enhance the stability of cathode materials. For instance, Li0.53Na0.03MnO2 with a pseudo‐spinel structure displays an increasing discharge capacity, which reaches 200 mAh g−1 in the 50th cycle.[\\n##UREF##82##\\n93\\n##\\n] Up to now, various doping elements have been found to improve the electrochemical properties of layered LiMnO2, including Na, Cu, Mg, Zn, Fe, Ni, Al, Co, Cr, Y, In, S, Ti, V, Nb, Ru, B, F, BO3\\n3−, PO4\\n3−and SiO3\\n3−.
Transition metal elements usually occupy the Mn site of lithium manganese oxide to improve electrochemical performance. The ionic radius of the doping Cu2+ (0.072 nm) is larger than that of Mn3+, which is beneficial for faster mobility of Li+ in bulk, contributing to the decreased charge transfer impedance and Warburg impedance of LiMnO2. Such lower resistance has an important role in improving high‐rate capability by 65.5%.[\\n##UREF##83##\\n94\\n##\\n] Unlike the doping of transition metal elements, the Mg may occupy both Li and Mn sites. During the charge/discharge cycling, the presence of Mg at the Li sites is beneficial for maintaining the stability of the structure but reduces the electrochemical extraction of lithium ions.[\\n##UREF##84##\\n95\\n##\\n]\\n
\",\n \"
Suresh et al. investigated the doping effect of Zn and Fe elements using the ion‐exchange method.[\\n##UREF##85##\\n96\\n##\\n] It was only 5% Zn/Fe (LiMn0.95Zn0.05O2/LiMn0.95Fe0.05O2) substituted into LiMnO2 that could exhibit a better discharge capacity of 180 and 193 mAh g−1, respectively. The substitution of Zn can improve the retention of capacity. However, increasing the content of Zn leads to the capacity fading due to decreasing of electrochemically active Mn3+. The decreased intensity of Mn3+/Mn4+ redox peak in the cyclic voltammograms (CV) curves confirms it.
\",\n \"Mixed Bivalent and Trivalent Cations\",\n \"
Quine et al. reported that Li\\nx\\nMn0.95Ni0.05O2 with the O3 (α‐NaFeO2) structure formed via an ion‐exchange route. Li\\nx\\nMn0.95Ni0.05O2 delivered a high capacity of 220 mAh g−1 above 2.5 V.[\\n##UREF##86##\\n97\\n##\\n] Inevitably, a phase transition to spinel occurred after the cycling. In that case, Ni substituted in the layered LiMnO2 was in a state of Ni2+ thus accompanied by a reaction from Mn3+ to Mn4+. Drawing support from the x‐ray absorption spectroscopy (XAS) spectrum, Wadati et al. pointed out that Li deficiencies could not be created by the Ni2+ valence and the change from Mn3+ to Mn4+.[\\n##UREF##87##\\n98\\n##\\n] However, the Ni3+ was observed in the CV curves, corresponding to anodic peak of 3.05 V.[\\n##UREF##84##\\n95\\n##\\n] In addition, when 5% Ni is substituted, the compound provides the highest capacity of 220 mAh g−1 but is followed by a rapid capacity decay on repeated cycling. The high Ni contents can provide stable capacity, due to the decrease of Mn3+ contents, while the loss of capacity is inevitable. Nahm and co‐workers found that O2‐Li0.7[Ni1/6Mn5/6]O2 instead of Li0.7[Li1/6Mn5/6]O2 exhibited no phase change after 30 cycles.[\\n##UREF##88##\\n99\\n##\\n] In the first cycle, this sample delivered a discharge capacity of 198 mAh g−1 between the voltage of 4.6 and 2.0 V. Meanwhile, it exhibited a 96% capacity retention after 25 cycles at 1/3 C. Therefore, Ni doping can exert a positive effect on the stability of the structure and the retention of capacity.
\",\n \"Trivalent Cations\",\n \"
In the 1990s and early 2000s, interests in modifying layered LiMnO2 were focused more on the solid solution formed between LiMnO2 and LiMO2 analogues (LiAlO2, LiCoO2, and LiCrO2). Although pure LiAlO2 is electrochemically inert, the solid solution consisting of LiAlO2 and other lithiated transition‐metal oxides probably possesses a high intercalation potential, leading to high energy density.[\\n##UREF##89##\\n100\\n##\\n] And two effects can explain why Al3+ doping can stabilize the monoclinic phase:1) AlO6 octahedra is not distorted because of non‐J‐T Al3+; 2) the smaller size of Al3+ (0.054 nm) has advantages in building stable transition metal‐oxygen bond.[\\n##UREF##90##\\n101\\n##, ##UREF##91##\\n102\\n##\\n] Chiang et al. indicated that the Al‐doped LiMnO2 contained the monoclinic and orthorhombic ordered rock‐salt structures, and exhibited better cycling performance and higher discharge capacities under the test conditions of elevated temperatures.[\\n##UREF##92##\\n103\\n##\\n] But m‐Li\\nx\\nAl0.05Mn0.95O2 showed a higher discharge capacity of 188 mAh g−1 as well as higher energy densities of 602 Wh kg−1 than o‐Li\\nx\\nAl0.05Mn0.95O2 (146 mAh g−1 and 465 Wh kg−1) at 55 °C. The doping of an element not only directly improves the electrochemical performance of materials, but also affects the structure and morphology of materials. Regan et al. elucidated the impacts of Al doping on the structures and surface properties of material[\\n##UREF##93##\\n104\\n##\\n] The existence of Al favors on Mn‐Li inversion in alternating slabs of the LiAl\\nx\\nMn1‐\\n\\nx\\nO2. Moreover, it is manifested by XPS that Al is uniformly incorporated in the whole grains up to the limit of its solubility. And no aggregation of Al is observed on the surface. The group of Yang described the facile hydrothermal synthesis of o‐LiMn1‐\\n\\nx\\nAl\\nx\\nO2 and investigated the changes in the morphology with varying Al content in the layer.[\\n##UREF##94##\\n105\\n##\\n] The results indicate that Al3+ in a small content contributes to growth in the b‐axis direction (i.e., (010) direction) and formation of cuboid crystals, but the growth is inhibited and changes to cubic crystals with a high content of Al3+. In 1999, Robertson prepared Co‐substituted Li(Mn1‐\\n\\ny\\nCo\\ny\\n)O2 in the range 0≤y≤0.5 using a solution‐based route with the assistance of ion exchange.[\\n##UREF##95##\\n106\\n##\\n] On the one hand, it pointed out that Co entered the layered LiMnO2 as Co3+ rather than Co2+. On the other hand, it indicated that higher content of Co caused somewhat lower capacities, and the best amount of doping was 2.5%.[\\n##UREF##96##\\n107\\n##, ##UREF##97##\\n108\\n##\\n] The LiMn1‐\\n\\ny\\nCo\\ny\\nO2 from the hydrothermal synthesis gave different optimal doping content (10%).[\\n##UREF##98##\\n109\\n##\\n] Doping of Al, Co, and Cr makes sense in stabilizing LiMnO2 structure and improving cycle retention. In addition, all doped compounds show contracted crystal lattices by comparison with nonsubstituted LiMnO2 due to a relatively small radius of doping cations. Although LiMn1‐yCoyO2 and LiMn1‐yAlyO2 undergo a spinel phase transition at 4 V,[\\n##UREF##99##\\n110\\n##, ##UREF##100##\\n111\\n##\\n] this has almost no harmful effect on cycling performance and even increases discharge capacity.[\\n##UREF##101##\\n112\\n##\\n] But low levels of Cr3+‐substituted materials show insignificant transformation in cycling.[\\n##UREF##102##\\n113\\n##\\n] The key points of Cr doping depend on 1) forming a continuous solid solution in LiMnO2 across the whole range of composition from LiMnO2 to LiCrO2;[\\n##UREF##103##\\n114\\n##\\n] 2) more regular local coordination symmetry around Cr3+ than Mn3+; 3) higher coordination between Cr3+ and oxygen than Mn3+.[\\n##UREF##104##\\n115\\n##\\n] Furthermore, with the help of 6Li magic‐angle spinning NMR spectroscopy, it has been found that Cr3+ doping can partially disrupt the Mn‐Mn antiferromagnetic correlations, which is important for the stabilization of the layered structure over the orthorhombic structure.[\\n##UREF##103##\\n114\\n##, ##UREF##105##\\n116\\n##\\n] Alternatively, Pang observed that even though Cr doping into o‐LiMnO2 brought a change in the topography from orthorhombic to monoclinic geometry employing Pechini's synthesis method and XRD patterns,[\\n##UREF##106##\\n117\\n##\\n] it does not matter on improving cycling performance and reversible capacity. Xiao et al. reported the rheological phase method to prepare m‐LiMn1‐\\n\\nx\\nCr\\nx\\nO2,[\\n##UREF##107##\\n118\\n##\\n] forming ultrafine spherical particles with the size of 60–200 nm. Relative to the preparation through solid‐state reaction at the higher temperature of 1000 °C, LiMn0.85Cr0.15O2 by rheological phase method calcined at 800 °C yields a much higher initial discharge capacity (180 mAh g−1) and capacity retention as well (94% after 40 cycles).
\",\n \"
Y3+ as a substituent in place of Mn3+ in LiMnO2 was first explored by Zong and partners.[\\n##UREF##108##\\n119\\n##\\n] There is no 4 V plateau associated with spinel LiMn2O4 in the CV and charge/discharge curves of the Li/LiMn0.98Y0.02O2 cell, which can be attributed to the pillaring effect of Y3+. In addition, doping with a low content of Fe is beneficial for capacity and cycling stability.[\\n##UREF##85##\\n96\\n##\\n] Whereas, Myung found o‐LiMnO2 of Fe substitution by a hydrothermal reaction obtained much lower capacity than that of undoped one,[\\n##UREF##109##\\n120\\n##\\n] which is down to quasi‐reversible Fe4+/Fe3+ redox couple and gives unimpressive battery performance. The introduction of Ti3+ into o‐LiMnO2 is an effective attempt to moderate the phase evolution and stabilize the structure.[\\n##UREF##110##\\n121\\n##\\n] Unfortunately, the secondary phase m‐Li2MnO3 in LiMnO2 has a low conductivity. By doping Ti3+ into LiMnO2, c‐LiTiO2 with better electrical conductivity (ca. 10−6 S cm−1) can replace above mentioned secondary phase. And Ti─O bonds are stronger than Mn‐O bonds, which militates in favor of improved structural stability of o‐LiMn1‐\\n\\nx\\nTi\\nx\\nO2. In addition, B‐doped m‐LiMnO2 was prepared by carbothermal reduction using LiOH and MnO2 as reactants under the argon atmosphere.[\\n##UREF##47##\\n56\\n##\\n] Even though the transformation from layer to spinel cannot be suppressed by B‐doping, the cycle performance and Coulombic efficiency are improved. 5% B‐doping (m‐LiB0.05Mn0.95O2) gives the best electrochemical performance among B‐doped samples with different ratios, particularly at elevated temperatures (60 °C). The average Coulombic efficiency increases from 69.7% to 99.1% at 60 °C.
\",\n \"Anions Doping\",\n \"
Aside from cations doping, doping anions is another feasible approach to modify the LiMnO2 cathode. The S‐doped LiMnO2 can deliver a discharge capacity of 220 mAh g−1 after 50 cycles with a voltage of 2.0–4.6 V.[\\n##UREF##111##\\n122\\n##\\n] The F substitution can stabilize the host structure due to high electronegativity, showing better cycle performance compared with F‐free LiMnO2.[\\n##UREF##112##\\n123\\n##\\n] Because the S2− (0.184 nm) and F− (0.136 nm) are greater than the O2− (0.132 nm), the lattice constant of LiMnO2 doped with S2 and F− increases. However, above mentioned S and F‐doping cannot ultimately suppress the transformation from layer to spinel.
\",\n \"
Larger polyanions (BO3\\n3−, PO4\\n3−and SiO3\\n3−) in place of O2− ions in nonstoichiometric Li\\nx\\nMnO2 can generate an open 3D framework, which enhances the mobility of Li+ ions. Decreased Warburg impedance is also observed, which is reflected in the improved electrochemical properties of the high‐rate charge/discharge capability. Unfortunately, polyanion doping presents increased charge‐transfer resistance due to the poor electrochemical activity of polyanions relative to cations. Surprisingly, although the discharge capacity of Li\\nx\\nMnO1.99X0.01 (X = BO3\\n3−, PO4\\n3−and SiO3\\n3−) has decreased, the cycling ability has been improved.[\\n##UREF##83##\\n94\\n##\\n]\\n
\",\n \"Co‐Doping\",\n \"
Furthermore, doping multiple elements into LiMnO2 endows electrodes with a synergistic effect. Suresh doped 5% Ni and 5% Fe into LiMnO2,[\\n##UREF##113##\\n124\\n##\\n] forming a pure and homogeneous phase with a layered structure. The low content of Ni and F exhibited a discharge capacity of 250 mAh g−1 at the rate of 0.1 C, and improved the cycling stability. The co‐doping of cations and anions, such as Li+ and F− ions, into o‐LiMnO2 has been realized through a solid‐state reaction.[\\n##UREF##114##\\n125\\n##\\n] Scanning electron microscopy characterization indicates o‐Li1.07Mn0.93O1.92F0.08 presents a smooth surface even after cycling. Co‐doping of Li+ and F− ions reduces the contact area with the electrolyte as well as suppresses the reaction of the Mn dissolution. Eventually, the products improved cycling stability and rate capability at a higher temperature (55 °C). However, doped LiMnO2 with multiple elements still shows some reserved disadvantages, such as low specific capacity.
\",\n \"
Su investigated In3+ and S2− co‐doped o‐LiMnO2 synthesized via the hydrothermal method.[\\n##UREF##115##\\n126\\n##\\n] Compared to pristine and single doping (In or S), dual doping reduced the crystallinity of LiMnO2. Meanwhile, In and S dual doping exhibited the partial structural transformation from orthorhombic to spinel after 10 cycles at 50 mA g−1, but excellent cycle performance was obtained at various discharge current densities. In brief, In and S dual doping provides a great method to improve the rate performance of LiMnO2 cathode.
\",\n \"Theoretical Calculations on Doping Effect\",\n \"
As a quantum mechanical method for the study of the electronic structure of multi‐electron systems, density functional theory (DFT) is very effective in offering a deeper elaboration of elemental doping. An ab initio study pointed out that 10% Co‐substitution makes the antiferromagnetic spin orthorhombic structure more stable than the monoclinic structure, and 60% substitution helps stabilize the disordered local moment (DLM) rhombohedral layered structure over the ferromagnetic orthorhombic phase and offers easy migration of lithium during intercalation.[\\n##UREF##116##\\n127\\n##\\n]\\n
\",\n \"
Prasad et al. reported their first‐principles calculations of doped rhombohedral LiMnO2 against J–T distortion.[\\n##UREF##117##\\n128\\n##\\n] All calculations indicate that, for dopants, their oxidation state is the first significant factor for effectively stabilizing the rhombohedral structure, and then for a certain oxidation state, the t2g or eg subshell filling is the second. In addition, results show that divalent dopants (such as Mg, Zn) are more effective in the suppression of the J–T distortion than trivalent dopants because each divalent dopant can remove two Mn3+ ions from the sublattice.[\\n##UREF##118##\\n129\\n##\\n] Based on generalized gradient approximation, it is found that divalent dopants (Mg and Co) destabilized the monoclinic structure than the trivalent dopant (Fe).[\\n##UREF##119##\\n130\\n##\\n] Using the hybrid eigenvector‐following and DFT approach, Grey groups studied the effect of trivalent dopants on the Mn migration leading to spinel transformation, and found that dopants with a small ionic radius (Al3+ and Cr3+) can raise the migration barrier of Mn, but only Cr3+ does not move to tetrahedral sites within the Li layer.[\\n##UREF##120##\\n131\\n##\\n] Kong et al. investigated the effects of ten cationic (Mg, Ti, V, Nb, Fe, Ru, Co, Ni, Cu, and Al) and two anionic (N and F) dopants of LiMnO2 using ab initio DFT simulations.[\\n##UREF##121##\\n132\\n##\\n] The findings indicate that Mg, Ti, V, Nb, Ru as well as F can effectively reduce the redox potential, Ti, V, Nb, and Ru can increase the hole conductivity to inhibit the formation of the hole polaron. However, only Ni can decrease the diffusion batteries of Li+ by 0.23 V and only the N‐doped phase is thermodynamically unstable. When doping at the Li site, it shows unfavorable thermodynamics than Mn‐site doped configurations.[\\n##UREF##122##\\n133\\n##\\n] Similarly, Khang Hoang indicates that substitutes (Al, Fe) are more favorable at the Mn site.[\\n##UREF##123##\\n134\\n##\\n]\\n
\",\n \"
To summarize, a systematical comparison of ionic radius and electrochemical performance of different elements doped into LiMnO2 is presented in Table\\n##TAB##0##\\n1\\n##. Element doping is effective in stabilizing structures and improving the electrochemical performance of LiMnO2. To one degree or another, it ameliorates the adverse effects of transformation from a layer to a spinel crystal structure. For Co and Cr doping, it shows good capacity retention upon cycling. However, element doping in LiMnO2 still suffers from some limitations. For instance, it is inevitable for Al‐doped LiMnO2 to convert into a spinel phase at 4 V. Keeping the high capacity retention for long cycles is still the biggest problem. Decreasing the amount of Li also needs to be solved during the process of electrochemical extraction, when a dopant occurs at the Li site
\",\n \"Surface Coating\",\n \"
Element doping is particularly effective in stabilizing the bulk structure, while coating makes great sense to protect the surface of electrode materials for resisting manganese dissolution into the electrolyte and impeding surface reactions.
\",\n \"
Al2O3, an ionic and electronic insulation, is often used as a coating for the cathode to prevent active electrode materials from being corroded by electrolytes. Cho et al. confirmed that the capacity loss of the Al2O3‐coated o‐LiMnO2 (only 2% after 50 cycles) was significantly lower than that of the bare one (35% loss).[\\n##UREF##124##\\n135\\n##\\n] Most significantly, there are no Li2Mn2O4 phases in the Al2O3‐coated electrode during the process of cycling compared with the uncoated one, implying that the protective coating layer can keep the lattice stable and suppress the J–T distortion. However, the LiMn1‐\\n\\nx\\nAl\\nx\\nO2 solid solution or LiAlO2 may be formed during the process of coating Al2O3 with sol‐gel and heating treatment.[\\n##UREF##125##\\n136\\n##\\n] For the materials prepared at high temperatures (600 °C and 700 °C), the uniform formation of the LiMn1‐\\n\\nx\\nAl\\nx\\nO2 solid solution across the particle instead of staying on the surface may bring the rapid capacity decrease. The LiAlO2 peak can be observed from the XRD pattern, indicating that the Al2O3 gel solution would react with the o‐LiMnO2 on the surface of materials. Likewise, similar effects can be observed in CoO‐coated o‐LiMnO2.[\\n##UREF##126##\\n137\\n##\\n] Even if CoO‐coated LiMnO2 exhibits a much lower initial discharge capacity (127 mAh g−1) compared to that of the bare one (162 mAh g−1), the former presents rapidly increasing capacity (185 mAh g−1 after 15–20 cycles) and just 12% capacity decay after 50 cycles. A significant amount of Co atoms appears in the range of 2‐µm range near the surface than in the bulk, which is a sign of solid solution formation on the surface. Thanks to the presence of solid solutions, the electrochemical stability of CoO‐coated LiMnO2 has been improved at 55 °C. In addition, the disappearance of the Li2MnO3 impurity phase after Al2O3/CoO coating implies that the coating layer lies on the surface of LiMnO2.\\n[\\n##UREF##127##\\n138\\n##\\n] It may be a reference for the removal of Li2MnO3 impurity that occurred in the synthesis of related lithium‐manganese oxygen cathodes. There are many similar effects in Al2O3 and CoO‐coated o‐LiMnO2. However, only CoO‐coated LiMnO2 displays an additional voltage plateau at 2 V probably causing capacity decay. This phenomenon illustrates the structural disorder of Al2O3/CoO coating is different.
\",\n \"
Lithium boron oxide (LBO) with good ionic conductivity has been successfully used to coat spinel and binary/ternary layered cathode materials for improving cycling stability. There is no doubt that above mentioned cycling stability is also observed in the LBO‐modified o‐LiMnO2. Nagasubramanian et al. studied the cycling performance of the LBO‐coated o‐LiMnO2 and its potential explanation.[\\n##UREF##128##\\n139\\n##\\n] During the first charge‐discharge, the intercalation/deintercalation behavior of Li+ is considered to be irreversible, which can be attributed to the structure transformation from the layered to the spinel. But as the phase transition is completed during cycles, the maximum capacity is raised to 189 mAh g−1 and the electrode retains 91% of the maximum capacities after 70 cycles, while only 77% of capacity can be obtained for uncoated one. By observing the contribution of discharge capacity from different platforms (3 V and 4 V) in the charge/discharge curves before and after coating, it can be seen that the major capacity loss is occurring from the 3 V region. Coupled with significantly reduced charge‐transfer resistance in the corresponding voltage range, it is obvious that LBO is beneficial for Li+ inserting into the octahedral voids in the LiMn2O4 structure, resulting in less capacity loss.
\",\n \"
In recent years, electrode materials with core‐shell structures have been aggressively developed to resolve the problem of low capacity and poor cyclic instability.[\\n##REF##21879116##\\n140\\n##\\n] Generally speaking, the shell can be formed by coating and acts as a protection layer to make better overall performance of the materials. Guo et al. confirm that designing an o‐LiMnO2@Li2CO3 nanosheet array cathode with a core‐shell structure presents better electrochemical behavior.[\\n##REF##27270124##\\n141\\n##\\n] Intriguingly, the o‐LiMnO2@Li2CO3 electrode shows 80% and 79% initial capacity after 400 cycles at 2C at 20 °C and 60 °C, respectively (Figure\\n##FIG##6##\\n7a## and ##FIG##5##6b##), yet the control electrode without Li2CO3 coating at 0.5 C only exhibits capacity retention of 18% at 20 °C and less than 1% at 60 °C after 400 cycles (Figure ##FIG##6##7c##). The reasons for the excellent cycling performance of o‐LiMnO2@Li2CO3 nanosheet array electrode can be explained in Figure ##FIG##6##7d–f##. First, the outer layer of Li2CO3 on the o‐LiMnO2 surface can be confirmed by high resolution transmission electron microscope (HRTEM) (Figure ##FIG##6##7d##). The outer layer can significantly minimize or avoid adverse reactions (o‐LMO dissolution and oxygen release) (Figure ##FIG##6##7e##). The Li2CO3 is one of the parts of the SEI layer, the Li2CO3 layer is beneficial to electrochemical stability at elevated temperatures. Second, the nanosheet array structure is not only equipped with a 1D pathway for fast electron transport but also shortens the diffusion length of lithium, which ensures sufficient contact of active material with electrolyte (Figure ##FIG##6##7f##). Huang et al. induced two different core‐shell microstructures of Li‐Mn‐O materials by heating treatment (Figure\\n##FIG##7##\\n8a##).[\\n##UREF##129##\\n142\\n##\\n] The phase transformation is summarized in detail in Figure ##FIG##7##8b##, there are three stages at 300 °C and 700 °C, corresponding to two transformations. TGA analysis was provided in Figure ##FIG##7##8c##. When annealed to 350°C, the oxygen on the surface of Li1.11Mn0.76O2 (LMO) is released and the lithium diffuses to form Li2O. Thus, the lithium vacancies are occupied by Mn to form spinel, resulting in a layered core and spinel shell. As the annealing proceeds to higher temperatures, the internal Li+ diffuses to the external spinel, forming a LiMnO2 shell. That is, a LiMnO2 shell wrapped around a spinel core is formed at 750 °C. The cycling performance and rate capability of these two core‐shell structural materials are shown in Figure ##FIG##7##8d,e##. Notably, the annealing‐induced synthesis method ensures the homogeneity of the core‐shell structure. Compared with conventional coating methods, this approach presents atomic‐level contacts between the cores and shells.
\",\n \"
In brief, modification by coating has resulted in a relatively feasible solution of optimizing the electrode surface to improve electrochemical performance. For instance, the coating layer reduces phase transformation (from the layer to spinel), decreases capacity loss, facilitates Li+ transfer, and increases capacities. Particularly, the coating delivers high capacity retention even at elevated temperatures, which may be attributed to impeding the active materials touch with the electrolyte solution, thus the side effects of LiMnO2 are minimized during cycling, especially manganese dissolution. Maybe coating is just like eggshells, keeping the active material safe in its role. But how to get the depth of the coating to hit the spot in the process of material synthesis still needs further study. Additionally, research on LiMnO2 coating appears to be slightly less than other cathodes (such as LiCoO2 and LiMn2O4). Co‐coating can be considered as a new modification method to improve the electrochemical performance of LiMnO2, due to its synergistic effect.
\",\n \"Composites Designing\",\n \"
To break through the bottleneck of imperfect LiMnO2 cathode, introducing other favorable materials to fabricate composites has been investigated in recent years. These materials are conducive to the electrochemical performance of electrodes, especially for carbon materials. LiMnO2‐carbon composites are usually prepared by mixing carbon materials (carbon nanotubes (CNTs), reduced graphene oxide (rGO) and graphene nanoplatelet (GNP)) with prepared LiMnO2 or with raw material for the preparation of LiMnO2. The carbon materials in LiMnO2‐carbon composites can offer high electronic conductivity and fast Li+ diffusion, which contributes to enhanced specific capacity, improved capacity retention and reduced electrochemical impedance relative to pristine LiMnO2.[\\n##UREF##130##\\n143\\n##, ##UREF##131##\\n144\\n##\\n]\\n
\",\n \"
\\nFigure\\n##FIG##8##\\n9\\n## is the scheme and scanning electron microscope (SEM) images of orthorhombic LiMnO2/CNTs nanocomposites.[\\n##UREF##132##\\n145\\n##, ##UREF##133##\\n146\\n##\\n] As schematically illustrated in Figure ##FIG##8##9a##, o‐LiMnO2/CNTs composites were prepared by a one‐step dynamic hydrothermal way. And the corresponding SEM images show that LiMnO2 particles are “confined” among CNTs (Figure ##FIG##8##9b,c##).
\",\n \"
Figure ##FIG##8##9a## is the prepared scheme of o‐LiMnO2/CNTs composites through a one‐step dynamic hydrothermal way, and the corresponding SEM images show that LiMnO2 particles are “confined” among CNTs (Figure ##FIG##8##9b,c##). The composite with 5 wt% CNTs brings about the optimum specific capacity of 204.9 mAh g−1. The o‐LiMnO2‐MWCNTs nanocomposites, where CNTs instead of carbon black worked as an additive material, display the intricate network in Figure ##FIG##8##9d##. The addition of CNTs reduces the charge transfer resistance from 190 to 105 kΩ. The 3D network provided by CNTs increases electronic conductivity of the samples and facilitates the Li+ diffusion across the interface of electrolyte. Tian et al. synthesized LiMnO2@rGO composites via a one‐pot hydrothermal route at 200 °C for 12 h,[\\n##REF##33043900##\\n147\\n##\\n] in which LiMnO2 nanoparticles were uniformly anchored onto rGO nanosheets. It is rewarding to note that the rGO with superior conductivity provides a stable conductive net, thus the first discharge capacity of LiMnO2@rGO could be improved to 175 mAh g−1, as well as capacity retention increased from 53.53% to 80.97% after 100 cycles.
\",\n \"
Although manganese‐based cathode materials are of great significance for the sustainability of LIBs, the J–T distortion of Mn3+ has become a bottleneck to improving their structural stability. Zhu et al first proposed a nice idea to suppress the J–T distortion of Mn3+ using interfacial orbital ordering.[\\n##UREF##134##\\n148\\n##\\n] As shown in Figure\\n##FIG##9##\\n10a,b##, the in situ electrochemical conversion from Mn3O4 was used to prepare heterostructured spinel‐layered LiMnO2 (SPL‐LMO). Layered domains within the spinel matrix and the orientation of orbitals at the spinel‐layered interface can be seen in Figure ##FIG##9##10c–e##, where MnO6 of the layered and spinel phases is linked with an included angle of 83.8°. Significantly, the SPL‐LMO materials exhibit superior rate performance (Figure ##FIG##9##10f##) as well as cycling stability of the full cell at a current density of 1.0 A g−1 for 1000 cycles (Figure ##FIG##9##10g##), which is superior to layered or spinel LiMnO2 cathodes reported previously.
\",\n \"Compatible Electrolyte\",\n \"
The electrolyte is one of the most important components of the battery, as it endows with the ability to establish ionic conductive channels and block the electronic conductivity between the cathode and anode. The development of electrolytes for LiMnO2 has been underway for several years.
\",\n \"
In 1997, the group of Davidson researched the electrochemistry of LiMnO2 over the range of 1.0‐4.6 V with a variety of electrolytes.[\\n##UREF##135##\\n149\\n##\\n] Li/LiMnO2 cells exhibit a higher discharge capacity in 1 M LiClO4 ethylene carbonate (EC)/dimethyl carbonate (DMC) electrolyte (257 mAh g−1) than that of 1 M LiPF6 EC/DMC electrolyte (220 mAh g−1) between 1.0 and 4.4 V. In addition, the voltage range also has a significant effect on the charge and discharge capacity. For example, with 1 M LiClO4 in propylene carbonate (PC)/EC, charge and discharge capacities are 253 and 250 mAh g−1 over the range of 1.0–4.4 V, respectively. But when charging to 4.2 V, it displays only the charge capacity of 228 mAh g−1. However, they give no further explanation. Thus, substantial differences occur when varying the electrolytes, which also needs to adjust the voltage range for better compatibility.
\",\n \"
Based on 1, 3‐dioxolane‐LiAsF6 solutions, Tadiran developed the LiMnO2 rechargeable battery with excellent electrochemical performance.[\\n##UREF##136##\\n150\\n##\\n] In 1, 3‐dioxolane‐LiAsF6 solutions, the surface film consisting of Li alkoxides, Li formate, LiF and Li\\nx\\nAsF\\ny\\n is formed on the Li anode, which induces uniform and smooth Li deposition. Thus, the smooth morphology of Li deposition provides a high cycle life.
\",\n \"
The layered lithium transition metal oxides tend to release oxygen in a highly delithiated state, possibly accompanied by an exothermic reaction with the electrolyte. These could be the most serious threat to the safety of batteries. Reassuringly, when o‐LiMnO2 cathode contacts with 1 M LiPF6 solution in 1:1:1 wt.% EC/diethyl carbonate (DEC)/DMC solvent, DSC measurements show a much lower heat effect during the first process compared with other cathode materials.[\\n##UREF##137##\\n151\\n##\\n]\\n
\",\n \"
Solid polymer electrolyte (SPE) has the advantage of excellent processability and flexible tunability. All of these advantages make it particularly valuable in designing and developing high‐energy, long‐life solid state lithium batteries. Lithium polymer batteries (LPBs) involved LiMnO2 cathode have been investigated in recent years, which relates to SPE such as polyethylene oxide (PEO), polyvinylidene fluoride‐hexafluoropropylene (PVDF‐HFP), and polyvinylidene fluoride (PVDF). Xia et al. evaluated the thermal stability of LiMn2O4 and LiMnO2 in PEO polymer electrolyte. DSC curves show that delithiated LiMn2O4 decomposes at 230 °C, while the decomposition of LiMnO2 with partial charge occurs at 350 °C. It is worth noting that the LiMnO2 cathode displays much higher thermal stability than that of LiMn2O4 when the PEO polymer is used as an electrolyte.[\\n##UREF##138##\\n152\\n##\\n] Gu's group obtained PVDF‐HFP‐PC10EC10LiClO4 as SPE to fabricate LiMnO2/SPE/Li battery. First, they prepared a mixed solution consisting of LiClO4, EC, PC, and 25 wt.% PVDF, and then casting and quickly heating it at 110 °C for 10 min.[\\n##UREF##139##\\n153\\n##\\n] Compared with LiMnO2/Li battery with the 1 M LiPF6 in EC/DMC (EC:DMC = 1:1) liquid electrolyte, LiMnO2/SPE/Li battery shows a greater discharge capacity of 124 mAh g−1.[\\n##UREF##140##\\n154\\n##\\n] In addition, LiMnO2‐PAn‐DMcT composite cathode was prepared by mixing LiMnO2 powder with polyaniline (PAn), 2,5‐dimercapto‐1,3,4‐thiadiazole (DMcT) and carbon in N‐mehtylpyrrolidinone (NMP).[\\n##UREF##141##\\n155\\n##\\n] LiMnO2‐PAn‐DMcT composite cathode can increase the capacity of lithium polymer battery. In addition, PVDF‐based SPE has been intensively studied due to its high mechanical strength and good thermal stability. In 2021, Fouladvand et al. prepared a PVDF/SGO polymer electrolyte by introducing sulfonated graphene oxide (SGO) as an effective nanofiller. LiMnO2 cathode matched with PVDF/SGO polymer electrolyte obtains a high discharge capacity of 204 mAh g−1 at 0.1 C.[\\n##UREF##142##\\n156\\n##\\n]\\n
\",\n \"
All solid‐state batteries (ASSBs) are the strong contender for the new energy industry due to their longer cycle life, higher energy density and safety compared to conventional liquid batteries. And further research into competitive high energy‐density ASSB is focused on small‐size cathode composite material consisting of active material, solid state electrolyte (SSE) and conductive additive. In order to enhance the charge transfer of Li+, active material must bond strongly with SSE by high temperature annealing. Rumpel et al. investigated the thermal stabilities of LiMnO2 and LiMnPO4 (LMP) in ASSB, which was assembled with the ceramic electrolyte Li1.3Al0.3Ti1.7(PO4)3 (LATP).[\\n##UREF##143##\\n157\\n##\\n] Unfortunately, LiMnO2 decomposes into Mn3O4 and LiMn2O4 at <500 °C because of the disproportionation of Mn3+. In contrast, LATP remains thermally stable even under 800 °C in an argon atmosphere.
\",\n \"
In conclusion, LiMnO2 as a cathode can be used in various batteries, including non‐aqueous batteries, LPBs, and ASSBs. To facilitate the commercialization of LiMnO2 cathode, the design of electrolytes is of great significance. Thus, the application of LiMnO2 cathode can be researched in more fields.
J.M. and T.L. contributed equally to this work. This work was supported by the National Natural Science Foundation (22379114, 51972235), the Natural Science Foundation of Shanghai (22ZR1465600), Innovation Program of Shanghai Municipal Education Commission (2023ZKZD32), and the Fundamental Research Funds for the Central Universities.
","
\nJin Ma is currently a Master's student at School of Chemical Sciences and Engineering, Tongji University. Her current research focuses on manganese‐based cathode materials for lithium‐ion batteries.
","
\nTingting Liu received her M.S. degree from Ningbo University in 2020 and she is now pursuing her Ph.D. degree at Tongji University. Her current research focuses on cathode materials for rechargeable lithium‐ion batteries.
","
\nJie Ma is a Professor in College of Environmental Science and Engineering at Tongji University. He received an M.S. degree from Hefei University of Technology in 2005, a Ph.D. degree from Shanghai Jiaotong University in 2009, and was a visiting scholar in Department of Chemical & Biomolecular Engineering at the University of Akron in 2015–2016. Ma's current research focuses on nanomaterial innovations and electrochemical technology for sustainable environment. He has published 150+ journal papers, with a total citation of 9000+ times and an h‐index of 53.
","
\nChi Zhang is a specially‐appointed tenured professor at Tongji University. He received his Ph.D. degree from Nanjing University, and worked at Nagoya University as Research Fellow of Japanese Society for the Promotion of Science and Technical University of Munich as Research Fellow of Alexander von Humboldt Foundation. Zhang's scientific interest is the development of advanced functional materials for optical and energy applications. He has published over 450 SCI papers including Advanced Materials, Journal of the American Chemical Society and Angewandte Chemie International Edition.
","
\nJinhu Yang is a specially‐appointed tenured professor at Tongji University. He received his Ph.D. degree from Peking University in 2005. Then, he worked at The Hong Kong University of Science and Technology as a Research Associate from 2005 to 2006, the University of Tokyo as a Foreign Researcher supported by JSPS organization from 2006 to 2008, and Munich University as a Humboldt Research Fellow from 2009 to 2011. His current research focuses on the design and fabrication of advanced electrode materials for rechargeable batteries, supercapacitors, and electrocatalysis.
"],"string":"[\n \"Acknowledgements\",\n \"
J.M. and T.L. contributed equally to this work. This work was supported by the National Natural Science Foundation (22379114, 51972235), the Natural Science Foundation of Shanghai (22ZR1465600), Innovation Program of Shanghai Municipal Education Commission (2023ZKZD32), and the Fundamental Research Funds for the Central Universities.
\",\n \"
\\nJin Ma is currently a Master's student at School of Chemical Sciences and Engineering, Tongji University. Her current research focuses on manganese‐based cathode materials for lithium‐ion batteries.
\",\n \"
\\nTingting Liu received her M.S. degree from Ningbo University in 2020 and she is now pursuing her Ph.D. degree at Tongji University. Her current research focuses on cathode materials for rechargeable lithium‐ion batteries.
\",\n \"
\\nJie Ma is a Professor in College of Environmental Science and Engineering at Tongji University. He received an M.S. degree from Hefei University of Technology in 2005, a Ph.D. degree from Shanghai Jiaotong University in 2009, and was a visiting scholar in Department of Chemical & Biomolecular Engineering at the University of Akron in 2015–2016. Ma's current research focuses on nanomaterial innovations and electrochemical technology for sustainable environment. He has published 150+ journal papers, with a total citation of 9000+ times and an h‐index of 53.
\",\n \"
\\nChi Zhang is a specially‐appointed tenured professor at Tongji University. He received his Ph.D. degree from Nanjing University, and worked at Nagoya University as Research Fellow of Japanese Society for the Promotion of Science and Technical University of Munich as Research Fellow of Alexander von Humboldt Foundation. Zhang's scientific interest is the development of advanced functional materials for optical and energy applications. He has published over 450 SCI papers including Advanced Materials, Journal of the American Chemical Society and Angewandte Chemie International Edition.
\",\n \"
\\nJinhu Yang is a specially‐appointed tenured professor at Tongji University. He received his Ph.D. degree from Peking University in 2005. Then, he worked at The Hong Kong University of Science and Technology as a Research Associate from 2005 to 2006, the University of Tokyo as a Foreign Researcher supported by JSPS organization from 2006 to 2008, and Munich University as a Humboldt Research Fellow from 2009 to 2011. His current research focuses on the design and fabrication of advanced electrode materials for rechargeable batteries, supercapacitors, and electrocatalysis.
\"\n]"},"figure":{"kind":"list like","value":["
a) Three main types of cathode materials commercialized for lithium‐ion batteries. Reproduced under terms of the CC‐BY license.[\n##REF##24202440##\n7\n##\n] Copyright 2014, M. Saiful Islam and Craig A. J. Fisher, published by Royal Society of Chemistry. Reproduced with permission.[\n##UREF##3##\n8\n##\n] Copyright 2017, American Chemical Society. b) Crustal content (marked in blue font) of metal element involved in commercialized cathode materials for LIBs. c) Radar maps for cathode materials commercialized for lithium‐ion batteries.
","
a) LiMn2O4 spinel crystal structure and Li+ ions diffuse rapidly between face‐sharing octahedral 16c and tetrahedral 8a sites. Reproduced with permission.[\n##UREF##19##\n26\n##\n] Copyright 2020, Springer Nature. Crystal structure of b) o‐LiMnO2, c) m‐LiMnO2. Reproduced with permission.[\n##UREF##22##\n29\n##\n] Copyright 2021, Elsevier. d) Metastable and cation‐disordered rocksalt‐type LiMnO2. Reproduced with permission.[\n##UREF##1##\n5\n##\n] Copyright 2018, Royal Society of Chemistry
","
a) The molecular orbital energy diagram of the octahedral MnO6 and the electronic orbitals of Mn2+/Mn3+/Mn4+ ions. b) A schematic of the octahedral MnO6 before and after the J–T distortion. Reproduced with permission.[\n##UREF##35##\n43\n##\n] Copyright 2021, Elsevier. c) XRD pattern evolution of the o‐LiMnO2 electrodes after different cycles. Reproduced with permission.[\n##UREF##36##\n45\n##\n] Copyright 2007, Springer Nature. d) Charge and discharge characteristics of the o‐LiMnO2 for 30 cycles within the voltage range of 2–4.5 V. Reproduced with permission.[\n##UREF##39##\n48\n##\n] Copyright 2004, Elsevier.
","
a) The supposed mechanism of the function of EDTA‐2Na in the hydrothermal reaction system to eliminate the Li2MnO3 and form pure o‐LiMnO2. XRD patterns of the resulting LiMnO2 samples at b) different EDTA‐2Na concentrations at 200 °C and c) different hydrothermal temperatures at 0.15 M EDTA‐2Na. Reproduced with permission.[\n##UREF##69##\n79\n##\n] Copyright 2020, Elsevier.
","
a) SEM images of o‐LiMnO2 nanoplates. Reproduced with permission.[\n##UREF##70##\n81\n##\n] Copyright 2009, Elsevier. b) TEM images of LiMnO2 powders. Reproduced with permission.[\n##UREF##71##\n82\n##\n] Copyright 2010, Elsevier. c) SEM images of the LiMnO2 nanorods. Reproduced with permission.[\n##UREF##72##\n83\n##\n] Copyright 2016, Elsevier. d) Schematic illustration of the preparation of the final LiMnO2 microcubes (v and vi). Reproduced with permission.[\n##UREF##73##\n84\n##\n] Copyright 2013, Springer.
","
a) A scheme showing the formation of o‐LiMnO2 nanorod. b) SEM micrograph of sample powders with different hydrothermal times. i) the MnOOH precursor, ii) 2 h incorporation, iii) 3 h incorporation, and iv) 4 h incorporation). Reproduced with permission.[\n##UREF##74##\n85\n##\n] Copyright 2007, Elsevier.
","
Cycling performance of the o‐LMO@Li2CO3 nanosheet array cathode at 2 C at a) 20 °C and b) 60 °C. c) Cycling performance of the uncoated o‐LMO cathode at 0.5 C. d) High‐resolution TEM image of o‐LMO@Li2CO3 nanosheet array electrode. e) Proposed interfacial change of the bare o‐LMO electrode and that coated with a Li2CO3 layer during charge/discharge process. f) Pathway of electron transport in the o‐LMO@Li2CO3 nanosheet array electrode. Reproduced with permission.[\n##REF##27270124##\n141\n##\n] Copyright 2016, American Chemical Society.
","
a) Schematic illustration of core‐shell structural architecture with the annealing treatment. b) The evolution of the phase composition with the temperature based on Rietveld refinements of the corresponding XRD patterns. c) TGA analysis of pristine LMO. d) Cycling stability at 25 °C and 50 mA g−1 and e) rate capability at a different current density from 10 to 1000 mA g−1 for the samples Li2MnO3, LMO, LMO‐PA450, and LMO‐PA750. Reproduced with permission.[\n##UREF##129##\n142\n##\n] Copyright 2022, Elsevier.
","
a) One‐step dynamic hydrothermal synthesis of o‐LiMnO2/CNTs composites. SEM images of b) 1%CNT‐LMO, c) 5%CNT‐LMO with one‐step dynamic hydrothermal synthesis. Reproduced with permission.[\n##UREF##132##\n145\n##\n] Copyright 2021, Elsevier. d) SEM image of o‐LiMnO2‐MWCNTs. Reproduced with permission.[\n##UREF##133##\n146\n##\n] Copyright 2019, Elsevier.
","
a) Synthesis of SPL‐LMO (CE, counter electrode; RE, reference electrode; WE, working electrode). b) Synthesis of SPL‐LMO/Mn3O4. c) HAADF‐STEM image for the SPL‐LMO nanoparticle. Scale bar, 5 nm. d) HAADF‐STEM image and e) ABF‐STEM image at the spinel‐layered interface for the SPL‐LMO sample along the [010] zone axis. Scale bar, 1 nm. f) Rate capability and cycle performance of the SPL‐LMO cathode. g) Cycle performance of the SPL‐LMO//graphite full cell. Reproduced with permission.[\n##UREF##134##\n148\n##\n] Copyright 2021, Springer Nature.
"],"string":"[\n \"
a) Three main types of cathode materials commercialized for lithium‐ion batteries. Reproduced under terms of the CC‐BY license.[\\n##REF##24202440##\\n7\\n##\\n] Copyright 2014, M. Saiful Islam and Craig A. J. Fisher, published by Royal Society of Chemistry. Reproduced with permission.[\\n##UREF##3##\\n8\\n##\\n] Copyright 2017, American Chemical Society. b) Crustal content (marked in blue font) of metal element involved in commercialized cathode materials for LIBs. c) Radar maps for cathode materials commercialized for lithium‐ion batteries.
\",\n \"
a) LiMn2O4 spinel crystal structure and Li+ ions diffuse rapidly between face‐sharing octahedral 16c and tetrahedral 8a sites. Reproduced with permission.[\\n##UREF##19##\\n26\\n##\\n] Copyright 2020, Springer Nature. Crystal structure of b) o‐LiMnO2, c) m‐LiMnO2. Reproduced with permission.[\\n##UREF##22##\\n29\\n##\\n] Copyright 2021, Elsevier. d) Metastable and cation‐disordered rocksalt‐type LiMnO2. Reproduced with permission.[\\n##UREF##1##\\n5\\n##\\n] Copyright 2018, Royal Society of Chemistry
\",\n \"
a) The molecular orbital energy diagram of the octahedral MnO6 and the electronic orbitals of Mn2+/Mn3+/Mn4+ ions. b) A schematic of the octahedral MnO6 before and after the J–T distortion. Reproduced with permission.[\\n##UREF##35##\\n43\\n##\\n] Copyright 2021, Elsevier. c) XRD pattern evolution of the o‐LiMnO2 electrodes after different cycles. Reproduced with permission.[\\n##UREF##36##\\n45\\n##\\n] Copyright 2007, Springer Nature. d) Charge and discharge characteristics of the o‐LiMnO2 for 30 cycles within the voltage range of 2–4.5 V. Reproduced with permission.[\\n##UREF##39##\\n48\\n##\\n] Copyright 2004, Elsevier.
\",\n \"
a) The supposed mechanism of the function of EDTA‐2Na in the hydrothermal reaction system to eliminate the Li2MnO3 and form pure o‐LiMnO2. XRD patterns of the resulting LiMnO2 samples at b) different EDTA‐2Na concentrations at 200 °C and c) different hydrothermal temperatures at 0.15 M EDTA‐2Na. Reproduced with permission.[\\n##UREF##69##\\n79\\n##\\n] Copyright 2020, Elsevier.
\",\n \"
a) SEM images of o‐LiMnO2 nanoplates. Reproduced with permission.[\\n##UREF##70##\\n81\\n##\\n] Copyright 2009, Elsevier. b) TEM images of LiMnO2 powders. Reproduced with permission.[\\n##UREF##71##\\n82\\n##\\n] Copyright 2010, Elsevier. c) SEM images of the LiMnO2 nanorods. Reproduced with permission.[\\n##UREF##72##\\n83\\n##\\n] Copyright 2016, Elsevier. d) Schematic illustration of the preparation of the final LiMnO2 microcubes (v and vi). Reproduced with permission.[\\n##UREF##73##\\n84\\n##\\n] Copyright 2013, Springer.
\",\n \"
a) A scheme showing the formation of o‐LiMnO2 nanorod. b) SEM micrograph of sample powders with different hydrothermal times. i) the MnOOH precursor, ii) 2 h incorporation, iii) 3 h incorporation, and iv) 4 h incorporation). Reproduced with permission.[\\n##UREF##74##\\n85\\n##\\n] Copyright 2007, Elsevier.
\",\n \"
Cycling performance of the o‐LMO@Li2CO3 nanosheet array cathode at 2 C at a) 20 °C and b) 60 °C. c) Cycling performance of the uncoated o‐LMO cathode at 0.5 C. d) High‐resolution TEM image of o‐LMO@Li2CO3 nanosheet array electrode. e) Proposed interfacial change of the bare o‐LMO electrode and that coated with a Li2CO3 layer during charge/discharge process. f) Pathway of electron transport in the o‐LMO@Li2CO3 nanosheet array electrode. Reproduced with permission.[\\n##REF##27270124##\\n141\\n##\\n] Copyright 2016, American Chemical Society.
\",\n \"
a) Schematic illustration of core‐shell structural architecture with the annealing treatment. b) The evolution of the phase composition with the temperature based on Rietveld refinements of the corresponding XRD patterns. c) TGA analysis of pristine LMO. d) Cycling stability at 25 °C and 50 mA g−1 and e) rate capability at a different current density from 10 to 1000 mA g−1 for the samples Li2MnO3, LMO, LMO‐PA450, and LMO‐PA750. Reproduced with permission.[\\n##UREF##129##\\n142\\n##\\n] Copyright 2022, Elsevier.
\",\n \"
a) One‐step dynamic hydrothermal synthesis of o‐LiMnO2/CNTs composites. SEM images of b) 1%CNT‐LMO, c) 5%CNT‐LMO with one‐step dynamic hydrothermal synthesis. Reproduced with permission.[\\n##UREF##132##\\n145\\n##\\n] Copyright 2021, Elsevier. d) SEM image of o‐LiMnO2‐MWCNTs. Reproduced with permission.[\\n##UREF##133##\\n146\\n##\\n] Copyright 2019, Elsevier.
\",\n \"
a) Synthesis of SPL‐LMO (CE, counter electrode; RE, reference electrode; WE, working electrode). b) Synthesis of SPL‐LMO/Mn3O4. c) HAADF‐STEM image for the SPL‐LMO nanoparticle. Scale bar, 5 nm. d) HAADF‐STEM image and e) ABF‐STEM image at the spinel‐layered interface for the SPL‐LMO sample along the [010] zone axis. Scale bar, 1 nm. f) Rate capability and cycle performance of the SPL‐LMO cathode. g) Cycle performance of the SPL‐LMO//graphite full cell. Reproduced with permission.[\\n##UREF##134##\\n148\\n##\\n] Copyright 2021, Springer Nature.
\"\n]"},"table":{"kind":"list like","value":["
Comparison of ionic radius and electrochemical performance of different elements doped into LiMnO2.
Doping ions
Ionic radius [nm]
Cathode
Voltage range [V vs Li/Li+]
Initial discharge capacity [mAh g−1]/current density
Discharge capacity [mAh g−1]/retention/cycles
Reference
Na+\n
0.102
Li0.53Na0.03MnO2\n
2.4–4.5
180/0.2 C
200/111.1%/50
[##UREF##82##93##]
Cu2+\n
0.072
Li0.75Mn0.99Cu0.012O2\n
2.0–4.3
219/60 mA g−1\n
≈195/89%/50
[##UREF##83##94##]
Mg2+\n
0.072
LiMn0.95Mg0.05O2\n
2.0–4.5
150/1/7 C
≈150/100%/30
[##UREF##84##95##]
Zn2+\n
0.074
LiMn0.95Zn0.05O2\n
2.0–4.5
185/–
≈178/96.2%/30
[##UREF##85##96##]
Ni2+/Ni3+\n
0.069/0.056
LiMn0.95Ni0.05O2\n
2.4–4.8
220/25 mA g−1\n
≈215/97.7%/50
[##UREF##86##97##]
LiMn0.95Ni0.05O2\n
2.0–4.5
220/1/7 C
≈180/81.8%/30
[##UREF##84##95##]
Li0.7[Ni1/6Mn5/6]O2\n
2.0–4.8
198/1/3 C
190/96%/.25
[##UREF##88##99##]
Al3+\n
0.054
\n
m‐Li\nx\nAl0.05Mn0.95O2\n
\n
o‐Li\nx\nAl0.05Mn0.95O2\n
\n
\n
2.0–4.4
\n
2.0–4.4
\n
\n
≈150/26.1 mA g−1\n
\n
≈25/75 mA g−1\n
\n
\n
188/125.3%/50
\n
146/636.6%/100
\n
\n
[##UREF##92##103##]
\n
[##UREF##92##103##]
\n
Li0.93Mn0.96Al0.04O2\n
2.0–4.3
175/0.1 C
145/82.9%/25
[##UREF##94##105##]
Co3+\n
0.055
Li0.9Mn0.9Co0.1O2\n
2.6–4.8
210/0.1 mA cm−2\n
162/77.1%/50
[##UREF##86##97##]
Li\nx\nMn0.975Co0.025O2\n
2.4–4.6
200/25 mA g−1\n
188/94%/100
[##UREF##96##107##]
LiMn0.9Co0.1O2\n
2.0–4.3
71/45 mA g−1\n
170/239.1%/100
[##UREF##98##109##]
Cr3+\n
0.061
LiMn0.9Cr0.1O2\n
2.5–4.5
200/0.1 C
200/100%/30
[##UREF##106##117##]
\n
R‐LiMn0.85Cr0.15O2\n
\n
S‐LiMn0.85Cr0.15O2\n
\n
\n
2.0–4.4
\n
2.0–4.4
\n
\n
180/50 mA g−1\n
\n
144/50 mA g−1\n
\n
\n
169/93.9%/40
\n
121/84%/40
\n
\n
[##UREF##107##118##]
\n
[##UREF##107##118##]
\n
Y3+\n
0.090
LiMn0.98Y0.02O2\n
2.0–4.4
191/25 mA g−1\n
173/90.6%/20
[##UREF##108##119##]
Fe3+\n
0.055
LiMn0.95Fe0.05O2\n
2.0–4.5
200/–
≈180/90%/30
[##UREF##85##96##]
LiMn0.95Fe0.05O2\n
2.0–4.3
≈60/22.5 mA g−1\n
≈68/113.3%/50
[##UREF##109##120##]
Ti3+\n
0.076
LiMn0.95Ti0.05O2\n
2.0–4.5
≈32/0.2 C
≈132/412.5%/60
[##UREF##110##121##]
B3+\n
0.023
m‐LiMn0.95B0.05O2\n
2.0–4.5
≈152/50 mA g−1\n
150/98.7%/100
[##UREF##47##56##]
Bi3+\n
0.096
Li0.75Mn0.99Bi0.011O2\n
2.0–4.3
242/60 mA g−1\n
≈175/72.3%/50
[##UREF##83##94##]
S2−\n
0.184
Li0.56MnO1.98S0.02\n
2.0–4.6
170/0.4 mA cm−2\n
220/117.6%/50
[##UREF##111##122##]
F−\n
0.136
Li0.86MnO1.98F0.02\n
2.0–4.3
129/50 mA g−1\n
210/162.8%/50
[##UREF##112##123##]
BO3\n3−\n
–
Li0.64MnO1.991(BO3)0.009\n
2.0–4.3
≈209/60 mA g−1\n
≈163/78%/50
[##UREF##83##94##]
PO4\n3−\n
–
Li0.67MnO1.993(PO3)0.007\n
2.0–4.3
≈221/60 mA g−1\n
≈137/62%/50
[##UREF##83##94##]
SiO3\n3−\n
–
Li0.65MnO1.988(SiO3)0.012\n
2.0–4.3
≈173/60 mA g−1\n
≈162/93.6%/50
[##UREF##83##94##]
John Wiley & Sons, Ltd."],"string":"[\n \"
Comparison of ionic radius and electrochemical performance of different elements doped into LiMnO2.
Doping ions
Ionic radius [nm]
Cathode
Voltage range [V vs Li/Li+]
Initial discharge capacity [mAh g−1]/current density
Carbapenem‐resistant Enterobacterales (CRE) has been categorized as the highest priority pathogens for treatment by the World Health Organization.[\n##REF##30423057##\n1\n##\n] Carbapenemase is the main resistance determinant of CRE that renders bacterial resistance to nearly all β‐lactams antibiotics, including carbapenems.[\n##REF##22000347##\n2\n##\n] New Delhi Metallo‐β‐lactamases (NDMs) are one of the most prevalent carbapenemases and have spread over 70 countries in clinical settings since their discovery in 2009.[\n##REF##30700432##\n3\n##\n] In particular, the NDMs‐producing CRE can trigger multiple types of severe infection (e.g., pneumonia, septicemia, and abscesses), and kill almost half of infected in‐patients.[\n##REF##21623026##\n4\n##\n] NDMs is a Zn(II)‐dependent periplasmic enzyme that activates nucleophilic water to destroy the β‐lactam ring of carbapenems, thus resulting in poor clinical outcomes.[\n##REF##21507902##\n5\n##\n] Considering the existing antibiotic treatment failure combined with new antibiotics void,[\n##REF##35203785##\n6\n##\n] NDMs‐producing Enterobacterales leaves clinicians with few choices from the antibiotic pipeline.
","
To date, an economical and effective strategy for tackling NDMs producers is to revitalize existing antibiotics using antibiotic adjuvants.[\n##REF##31388008##\n7\n##\n] They are usually NDM inhibitors that decrease enzymatic function via kicking out the crucial Zn(II) cofactors, binding with amino acid residue of active sites, or mimicking the NDMs substrates.[\n##REF##30959050##\n8\n##\n] Among them, inhibitors with Zn(II) deprivation action, such as ethylenediamine‐N,N,N′,N′‐tetraacetate (EDTA), aspergillomarasmine A or bismuth (Bi(III)) compounds, have garnered more attention under their great potential in restoring the susceptibility of NDMs producers to carbapenems.[\n##REF##24965651##\n9\n##\n] However, such inhibitors indiscriminately displace Zn(II) from commensal bacteria and mammalian cells, therefore impairing many biological functions and triggering high off‐target toxicity in vivo application.[\n##REF##24965651##\n9b\n##\n] Additionally, the bacterial outer membrane has been recognized as an impermeable barrier, which hindered intracellular antibiotic and adjuvant accumulation.[\n##REF##30022160##\n10\n##\n] Recently, Nanotechnology has been promising for antibiotic adjuvant development due to its ability to control the loading, delivery, and release of antibiotics and to enhance the antibacterial potency.[\n##UREF##0##\n11\n##\n] However, spatial and temporal control remains an unresolved obstacle for nanoparticle‐constituent adjuvants due to off‐target distributions, systemic delivery, and limited modulatory effects.
","
Bismuth compound has been shown to irreversibly inactive NDMs via replacing zinc ions in the NDMs active site.[\n##REF##24965651##\n9b\n##\n] Developing a bismuth‐based nanoadjuvant that simultaneously overcome bacterial membrane barrier and precisely inactivate NDMs provides a new opportunity to reverse carbapenems resistance in NDMs producer. However, existing bismuth‐based nanoparticles are faced with a series of shortcomings: the lower loading capacity for ions, uncontrolled release of ions, and synthetic complexity.[\n##REF##16444262##\n12\n##\n] Here, bismuth potassium citrate (BPC) granule, a low‐price stomach medicine (<1 China Yuan/g), was directly converted into high‐security bismuth nanoclusters (BiNCs) via UV irradiation. BiNCs was found to not only be safe for use in vivo, but also have high bismuth ion loading, ROS‐responsive dissociation, and antibiotic adsorption capabilities. These characteristics are expected to endow BiNCs with an excellent targeted‐NDMs inhibitor.
","
Selectively overcoming bacterial outer membrane barrier that delivers BiNCs into bacterial periplasm is another tricky problem. Liposome fusion‐based transport (LiFT) strategy features direct drugs delivery into cells via a vesicle‐cell fusion process, providing a robust tool for breaking outer membrane barrier.[\n##UREF##1##\n13\n##\n] Although various membrane fusion liposome (MFL) has been developed, most of them was applied into mammalian cell transportation.[\n##REF##34094837##\n14\n##\n] A deeper excavation and application is urgent in bacterial transportation. Previous studies found that liposome consisting of L‐α‐phosphatidylcholine (EggPC) and cholesterol can fuse with outer membrane of Gram‐negative bacteria,[\n##UREF##2##\n15\n##\n] in which abundant EggPC provides a moderate phase transition temperature to maintain the fluidity of the lipid shell similar to bacterial membrane for fusion. Additionally, rational designing a bacterial anchored‐MFL further assists in dehydrating the gap between the lipid shell and bacterial membrane, accelerating fusion process. More iomportantly, membrane fusion could change membrane permeability, and then induce the initiation of ROS‐related signal pathway,[\n##REF##25962045##\n16\n##\n] providing a clue for intracellular bismuth ions release. Hence, pathogen‐targeted LiFT strategy would have a significant potential to selectively overcoming bacterial outer membrane barrier.
","
Herein, we designed a pathogen‐primed liposomal antibiotic booster for eradicating NDMs‐producing CRE via specifically inactivating periplasmic NDMs and then potentiating the efficiency of meropenem, a broad‐spectrum carbapenem (Scheme ##FIG##0##\n1\n##). High‐security bismuth nanoclusters (BiNCs) act as the core for meropenem loading. The fusion‐type liposome made of L‐α‐phosphatidylcholine (EggPC), cholesterol, and DSPE‐PEG‐maltodextrin (M‐MFL) is used as targeting shell for wrapping the meropenem‐loaded BiNCs (MB). Owing to bacteria‐specific maltodextrin transport pathway, the antibiotic booster could selectively target on pathogen with the assistance of maltodextrin corona. After successfully anchoring to pathogen surface, a rapid membrane fusion behavior between liposome and bacteria broke membrane barrier for direct and efficient intracellular MB accumulation, activated intracellular ROS amplification and then triggered the intracellular‐specific release of Bi(III) and meropenem. Released Bi(III) irreversibly inhibited NDMs via displacing Zn(II) in NDMs active sites, preventing meropenem hydrolyzing. Mice infection models revealed that the antibiotic booster restores meropenem efficacy against clinical NDMs‐producing pathogen. Taken together, we developed a nanoadjuvant‐platform for re‐potentiating meropenem activity with high specificity and effectiveness, to address the severe infections caused by NDMs‐producing CRE.
"],"string":"[\n \"Introduction\",\n \"
Carbapenem‐resistant Enterobacterales (CRE) has been categorized as the highest priority pathogens for treatment by the World Health Organization.[\\n##REF##30423057##\\n1\\n##\\n] Carbapenemase is the main resistance determinant of CRE that renders bacterial resistance to nearly all β‐lactams antibiotics, including carbapenems.[\\n##REF##22000347##\\n2\\n##\\n] New Delhi Metallo‐β‐lactamases (NDMs) are one of the most prevalent carbapenemases and have spread over 70 countries in clinical settings since their discovery in 2009.[\\n##REF##30700432##\\n3\\n##\\n] In particular, the NDMs‐producing CRE can trigger multiple types of severe infection (e.g., pneumonia, septicemia, and abscesses), and kill almost half of infected in‐patients.[\\n##REF##21623026##\\n4\\n##\\n] NDMs is a Zn(II)‐dependent periplasmic enzyme that activates nucleophilic water to destroy the β‐lactam ring of carbapenems, thus resulting in poor clinical outcomes.[\\n##REF##21507902##\\n5\\n##\\n] Considering the existing antibiotic treatment failure combined with new antibiotics void,[\\n##REF##35203785##\\n6\\n##\\n] NDMs‐producing Enterobacterales leaves clinicians with few choices from the antibiotic pipeline.
\",\n \"
To date, an economical and effective strategy for tackling NDMs producers is to revitalize existing antibiotics using antibiotic adjuvants.[\\n##REF##31388008##\\n7\\n##\\n] They are usually NDM inhibitors that decrease enzymatic function via kicking out the crucial Zn(II) cofactors, binding with amino acid residue of active sites, or mimicking the NDMs substrates.[\\n##REF##30959050##\\n8\\n##\\n] Among them, inhibitors with Zn(II) deprivation action, such as ethylenediamine‐N,N,N′,N′‐tetraacetate (EDTA), aspergillomarasmine A or bismuth (Bi(III)) compounds, have garnered more attention under their great potential in restoring the susceptibility of NDMs producers to carbapenems.[\\n##REF##24965651##\\n9\\n##\\n] However, such inhibitors indiscriminately displace Zn(II) from commensal bacteria and mammalian cells, therefore impairing many biological functions and triggering high off‐target toxicity in vivo application.[\\n##REF##24965651##\\n9b\\n##\\n] Additionally, the bacterial outer membrane has been recognized as an impermeable barrier, which hindered intracellular antibiotic and adjuvant accumulation.[\\n##REF##30022160##\\n10\\n##\\n] Recently, Nanotechnology has been promising for antibiotic adjuvant development due to its ability to control the loading, delivery, and release of antibiotics and to enhance the antibacterial potency.[\\n##UREF##0##\\n11\\n##\\n] However, spatial and temporal control remains an unresolved obstacle for nanoparticle‐constituent adjuvants due to off‐target distributions, systemic delivery, and limited modulatory effects.
\",\n \"
Bismuth compound has been shown to irreversibly inactive NDMs via replacing zinc ions in the NDMs active site.[\\n##REF##24965651##\\n9b\\n##\\n] Developing a bismuth‐based nanoadjuvant that simultaneously overcome bacterial membrane barrier and precisely inactivate NDMs provides a new opportunity to reverse carbapenems resistance in NDMs producer. However, existing bismuth‐based nanoparticles are faced with a series of shortcomings: the lower loading capacity for ions, uncontrolled release of ions, and synthetic complexity.[\\n##REF##16444262##\\n12\\n##\\n] Here, bismuth potassium citrate (BPC) granule, a low‐price stomach medicine (<1 China Yuan/g), was directly converted into high‐security bismuth nanoclusters (BiNCs) via UV irradiation. BiNCs was found to not only be safe for use in vivo, but also have high bismuth ion loading, ROS‐responsive dissociation, and antibiotic adsorption capabilities. These characteristics are expected to endow BiNCs with an excellent targeted‐NDMs inhibitor.
\",\n \"
Selectively overcoming bacterial outer membrane barrier that delivers BiNCs into bacterial periplasm is another tricky problem. Liposome fusion‐based transport (LiFT) strategy features direct drugs delivery into cells via a vesicle‐cell fusion process, providing a robust tool for breaking outer membrane barrier.[\\n##UREF##1##\\n13\\n##\\n] Although various membrane fusion liposome (MFL) has been developed, most of them was applied into mammalian cell transportation.[\\n##REF##34094837##\\n14\\n##\\n] A deeper excavation and application is urgent in bacterial transportation. Previous studies found that liposome consisting of L‐α‐phosphatidylcholine (EggPC) and cholesterol can fuse with outer membrane of Gram‐negative bacteria,[\\n##UREF##2##\\n15\\n##\\n] in which abundant EggPC provides a moderate phase transition temperature to maintain the fluidity of the lipid shell similar to bacterial membrane for fusion. Additionally, rational designing a bacterial anchored‐MFL further assists in dehydrating the gap between the lipid shell and bacterial membrane, accelerating fusion process. More iomportantly, membrane fusion could change membrane permeability, and then induce the initiation of ROS‐related signal pathway,[\\n##REF##25962045##\\n16\\n##\\n] providing a clue for intracellular bismuth ions release. Hence, pathogen‐targeted LiFT strategy would have a significant potential to selectively overcoming bacterial outer membrane barrier.
\",\n \"
Herein, we designed a pathogen‐primed liposomal antibiotic booster for eradicating NDMs‐producing CRE via specifically inactivating periplasmic NDMs and then potentiating the efficiency of meropenem, a broad‐spectrum carbapenem (Scheme ##FIG##0##\\n1\\n##). High‐security bismuth nanoclusters (BiNCs) act as the core for meropenem loading. The fusion‐type liposome made of L‐α‐phosphatidylcholine (EggPC), cholesterol, and DSPE‐PEG‐maltodextrin (M‐MFL) is used as targeting shell for wrapping the meropenem‐loaded BiNCs (MB). Owing to bacteria‐specific maltodextrin transport pathway, the antibiotic booster could selectively target on pathogen with the assistance of maltodextrin corona. After successfully anchoring to pathogen surface, a rapid membrane fusion behavior between liposome and bacteria broke membrane barrier for direct and efficient intracellular MB accumulation, activated intracellular ROS amplification and then triggered the intracellular‐specific release of Bi(III) and meropenem. Released Bi(III) irreversibly inhibited NDMs via displacing Zn(II) in NDMs active sites, preventing meropenem hydrolyzing. Mice infection models revealed that the antibiotic booster restores meropenem efficacy against clinical NDMs‐producing pathogen. Taken together, we developed a nanoadjuvant‐platform for re‐potentiating meropenem activity with high specificity and effectiveness, to address the severe infections caused by NDMs‐producing CRE.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results","The synthesis and characterization of bismuth nanoclusters (BiNCs)","
\nFigure ##FIG##1##\n1a## illustrates the preparation approach of BiNCs. A clinically available stomach medicine BPC (oral bismuth potassium citrate granules) was synthesized into BiNCs via a one‐step UV irradiation method.[\n##UREF##3##\n17\n##\n] The citrate and carboxymethyl cellulose (CMC) used in the synthesis are auxiliary materials in BPC, thereby avoiding the use of harmful reagents and residues of by‐products throughout the synthesis. The buffer containing BPC appeared colorless, whereas BiNCs appeared dark black (Figure ##SUPPL##0##S1##). Dynamic light scattering revealed that the hydrodynamic diameter of BiNCs was 21.04 ± 0.99 nm, and the zeta potential was −57.8 ± 4.27 mV (Figure ##FIG##1##1b##). TEM images showed the spherical morphology of the BiNCs with a typical lattice structure and a spacing of about 0.244 nm with a homogeneous size (Figure ##FIG##1##1c and d##). The peaks at 4f7 and 4f5 in X‐ray photoelectron spectroscopy (XPS) indicated the characteristic peaks of bismuth element (Figure ##FIG##1##1e##). The TEM elemental mappings also showed the uniform distribution of Bi elements in BiNCs (Figure ##FIG##1##1f and g##). Afterward, the X‐ray powder diffraction (XRD) pattern verified that the as‐prepared BiNCs were typical bismuth phases (Figure ##FIG##1##1h##). All these results confirmed the successful preparation of BiNCs.
","
Notably, we identified a reactive oxygen species (ROS)‐related Bi(III) release in BiNCs, which was enhanced with the increasing of H2O2 concentration (P < 0.01, Figure ##FIG##1##1i##). Morphological and hydrodynamic diameter changes of BiNCs in the presence of H2O2 also reflected ROS‐responsive disintegration of BiNCs (Figure ##FIG##1##1j##, Figure ##SUPPL##0##S2##). BiNCs also presented excellent photoacoustic (PA) imaging properties (Figure ##FIG##1##1k##), promising to realize visualization of pathogen tracing in vivo. We further verified BiNCs can respond to ROS to resensitize NDMs producers specifically toward meropenem (MEM) in a clinical NDM‐1‐producing E. coli isolate EC1322 recovered from peritoneal drainage fluid (Table ##SUPPL##0##S1##, Figure ##SUPPL##0##S3##). Compared with the BiNCs‐MEM group, the BiNCs‐MEM‐H2O2 group exhibited synergistic growth inhibition toward EC1322 with a fractional inhibitory concentration index (FICI) of 0.375 (Figure ##FIG##2##\n2a‐c##), and the bacterial amounts plummeted (P < 0.01) and their outgrowth was blocked throughout 4 h exposure (Figure ##FIG##2##2d##). While H2O2 itself, even at 400 µM, showed no growth inhibition and no synergistic interaction with MEM toward EC1322 (Figure ##SUPPL##0##S4##). Additionally, BiNCs could reduce the minimal inhibitory concentration (MIC) values of MEM toward NDM‐1 producer but not NDM‐1 negative strain in the presence of H2O2 (Figure ##SUPPL##0##S5##).
","
We further used an engineering E. coli BL21 expressing periplasmic NDM‐1 to demonstrate ROS‐powered Bi(III) release from BiNCs inactivating NDMs. Compared with single BiNCs treatment, H2O2‐pretreated BiNCs presented a more significantly obstructive effect on the hydrolysis rate of MEM in BL21 (Figure ##FIG##2##2e##). The NDM‐1 activity decreased as the 200 µM H2O2‐pretreated BiNCs concentration escalated (IC50 = 0.046 mg/mL), ultimately leading to the inhibition of ∼80% activities of NDM‐1 (Figure ##FIG##2##2f## and Figure ##SUPPL##0##S6##). Enzyme kinetics analysis revealed that the apparent Vmax of NDM‐1 decreased from 10.11 to 2.53 µM/s when 200 µM H2O2 – pretreated BiNCs concentration increased from 0 to 100 µg/mL, and a typical non‐competitive or an irreversible inhibition was observed according to the relevant Line‐weaver Burk plot (Figure ##FIG##2##2g##). Next, we found that the appearance of an absorption band at 340 nm after incubating apo‐NDM‐1 (lack of Zn(II)) with H2O2‐pretreated BiNCs (Figure ##SUPPL##0##S7##), which is characteristic for Bi–S ligand‐to‐metal charge transfer (LMCT) band, suggesting that the Bi(III) from BiNCs could bound to NDM‐1. Moreover, ICP‐MS results further revealed that the addition of increasing amounts of Bi(III) resulted in a Zn(II) removal in NDM‐1, accompanied by Bi(III) bound to NDM‐1 (Figure ##FIG##2##2h## and Figure ##SUPPL##0##S8##). Then, the affinities of Bi(III) from H2O2‐pretreated BiNCs to NDM‐1 were closely examined by isothermal titration calorimetry (ITC), unveiling that Bi(III) rather than BiNCs have a high affinity to NDM‐1 (Figure ##FIG##2##2i and j##), and the cellular thermal shift assay further reflecting the binding of intracellular BiNCs‐released Bi(III) to NDM‐1 in intact cells (Figure ##FIG##2##2k##). Together, Bi(III) released from BiNCs in response to ROS competitively replaces the Zn(II) to bind to NDM‐1, thereby hampering the activity of NDM‐1 and leading to carbapenem resistance reversal of NDM‐1‐producing E. coli. (Figure ##FIG##2##2l##).
","Fabrication and Characterization of Pathogen‐Primed Liposomal Antibiotic Booster","
To integrate pathogen‐targeting, precise intracellular delivery, and site‐specific release of drug capabilities, we produced pathogen‐targeting liposomal antibiotic booster (M‐MFL@MB) using a maltodextrin‐cloaked membrane‐fusion liposome (M‐MFL) as a shell and meropenem‐loaded BiNCs (MB) as a core (Figure ##FIG##3##\n3a##). In this system, MEM loaded into BiNCs with a 50% loading yield using an optimal input of 3.2 wt% (Figure ##SUPPL##0##S9##). Maltodextrin (MA)‐PEG‐DSPE successfully anchored to membrane‐fusion liposome (MFL) consisting of L‐α‐phosphatidylcholine (EggPC) and cholesterol via a phospholipids fusion,[\n##REF##30657302##\n18\n##\n] and the modification rate of MA was counted as 0.52 mg/mL. Then, MB was stored into the lumen of M‐MFL with a 29.5% loading yield (Figure ##SUPPL##0##S9##). M‐MFL@MB (166 nm) was slightly larger than M‐MFL (154 nm) and much larger than naked MB (21 nm). M‐MFL@MB possessed an equivalent surface charge (Figure ##FIG##3##3b##), a uniform and spherical structure with a unilamellar membrane coating, which is similar to that of M‐MFL (Figure ##FIG##3##3c##). Cryo‐TEM revealed the successful cloaking of MB into M‐MFL, reflected by a deeper image lining degree from M‐MFL to M‐MFL@MB (Figure ##FIG##3##3c##). M‐MFL protected MB from disaggregation under physiological (H2O2 concentration = 10 µM) and infectious (H2O2 concentration = 100 µM) microenvironment [\n##REF##31218078##\n19\n##\n] (Figure ##FIG##3##3d and e##). Once the shell is removed, MB could rapidly release MEM and Bi(III) in an H2O2 concentration‐dependent manner, 73% MEM and 80% Bi(III) were released under the 200 µM H2O2 treatment.
","
As a major microbial carbon source, maltodextrin (MA) is selectively internalized into bacterial cells through bacterial‐specific maltodextrin transporter, but hardly enters mammalian cells.[\n##REF##21765397##\n20\n##\n] As expected, MA corona endowed M‐MFL@MB with a more distinguished bacteria adhesion property than that of MFL@MB (Figure ##FIG##3##3f##). We further imaged M‐MFL@MB and MFL@MB in coculture with green fluorescence protein (GFP)‐expressing E.coli and mononuclear macrophages (RAW264.7 cells) (Figure ##FIG##3##3g##), and observed M‐MFL@MB targeted E.coli but not RAW264.7 cells (Figure ##FIG##3##3h##). The line‐scan profiles also denoted the specific co‐localization of M‐MFL@MB and bacteria. Whilst MFL@MB did not present differential targeting to E.coli and RAW264.7 cells (Figure ##FIG##3##3i##). Additionally, liposomal antibiotic booster also presented an excellent immune escape capability via an obvious reduction of endocytosis by monocyte‐macrophage (Figure ##SUPPL##0##S10a and b##).
","In Situ Bacterial Membrane Fusion, Site‐Specific Drug Transport, and Intracellular ROS Burst","
M‐MFL formulation was first examined for its fusion capability with GFP‐expressing E. coli (Figure ##FIG##4##\n4a and c##). The intensity of red fluorescence (MFL) in M‐MFL group was higher than that in MFL group, implying MA‐mediated pathogen targeting promotes fusion activity by accelerating bacterial adhesion. Meanwhile, flow cytometer analysis demonstrated the successful fusion (Figure ##SUPPL##0##S11##). Compared with common liposomes constituted by soybean lecithin and cholesterol, M‐MFL exhibited a prominent membrane fusion activity due to a more similar ingredient with bacterial membrane and a higher lipid fluidity [\n##REF##27725960##\n21\n##\n] (Figure ##SUPPL##0##S12##). Förster resonance energy transfer (FRET) assay further demonstrated the specific bacterial OM fusion of M‐MFL, which was observed successfully fused with E. coli but hardly with platelets (Figure ##SUPPL##0##S13##). We then demonstrated membrane fusion strategy could promote intracellular accumulation of drugs. CLSM and flow cytometry reflected that more Cy5 (substituting MB) was located inside the bacteria in M‐MFL group compared with the other groups (Figure ##FIG##4##4b and c##; Figure ##SUPPL##0##S14##). Bio‐TEM assay showed that MB reached inside bacteria with the aid of MFL (Figure ##FIG##4##4d##). TEM elemental mappings and ICP‐MS assay further revealed the presence of abundant bismuth inside bacteria in M‐MFL@MB and MFL@MB groups (Figure ##FIG##4##4d and e##).
","
ROS can trigger the dissociation of MB to release MEM and Bi(III). However, the level of ROS, especially H2O2, inherent within the bacteria is less than 10 µM, only triggering little drug release (Figure ##FIG##3##3d and e##). We identified that MFL‐mediated membrane fusion strategies could endogenously trigger intracellular ROS production (Figure ##FIG##4##4f## and Figure ##SUPPL##0##S15‐16a##). Even at 30 min, the ROS level in M‐MFL and M‐MFL@MB groups still existed steadily with no significant decreasing trend (Figure ##SUPPL##0##S16b##), indicating that membrane fusion strategy initiated a rapid and stable ROS burst inside bacteria. The H2O2 produced inside bacteria was dependent on M‐MFL concentration, and E.coli incubated with 1.8 mg/mL M‐MFL produced almost 100 µM H2O2, which is adequate for MB disintegration (Figure ##FIG##4##4g##). M‐MFL could result in the generation of a variety of bacterial ROS species, including O2−•, 1O2, •OH, all of which have high oxidative activity for catalyzing intracellular MB disintegration. In addition, M‐MFL‐mediated membrane fusion strengthened the permeability of outer membrane and inner membrane in E.coli due to the impaired integrity of bacterial membrane (Figure ##SUPPL##0##S17a and b##). Enhanced membrane permeability would trigger a change in intracellular osmotic pressure, leading to intra‐ and extracellular ions (e.g., Na+ and Cl−) homeostasis disrupted and then resulted in membrane potential depolarization, which acts as a stimulation trigger, and lastly endogenously activates intracellular ROS amplification (Figure ##SUPPL##0##S17c##).[\n##REF##25962045##\n16\n##\n] Together, liposomal antibiotic booster can serve as potent NDMs inactivator to reverse MEM resistance by a cascading process: selectivity targeting over bacteria, breaking membrane barrier, specifically accumulating intracellular drugs, endogenously activating ROS amplification for intracellular‐specific release of Bi(III) and MEM (Figure ##FIG##4##4i and j##). Thus, M‐MFL@MB prevents MEM from hydrolyzing in NDM‐1 producer more effectively, compared with MEM, MB and M‐MFL@MEM (P < 0.01, Figure ##SUPPL##0##S18##).
","Liposomal Antibiotic Booster Resensitizes NDM‐1‐Producing <italic toggle=\"yes\">E. Coli</italic> to MEM In Vitro","
The bactericide of targeting liposomal antibiotic booster was evaluated using six NDM‐1‐producing clinical E. coli isolates (Table ##SUPPL##0##S1##, Figure ##SUPPL##0##S3##). These strains showed much higher MICs (> 64 to 2 µg/mL) in individual MEM, BiNCs, MB, M‐MFL and M‐MFL@MEM groups, respectively (Figure ##FIG##5##\n5a## and Figure ##SUPPL##0##S19##). Whilst M‐MFL@MB exhibited a concentrate‐dependent inhibitory effect (8 to 2 µg/mL) on all tested isolates (Figure ##FIG##5##5b##), indicating that M‐MFL@MB could reverse MEM resistance in NDM producers. Time‐dependent killing of EC1322 showed M‐MFL@MB had excellent bactericidal activity against NDMs producer (Figure ##FIG##5##5c##), also reflected by SEM results (Figure ##FIG##5##5d##). Additionally, M‐MFL@MB destructed more exhaustive bacterial structure than other groups (Figure ##FIG##5##5d##). These results suggested that targeting liposomal antibiotic boosters could resensitize NDM‐1‐producing E. coli to MEM in vitro. Notably, the antibiotic booster also showed synergies with ceftazidime (β‐lactam antibiotics) against NDM‐1‐producing E. coli isolates, but not with ciprofloxacin (quinolones) and colistin (polypeptide antibiotics), as shown in Fig. ##SUPPL##0##S20 and S21##. The result implied that antibiotic booster mainly induces NDM‐1 inactivation, lastly reversing NDMs producer resistance against β‐lactam antibiotics.
","Liposomal Antibiotic Booster Targets Bacterial Infectious Sites and Restores Meropenem Efficacy In Vivo","
The targeting capability of M‐MFL@MB was evaluated in zebrafish infected with GFP‐expressing E. coli (Figure ##FIG##6##\n6a##). Compared with MFL group, M‐MFL group showed a more pronounced red fluorescence (RhB) trapped inside the bacteria with green fluorescence in zebrafish, demonstrating the M‐MFL can effectively recognize and adhere to the bacteria under the assist of MA (Figure ##FIG##6##6b1 and b2##). Whilst M‐MFL cannot retained inside the healthy zebrafish (Figure ##FIG##6##6b3##). Moreover, MA crown could distinguish bacterial infection from inflammation in a mouse bacterial and inflammatory co‐infection model. IVIS imaging showed more M‐MFL could accumulate effectively and specifically into the bacterial infectious tissue rather than inflammatory tissue (Figure ##SUPPL##0##S22a‐e##), and ex vivo tissues imaging further verified the MA‐mediated specific bacterial targeting ability of M‐MFL (Figure ##FIG##6##6c and d##). The mouse lung infection model further revealed that M‐MFL could facilitate drugs accumulation at infectious sites, as RhB‐labelled M‐MFL@Cy5, not MFL@Cy5, could be gradually accumulated into infected lung, and presented an excellent co‐location with bacteria in infected tissues (Figure ##FIG##6##6e and f##).
","
Pharmacokinetic analysis revealed higher concentrations of M‐MFL@MB (86.32‐15.67 µg/mL) in plasma compared with the free MEM (81.85‐5.25 µg/mL) at the indicating time points (Figure ##SUPPL##0##S23##). Compared with free MEM, the area‐under‐the‐curve (AUC0–∞), half‐life time (t1/2), and mean residence time (MRT0–∞) were distinctly improved in M‐MFL@MB‐injected mice. The blood clearance (CL) rate in M‐MFL@MB group has dropped nearly 10 times compared with MEM group (Figure ##FIG##6##6g##). An excellent photoacoustic imaging ability of M‐MFL@MB was also observed similar to BiNCs. The good PA imaging property jointing with specific targeting ability over bacterial infectious sites endowed M‐MFL@MB with a prominent diagnostic performance (Figure ##FIG##6##6h and i##).
","
A panel of biosafety evaluation assays supported the excellent biocompatibility and low toxicity profiles of M‐MFL@MB (Figures ##SUPPL##0##S24–S27##). The bismuth metabolism in vivo showed that the liver and spleen are dominant organs for its accumulation and metabolism, which may be mainly due to RES absorption (Figure ##SUPPL##0##S28##).[\n##REF##16444262##\n12b\n##\n] A mouse lung infection model (Figure ##FIG##7##\n7a##) revealed a remarkable decrease of clinical EC1322 isolates on lung tissue in M‐MFL@MB group (Figure ##FIG##8##\n8b##). The body temperature, severed as an important indicator of pneumonia recovery,[\n##REF##15127194##\n22\n##\n] had the least change in M‐MFL@MB group (Figure ##FIG##7##7c##). Additionally, the obvious decrease trends of three bacterial infectious biomarkers, c‐reactive protein (CRP), serum amyloid A (SAA) and procalcitonin (PCT) were observed after treatment in M‐MFL@MB groups, reflecting the pneumonia control after therapy (Figure ##FIG##7##7d and e##). Hematoxylin‐Eosin (HE) staining further confirmed the recovery of pneumonia mice after M‐MFL@MB treatment (Figure ##FIG##7##7f##).
","
We further investigated the systemic therapeutic effect of M‐MFL@MB on a murine sepsis model prepared by the intraperitoneal injection of EC1322 (106 CFU) (Figure ##FIG##7##7g##). Neither MEM nor other treatments protected any of the septic mice from death within 96 hours, while 80% septic mice were rescued in M‐MFL@MB group (Figure ##FIG##7##7h##). The bacterial load in the liver, spleen, kidney, and blood in M‐MFL@MB‐treated septic mice was reduced compared with that in the other therapeutic formulation groups. Particularly, M‐MFL@MB treatment resulted in nearly 104, 104, 103, and 105 bacterial reductions in the liver, spleen, kidney, and blood compared with MEM group, respectively (P < 0.001) (Figure ##FIG##7##7i##). The stable body temperature and the decreased trends of clinically related infectious indicators were also observed after M‐MLF@MB treatment (Figure ##FIG##7##7j and k##). Together with the dramatic decrease in leukocyte infiltration and relatively normal organizational structure in M‐MFL@MB group compared with other treatments (Figure ##FIG##8##8l##), the in vitro antimicrobial activity of M‐MFL@MB could be converted into in vivo efficacy.
","Liposomal Antibiotic Booster Limits Resistance Dissemination by Blocking OMVs Secretion","
Bacterial outer membrane vesicles (OMVs) can act as vehicle for transferring NDM‐1 protein and bla\nNDM‐1 gene among different pathogens, resulting in resistance spreading.[\n##REF##34579567##\n23\n##\n] Prevention of OMVs secretion is therefore a feasible strategy for blocking resistance spreading. Inspired by membrane fusion as an effective way of membrane perturbation via fusing with outer membrane,[\n##UREF##2##\n15\n##\n] we evaluated the interference role of M‐MFL‐induced membrane fusion behavior on OMVs secretion in a clinical NDM‐1 producing E. coli isolate (EC1429). The morphology of OMVs upon different treatments was not affected (Figure ##FIG##8##8a##). However, the number of OMVs in M‐MFL group was significantly decreased (Figure ##FIG##8##8a–c##). Compared with other groups, an obvious reduction of NDM‐1 in total protein was found in M‐MFL group (Figure ##FIG##8##8d##). The decrease in enzymatic activity of NDM‐1 also paralleled the decrease in NDM‐1 levels (Figure ##FIG##8##8e##). M‐MFL‐treated E. coli secret the OMVs containing the lower level of bla\nNDM‐1 gene abundance, whereas untreated and lipo‐treated E. coli had a negligible impact on the level of bla\nNDM‐1 inside OMVs (Figure ##FIG##8##8f## and Figure ##SUPPL##0##S29##). RNA‐seq further revealed the effects of M‐MFL on the RNA expression in EC1429, and 108 differentially expressed genes (DEG) between the control sample and M‐MFL treated sample were verified (Figure ##FIG##8##8g##). The M‐MFL treatment significantly down‐regulated the biosynthetic and metabolic process of EC1349 (Figure ##FIG##8##8h##), including fatty acid biosynthetic process, rRNA methylation, carbohydrate derivative catabolic process and regulation of cellular protein metabolic process, which are closely related to the composition and secretion of OMVs.[\n##REF##32722322##\n24\n##\n] Above all, liposomal antibiotic booster plays a positive role in blocking resistance dissemination by decreasing both the NDM‐1 production and OMV secretion.
"],"string":"[\n \"Results\",\n \"The synthesis and characterization of bismuth nanoclusters (BiNCs)\",\n \"
\\nFigure ##FIG##1##\\n1a## illustrates the preparation approach of BiNCs. A clinically available stomach medicine BPC (oral bismuth potassium citrate granules) was synthesized into BiNCs via a one‐step UV irradiation method.[\\n##UREF##3##\\n17\\n##\\n] The citrate and carboxymethyl cellulose (CMC) used in the synthesis are auxiliary materials in BPC, thereby avoiding the use of harmful reagents and residues of by‐products throughout the synthesis. The buffer containing BPC appeared colorless, whereas BiNCs appeared dark black (Figure ##SUPPL##0##S1##). Dynamic light scattering revealed that the hydrodynamic diameter of BiNCs was 21.04 ± 0.99 nm, and the zeta potential was −57.8 ± 4.27 mV (Figure ##FIG##1##1b##). TEM images showed the spherical morphology of the BiNCs with a typical lattice structure and a spacing of about 0.244 nm with a homogeneous size (Figure ##FIG##1##1c and d##). The peaks at 4f7 and 4f5 in X‐ray photoelectron spectroscopy (XPS) indicated the characteristic peaks of bismuth element (Figure ##FIG##1##1e##). The TEM elemental mappings also showed the uniform distribution of Bi elements in BiNCs (Figure ##FIG##1##1f and g##). Afterward, the X‐ray powder diffraction (XRD) pattern verified that the as‐prepared BiNCs were typical bismuth phases (Figure ##FIG##1##1h##). All these results confirmed the successful preparation of BiNCs.
\",\n \"
Notably, we identified a reactive oxygen species (ROS)‐related Bi(III) release in BiNCs, which was enhanced with the increasing of H2O2 concentration (P < 0.01, Figure ##FIG##1##1i##). Morphological and hydrodynamic diameter changes of BiNCs in the presence of H2O2 also reflected ROS‐responsive disintegration of BiNCs (Figure ##FIG##1##1j##, Figure ##SUPPL##0##S2##). BiNCs also presented excellent photoacoustic (PA) imaging properties (Figure ##FIG##1##1k##), promising to realize visualization of pathogen tracing in vivo. We further verified BiNCs can respond to ROS to resensitize NDMs producers specifically toward meropenem (MEM) in a clinical NDM‐1‐producing E. coli isolate EC1322 recovered from peritoneal drainage fluid (Table ##SUPPL##0##S1##, Figure ##SUPPL##0##S3##). Compared with the BiNCs‐MEM group, the BiNCs‐MEM‐H2O2 group exhibited synergistic growth inhibition toward EC1322 with a fractional inhibitory concentration index (FICI) of 0.375 (Figure ##FIG##2##\\n2a‐c##), and the bacterial amounts plummeted (P < 0.01) and their outgrowth was blocked throughout 4 h exposure (Figure ##FIG##2##2d##). While H2O2 itself, even at 400 µM, showed no growth inhibition and no synergistic interaction with MEM toward EC1322 (Figure ##SUPPL##0##S4##). Additionally, BiNCs could reduce the minimal inhibitory concentration (MIC) values of MEM toward NDM‐1 producer but not NDM‐1 negative strain in the presence of H2O2 (Figure ##SUPPL##0##S5##).
\",\n \"
We further used an engineering E. coli BL21 expressing periplasmic NDM‐1 to demonstrate ROS‐powered Bi(III) release from BiNCs inactivating NDMs. Compared with single BiNCs treatment, H2O2‐pretreated BiNCs presented a more significantly obstructive effect on the hydrolysis rate of MEM in BL21 (Figure ##FIG##2##2e##). The NDM‐1 activity decreased as the 200 µM H2O2‐pretreated BiNCs concentration escalated (IC50 = 0.046 mg/mL), ultimately leading to the inhibition of ∼80% activities of NDM‐1 (Figure ##FIG##2##2f## and Figure ##SUPPL##0##S6##). Enzyme kinetics analysis revealed that the apparent Vmax of NDM‐1 decreased from 10.11 to 2.53 µM/s when 200 µM H2O2 – pretreated BiNCs concentration increased from 0 to 100 µg/mL, and a typical non‐competitive or an irreversible inhibition was observed according to the relevant Line‐weaver Burk plot (Figure ##FIG##2##2g##). Next, we found that the appearance of an absorption band at 340 nm after incubating apo‐NDM‐1 (lack of Zn(II)) with H2O2‐pretreated BiNCs (Figure ##SUPPL##0##S7##), which is characteristic for Bi–S ligand‐to‐metal charge transfer (LMCT) band, suggesting that the Bi(III) from BiNCs could bound to NDM‐1. Moreover, ICP‐MS results further revealed that the addition of increasing amounts of Bi(III) resulted in a Zn(II) removal in NDM‐1, accompanied by Bi(III) bound to NDM‐1 (Figure ##FIG##2##2h## and Figure ##SUPPL##0##S8##). Then, the affinities of Bi(III) from H2O2‐pretreated BiNCs to NDM‐1 were closely examined by isothermal titration calorimetry (ITC), unveiling that Bi(III) rather than BiNCs have a high affinity to NDM‐1 (Figure ##FIG##2##2i and j##), and the cellular thermal shift assay further reflecting the binding of intracellular BiNCs‐released Bi(III) to NDM‐1 in intact cells (Figure ##FIG##2##2k##). Together, Bi(III) released from BiNCs in response to ROS competitively replaces the Zn(II) to bind to NDM‐1, thereby hampering the activity of NDM‐1 and leading to carbapenem resistance reversal of NDM‐1‐producing E. coli. (Figure ##FIG##2##2l##).
\",\n \"Fabrication and Characterization of Pathogen‐Primed Liposomal Antibiotic Booster\",\n \"
To integrate pathogen‐targeting, precise intracellular delivery, and site‐specific release of drug capabilities, we produced pathogen‐targeting liposomal antibiotic booster (M‐MFL@MB) using a maltodextrin‐cloaked membrane‐fusion liposome (M‐MFL) as a shell and meropenem‐loaded BiNCs (MB) as a core (Figure ##FIG##3##\\n3a##). In this system, MEM loaded into BiNCs with a 50% loading yield using an optimal input of 3.2 wt% (Figure ##SUPPL##0##S9##). Maltodextrin (MA)‐PEG‐DSPE successfully anchored to membrane‐fusion liposome (MFL) consisting of L‐α‐phosphatidylcholine (EggPC) and cholesterol via a phospholipids fusion,[\\n##REF##30657302##\\n18\\n##\\n] and the modification rate of MA was counted as 0.52 mg/mL. Then, MB was stored into the lumen of M‐MFL with a 29.5% loading yield (Figure ##SUPPL##0##S9##). M‐MFL@MB (166 nm) was slightly larger than M‐MFL (154 nm) and much larger than naked MB (21 nm). M‐MFL@MB possessed an equivalent surface charge (Figure ##FIG##3##3b##), a uniform and spherical structure with a unilamellar membrane coating, which is similar to that of M‐MFL (Figure ##FIG##3##3c##). Cryo‐TEM revealed the successful cloaking of MB into M‐MFL, reflected by a deeper image lining degree from M‐MFL to M‐MFL@MB (Figure ##FIG##3##3c##). M‐MFL protected MB from disaggregation under physiological (H2O2 concentration = 10 µM) and infectious (H2O2 concentration = 100 µM) microenvironment [\\n##REF##31218078##\\n19\\n##\\n] (Figure ##FIG##3##3d and e##). Once the shell is removed, MB could rapidly release MEM and Bi(III) in an H2O2 concentration‐dependent manner, 73% MEM and 80% Bi(III) were released under the 200 µM H2O2 treatment.
\",\n \"
As a major microbial carbon source, maltodextrin (MA) is selectively internalized into bacterial cells through bacterial‐specific maltodextrin transporter, but hardly enters mammalian cells.[\\n##REF##21765397##\\n20\\n##\\n] As expected, MA corona endowed M‐MFL@MB with a more distinguished bacteria adhesion property than that of MFL@MB (Figure ##FIG##3##3f##). We further imaged M‐MFL@MB and MFL@MB in coculture with green fluorescence protein (GFP)‐expressing E.coli and mononuclear macrophages (RAW264.7 cells) (Figure ##FIG##3##3g##), and observed M‐MFL@MB targeted E.coli but not RAW264.7 cells (Figure ##FIG##3##3h##). The line‐scan profiles also denoted the specific co‐localization of M‐MFL@MB and bacteria. Whilst MFL@MB did not present differential targeting to E.coli and RAW264.7 cells (Figure ##FIG##3##3i##). Additionally, liposomal antibiotic booster also presented an excellent immune escape capability via an obvious reduction of endocytosis by monocyte‐macrophage (Figure ##SUPPL##0##S10a and b##).
\",\n \"In Situ Bacterial Membrane Fusion, Site‐Specific Drug Transport, and Intracellular ROS Burst\",\n \"
M‐MFL formulation was first examined for its fusion capability with GFP‐expressing E. coli (Figure ##FIG##4##\\n4a and c##). The intensity of red fluorescence (MFL) in M‐MFL group was higher than that in MFL group, implying MA‐mediated pathogen targeting promotes fusion activity by accelerating bacterial adhesion. Meanwhile, flow cytometer analysis demonstrated the successful fusion (Figure ##SUPPL##0##S11##). Compared with common liposomes constituted by soybean lecithin and cholesterol, M‐MFL exhibited a prominent membrane fusion activity due to a more similar ingredient with bacterial membrane and a higher lipid fluidity [\\n##REF##27725960##\\n21\\n##\\n] (Figure ##SUPPL##0##S12##). Förster resonance energy transfer (FRET) assay further demonstrated the specific bacterial OM fusion of M‐MFL, which was observed successfully fused with E. coli but hardly with platelets (Figure ##SUPPL##0##S13##). We then demonstrated membrane fusion strategy could promote intracellular accumulation of drugs. CLSM and flow cytometry reflected that more Cy5 (substituting MB) was located inside the bacteria in M‐MFL group compared with the other groups (Figure ##FIG##4##4b and c##; Figure ##SUPPL##0##S14##). Bio‐TEM assay showed that MB reached inside bacteria with the aid of MFL (Figure ##FIG##4##4d##). TEM elemental mappings and ICP‐MS assay further revealed the presence of abundant bismuth inside bacteria in M‐MFL@MB and MFL@MB groups (Figure ##FIG##4##4d and e##).
\",\n \"
ROS can trigger the dissociation of MB to release MEM and Bi(III). However, the level of ROS, especially H2O2, inherent within the bacteria is less than 10 µM, only triggering little drug release (Figure ##FIG##3##3d and e##). We identified that MFL‐mediated membrane fusion strategies could endogenously trigger intracellular ROS production (Figure ##FIG##4##4f## and Figure ##SUPPL##0##S15‐16a##). Even at 30 min, the ROS level in M‐MFL and M‐MFL@MB groups still existed steadily with no significant decreasing trend (Figure ##SUPPL##0##S16b##), indicating that membrane fusion strategy initiated a rapid and stable ROS burst inside bacteria. The H2O2 produced inside bacteria was dependent on M‐MFL concentration, and E.coli incubated with 1.8 mg/mL M‐MFL produced almost 100 µM H2O2, which is adequate for MB disintegration (Figure ##FIG##4##4g##). M‐MFL could result in the generation of a variety of bacterial ROS species, including O2−•, 1O2, •OH, all of which have high oxidative activity for catalyzing intracellular MB disintegration. In addition, M‐MFL‐mediated membrane fusion strengthened the permeability of outer membrane and inner membrane in E.coli due to the impaired integrity of bacterial membrane (Figure ##SUPPL##0##S17a and b##). Enhanced membrane permeability would trigger a change in intracellular osmotic pressure, leading to intra‐ and extracellular ions (e.g., Na+ and Cl−) homeostasis disrupted and then resulted in membrane potential depolarization, which acts as a stimulation trigger, and lastly endogenously activates intracellular ROS amplification (Figure ##SUPPL##0##S17c##).[\\n##REF##25962045##\\n16\\n##\\n] Together, liposomal antibiotic booster can serve as potent NDMs inactivator to reverse MEM resistance by a cascading process: selectivity targeting over bacteria, breaking membrane barrier, specifically accumulating intracellular drugs, endogenously activating ROS amplification for intracellular‐specific release of Bi(III) and MEM (Figure ##FIG##4##4i and j##). Thus, M‐MFL@MB prevents MEM from hydrolyzing in NDM‐1 producer more effectively, compared with MEM, MB and M‐MFL@MEM (P < 0.01, Figure ##SUPPL##0##S18##).
\",\n \"Liposomal Antibiotic Booster Resensitizes NDM‐1‐Producing <italic toggle=\\\"yes\\\">E. Coli</italic> to MEM In Vitro\",\n \"
The bactericide of targeting liposomal antibiotic booster was evaluated using six NDM‐1‐producing clinical E. coli isolates (Table ##SUPPL##0##S1##, Figure ##SUPPL##0##S3##). These strains showed much higher MICs (> 64 to 2 µg/mL) in individual MEM, BiNCs, MB, M‐MFL and M‐MFL@MEM groups, respectively (Figure ##FIG##5##\\n5a## and Figure ##SUPPL##0##S19##). Whilst M‐MFL@MB exhibited a concentrate‐dependent inhibitory effect (8 to 2 µg/mL) on all tested isolates (Figure ##FIG##5##5b##), indicating that M‐MFL@MB could reverse MEM resistance in NDM producers. Time‐dependent killing of EC1322 showed M‐MFL@MB had excellent bactericidal activity against NDMs producer (Figure ##FIG##5##5c##), also reflected by SEM results (Figure ##FIG##5##5d##). Additionally, M‐MFL@MB destructed more exhaustive bacterial structure than other groups (Figure ##FIG##5##5d##). These results suggested that targeting liposomal antibiotic boosters could resensitize NDM‐1‐producing E. coli to MEM in vitro. Notably, the antibiotic booster also showed synergies with ceftazidime (β‐lactam antibiotics) against NDM‐1‐producing E. coli isolates, but not with ciprofloxacin (quinolones) and colistin (polypeptide antibiotics), as shown in Fig. ##SUPPL##0##S20 and S21##. The result implied that antibiotic booster mainly induces NDM‐1 inactivation, lastly reversing NDMs producer resistance against β‐lactam antibiotics.
\",\n \"Liposomal Antibiotic Booster Targets Bacterial Infectious Sites and Restores Meropenem Efficacy In Vivo\",\n \"
The targeting capability of M‐MFL@MB was evaluated in zebrafish infected with GFP‐expressing E. coli (Figure ##FIG##6##\\n6a##). Compared with MFL group, M‐MFL group showed a more pronounced red fluorescence (RhB) trapped inside the bacteria with green fluorescence in zebrafish, demonstrating the M‐MFL can effectively recognize and adhere to the bacteria under the assist of MA (Figure ##FIG##6##6b1 and b2##). Whilst M‐MFL cannot retained inside the healthy zebrafish (Figure ##FIG##6##6b3##). Moreover, MA crown could distinguish bacterial infection from inflammation in a mouse bacterial and inflammatory co‐infection model. IVIS imaging showed more M‐MFL could accumulate effectively and specifically into the bacterial infectious tissue rather than inflammatory tissue (Figure ##SUPPL##0##S22a‐e##), and ex vivo tissues imaging further verified the MA‐mediated specific bacterial targeting ability of M‐MFL (Figure ##FIG##6##6c and d##). The mouse lung infection model further revealed that M‐MFL could facilitate drugs accumulation at infectious sites, as RhB‐labelled M‐MFL@Cy5, not MFL@Cy5, could be gradually accumulated into infected lung, and presented an excellent co‐location with bacteria in infected tissues (Figure ##FIG##6##6e and f##).
\",\n \"
Pharmacokinetic analysis revealed higher concentrations of M‐MFL@MB (86.32‐15.67 µg/mL) in plasma compared with the free MEM (81.85‐5.25 µg/mL) at the indicating time points (Figure ##SUPPL##0##S23##). Compared with free MEM, the area‐under‐the‐curve (AUC0–∞), half‐life time (t1/2), and mean residence time (MRT0–∞) were distinctly improved in M‐MFL@MB‐injected mice. The blood clearance (CL) rate in M‐MFL@MB group has dropped nearly 10 times compared with MEM group (Figure ##FIG##6##6g##). An excellent photoacoustic imaging ability of M‐MFL@MB was also observed similar to BiNCs. The good PA imaging property jointing with specific targeting ability over bacterial infectious sites endowed M‐MFL@MB with a prominent diagnostic performance (Figure ##FIG##6##6h and i##).
\",\n \"
A panel of biosafety evaluation assays supported the excellent biocompatibility and low toxicity profiles of M‐MFL@MB (Figures ##SUPPL##0##S24–S27##). The bismuth metabolism in vivo showed that the liver and spleen are dominant organs for its accumulation and metabolism, which may be mainly due to RES absorption (Figure ##SUPPL##0##S28##).[\\n##REF##16444262##\\n12b\\n##\\n] A mouse lung infection model (Figure ##FIG##7##\\n7a##) revealed a remarkable decrease of clinical EC1322 isolates on lung tissue in M‐MFL@MB group (Figure ##FIG##8##\\n8b##). The body temperature, severed as an important indicator of pneumonia recovery,[\\n##REF##15127194##\\n22\\n##\\n] had the least change in M‐MFL@MB group (Figure ##FIG##7##7c##). Additionally, the obvious decrease trends of three bacterial infectious biomarkers, c‐reactive protein (CRP), serum amyloid A (SAA) and procalcitonin (PCT) were observed after treatment in M‐MFL@MB groups, reflecting the pneumonia control after therapy (Figure ##FIG##7##7d and e##). Hematoxylin‐Eosin (HE) staining further confirmed the recovery of pneumonia mice after M‐MFL@MB treatment (Figure ##FIG##7##7f##).
\",\n \"
We further investigated the systemic therapeutic effect of M‐MFL@MB on a murine sepsis model prepared by the intraperitoneal injection of EC1322 (106 CFU) (Figure ##FIG##7##7g##). Neither MEM nor other treatments protected any of the septic mice from death within 96 hours, while 80% septic mice were rescued in M‐MFL@MB group (Figure ##FIG##7##7h##). The bacterial load in the liver, spleen, kidney, and blood in M‐MFL@MB‐treated septic mice was reduced compared with that in the other therapeutic formulation groups. Particularly, M‐MFL@MB treatment resulted in nearly 104, 104, 103, and 105 bacterial reductions in the liver, spleen, kidney, and blood compared with MEM group, respectively (P < 0.001) (Figure ##FIG##7##7i##). The stable body temperature and the decreased trends of clinically related infectious indicators were also observed after M‐MLF@MB treatment (Figure ##FIG##7##7j and k##). Together with the dramatic decrease in leukocyte infiltration and relatively normal organizational structure in M‐MFL@MB group compared with other treatments (Figure ##FIG##8##8l##), the in vitro antimicrobial activity of M‐MFL@MB could be converted into in vivo efficacy.
Bacterial outer membrane vesicles (OMVs) can act as vehicle for transferring NDM‐1 protein and bla\\nNDM‐1 gene among different pathogens, resulting in resistance spreading.[\\n##REF##34579567##\\n23\\n##\\n] Prevention of OMVs secretion is therefore a feasible strategy for blocking resistance spreading. Inspired by membrane fusion as an effective way of membrane perturbation via fusing with outer membrane,[\\n##UREF##2##\\n15\\n##\\n] we evaluated the interference role of M‐MFL‐induced membrane fusion behavior on OMVs secretion in a clinical NDM‐1 producing E. coli isolate (EC1429). The morphology of OMVs upon different treatments was not affected (Figure ##FIG##8##8a##). However, the number of OMVs in M‐MFL group was significantly decreased (Figure ##FIG##8##8a–c##). Compared with other groups, an obvious reduction of NDM‐1 in total protein was found in M‐MFL group (Figure ##FIG##8##8d##). The decrease in enzymatic activity of NDM‐1 also paralleled the decrease in NDM‐1 levels (Figure ##FIG##8##8e##). M‐MFL‐treated E. coli secret the OMVs containing the lower level of bla\\nNDM‐1 gene abundance, whereas untreated and lipo‐treated E. coli had a negligible impact on the level of bla\\nNDM‐1 inside OMVs (Figure ##FIG##8##8f## and Figure ##SUPPL##0##S29##). RNA‐seq further revealed the effects of M‐MFL on the RNA expression in EC1429, and 108 differentially expressed genes (DEG) between the control sample and M‐MFL treated sample were verified (Figure ##FIG##8##8g##). The M‐MFL treatment significantly down‐regulated the biosynthetic and metabolic process of EC1349 (Figure ##FIG##8##8h##), including fatty acid biosynthetic process, rRNA methylation, carbohydrate derivative catabolic process and regulation of cellular protein metabolic process, which are closely related to the composition and secretion of OMVs.[\\n##REF##32722322##\\n24\\n##\\n] Above all, liposomal antibiotic booster plays a positive role in blocking resistance dissemination by decreasing both the NDM‐1 production and OMV secretion.
Novel treatments against NDMs‐positive Enterobacterales, which exhibited multidrug‐resistant (MDR) or extensively drug‐resistant (XDR) profiles, are urgently needed in clinical practice since colistin and tigecycline were largely compromised due to the emerging and spreading of mobile colistin resistance gene mcr‐1 and tigecycline resistance genes tet(X3) and tet(X4).[\n##REF##26603172##\n25\n##\n] Given that few antimicrobials against Gram‐negative pathogens for entering clinical trials, developing NDMs inhibitors to restore carbapenem activity is a promising strategy. However, the structural diversity in active sites of metallo‐β‐lactamases (MBLs) restricted the development of effective MBL inhibitors.[\n##UREF##4##\n26\n##\n] As a common active site shared by different types of MBLs (e.g., NDM, IMP, and VIM), Zn(II) is an ideal target for MBL inhibitors. Based on Zn(II)‐binding inhibition mode, two types of MBL inhibitors, metal‐depriving compounds (AMA) and metal ion [Bi(III)] replacing compounds, have been demonstrated.[\n##REF##24965651##\n9\n##, ##REF##24965651##\n27\n##\n] In the development of clinically useful inhibitors, however, these small‐molecule MBL inhibitors are faced with challenges of metalloenzyme selectivity in vivo and efficient intracellular accumulation.[\n##UREF##5##\n28\n##\n] Recently, nanomaterial‐based therapeutics with unique advantages in antibacterial effects attracted more attention.[\n##REF##33024312##\n29\n##\n] It can be served as drug carrier to augment the potency of antibiotics or be used instead of antibiotics to exert entirely new antibacterial actions. Herein, we combined the high NDMs inhibition efficacy of Bi(III) and the advantages of pathogen targeting and membrane barrier breakthrough of nanomaterial‐based therapeutics to design and construct M‐MFL@MB, a liposomal antibiotic booster that can effectively target pathogens and achieve the intracellular co‐delivery of meropenem and Bi(III) through membrane fusion.
","
Lipopolysaccharide‐coated outer membrane of Gram‐negative bacteria was considered as a barrier for compounds crossing. Small molecular MBL inhibitors traverse outer membrane mainly through narrow β‐barrel porins (eg, OmpF and OmpC).[\n##REF##31844257##\n30\n##\n] Thus, the cellular accumulation of NDMs inhibitors is an important factor affecting their effectiveness in rescuing carbapenem activity.[\n##UREF##5##\n28\n##\n] To address this challenge, we chose liposomes to carry adjuvants and antibiotics to break through the out‐membrane barrier via membrane fusion. To date, abundant advantages of commercial liposomes such as mature production and high biocompatibility have been described,[\n##REF##31918220##\n31\n##\n] and these liposome has been successfully used in clinical or preclinical practice for improving the delivery efficiency of antibiotics or antitumor drugs to disease sites.[\n##REF##19010847##\n32\n##\n] However, understanding of the capability of bacterial outer membrane penetrability of liposome remains limited. Here, we introduced the targeting membrane fusion liposomes that can effectively reduce in vivo off‐target toxicity of inhibitors and help inhibitors and antibiotics cross the barrier of bacterial outer membrane. Moreover, we found, for the first time, that liposome‐mediated membrane fusion could endogenously activate bacterial intracellular ROS amplification, providing a self‐activated “key” for Bi(III) release into bacterial periplasm, leading to an in‐situ Bi(III)‐mediated Zn(II) deprivation. Additionally, membrane fusion strategy also slowed down NDMs‐related resistance dissemination by decreasing the secretion of bacterial OMVs. Taken together, this strategy improves the effect of Bi(III) on accurately inactivating NDM‐1 in vivo and reduces its off‐target toxicity, therefore is a promising approach for its possible application in clinical settings.
","
We acknowledged few limitations existed in this study. First, the visible light‐mediated decomposition of BiNCs needs to be further addressed. Second, more accurate ROS burst mechanisms originating from membrane fusion liposome need to be further elucidated. Third, more evaluations in larger preclinical such as nonhuman primates, are needed to be conducted to advance clinical translation. Nevertheless, all ingredients in the M‐MFL@MB, including liposome, maltodextrin and gastric drug, were FDA‐approved and easily obtained at the kilogram level, which is expected to promote the clinical translation of antibiotic booster.
","
In summary, we developed a pathogen‐primed liposomal antibiotic booster, M‐MFL@MB (maltodextrin‐cloaked membrane fusion liposome‐encapsulated meropenem‐loaded BiNCs) for reviving carbapenem efficiency in NDMs‐producing clinical E. coli isolates in vitro and in vivo. M‐MFL@MB decreased the mortality of infected mice via its pathogen‐targeting, physical barrier breaks, and ROS‐responsive Bi(III)‐mediated Zn(II) removal. Additionally, membrane fusion strategy mediated by M‐MFL decreased the secretion of bacterial OMVs and slowed down the resistance spreading. Our work offered a potential nano‐adjuvant platform for repurposing carbapenems potency and curing NDMs‐producer infections.
"],"string":"[\n \"Discussion\",\n \"
Novel treatments against NDMs‐positive Enterobacterales, which exhibited multidrug‐resistant (MDR) or extensively drug‐resistant (XDR) profiles, are urgently needed in clinical practice since colistin and tigecycline were largely compromised due to the emerging and spreading of mobile colistin resistance gene mcr‐1 and tigecycline resistance genes tet(X3) and tet(X4).[\\n##REF##26603172##\\n25\\n##\\n] Given that few antimicrobials against Gram‐negative pathogens for entering clinical trials, developing NDMs inhibitors to restore carbapenem activity is a promising strategy. However, the structural diversity in active sites of metallo‐β‐lactamases (MBLs) restricted the development of effective MBL inhibitors.[\\n##UREF##4##\\n26\\n##\\n] As a common active site shared by different types of MBLs (e.g., NDM, IMP, and VIM), Zn(II) is an ideal target for MBL inhibitors. Based on Zn(II)‐binding inhibition mode, two types of MBL inhibitors, metal‐depriving compounds (AMA) and metal ion [Bi(III)] replacing compounds, have been demonstrated.[\\n##REF##24965651##\\n9\\n##, ##REF##24965651##\\n27\\n##\\n] In the development of clinically useful inhibitors, however, these small‐molecule MBL inhibitors are faced with challenges of metalloenzyme selectivity in vivo and efficient intracellular accumulation.[\\n##UREF##5##\\n28\\n##\\n] Recently, nanomaterial‐based therapeutics with unique advantages in antibacterial effects attracted more attention.[\\n##REF##33024312##\\n29\\n##\\n] It can be served as drug carrier to augment the potency of antibiotics or be used instead of antibiotics to exert entirely new antibacterial actions. Herein, we combined the high NDMs inhibition efficacy of Bi(III) and the advantages of pathogen targeting and membrane barrier breakthrough of nanomaterial‐based therapeutics to design and construct M‐MFL@MB, a liposomal antibiotic booster that can effectively target pathogens and achieve the intracellular co‐delivery of meropenem and Bi(III) through membrane fusion.
\",\n \"
Lipopolysaccharide‐coated outer membrane of Gram‐negative bacteria was considered as a barrier for compounds crossing. Small molecular MBL inhibitors traverse outer membrane mainly through narrow β‐barrel porins (eg, OmpF and OmpC).[\\n##REF##31844257##\\n30\\n##\\n] Thus, the cellular accumulation of NDMs inhibitors is an important factor affecting their effectiveness in rescuing carbapenem activity.[\\n##UREF##5##\\n28\\n##\\n] To address this challenge, we chose liposomes to carry adjuvants and antibiotics to break through the out‐membrane barrier via membrane fusion. To date, abundant advantages of commercial liposomes such as mature production and high biocompatibility have been described,[\\n##REF##31918220##\\n31\\n##\\n] and these liposome has been successfully used in clinical or preclinical practice for improving the delivery efficiency of antibiotics or antitumor drugs to disease sites.[\\n##REF##19010847##\\n32\\n##\\n] However, understanding of the capability of bacterial outer membrane penetrability of liposome remains limited. Here, we introduced the targeting membrane fusion liposomes that can effectively reduce in vivo off‐target toxicity of inhibitors and help inhibitors and antibiotics cross the barrier of bacterial outer membrane. Moreover, we found, for the first time, that liposome‐mediated membrane fusion could endogenously activate bacterial intracellular ROS amplification, providing a self‐activated “key” for Bi(III) release into bacterial periplasm, leading to an in‐situ Bi(III)‐mediated Zn(II) deprivation. Additionally, membrane fusion strategy also slowed down NDMs‐related resistance dissemination by decreasing the secretion of bacterial OMVs. Taken together, this strategy improves the effect of Bi(III) on accurately inactivating NDM‐1 in vivo and reduces its off‐target toxicity, therefore is a promising approach for its possible application in clinical settings.
\",\n \"
We acknowledged few limitations existed in this study. First, the visible light‐mediated decomposition of BiNCs needs to be further addressed. Second, more accurate ROS burst mechanisms originating from membrane fusion liposome need to be further elucidated. Third, more evaluations in larger preclinical such as nonhuman primates, are needed to be conducted to advance clinical translation. Nevertheless, all ingredients in the M‐MFL@MB, including liposome, maltodextrin and gastric drug, were FDA‐approved and easily obtained at the kilogram level, which is expected to promote the clinical translation of antibiotic booster.
\",\n \"
In summary, we developed a pathogen‐primed liposomal antibiotic booster, M‐MFL@MB (maltodextrin‐cloaked membrane fusion liposome‐encapsulated meropenem‐loaded BiNCs) for reviving carbapenem efficiency in NDMs‐producing clinical E. coli isolates in vitro and in vivo. M‐MFL@MB decreased the mortality of infected mice via its pathogen‐targeting, physical barrier breaks, and ROS‐responsive Bi(III)‐mediated Zn(II) removal. Additionally, membrane fusion strategy mediated by M‐MFL decreased the secretion of bacterial OMVs and slowed down the resistance spreading. Our work offered a potential nano‐adjuvant platform for repurposing carbapenems potency and curing NDMs‐producer infections.
Infections caused by Enterobacterales producing New Delhi Metallo‐β‐lactamases (NDMs), Zn(II)‐dependent enzymes hydrolyzing carbapenems, are difficult to treat. Depriving Zn(II) to inactivate NDMs is an effective solution to reverse carbapenems resistance in NDMs‐producing bacteria. However, specific Zn(II) deprivation and better bacterial outer membrane penetrability in vivo are challenges. Herein, authors present a pathogen‐primed liposomal antibiotic booster (M‐MFL@MB), facilitating drugs transportation into bacteria and removing Zn(II) from NDMs. M‐MFL@MB introduces bismuth nanoclusters (BiNCs) as a storage tank of Bi(III) for achieving ROS‐initiated Zn(II) removal. Inspired by bacteria‐specific maltodextrin transport pathway, meropenem‐loaded BiNCs are camouflaged by maltodextrin‐cloaked membrane fusion liposome to cross the bacterial envelope barrier via selectively targeting bacteria and directly outer membrane fusion. This fusion disturbs bacterial membrane homeostasis, then triggers intracellular ROS amplification, which activates Bi(III)‐mediated Zn(II) replacement and meropenem release, realizing more precise and efficient NDMs producer treatment. Benefiting from specific bacteria‐targeting, adequate drugs intracellular accumulation and self‐activation Zn(II) replacement, M‐MFL@MB rescues all mice infected by NDM producer without systemic side effects. Additionally, M‐MFL@MB decreases the bacterial outer membrane vesicles secretion, slowing down NDMs producer's transmission by over 35 times. Taken together, liposomal antibiotic booster as an efficient and safe tool provides new strategy for tackling NDMs producer‐induced infections.
","
A pathogen‐primed liposomal antibiotic booster is contructed than can revive carbapenem efficiency in NDMs‐producing clinical E. coli isolates in vitro and in vivo, via a cascading process: pathogen‐targeting, membrane fusion‐induced physical barrier breaks, and ROS‐responsive Bi(III)‐mediated Zn(II) removal.\n\n
Infections caused by Enterobacterales producing New Delhi Metallo‐β‐lactamases (NDMs), Zn(II)‐dependent enzymes hydrolyzing carbapenems, are difficult to treat. Depriving Zn(II) to inactivate NDMs is an effective solution to reverse carbapenems resistance in NDMs‐producing bacteria. However, specific Zn(II) deprivation and better bacterial outer membrane penetrability in vivo are challenges. Herein, authors present a pathogen‐primed liposomal antibiotic booster (M‐MFL@MB), facilitating drugs transportation into bacteria and removing Zn(II) from NDMs. M‐MFL@MB introduces bismuth nanoclusters (BiNCs) as a storage tank of Bi(III) for achieving ROS‐initiated Zn(II) removal. Inspired by bacteria‐specific maltodextrin transport pathway, meropenem‐loaded BiNCs are camouflaged by maltodextrin‐cloaked membrane fusion liposome to cross the bacterial envelope barrier via selectively targeting bacteria and directly outer membrane fusion. This fusion disturbs bacterial membrane homeostasis, then triggers intracellular ROS amplification, which activates Bi(III)‐mediated Zn(II) replacement and meropenem release, realizing more precise and efficient NDMs producer treatment. Benefiting from specific bacteria‐targeting, adequate drugs intracellular accumulation and self‐activation Zn(II) replacement, M‐MFL@MB rescues all mice infected by NDM producer without systemic side effects. Additionally, M‐MFL@MB decreases the bacterial outer membrane vesicles secretion, slowing down NDMs producer's transmission by over 35 times. Taken together, liposomal antibiotic booster as an efficient and safe tool provides new strategy for tackling NDMs producer‐induced infections.
\",\n \"
A pathogen‐primed liposomal antibiotic booster is contructed than can revive carbapenem efficiency in NDMs‐producing clinical E. coli isolates in vitro and in vivo, via a cascading process: pathogen‐targeting, membrane fusion‐induced physical barrier breaks, and ROS‐responsive Bi(III)‐mediated Zn(II) removal.\\n\\n
\"\n]"},"body":{"kind":"list like","value":["Conflict of Interest","
The authors declare no conflict of interest.
","Author Contributions","
S.W., Y.W., and Y.W., contributed equally to this work. S.X.W., J.J.S. and S.S.Q. performed conceptualization, S.X.W. and Y.B.W. performed methodology, Y.B.W. and J.J.S. performed investigation, S.X.W., D.J.L., and Y.B.W. performed visualization, J.J.S., S.S.Q., Y.W., and D.J.L. performed funding acquisition, Z.Z.Z., J.Z.S., and S.S.Q. performed supervision, S.X.W., Y.B.W., and Y.M.W., wrote the original draft, S.X.W., Y.W., J.J.S., S.S.Q., and J.Z.S. wrote, reviewed and edited the manuscripts.
","Supporting information"],"string":"[\n \"Conflict of Interest\",\n \"
The authors declare no conflict of interest.
\",\n \"Author Contributions\",\n \"
S.W., Y.W., and Y.W., contributed equally to this work. S.X.W., J.J.S. and S.S.Q. performed conceptualization, S.X.W. and Y.B.W. performed methodology, Y.B.W. and J.J.S. performed investigation, S.X.W., D.J.L., and Y.B.W. performed visualization, J.J.S., S.S.Q., Y.W., and D.J.L. performed funding acquisition, Z.Z.Z., J.Z.S., and S.S.Q. performed supervision, S.X.W., Y.B.W., and Y.M.W., wrote the original draft, S.X.W., Y.W., J.J.S., S.S.Q., and J.Z.S. wrote, reviewed and edited the manuscripts.
The authors acknowledge the Modern Analysis and Computing Center of Zhengzhou University for the technical assistance. Additionally, they would like to thank Wang Hui Quan from Shiyanjia Lab (www.shiyanjia.com) for the test assay. Special thanks go to: National Science Foundation for Distinguished Young Scholars 82222067 (J.J.S), National Natural Science Foundation of China 81874304 (J.J.S), National Natural Science Foundation of China 82073395 (Y.W), Innovation Talent Support Program of Henan Province 19HASTIT006 (J.J.S), Postdoctoral Science Foundation of China 2018T110745 (D.J.L), Training Plan for Young Backbone Teachers in Colleges and Universities in Henan 2021GGJS016 (S.S.Q). All animal studies were carried out following the guidelines of the Regional Ethics Committee for Animal Experiments and the Care Regulations approved by the Institutional Animal Care and Use Committee of Zhengzhou University. The license number of the experimental animal is No.410975211100031648.
","Data Availability Statement","
Research data are not shared.
"],"string":"[\n \"Acknowledgements\",\n \"
The authors acknowledge the Modern Analysis and Computing Center of Zhengzhou University for the technical assistance. Additionally, they would like to thank Wang Hui Quan from Shiyanjia Lab (www.shiyanjia.com) for the test assay. Special thanks go to: National Science Foundation for Distinguished Young Scholars 82222067 (J.J.S), National Natural Science Foundation of China 81874304 (J.J.S), National Natural Science Foundation of China 82073395 (Y.W), Innovation Talent Support Program of Henan Province 19HASTIT006 (J.J.S), Postdoctoral Science Foundation of China 2018T110745 (D.J.L), Training Plan for Young Backbone Teachers in Colleges and Universities in Henan 2021GGJS016 (S.S.Q). All animal studies were carried out following the guidelines of the Regional Ethics Committee for Animal Experiments and the Care Regulations approved by the Institutional Animal Care and Use Committee of Zhengzhou University. The license number of the experimental animal is No.410975211100031648.
\",\n \"Data Availability Statement\",\n \"
Research data are not shared.
\"\n]"},"figure":{"kind":"list like","value":["
Schemes for construction of targeting liposomal antibiotic booster for targeted periplasmic NDMs inactivation via Bi(III)‐mediated Zn(II) removal. The meropenem‐loaded bismuth nanoclusters (MB) are encapsulated in the maltodextrin‐cloaked membrane fusion liposome and precisely delivered to the infectious sites with the assistance of bacteria‐specific maltodextrin transport pathway, where the liposome fuses with bacterial outer membrane, facilitating the periplasmic translocation of MB and intracellular ROS burst. Meanwhile, endogenous ROS amplification triggers the intracellular‐specific release of Bi(III) and meropenem. Released Bi(III) irreversibly inactive NDMs via displacing Zn(II) from NDMs active sites, thus protecting meropenem from hydrolyzation.
","
a) The scheme presenting the synthesis process of Bismuth‐Nano‐Clusters (BiNCs) and ROS‐responsive Bi(III) release from BiNCs. b) Hydrodynamic diameter distribution and zeta potential distribution (inset) obtained for BiNCs. c) Representative TEM image of BiNCs. Scale bar, 10 nm. d) A representative high‐resolution TEM image of BiNCs. Scale bar, 2.5 nm. e) XPS diffraction spectrum of BiNCs. f) STEM‐HAADF image and corresponding EDS elemental mappings of Bi, O, P, N, and C in BiNCs. g) EDS spectrum for bismuth element analysis of BiNCs. h) X‐ray diffraction (XRD) analysis of BiNCs. i) In vitro Bi(III) release from BiNCs against different levels of H2O2. j) Representative TEM images of BiNCs after 24 h incubation with different levels of H2O2. Scale bar, 100 nm. k) PA response to different concentrations of BiNCs (0.1, 0.3, 0.6, 0.9, 1.2, and 1.5 mg/mL) (inset: PA imaging of BiNCs). Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a, b) Representative heat plots of microdilution checkerboard assays for the combination of BiNCs and meropenem in the absence (a) or presence (b) of H2O2 against EC1322. c) Isobolograms of the combination of BiNCs and meropenem in the absence or presence of H2O2 against EC1322. The black dotted line shows the ideal isobole, where drugs act additively and independently. Data points below this line reveal synergism. d) Time‐kill curves for meropenem or BiNCs monotherapy, or their combination therapy in the absence or presence of H2O2 against EC1322 during 6 h incubation. The concentrations of meropenem, BiNCs, and H2O2 are used at 8 µg/mL, 125 µg/mL, and 200 µM, respectively. e) Hydrolytic effects of the BiNCs or BiNCs+H2O2 pre‐treated NDM‐1‐producing E. coli BL21 on meropenem (n = 3). f) Inhibition of NDM‐1 activity by Bi(III) from 200 µM H2O2‐pretreated BiNCs with IC50 of 0.046 mg/mL (n = 3). g) Double reciprocal plot of substrate‐dependent enzyme kinetics on inhibition of NDM‐1 activity by Bi(III) from 200 µM H2O2‐pretreated BiNCs, reflecting that Bi(III) (released from BiNCs) inhibited NDM‐1 via either a non‐competitive or an irreversible inhibition mode. h) Zn(II) content in Zn2‐NDM‐1 and the supernatant after being treated with different concentrations of Bi(III) from H2O2‐pretreated BiNCs by equilibrium dialysis, respectively. The metal content was determined by ICP‐MS. i, j) ITC thermograms for the binding of Bi(III) from H2O2‐pretreated BiNCs (i) or BiNCs (j) to NDM‐1. The downward peaks indicate an exothermic process. k) Cellular thermal shift assay demonstrating the binding of Bi(III) to NDM‐1 in NDM‐producing E. coli BL21. NDM‐1 melting temperature was shifted from 68.1° to 56.8 °C for control and BiNCs‐H2O2 combination group, respectively. The images show the western blotting result. l Schematic diagram of the action mechanism of BiNCs under non‐oxidative stress and oxidative stress on NDM‐1‐producing E. coli. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a) The schematic illustration exhibiting the preparation process of maltodextrin‐decorated membrane fusion liposome that wraps meropenem‐loaded BiNCs (M‐MFL@MB) and the highly specific bacterial targeting mechanism. b) Hydrodynamic diameter and ζ potential of BiNCs, meropenem‐loaded BiNCs (MB), maltodextrin‐decorated membrane fusion liposome (M‐MFL) and M‐MFL@MB (n = 3). c) Representative TEM and cryo‐TEM (inset) images of M‐MFL (left) and M‐MFL@MB (right). Scale bar, 100 nm. d, e) In vitro MEM (d) and Bi(III) (e) release curves of MEM@BiNCs (MB) (upper) and M‐MFL@MB (lower) in PBS containing different concentrations of H2O2 (1, 10, 100 and 200 µM), (n = 3). f) Representative pseudo‐color SEM images of EC1322 after incubation with PBS, M‐MFL@MB, and MFL@MB, respectively. Scale bar, 1 µm. g) The experimental scheme of coculture experiments for verifying the capability of maltodextrin‐mediated specifical targeting to bacteria, not mammalian cells. h, i) Confocal images of mononuclear macrophages (RAW 264.7 cells) cocultured with GFP‐expressing E. coli and imaged after labeling with M‐MFL@MB (h) or MFL@MB (i), respectively. Scale bar, 5 µm. The nucleus of RAW 264.7 cells, E. coli, and MFL were labeled with DAPI (blue), Green fluorescent protein (green), and Rhodamine (red), respectively. Plot profiles corresponding to white lines are shown on the right. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a, b) Representative confocal images visualize the membrane fusion interaction between M‐MFL or MFL with E. coli (a), and intracellular drug delivery of M‐MFL or MFL into E.coli (b). M‐MFL and MFL were labeled with fluorescent dye Rhodamine (red), Cy5 fluorescent dye (pink) was loaded into M‐MFL or MFL for substituting MB, and the E. coli could express GFP (green fluorescent protein, green). The control group was incubated with PBS. Scale bar, 100 nm. c) The corresponding fluorescence semi‐quantitative analysis (n = 3), shows membrane fusion and intracellular delivery efficiency of M‐MFL and MFL, respectively. d) Bio‐TEM images and the bismuth element mapping of different nanoparticles‐treated EC1322. Untreated B16‐F10 cells were used as control. Scar bar: 1 µm; Red arrows pointed to BiNCs. e) ICP‐MS analyzes the effect of different treatments on intracellular Bi(III) accumulation of E. coli (n = 3). f) Intracellular ROS level after being treated with different nanoparticles was monitored by detection of DCFH‐DA fluorescence intensity using confocal imaging. g) ESR spectrum of M‐MFL‐treated E. coli, the untreated E.coli was used as control. Scale bar, 30 µm. h) Determination of H2O2 amount in NDM‐1‐EC1322 after different concentrations of M‐MFL treatment (n = 3). i) Hydrolytic effects of the EC1322 on meropenem after different treatments, including MEM, MEM@BiNCs, M‐MFL@MEM and M‐MFL@MB. n = 3. j) Schematic diagram of the action mechanism of targeting liposomal antibiotic booster on NDM‐1 producers. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a) Measurement of bacterial colony‐forming units, obtained from six clinical isolates of NDM‐1‐producing E. coli treated with different concentrations of meropenem (MEM), MB, M‐MFL@MEM and M‐MFL@MB, respectively (n = 6). b) MIC of MEM, MB, M‐MFL@MEM and M‐MFL@MB in six clinical isolates of NDM‐1‐positive E. coli (n = 6). c) Time‐kill curves for MEM, BiNCs, M‐MFL, MB, M‐MFL@MEM, and M‐MFL@MB against EC1322 during 6 h incubation, respectively (n = 3). The concentrations of MEM were 8 µg/mL in those groups. The concentrations of BiNCs and M‐MFL were about 53.33 and 177.77 µg/mL, respectively. d) Representative SEM images of EC1322 after treatment with different nanoparticles for 1 h and 6 h, respectively. Scale bar, 0.5 µm. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a) Experimental scheme of the bacterial‐infected zebrafish model for demonstrating maltodextrin‐mediated adhesion ability. b) Lateral views of the whole bacteria‐infected zebrafish after DiI‐labeled M‐MFL treatment (b1). The bacterial‐infected zebrafish incubated with the DiI‐ labeled MFLipo treatment (b2) and the healthy zebrafish incubated with DiI‐labeled M‐MFL (b3) were used as the control. c, d) Ex‐vivo tissue NIR FL images I and fluorescence semi‐quantitative analysis (d) of mice model of dual infection with LPS and E. coli at 22 h post‐injection with M‐MFL@IR783, MFL@IR783, or IR783, respectively (n = 3). e, f) Representative CLSM images I and fluorescence semi‐quantitative analysis (f) of MFL, M‐MFL distribution at different time points after intravenous injection in the E. coli infected lung tissues, respectively. The nanoliposome was stained with rhodamine (red). The encapsulated drugs were replaced by cy5 (pink). The nucleus of lung tissues was stained with DAPI (blue). Scar bar, 100 µm. h, i) PA imaging (h) and PA response value (i) of E. coli‐infected mice model at different time points after intravenous injection with BiNCs and M‐MFL@MB, respectively. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a) Schematic diagram of the infection, treatment used in mice with pneumonia. b, c) Bacterial loads (b) and mouse body temperature changes (c) in the pneumonia mice model after different nanoformulations treatment (n = 6). d) Experimental roadmap for detecting inflammation‐related indicators (CRP, SAA and PCT) of mice with pneumonia. e) CRP, SAA and PCT level in the pneumonia mice model after different treatments (n = 6). f) HE staining of infected lung tissues in the pneumonia mice model after different treatments. Scar bar, 100 µm. g) Schematic diagram of the infection, treatment used in mice with sepsis. h, i, j) Survival curves (h), bacterial loads (i) and body temperature changes (j) in the sepsis mice model after different treatments, n = 6. k) CRP, SAA and PCT level in the sepsis mice model after different treatments (n = 6). l) HE staining of infected tissues in the sepsis mice model subjected to different treatments. Scar bar, 100 µm. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
","
a) an NTA analysis and representative TEM images (inset) of E. coli OMVs extracted from E. coli incubated with Lipo and M‐MFL, respectively. The OMVs extracted from untreated E. coli were used as control. Scar bar, 100 nm. b) Statistics of size and concentration of E. coli OMVs extracted from E. coli incubated with different nanoparticles. b, c, d) the measurement of total protein content (b, n = 3), NDM‐1 protein content (c, n = 3) and enzymatic activity of NDM‐1 (d, n = 3) of E. coli OMVs extracted from E. coli incubated with Lipo and M‐MFL, respectively. The OMVs extracted from untreated E. coli were used as control. e) Absolute copy number of bla\nNDM‐1 gene in E. coli OMVs extracted from E. coli incubated with Lipo and M‐MFL, respectively. The OMVs extracted from untreated E. coli were used as control. n = 3. f) The MIC of EC15922 after incubation with different E. coli OMVs (n = 6). g) Clustering heat map of differentially expressed genes (DEGs) between M‐MFL‐treated E. coli and untreated E. coli. The abscissa is the sample name, and the ordinate is the normalized value of the DEGs. The redder the color, the higher the expression level, and the bluer the expression level, the lower the expression level. h) KEGG down‐regulated pathway enrichment analysis of differentially expressed genes between M‐MFL and control treatment group. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
"],"string":"[\n \"
Schemes for construction of targeting liposomal antibiotic booster for targeted periplasmic NDMs inactivation via Bi(III)‐mediated Zn(II) removal. The meropenem‐loaded bismuth nanoclusters (MB) are encapsulated in the maltodextrin‐cloaked membrane fusion liposome and precisely delivered to the infectious sites with the assistance of bacteria‐specific maltodextrin transport pathway, where the liposome fuses with bacterial outer membrane, facilitating the periplasmic translocation of MB and intracellular ROS burst. Meanwhile, endogenous ROS amplification triggers the intracellular‐specific release of Bi(III) and meropenem. Released Bi(III) irreversibly inactive NDMs via displacing Zn(II) from NDMs active sites, thus protecting meropenem from hydrolyzation.
\",\n \"
a) The scheme presenting the synthesis process of Bismuth‐Nano‐Clusters (BiNCs) and ROS‐responsive Bi(III) release from BiNCs. b) Hydrodynamic diameter distribution and zeta potential distribution (inset) obtained for BiNCs. c) Representative TEM image of BiNCs. Scale bar, 10 nm. d) A representative high‐resolution TEM image of BiNCs. Scale bar, 2.5 nm. e) XPS diffraction spectrum of BiNCs. f) STEM‐HAADF image and corresponding EDS elemental mappings of Bi, O, P, N, and C in BiNCs. g) EDS spectrum for bismuth element analysis of BiNCs. h) X‐ray diffraction (XRD) analysis of BiNCs. i) In vitro Bi(III) release from BiNCs against different levels of H2O2. j) Representative TEM images of BiNCs after 24 h incubation with different levels of H2O2. Scale bar, 100 nm. k) PA response to different concentrations of BiNCs (0.1, 0.3, 0.6, 0.9, 1.2, and 1.5 mg/mL) (inset: PA imaging of BiNCs). Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a, b) Representative heat plots of microdilution checkerboard assays for the combination of BiNCs and meropenem in the absence (a) or presence (b) of H2O2 against EC1322. c) Isobolograms of the combination of BiNCs and meropenem in the absence or presence of H2O2 against EC1322. The black dotted line shows the ideal isobole, where drugs act additively and independently. Data points below this line reveal synergism. d) Time‐kill curves for meropenem or BiNCs monotherapy, or their combination therapy in the absence or presence of H2O2 against EC1322 during 6 h incubation. The concentrations of meropenem, BiNCs, and H2O2 are used at 8 µg/mL, 125 µg/mL, and 200 µM, respectively. e) Hydrolytic effects of the BiNCs or BiNCs+H2O2 pre‐treated NDM‐1‐producing E. coli BL21 on meropenem (n = 3). f) Inhibition of NDM‐1 activity by Bi(III) from 200 µM H2O2‐pretreated BiNCs with IC50 of 0.046 mg/mL (n = 3). g) Double reciprocal plot of substrate‐dependent enzyme kinetics on inhibition of NDM‐1 activity by Bi(III) from 200 µM H2O2‐pretreated BiNCs, reflecting that Bi(III) (released from BiNCs) inhibited NDM‐1 via either a non‐competitive or an irreversible inhibition mode. h) Zn(II) content in Zn2‐NDM‐1 and the supernatant after being treated with different concentrations of Bi(III) from H2O2‐pretreated BiNCs by equilibrium dialysis, respectively. The metal content was determined by ICP‐MS. i, j) ITC thermograms for the binding of Bi(III) from H2O2‐pretreated BiNCs (i) or BiNCs (j) to NDM‐1. The downward peaks indicate an exothermic process. k) Cellular thermal shift assay demonstrating the binding of Bi(III) to NDM‐1 in NDM‐producing E. coli BL21. NDM‐1 melting temperature was shifted from 68.1° to 56.8 °C for control and BiNCs‐H2O2 combination group, respectively. The images show the western blotting result. l Schematic diagram of the action mechanism of BiNCs under non‐oxidative stress and oxidative stress on NDM‐1‐producing E. coli. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a) The schematic illustration exhibiting the preparation process of maltodextrin‐decorated membrane fusion liposome that wraps meropenem‐loaded BiNCs (M‐MFL@MB) and the highly specific bacterial targeting mechanism. b) Hydrodynamic diameter and ζ potential of BiNCs, meropenem‐loaded BiNCs (MB), maltodextrin‐decorated membrane fusion liposome (M‐MFL) and M‐MFL@MB (n = 3). c) Representative TEM and cryo‐TEM (inset) images of M‐MFL (left) and M‐MFL@MB (right). Scale bar, 100 nm. d, e) In vitro MEM (d) and Bi(III) (e) release curves of MEM@BiNCs (MB) (upper) and M‐MFL@MB (lower) in PBS containing different concentrations of H2O2 (1, 10, 100 and 200 µM), (n = 3). f) Representative pseudo‐color SEM images of EC1322 after incubation with PBS, M‐MFL@MB, and MFL@MB, respectively. Scale bar, 1 µm. g) The experimental scheme of coculture experiments for verifying the capability of maltodextrin‐mediated specifical targeting to bacteria, not mammalian cells. h, i) Confocal images of mononuclear macrophages (RAW 264.7 cells) cocultured with GFP‐expressing E. coli and imaged after labeling with M‐MFL@MB (h) or MFL@MB (i), respectively. Scale bar, 5 µm. The nucleus of RAW 264.7 cells, E. coli, and MFL were labeled with DAPI (blue), Green fluorescent protein (green), and Rhodamine (red), respectively. Plot profiles corresponding to white lines are shown on the right. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a, b) Representative confocal images visualize the membrane fusion interaction between M‐MFL or MFL with E. coli (a), and intracellular drug delivery of M‐MFL or MFL into E.coli (b). M‐MFL and MFL were labeled with fluorescent dye Rhodamine (red), Cy5 fluorescent dye (pink) was loaded into M‐MFL or MFL for substituting MB, and the E. coli could express GFP (green fluorescent protein, green). The control group was incubated with PBS. Scale bar, 100 nm. c) The corresponding fluorescence semi‐quantitative analysis (n = 3), shows membrane fusion and intracellular delivery efficiency of M‐MFL and MFL, respectively. d) Bio‐TEM images and the bismuth element mapping of different nanoparticles‐treated EC1322. Untreated B16‐F10 cells were used as control. Scar bar: 1 µm; Red arrows pointed to BiNCs. e) ICP‐MS analyzes the effect of different treatments on intracellular Bi(III) accumulation of E. coli (n = 3). f) Intracellular ROS level after being treated with different nanoparticles was monitored by detection of DCFH‐DA fluorescence intensity using confocal imaging. g) ESR spectrum of M‐MFL‐treated E. coli, the untreated E.coli was used as control. Scale bar, 30 µm. h) Determination of H2O2 amount in NDM‐1‐EC1322 after different concentrations of M‐MFL treatment (n = 3). i) Hydrolytic effects of the EC1322 on meropenem after different treatments, including MEM, MEM@BiNCs, M‐MFL@MEM and M‐MFL@MB. n = 3. j) Schematic diagram of the action mechanism of targeting liposomal antibiotic booster on NDM‐1 producers. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a) Measurement of bacterial colony‐forming units, obtained from six clinical isolates of NDM‐1‐producing E. coli treated with different concentrations of meropenem (MEM), MB, M‐MFL@MEM and M‐MFL@MB, respectively (n = 6). b) MIC of MEM, MB, M‐MFL@MEM and M‐MFL@MB in six clinical isolates of NDM‐1‐positive E. coli (n = 6). c) Time‐kill curves for MEM, BiNCs, M‐MFL, MB, M‐MFL@MEM, and M‐MFL@MB against EC1322 during 6 h incubation, respectively (n = 3). The concentrations of MEM were 8 µg/mL in those groups. The concentrations of BiNCs and M‐MFL were about 53.33 and 177.77 µg/mL, respectively. d) Representative SEM images of EC1322 after treatment with different nanoparticles for 1 h and 6 h, respectively. Scale bar, 0.5 µm. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a) Experimental scheme of the bacterial‐infected zebrafish model for demonstrating maltodextrin‐mediated adhesion ability. b) Lateral views of the whole bacteria‐infected zebrafish after DiI‐labeled M‐MFL treatment (b1). The bacterial‐infected zebrafish incubated with the DiI‐ labeled MFLipo treatment (b2) and the healthy zebrafish incubated with DiI‐labeled M‐MFL (b3) were used as the control. c, d) Ex‐vivo tissue NIR FL images I and fluorescence semi‐quantitative analysis (d) of mice model of dual infection with LPS and E. coli at 22 h post‐injection with M‐MFL@IR783, MFL@IR783, or IR783, respectively (n = 3). e, f) Representative CLSM images I and fluorescence semi‐quantitative analysis (f) of MFL, M‐MFL distribution at different time points after intravenous injection in the E. coli infected lung tissues, respectively. The nanoliposome was stained with rhodamine (red). The encapsulated drugs were replaced by cy5 (pink). The nucleus of lung tissues was stained with DAPI (blue). Scar bar, 100 µm. h, i) PA imaging (h) and PA response value (i) of E. coli‐infected mice model at different time points after intravenous injection with BiNCs and M‐MFL@MB, respectively. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a) Schematic diagram of the infection, treatment used in mice with pneumonia. b, c) Bacterial loads (b) and mouse body temperature changes (c) in the pneumonia mice model after different nanoformulations treatment (n = 6). d) Experimental roadmap for detecting inflammation‐related indicators (CRP, SAA and PCT) of mice with pneumonia. e) CRP, SAA and PCT level in the pneumonia mice model after different treatments (n = 6). f) HE staining of infected lung tissues in the pneumonia mice model after different treatments. Scar bar, 100 µm. g) Schematic diagram of the infection, treatment used in mice with sepsis. h, i, j) Survival curves (h), bacterial loads (i) and body temperature changes (j) in the sepsis mice model after different treatments, n = 6. k) CRP, SAA and PCT level in the sepsis mice model after different treatments (n = 6). l) HE staining of infected tissues in the sepsis mice model subjected to different treatments. Scar bar, 100 µm. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
\",\n \"
a) an NTA analysis and representative TEM images (inset) of E. coli OMVs extracted from E. coli incubated with Lipo and M‐MFL, respectively. The OMVs extracted from untreated E. coli were used as control. Scar bar, 100 nm. b) Statistics of size and concentration of E. coli OMVs extracted from E. coli incubated with different nanoparticles. b, c, d) the measurement of total protein content (b, n = 3), NDM‐1 protein content (c, n = 3) and enzymatic activity of NDM‐1 (d, n = 3) of E. coli OMVs extracted from E. coli incubated with Lipo and M‐MFL, respectively. The OMVs extracted from untreated E. coli were used as control. e) Absolute copy number of bla\\nNDM‐1 gene in E. coli OMVs extracted from E. coli incubated with Lipo and M‐MFL, respectively. The OMVs extracted from untreated E. coli were used as control. n = 3. f) The MIC of EC15922 after incubation with different E. coli OMVs (n = 6). g) Clustering heat map of differentially expressed genes (DEGs) between M‐MFL‐treated E. coli and untreated E. coli. The abscissa is the sample name, and the ordinate is the normalized value of the DEGs. The redder the color, the higher the expression level, and the bluer the expression level, the lower the expression level. h) KEGG down‐regulated pathway enrichment analysis of differentially expressed genes between M‐MFL and control treatment group. Data are presented as mean values ± SD, n = 3 biologically independent samples. Statistical significance was analyzed by the two‐tailed Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Urethral stricture, which is a narrowing process of urethra lumen caused by fibrosis of the urethra mucosa and the surrounding spongy corpus spongiosum, is often inevitable after urethral injury.[\n##UREF##0##\n1\n##, ##UREF##1##\n2\n##, ##REF##28118170##\n3\n##\n] It has been reported that 229 to 627 out of 100 000 males suffer from urethral stricture, but the treatment is still limited in the field of urology.[\n##REF##17437780##\n4\n##\n] Obviously, urethral stricture has a substantial impact on the quality of life in patients with high health costs.[\n##REF##17437780##\n4\n##\n] Urethroplasty procedures have been considered to be the most effective gold‐standard method, compared to certain micro‐invasive surgeries (e.g., urethral dilation and internal urethrotomy).[\n##REF##26631921##\n5\n##, ##REF##21176068##\n6\n##\n] A number of challenges indeed exist in the surgical treatment of urethral strictures, such as the use of substitute materials (autologous penile flap or oral mucosa).[\n##REF##27940192##\n7\n##\n] The autologous substitute materials for urethral reconstruction have limitations, which can cause damage at the harvesting site. Thus, suitable tissue engineering and alternative biomaterials have recently been intensively explored for more effective regenerative medicine in an attempt to overcome such problems of urethral reconstruction.[\n##REF##26631921##\n5\n##, ##REF##27940192##\n7\n##, ##REF##25689740##\n8\n##, ##REF##26941491##\n9\n##, ##REF##20477828##\n10\n##\n]\n
","
It is difficult to mimic an actual urethral structure with the dense mucosal layer and the loose submucosal layer for scarless regeneration. 3D fabrication of biologically functional components is certainly a promising technology, as it adopts the extracellular matrix (ECM) molecules and provides a biomimetic microenvironment for cells.[\n##REF##34133940##\n11\n##, ##REF##27315476##\n12\n##\n] With its emergence, the synthesis of scaffolds has attracted enormous attention owing to the programmable deposition of biocompatible materials.[\n##REF##25093879##\n13\n##, ##REF##31426033##\n14\n##\n] Currently, hydrogel biomaterials are routinely used as (bio)inks in extrusion‐based 3D (bio)printing thanks to the printability and cell‐laden capacity.[\n##REF##26561931##\n15\n##\n] In our previous study, we have shown that the fibrin hydrogels laden with urothelial cells (UCs) and smooth muscle cells (SMCs) could maintain sufficient cell viability and proliferation.[\n##REF##27940192##\n7\n##\n] Nonetheless, the mechanical properties of fibrin were too weak to function as the urethral scaffold. Thus, a new class of hydrogel biomaterials for urethral reconstruction is strongly necessary.
","
SA and Gel, as the natural hydrogel biomaterials that are biocompatible and printable, have been widely used in engineered organs.[\n##REF##22318897##\n16\n##, ##REF##27898010##\n17\n##, ##REF##27935198##\n18\n##\n] There are some unique characteristics for SA and Gel. The polymer chains of SA can be cross‐linked with Ca2+ to form a stable structure for mechanical support.[\n##UREF##2##\n19\n##\n] The microenvironment of urethra is filled with urine that has plenty of calcium ions.[\n##REF##23377289##\n20\n##\n] It is feasible to realize a second cross‐link of SA to reinforce the strength. Gel is a thermosensitive material that transforms from liquid phase to gel state via physical cross‐linking, and maintains the initial structural stability of the 3D‐printed scaffolds.[\n##REF##32948593##\n21\n##\n] Meanwhile, due to the arginine‐glycine‐aspartic acid (RGD) sequences intrinsic to the Gel chains, it is considered friendly to the enhanced cell attachment and growth.[\n##REF##26523399##\n22\n##, ##UREF##3##\n23\n##\n]\n
","
For 3D printing, the formulation of the inks is the decisive factor for the successful scaffold fabrication.[\n##REF##26561931##\n15\n##\n] Zhang et al. have designed the low‐density SA (0.8%) and Gel (4.1%) hybrid bioinks for 3D bioprinting.[\n##REF##31426033##\n14\n##\n] The results showed that the cell viability for the concentration of 0.8% SA was 84 ± 0.7%. Yet, the mechanical properties, printability, and shape fidelity of the scaffolds were insufficient. The cell viability of 2.3% SA was only 68 ± 1.3%, but the fidelity and mechanical properties of the scaffolds were improved due to the increased viscosity of SA. The optimal concentrations of printability ranged from 0.6 to 2.5%, depending on the viscosity of SA. Several other studies have also optimized the formulation of SA and Gel to satisfy the printability of bioinks and improve the viability of cells.[\n##REF##29304429##\n24\n##, ##REF##24157694##\n25\n##, ##UREF##4##\n26\n##, ##REF##31782460##\n27\n##\n]\n
","
On the other hand, to obtain the desired properties of bioinks, certain nanomaterials (e.g., inorganic and carbon nanomaterials) have been integrated with SA/Gel to prepare the nanocomposite bioinks.[\n##REF##33326888##\n28\n##, ##REF##29636985##\n29\n##\n] Graphene‐based materials are considered to be the reinforcement nanomaterials: the representative derivatives are GO and rGO, with attributes of favorable antibacterial properties,[\n##REF##23586616##\n30\n##, ##REF##32254787##\n31\n##\n] angiogenic potential,[\n##UREF##5##\n32\n##, ##REF##29428812##\n33\n##\n] mechanical strength,[\n##REF##33321630##\n34\n##, ##REF##33599084##\n35\n##\n] as well as low cytotoxicity.[\n##UREF##6##\n36\n##\n] Studies have suggested that regulating the surface oxygen content of rGO enhances the cell adhesion and proliferation due to the non‐covalent interactions to improve the surface adsorption of rGO.[\n##UREF##7##\n37\n##\n] In addition, Mukherjee et al. reported that rGO exhibited considerable angiogenic activity in a dose‐dependent manner.[\n##UREF##5##\n32\n##\n] Cell proliferation and wound healing were also promoted by the increased concentration of reactive oxygen species (ROS) due to the incorporation of rGO.[\n##REF##31534523##\n38\n##, ##REF##33684536##\n39\n##\n]\n
","
To our knowledge, there are still few studies on fabricating the urethral substitutes by the 3D‐printing technology that would function in the harsh urethral microenvironment. The ideal treatment on urethral stricture should exhibit the following characteristics: i) the match of the patch with the structure of urethral decellularized matrix (Figure ##SUPPL##0##S1##, Supporting Information); ii) angiogenesis and reconstruction of urethral functions; iii) inhibition of bioactive factors on urethral fibrosis and scar hyperplasia. In this study, we design a 3D‐printed SA/Gel/rGO patch, by a double‐freeze‐drying process and also cross‐linked by Ca2+ with enhanced stability, to characterize its anti‐fibrotic, angiogenic, and epitheliogenic properties in urine (Scheme\n##FIG##0##\n1\n##). Systematic experiments in vitro and in vivo were conducted to understand the unique features, which would demonstrate the therapeutic efficacy of the patch for urethral reconstruction.
"],"string":"[\n \"Introduction\",\n \"
Urethral stricture, which is a narrowing process of urethra lumen caused by fibrosis of the urethra mucosa and the surrounding spongy corpus spongiosum, is often inevitable after urethral injury.[\\n##UREF##0##\\n1\\n##, ##UREF##1##\\n2\\n##, ##REF##28118170##\\n3\\n##\\n] It has been reported that 229 to 627 out of 100 000 males suffer from urethral stricture, but the treatment is still limited in the field of urology.[\\n##REF##17437780##\\n4\\n##\\n] Obviously, urethral stricture has a substantial impact on the quality of life in patients with high health costs.[\\n##REF##17437780##\\n4\\n##\\n] Urethroplasty procedures have been considered to be the most effective gold‐standard method, compared to certain micro‐invasive surgeries (e.g., urethral dilation and internal urethrotomy).[\\n##REF##26631921##\\n5\\n##, ##REF##21176068##\\n6\\n##\\n] A number of challenges indeed exist in the surgical treatment of urethral strictures, such as the use of substitute materials (autologous penile flap or oral mucosa).[\\n##REF##27940192##\\n7\\n##\\n] The autologous substitute materials for urethral reconstruction have limitations, which can cause damage at the harvesting site. Thus, suitable tissue engineering and alternative biomaterials have recently been intensively explored for more effective regenerative medicine in an attempt to overcome such problems of urethral reconstruction.[\\n##REF##26631921##\\n5\\n##, ##REF##27940192##\\n7\\n##, ##REF##25689740##\\n8\\n##, ##REF##26941491##\\n9\\n##, ##REF##20477828##\\n10\\n##\\n]\\n
\",\n \"
It is difficult to mimic an actual urethral structure with the dense mucosal layer and the loose submucosal layer for scarless regeneration. 3D fabrication of biologically functional components is certainly a promising technology, as it adopts the extracellular matrix (ECM) molecules and provides a biomimetic microenvironment for cells.[\\n##REF##34133940##\\n11\\n##, ##REF##27315476##\\n12\\n##\\n] With its emergence, the synthesis of scaffolds has attracted enormous attention owing to the programmable deposition of biocompatible materials.[\\n##REF##25093879##\\n13\\n##, ##REF##31426033##\\n14\\n##\\n] Currently, hydrogel biomaterials are routinely used as (bio)inks in extrusion‐based 3D (bio)printing thanks to the printability and cell‐laden capacity.[\\n##REF##26561931##\\n15\\n##\\n] In our previous study, we have shown that the fibrin hydrogels laden with urothelial cells (UCs) and smooth muscle cells (SMCs) could maintain sufficient cell viability and proliferation.[\\n##REF##27940192##\\n7\\n##\\n] Nonetheless, the mechanical properties of fibrin were too weak to function as the urethral scaffold. Thus, a new class of hydrogel biomaterials for urethral reconstruction is strongly necessary.
\",\n \"
SA and Gel, as the natural hydrogel biomaterials that are biocompatible and printable, have been widely used in engineered organs.[\\n##REF##22318897##\\n16\\n##, ##REF##27898010##\\n17\\n##, ##REF##27935198##\\n18\\n##\\n] There are some unique characteristics for SA and Gel. The polymer chains of SA can be cross‐linked with Ca2+ to form a stable structure for mechanical support.[\\n##UREF##2##\\n19\\n##\\n] The microenvironment of urethra is filled with urine that has plenty of calcium ions.[\\n##REF##23377289##\\n20\\n##\\n] It is feasible to realize a second cross‐link of SA to reinforce the strength. Gel is a thermosensitive material that transforms from liquid phase to gel state via physical cross‐linking, and maintains the initial structural stability of the 3D‐printed scaffolds.[\\n##REF##32948593##\\n21\\n##\\n] Meanwhile, due to the arginine‐glycine‐aspartic acid (RGD) sequences intrinsic to the Gel chains, it is considered friendly to the enhanced cell attachment and growth.[\\n##REF##26523399##\\n22\\n##, ##UREF##3##\\n23\\n##\\n]\\n
\",\n \"
For 3D printing, the formulation of the inks is the decisive factor for the successful scaffold fabrication.[\\n##REF##26561931##\\n15\\n##\\n] Zhang et al. have designed the low‐density SA (0.8%) and Gel (4.1%) hybrid bioinks for 3D bioprinting.[\\n##REF##31426033##\\n14\\n##\\n] The results showed that the cell viability for the concentration of 0.8% SA was 84 ± 0.7%. Yet, the mechanical properties, printability, and shape fidelity of the scaffolds were insufficient. The cell viability of 2.3% SA was only 68 ± 1.3%, but the fidelity and mechanical properties of the scaffolds were improved due to the increased viscosity of SA. The optimal concentrations of printability ranged from 0.6 to 2.5%, depending on the viscosity of SA. Several other studies have also optimized the formulation of SA and Gel to satisfy the printability of bioinks and improve the viability of cells.[\\n##REF##29304429##\\n24\\n##, ##REF##24157694##\\n25\\n##, ##UREF##4##\\n26\\n##, ##REF##31782460##\\n27\\n##\\n]\\n
\",\n \"
On the other hand, to obtain the desired properties of bioinks, certain nanomaterials (e.g., inorganic and carbon nanomaterials) have been integrated with SA/Gel to prepare the nanocomposite bioinks.[\\n##REF##33326888##\\n28\\n##, ##REF##29636985##\\n29\\n##\\n] Graphene‐based materials are considered to be the reinforcement nanomaterials: the representative derivatives are GO and rGO, with attributes of favorable antibacterial properties,[\\n##REF##23586616##\\n30\\n##, ##REF##32254787##\\n31\\n##\\n] angiogenic potential,[\\n##UREF##5##\\n32\\n##, ##REF##29428812##\\n33\\n##\\n] mechanical strength,[\\n##REF##33321630##\\n34\\n##, ##REF##33599084##\\n35\\n##\\n] as well as low cytotoxicity.[\\n##UREF##6##\\n36\\n##\\n] Studies have suggested that regulating the surface oxygen content of rGO enhances the cell adhesion and proliferation due to the non‐covalent interactions to improve the surface adsorption of rGO.[\\n##UREF##7##\\n37\\n##\\n] In addition, Mukherjee et al. reported that rGO exhibited considerable angiogenic activity in a dose‐dependent manner.[\\n##UREF##5##\\n32\\n##\\n] Cell proliferation and wound healing were also promoted by the increased concentration of reactive oxygen species (ROS) due to the incorporation of rGO.[\\n##REF##31534523##\\n38\\n##, ##REF##33684536##\\n39\\n##\\n]\\n
\",\n \"
To our knowledge, there are still few studies on fabricating the urethral substitutes by the 3D‐printing technology that would function in the harsh urethral microenvironment. The ideal treatment on urethral stricture should exhibit the following characteristics: i) the match of the patch with the structure of urethral decellularized matrix (Figure ##SUPPL##0##S1##, Supporting Information); ii) angiogenesis and reconstruction of urethral functions; iii) inhibition of bioactive factors on urethral fibrosis and scar hyperplasia. In this study, we design a 3D‐printed SA/Gel/rGO patch, by a double‐freeze‐drying process and also cross‐linked by Ca2+ with enhanced stability, to characterize its anti‐fibrotic, angiogenic, and epitheliogenic properties in urine (Scheme\\n##FIG##0##\\n1\\n##). Systematic experiments in vitro and in vivo were conducted to understand the unique features, which would demonstrate the therapeutic efficacy of the patch for urethral reconstruction.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results and Discussion","rGO Characterizations and Ink Preparation","
Commercially available monolayer rGO powder was used. The rGO was dispersed in water according to the information provided by the manufacturer. The monolayer ratio of rGO powder was up to 80%, the diameters were 0.5–5 µm, and the thicknesses were 0.8–1.2 nm. The morphology of rGO was confirmed under scanning electron microscope (SEM) and transmission electron microscope (TEM) (Figure\n##FIG##1##\n1a,b##). The stock rGO dispersion (1 mg mL−1) was diluted to concentrations in a series of 0.02, 0.05, 0.1, and 0.2 mg mL−1, and all suspensions remained stable and homogeneous for several weeks upon evaluation. With the increasing concentration of rGO, the solution color changed from gray to dark (Figure ##FIG##1##1c##). The solutions were also examined under optical microscopy to show the good dispersion with no obvious aggregation for rGO (Figure ##FIG##1##1d–g##). The area distributions of the rGO were mostly at the 1–50 µm2 range (Figure ##FIG##1##1h–k##). The mean area rate (%) is from 4% to 28% (Figure ##SUPPL##0##S2##, Supporting Information). The results demonstrate that the rGO distributes evenly in the water solution, and most of the rGO particles do not adhere to each other.
","Characterizations of SA/Gel/rGO Hydrogels","
Raman spectroscopic analysis of SA/Gel/rGO shows two characteristic bands of G‐ and D‐bands (Figure\n##FIG##2##\n2a##). The D‐band was at ≈1342 cm−1 and the G‐band was at ≈1584 cm−1. The D‐band reflects the disorder between graphite lamellae due to the interference of sp2\n hybridization of carbon. The G‐band reflects the symmetry and degree of crystallization due to the sp2\n hybrid in‐plane stretching vibration of carbon crystals. ID/IG is the intensity ratio between the D‐ and G‐bands, suggesting the degree of defects in the graphite lamellae[\n##REF##33982723##\n40\n##, ##REF##34576956##\n41\n##\n]; the higher the intensity ratio is, the more defects the C atomic crystal has. Chakraborty reported that the ID/IG ratios of GO and rGO were 0.9 and 1.1, respectively.[\n##REF##35548324##\n42\n##\n] To verify the incorporation of rGO into the SA/Gel hydrogels, the composition of the SA/Gel/rGO composite was confirmed through Fourier‐transform infrared (FTIR) spectroscopy (Figure ##FIG##2##2b##). The SA hydroxyl bond (O─H) stretching was at ≈3445 cm−1, the CH2 group stretching was at ≈2935 cm−1, and the carbonyl bond (C═O) stretching of amide I was at 1632 cm−1. The characteristic bands of SA at ≈1632 and 1401 cm−1 are associated with the carboxyl (─COOH) bond and asymmetric and symmetric stretching peaks of carboxylate salt groups, respectively. The peak at 1542 cm−1 is the ─NH group stretching of amide II and the peak at 1238 cm−1 is the ─NH group stretching of amide III of Gel. The peak at 1177 cm−1 is the C─O─C asymmetric stretching. The peak at 1028 cm−1 is the C─O─H stretching of SA. The characteristic peaks of rGO at ≈2930 and 2854 cm−1 are associated with CH2 and CH3 groups, respectively. Interestingly, the strong absorption peaks at ≈2930 and 2854 cm−1 appeared with the increased concentration of rGO. As such, the FTIR spectra clearly indicated the presence of the rGO in the hybrid hydrogels.
","
The swelling behavior of a hydrogel construct plays a crucial role in its surface properties and mechanical integrity for biomaterial applications.[\n##REF##32310981##\n43\n##, ##REF##32500887##\n44\n##\n] The swelling behaviors of the various hydrogel formulations suggested that the swelling ratio increased when the concentration of SA was elevated (1%, 1.5%, and 2%) and the concentration of rGO was reduced (Figure ##FIG##2##2c##). The higher content of SA enhanced the hydrophilicity of the hydrogel network to improve the equilibrium swelling ratio of SA. For the positive effect of rGO concentration, it was likely due to the ability of rGO to interact with the hydrogen bonds of SA/Gel to act like a multi‐functional cross‐linking agent, therefore increasing the density of the cross‐linked network of the SA/Gel hydrogels at a higher concentration.
","
Overtime, the hydrogels would be subjected to hydrolysis and other forms of degradation after being immersed in the liquid environment. The degree of degradation was evaluated by the mass remaining (%) of the hydrogels over 21 days. The various degrees of degradation showed a significant weight reduction in the first 7 days (Figure ##FIG##2##2d##), which might be due to the hydrolysis of the hydrogels. The remaining mass percentages (%) of the hydrogels are ≈42.62 ± 1.98% to 55.58 ± 1.44% in the different groups. In the next few days, there was a slight decrease in the weights of the hydrogels, which might correspond to the reduced degradation of the cross‐linked structure. The total weights of hydrogels had no significant differences between the SA/Gel hydrogels with and without rGO, implying that the incorporation of rGO did not noticeably affect the degradation of the hydrogel formulations.
","Morphological Evaluations on the SA/Gel/rGO Hydrogels","
The colors of the SA/Gel/rGO hydrogels were observed to be darker with the increased concentration of rGO. The SEM images reveal the porous structures of the SA/Gel/rGO hydrogels with different concentrations of rGO (0, 0.02, 0.05, 0.1, and 0.2 mg mL−1) (Figure\n##FIG##3##\n3a–j##). As the rGO loading increases, the pore size decreases and the pore density increases, further indicating that a high rGO concentration led to a high cross‐linking density, resulting in smaller pore sizes in composite hydrogels (Figure ##FIG##3##3b–j##). The pore size of hydrogels is highly correlated with cell adhesion, growth and proliferation, and nutrient exchange in the matrix. Most importantly, the hydrogels with a porous structure could facilitate efficient biomolecule transport within the environment.[\n##REF##35548324##\n42\n##\n] However, it should be noted that the pore sizes obtained by SEM observations may not indicate the actual pore sizes of the hydrogels under the hydrated states.
","
From the distributions of the pore sizes for the SA/Gel/rGO hydrogels (Figure ##FIG##3##3k–o##), there were network structures with the pore sizes of <100 µm for all the hydrogels. The distributions of the pore sizes of the SA/Gel hydrogel were mainly in the range of 80–200 µm; the pore sizes of the SA/Gel/rGO hydrogels decreased with the increasing concentration of rGO (Figure ##FIG##3##3p##). The pore sizes of the SA/Gel and SA/Gel/rGO0.02 hydrogels were 92.82 ± 12.60 and 91.59 ± 11.6 µm, respectively . There was significant difference between SA/Gel/rGO0.05 (69.39 ± 7.97 µm) and SA/Gel/rGO0.1 (47.20 ± 7.26 µm), compared with the SA/Gel hydrogels. The pore size of SA/Gel/rGO0.2 was the smallest among all the hydrogels (36.29 ± 17.77 µm, ). These results showed that the incorporation of rGO indeed influenced the pore size of the hydrogels in a dose‐dependent pattern.
","Morphological and Mechanical Evaluations on SA/Gel/rGO Patches","
We 3D‐printed various SA/Gel scaffolds with different concentrations of rGO from 0 to 0.2 mg mL−1 (Figure\n##FIG##4##\n4a##). We used two freeze‐drying and one cross‐linking process to obtain the patches post‐printing. The gross morphologies were not significantly different except that the color changed from white to dark at the rGO concentration was increased. The 3D printer was an Organ Printing United system (Figure ##SUPPL##0##S3a##, Supporting Information). The mechanical properties of the SA/Gel/rGO patches are a valuable element for urethral repair and tissue regeneration. The tensile mechanical characteristics of the SA/Gel/rGO patches with different concentrations of rGO were evaluated by the stretching process of the patches (Figure ##SUPPL##0##S3b##, Supporting Information), the length of the patches that stretched between the upper and lower grips of the tensile machine could be adjusted. Then, each sample was stretched at 10 mm per minute until reaching its breaking points. All the samples exhibited the linear elastic behavior (Figure ##FIG##4##4b##). Compared with the patches containing rGO, the SA/Gel patch without rGO showed a lower modulus, 139.68 ± 0.144 KPa (Figure ##FIG##4##4c##). The tensile strengths of the SA/Gel patches at concentrations of 0, 0.02, 0.05, and 0.2 mg mL−1 of rGO were in the range of 139.87 ± 1.02–296.65 ± 1.11 KPa (Figure ##FIG##4##4d##). The tensile strength of the SA/Gel/rGO0.2 patch was higher (296.65 ±1.11 KPa) when the concentration of rGO was increased. The elevation of strain at break in the SA/Gel/rGO0.05 was 211.10 ± 1.34% (Figure ##FIG##4##4e##). Besides, the patches with appropriate elongation are flexible and elastic. Interestingly, the strain at break of SA/Gel/rGO0.2 patch was 137.77 ± 0.86% . These observations might be attributed to the SA/Gel patch to become fragile under the higher concentration of rGO. The SA/Gel/rGO patch was saturable and exhibits excellent mechanical properties to fit the requirement of urethral reconstruction. Interestingly, the rGO hybrid patches appeared to possess higher tensile strengths and Young's modulus. The rGO0.05 was an appropriate concentration to achieve the best mechanical performance among the groups examined.
","Characteristics of Cell Behaviors","
SA/Gel/rGO patches served as the templates for cell adhesion, and proliferation, thus superior cytocompatibility is required. The characteristics of cell survival and proliferation were evaluated through seeding fibroblasts on SA/Gel/rGO hydrogels containing different concentrations of rGO. The live and dead cells were stained with calcein AM and propidium iodides (PI) after 1 day, respectively. The fluorescence of living cells in SA/Gel/rGO hydrogels of 0.1 and 0.2 mg mL−1 of rGO was less (Figure\n##FIG##5##\n5a##). The increased number of dead cells might have been affected in part by the relatively high concentrations of rGO present. The cell morphologies were further observed by SEM, where fibroblasts were generally located within the pores of the 3D‐printed SA/Gel/rGO patches (Figure ##SUPPL##0##S4##, Supporting Information).
","
From the expression patterns of differentially expressed proteins (DEPs) by fibroblasts (cell density: 1 × 107) with the different concentrations of rGO (Figure ##FIG##5##5b##), the cells treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 were compared with the cells with no treatment. The culture time was 72 h. Some biological processes and pathways, which are correlated with tissue regeneration or tissue repair, were observed to be significantly enriched in these DEPs (Figures ##SUPPL##0##S5a,S6a##, and ##SUPPL##0##S7a##, Supporting Information). For example, it is reported that the transmembrane receptor protein tyrosine kinase signaling pathway has a role in promoting sprouting of the angiogenic endothelium.[\n##REF##20445537##\n45\n##\n] The Ras/MAPK signaling cascade plays an important role in zebrafish heart regeneration.[\n##UREF##8##\n46\n##\n] The cell proliferation process is required for cardiomyocytes regeneration.[\n##REF##28185170##\n47\n##\n] In addition, the cellular components related to platelet alpha granule, secretory veside, vesicle lumen, and endomembrane system were enriched in DEPs treated with rGO0.02 (Figure ##SUPPL##0##S5b##, Supporting Information). Similar situations were observed when treated with rGO0.05 and treated with rGO0.1 (Figures ##SUPPL##0##S6b## and ##SUPPL##0##S7b##, Supporting Information). In addition, in molecular functions, the growth factor binding, growth factor activity, receptor‐ligand activity, and signaling receptor activator were enriched (Figures ##SUPPL##0##S5c,S6c##, and ##SUPPL##0##S7c##, Supporting Information). Similarly, the enriched Ras and MAPK signaling pathways were also observed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figures ##SUPPL##0##S5d,S6d##, and ##SUPPL##0##S7d##, Supporting Information). The genes involved in these biological processes and pathways might contribute to angiogenesis mediated by rGO. The bubble graph was also shown for gene ontology enrichment treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 for the biological process, molecular process, and cellular components (Figure ##SUPPL##0##S8a–i##, Supporting Information).
","
Scratch assay is a standard for cell migration study in vitro. The migration of fibroblasts for the wound healing process is of great importance; after the injury, fibroblasts were used to proliferate and migrate from neighboring tissues into the wound area.[\n##REF##20824344##\n48\n##\n] To evaluate the potential of rGO in enhancing the migration of fibroblasts, a time‐dependent experiment was carried out (0–24 h). The results of the scratch assay showed that the migration ability of fibroblasts was greatly improved within 24 h after the treatment with rGO concentrations of 0.02 and 0.05 mg mL−1 (Figure ##FIG##5##5c##). With the increasing concentration of rGO, the proliferation of fibroblasts and wound healing increased first and then decreased, with a maximum migration rate of 61.18 ± 2.61% for rGO at 0.05 mg mL−1 (Figure ##FIG##5##5d##). Of note, when the concentration of rGO was set at 0.2 mg mL−1, the migration and proliferation of fibroblasts were significantly inhibited (26.39 ± 3.93%).
","
Cell counting kit‐8 (CCK‐8) assay was subsequently used to assess the cytotoxic effect of SA/Gel/rGO patches on the fibroblasts (Figure ##FIG##5##5e##). After 1 and 4 days of cell culture, the metabolic activities of cells on the SA/Gel/rGO patches exhibited inhibitory effects. The higher concentration of rGO revealed inhospitable environments to cell metabolic activities during this time point. However, after 7 days of cell culture, the cell metabolic activities on SA/Gel/rGO0.02 and SA/Gel/rGO0.05 became higher than the remaining patches close to that for the control patches without rGO. Consequently, it might be undesirably toxic to cells at the concentration of rGO above 0.05 mg mL−1, in alignment with the cell viability staining results. Some reports have suggested that GO concentration of 50 µg mL−1 on fibroblasts led to significant cytotoxicity, while rGO at a concentration higher than 0.1 µg mL−1 would be toxic to endothelial cells.[\n##UREF##5##\n32\n##, ##REF##21082807##\n49\n##\n] Our results were overall consistent with the previous studies.
","Urethrogram and Gross Morphology","
The safety and functions of the SA/Gel/rGO patches were further proven in clinically relevant rabbit urethral injury models. According to the results of the in vitro mechanical test and biocompatibility of the scaffold, the patches without rGO were selected as the control group, and the rGO concentration of 0.05 and 0.2 mg mL−1 was selected as the experimental group. The patches were sutured to the dorsal urethral defect, and the injury model of the urethra and the surgical process of repair are shown in Figure ##SUPPL##0##S9## (Supporting Information). The recovery of the repaired urethras was detected at 4 and 8 weeks after the operation. The 4‐ and 8‐week urethrogram of the SA/Gel group displayed narrow lumens because of scar formation (Figure\n##FIG##6##\n6a,d##). The SA/Gel/rGO0.05 (Figure ##FIG##6##6b,e##) groups showed wide lumens of urethras similar to the normal urethra (Figure ##FIG##6##6g##). In the 4‐week urethrogram, the SA/Gel/rGO0.2 groups had a narrower urethral lumen, the widths developed larger at 8 weeks (Figure ##FIG##6##6c,f##). The reason might be the inflammation‐related swelling of urethral tissue caused by rGO with high concentration. The blockage rate determines whether and how much urine can flow out. The blockage ratio of the urethras treated with the SA/Gel/rGO0.05 patches could approach that of normal lumens (Figure ##FIG##6##6i##). After the incision of the ventral urethral wall, the dorsal wall could be exposed. Gross morphology revealed that urethral tissue in the SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups was significantly better than that in the SA/Gel group. The SA/Gel group showed the growth of severe scar‐like tissue in the urethra (Figure ##FIG##6##6j,m##). At 8 weeks, both of SA/Gel/rGO0.05 (Figure ##FIG##6##6n##) and SA/Gel/rGO0.2 (Figure ##FIG##6##6o##) groups showed smooth epithelial layer similar to the normal urethra of rabbits (Figure ##FIG##6##6h##). However, SA/Gel/rGO0.2 group at 4 weeks (Figure ##FIG##6##6i##) revealed red and swollen mucosa compared with that in the SA/Gel/rGO0.2 group (Figure ##FIG##6##6k##), which is consistent with the lumen width in the urethrogram. Therefore, the treatment of urethral injury could be improved by choosing a low dose of rGO for future application.
","Histological Evaluations","
The histology analyses of urethral reconstruction at 4‐ and 8‐weeks post‐surgery were performed by hematoxylin and eosin (H&E) and Masson's trichrome staining. The SA/Gel patches with/without rGO showed various outcomes. Additionally, there was no significant infection during the experimental period, suggesting good biocompatibility of the patches. According to the histology, the urethras treated with the SA/Gel patches were associated with thinner/uncomplete urothelium layers, fewer blood vessels, and thicker submucosal tissue (Figure\n##FIG##7##\n7a–d##). Due to the lack of regenerated urothelium layers, and over‐deposited ECM, the urethra developed to urethral stricture. In contrast, the reconstructed urethra treated with the SA/Gel/rGO patches had the nearly normal urothelium layers, submucosal tissue, and blood vessels. The continuous and complete urothelium layers were formed on the lumen surfaces for the SA/Gel/rGO0.05 group after 4‐ and 8‐weeks post‐surgery. However, the SA/Gel/rGO0.2 patches could lead to a short period of swelling and inflammation in 4 weeks. These results suggested that rGO at a lower concentration (0.05 mg mL−1) could perform best function in the regeneration of urothelium layers and blood vessels, but not at a higher concentration (0.2 mg mL−1). The mucosal coverage ratios by the SA/Gel/rGO patches were measured (Figure ##FIG##7##7e,f##). The urethras treated with the SA/Gel/rGO0.05 patches had more significant coverages, compared with the other groups. The urethral collagen thicknesses were also measured after the wound healing (Figure ##FIG##7##7g,h##). The SA/Gel/rGO0.05 patch could induce a less collagen deposition under the mucosa, therefore developing a scar‐less wound healing. The histology showed that an optimal concentration of rGO would promote the regeneration of epithelial cells and decrease submucosal ECM deposition to inhibit urethral fibrosis.
","Immunofluorescence","
The distribution of cytokeratin was investigated based on the expression of epithelial cytokeratin AE1/AE3 (an important membrane surface protein marker of ECs) via immunofluorescence staining of the urethra. The expression level for AE1/AE3 on the SA/Gel/rGO0.05 at 4‐and 8‐weeks post‐surgery (4W: 16.70 ± 2.66 and 8W: 24.72 ± 2.81) is significantly higher than that in the other groups. Although the continuous urothelium layer is formed, the urothelium layer of the SA/Gel/rGO0.2 group is thinner than that of the SA/Gel/rGO0.05 group. The expression of cytokeratin in the SA/Gel group is low (4W: 8.19 ± 1.72 and 8W: 8.29 ± 1.65), there is no complete regeneration of a discontinuous urothelium layer (Figure\n##FIG##8##\n8a,e##). It suggests that the introduction of rGO has the potential to promote the regeneration of urothelium layers. Studies have shown that the significant pro‐angiogenic properties of rGO depend on its concentration.[\n##UREF##5##\n32\n##, ##REF##29892387##\n50\n##\n] In the results of fluorescence staining at 4‐weeks post‐surgery, the SA/Gel/rGO0.05 group showed a number of CD 31‐positive cells (the blood vessels labeled by CD 31), which were more than them in the SA/Gel and SA/Gel/rGO0.2 group. At the beginning process of urethral regeneration with inflammation, the vessels in tissue would be much more obvious that is consistent with the results.[\n##REF##12490959##\n51\n##\n] For SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups, the number of CD 31‐positive cells at 8 weeks (SA/Gel/rGO0.05: 14.10 ± 3.99 and SA/Gel/rGO0.2: 8.63 ± 1.23) are lower than them at 4 weeks (SA/Gel/rGO0.05: 15.35 ± 4.55 and SA/Gel/rGO0.2: 14.77 ± 3.07) (Figure ##FIG##8##8b,f##).
","
Different degrees of inflammation response at the injured sites would appear because of the transplantation of external scaffolds. CD 206 is used to label M2 macrophages.[\n##REF##31903147##\n52\n##\n] The results showed that the number of CD206‐positive cells in the SA/Gel group was the lowest. The number of CD206 positive cells in the SA/Gel/rGO0.05 group in 4‐and 8‐weeks post‐surgery (4W: 36.36 ± 3.54 and 8W: 40.74 ± 2.23) were significantly higher than them in the other groups, indicating that the lower level of local inflammatory response was caused by the rGO treated group (Figure ##FIG##8##8c,g##). This indicated that rGO appeared to promote the transition of M2 macrophages and inhibit the inflammatory response during the urethral repair. Meanwhile, the low inflammatory response might promote the proliferation and activation of fibroblasts. The positive expression of proliferating cell nuclear antigen (PCNA) was obviously increased in the SA/Gel/rGO0.05 group at 4‐and 8‐weeks post‐surgery (4W: 7.77 ± 0.25 and 8W: 8.46 ± 0.94) (Figure ##FIG##8##8d,h##).
","Flow Cytometric Analysis of RAW264.7 Surface Markers","
To evaluate the macrophages polarization status, the M1 surface markers (CD68, CD86) and M2 surface markers (CD206) in RAW 264.7 macrophages were further analyzed by flow cytometric (Figure\n##FIG##9##\n9a##). The results revealed that more than 90% of the cells strongly expressed surface antigens such as CD68. The inflammatory cell density was evaluated by CD86 staining. The CD86 inflammatory cell density in the 0.02rGO and 0.1rGO groups was no significant difference in the control group. However, as the amount of rGO increases, it leads to the increase of CD206‐positive macrophages. The expression of CD86 in M1 type was significantly increased by LPS + INF‐r, and the expression of CD206‐positive macrophages was significantly higher than that in the control group after the introduction of rGO (Figure ##FIG##9##9b##). The results of flow cytometry analysis showed that the addition of rGO promoted the polarization of macrophages to the M2 phenotype in vivo.
","qRT‐PCR Analysis of mRNA Levels of the Markers in Tissue and RAW 264.7 Cells","
The gene expression of Arg1, Fizz, and Ym1 in the 0.02rGO group and the 0.1rGO group was significantly higher than that in the control group, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the control group (Figure\n##FIG##10##\n10a##). At the mRNA level, rGO treatment was associated with significant increases in Arg1, Fizz, and Ym1 expression compared to the control and M1 groups, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the M1 group (Figure ##FIG##10##10b##). These results suggest that rGO may also have an inducing effect on the macrophage anti‐inflammatory phenotype in vivo. The expression of SA/Gel/rGO0.05 α‐SMA, COL1A1, COL3A1, CD206, CD31, TGF‐β2 was significantly higher than that in the control group. The expression of TGFBR1, β‐catenin, and VWF was no significant difference in the control group (Figure ##FIG##10##10c–i##). Overall, the low‐dose rGO effectively promoted epithelization and neovascularization while reducing inflammation. These results suggested that rGO can promote urethral healing and regeneration.
"],"string":"[\n \"Results and Discussion\",\n \"rGO Characterizations and Ink Preparation\",\n \"
Commercially available monolayer rGO powder was used. The rGO was dispersed in water according to the information provided by the manufacturer. The monolayer ratio of rGO powder was up to 80%, the diameters were 0.5–5 µm, and the thicknesses were 0.8–1.2 nm. The morphology of rGO was confirmed under scanning electron microscope (SEM) and transmission electron microscope (TEM) (Figure\\n##FIG##1##\\n1a,b##). The stock rGO dispersion (1 mg mL−1) was diluted to concentrations in a series of 0.02, 0.05, 0.1, and 0.2 mg mL−1, and all suspensions remained stable and homogeneous for several weeks upon evaluation. With the increasing concentration of rGO, the solution color changed from gray to dark (Figure ##FIG##1##1c##). The solutions were also examined under optical microscopy to show the good dispersion with no obvious aggregation for rGO (Figure ##FIG##1##1d–g##). The area distributions of the rGO were mostly at the 1–50 µm2 range (Figure ##FIG##1##1h–k##). The mean area rate (%) is from 4% to 28% (Figure ##SUPPL##0##S2##, Supporting Information). The results demonstrate that the rGO distributes evenly in the water solution, and most of the rGO particles do not adhere to each other.
\",\n \"Characterizations of SA/Gel/rGO Hydrogels\",\n \"
Raman spectroscopic analysis of SA/Gel/rGO shows two characteristic bands of G‐ and D‐bands (Figure\\n##FIG##2##\\n2a##). The D‐band was at ≈1342 cm−1 and the G‐band was at ≈1584 cm−1. The D‐band reflects the disorder between graphite lamellae due to the interference of sp2\\n hybridization of carbon. The G‐band reflects the symmetry and degree of crystallization due to the sp2\\n hybrid in‐plane stretching vibration of carbon crystals. ID/IG is the intensity ratio between the D‐ and G‐bands, suggesting the degree of defects in the graphite lamellae[\\n##REF##33982723##\\n40\\n##, ##REF##34576956##\\n41\\n##\\n]; the higher the intensity ratio is, the more defects the C atomic crystal has. Chakraborty reported that the ID/IG ratios of GO and rGO were 0.9 and 1.1, respectively.[\\n##REF##35548324##\\n42\\n##\\n] To verify the incorporation of rGO into the SA/Gel hydrogels, the composition of the SA/Gel/rGO composite was confirmed through Fourier‐transform infrared (FTIR) spectroscopy (Figure ##FIG##2##2b##). The SA hydroxyl bond (O─H) stretching was at ≈3445 cm−1, the CH2 group stretching was at ≈2935 cm−1, and the carbonyl bond (C═O) stretching of amide I was at 1632 cm−1. The characteristic bands of SA at ≈1632 and 1401 cm−1 are associated with the carboxyl (─COOH) bond and asymmetric and symmetric stretching peaks of carboxylate salt groups, respectively. The peak at 1542 cm−1 is the ─NH group stretching of amide II and the peak at 1238 cm−1 is the ─NH group stretching of amide III of Gel. The peak at 1177 cm−1 is the C─O─C asymmetric stretching. The peak at 1028 cm−1 is the C─O─H stretching of SA. The characteristic peaks of rGO at ≈2930 and 2854 cm−1 are associated with CH2 and CH3 groups, respectively. Interestingly, the strong absorption peaks at ≈2930 and 2854 cm−1 appeared with the increased concentration of rGO. As such, the FTIR spectra clearly indicated the presence of the rGO in the hybrid hydrogels.
\",\n \"
The swelling behavior of a hydrogel construct plays a crucial role in its surface properties and mechanical integrity for biomaterial applications.[\\n##REF##32310981##\\n43\\n##, ##REF##32500887##\\n44\\n##\\n] The swelling behaviors of the various hydrogel formulations suggested that the swelling ratio increased when the concentration of SA was elevated (1%, 1.5%, and 2%) and the concentration of rGO was reduced (Figure ##FIG##2##2c##). The higher content of SA enhanced the hydrophilicity of the hydrogel network to improve the equilibrium swelling ratio of SA. For the positive effect of rGO concentration, it was likely due to the ability of rGO to interact with the hydrogen bonds of SA/Gel to act like a multi‐functional cross‐linking agent, therefore increasing the density of the cross‐linked network of the SA/Gel hydrogels at a higher concentration.
\",\n \"
Overtime, the hydrogels would be subjected to hydrolysis and other forms of degradation after being immersed in the liquid environment. The degree of degradation was evaluated by the mass remaining (%) of the hydrogels over 21 days. The various degrees of degradation showed a significant weight reduction in the first 7 days (Figure ##FIG##2##2d##), which might be due to the hydrolysis of the hydrogels. The remaining mass percentages (%) of the hydrogels are ≈42.62 ± 1.98% to 55.58 ± 1.44% in the different groups. In the next few days, there was a slight decrease in the weights of the hydrogels, which might correspond to the reduced degradation of the cross‐linked structure. The total weights of hydrogels had no significant differences between the SA/Gel hydrogels with and without rGO, implying that the incorporation of rGO did not noticeably affect the degradation of the hydrogel formulations.
\",\n \"Morphological Evaluations on the SA/Gel/rGO Hydrogels\",\n \"
The colors of the SA/Gel/rGO hydrogels were observed to be darker with the increased concentration of rGO. The SEM images reveal the porous structures of the SA/Gel/rGO hydrogels with different concentrations of rGO (0, 0.02, 0.05, 0.1, and 0.2 mg mL−1) (Figure\\n##FIG##3##\\n3a–j##). As the rGO loading increases, the pore size decreases and the pore density increases, further indicating that a high rGO concentration led to a high cross‐linking density, resulting in smaller pore sizes in composite hydrogels (Figure ##FIG##3##3b–j##). The pore size of hydrogels is highly correlated with cell adhesion, growth and proliferation, and nutrient exchange in the matrix. Most importantly, the hydrogels with a porous structure could facilitate efficient biomolecule transport within the environment.[\\n##REF##35548324##\\n42\\n##\\n] However, it should be noted that the pore sizes obtained by SEM observations may not indicate the actual pore sizes of the hydrogels under the hydrated states.
\",\n \"
From the distributions of the pore sizes for the SA/Gel/rGO hydrogels (Figure ##FIG##3##3k–o##), there were network structures with the pore sizes of <100 µm for all the hydrogels. The distributions of the pore sizes of the SA/Gel hydrogel were mainly in the range of 80–200 µm; the pore sizes of the SA/Gel/rGO hydrogels decreased with the increasing concentration of rGO (Figure ##FIG##3##3p##). The pore sizes of the SA/Gel and SA/Gel/rGO0.02 hydrogels were 92.82 ± 12.60 and 91.59 ± 11.6 µm, respectively . There was significant difference between SA/Gel/rGO0.05 (69.39 ± 7.97 µm) and SA/Gel/rGO0.1 (47.20 ± 7.26 µm), compared with the SA/Gel hydrogels. The pore size of SA/Gel/rGO0.2 was the smallest among all the hydrogels (36.29 ± 17.77 µm, ). These results showed that the incorporation of rGO indeed influenced the pore size of the hydrogels in a dose‐dependent pattern.
\",\n \"Morphological and Mechanical Evaluations on SA/Gel/rGO Patches\",\n \"
We 3D‐printed various SA/Gel scaffolds with different concentrations of rGO from 0 to 0.2 mg mL−1 (Figure\\n##FIG##4##\\n4a##). We used two freeze‐drying and one cross‐linking process to obtain the patches post‐printing. The gross morphologies were not significantly different except that the color changed from white to dark at the rGO concentration was increased. The 3D printer was an Organ Printing United system (Figure ##SUPPL##0##S3a##, Supporting Information). The mechanical properties of the SA/Gel/rGO patches are a valuable element for urethral repair and tissue regeneration. The tensile mechanical characteristics of the SA/Gel/rGO patches with different concentrations of rGO were evaluated by the stretching process of the patches (Figure ##SUPPL##0##S3b##, Supporting Information), the length of the patches that stretched between the upper and lower grips of the tensile machine could be adjusted. Then, each sample was stretched at 10 mm per minute until reaching its breaking points. All the samples exhibited the linear elastic behavior (Figure ##FIG##4##4b##). Compared with the patches containing rGO, the SA/Gel patch without rGO showed a lower modulus, 139.68 ± 0.144 KPa (Figure ##FIG##4##4c##). The tensile strengths of the SA/Gel patches at concentrations of 0, 0.02, 0.05, and 0.2 mg mL−1 of rGO were in the range of 139.87 ± 1.02–296.65 ± 1.11 KPa (Figure ##FIG##4##4d##). The tensile strength of the SA/Gel/rGO0.2 patch was higher (296.65 ±1.11 KPa) when the concentration of rGO was increased. The elevation of strain at break in the SA/Gel/rGO0.05 was 211.10 ± 1.34% (Figure ##FIG##4##4e##). Besides, the patches with appropriate elongation are flexible and elastic. Interestingly, the strain at break of SA/Gel/rGO0.2 patch was 137.77 ± 0.86% . These observations might be attributed to the SA/Gel patch to become fragile under the higher concentration of rGO. The SA/Gel/rGO patch was saturable and exhibits excellent mechanical properties to fit the requirement of urethral reconstruction. Interestingly, the rGO hybrid patches appeared to possess higher tensile strengths and Young's modulus. The rGO0.05 was an appropriate concentration to achieve the best mechanical performance among the groups examined.
\",\n \"Characteristics of Cell Behaviors\",\n \"
SA/Gel/rGO patches served as the templates for cell adhesion, and proliferation, thus superior cytocompatibility is required. The characteristics of cell survival and proliferation were evaluated through seeding fibroblasts on SA/Gel/rGO hydrogels containing different concentrations of rGO. The live and dead cells were stained with calcein AM and propidium iodides (PI) after 1 day, respectively. The fluorescence of living cells in SA/Gel/rGO hydrogels of 0.1 and 0.2 mg mL−1 of rGO was less (Figure\\n##FIG##5##\\n5a##). The increased number of dead cells might have been affected in part by the relatively high concentrations of rGO present. The cell morphologies were further observed by SEM, where fibroblasts were generally located within the pores of the 3D‐printed SA/Gel/rGO patches (Figure ##SUPPL##0##S4##, Supporting Information).
\",\n \"
From the expression patterns of differentially expressed proteins (DEPs) by fibroblasts (cell density: 1 × 107) with the different concentrations of rGO (Figure ##FIG##5##5b##), the cells treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 were compared with the cells with no treatment. The culture time was 72 h. Some biological processes and pathways, which are correlated with tissue regeneration or tissue repair, were observed to be significantly enriched in these DEPs (Figures ##SUPPL##0##S5a,S6a##, and ##SUPPL##0##S7a##, Supporting Information). For example, it is reported that the transmembrane receptor protein tyrosine kinase signaling pathway has a role in promoting sprouting of the angiogenic endothelium.[\\n##REF##20445537##\\n45\\n##\\n] The Ras/MAPK signaling cascade plays an important role in zebrafish heart regeneration.[\\n##UREF##8##\\n46\\n##\\n] The cell proliferation process is required for cardiomyocytes regeneration.[\\n##REF##28185170##\\n47\\n##\\n] In addition, the cellular components related to platelet alpha granule, secretory veside, vesicle lumen, and endomembrane system were enriched in DEPs treated with rGO0.02 (Figure ##SUPPL##0##S5b##, Supporting Information). Similar situations were observed when treated with rGO0.05 and treated with rGO0.1 (Figures ##SUPPL##0##S6b## and ##SUPPL##0##S7b##, Supporting Information). In addition, in molecular functions, the growth factor binding, growth factor activity, receptor‐ligand activity, and signaling receptor activator were enriched (Figures ##SUPPL##0##S5c,S6c##, and ##SUPPL##0##S7c##, Supporting Information). Similarly, the enriched Ras and MAPK signaling pathways were also observed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figures ##SUPPL##0##S5d,S6d##, and ##SUPPL##0##S7d##, Supporting Information). The genes involved in these biological processes and pathways might contribute to angiogenesis mediated by rGO. The bubble graph was also shown for gene ontology enrichment treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 for the biological process, molecular process, and cellular components (Figure ##SUPPL##0##S8a–i##, Supporting Information).
\",\n \"
Scratch assay is a standard for cell migration study in vitro. The migration of fibroblasts for the wound healing process is of great importance; after the injury, fibroblasts were used to proliferate and migrate from neighboring tissues into the wound area.[\\n##REF##20824344##\\n48\\n##\\n] To evaluate the potential of rGO in enhancing the migration of fibroblasts, a time‐dependent experiment was carried out (0–24 h). The results of the scratch assay showed that the migration ability of fibroblasts was greatly improved within 24 h after the treatment with rGO concentrations of 0.02 and 0.05 mg mL−1 (Figure ##FIG##5##5c##). With the increasing concentration of rGO, the proliferation of fibroblasts and wound healing increased first and then decreased, with a maximum migration rate of 61.18 ± 2.61% for rGO at 0.05 mg mL−1 (Figure ##FIG##5##5d##). Of note, when the concentration of rGO was set at 0.2 mg mL−1, the migration and proliferation of fibroblasts were significantly inhibited (26.39 ± 3.93%).
\",\n \"
Cell counting kit‐8 (CCK‐8) assay was subsequently used to assess the cytotoxic effect of SA/Gel/rGO patches on the fibroblasts (Figure ##FIG##5##5e##). After 1 and 4 days of cell culture, the metabolic activities of cells on the SA/Gel/rGO patches exhibited inhibitory effects. The higher concentration of rGO revealed inhospitable environments to cell metabolic activities during this time point. However, after 7 days of cell culture, the cell metabolic activities on SA/Gel/rGO0.02 and SA/Gel/rGO0.05 became higher than the remaining patches close to that for the control patches without rGO. Consequently, it might be undesirably toxic to cells at the concentration of rGO above 0.05 mg mL−1, in alignment with the cell viability staining results. Some reports have suggested that GO concentration of 50 µg mL−1 on fibroblasts led to significant cytotoxicity, while rGO at a concentration higher than 0.1 µg mL−1 would be toxic to endothelial cells.[\\n##UREF##5##\\n32\\n##, ##REF##21082807##\\n49\\n##\\n] Our results were overall consistent with the previous studies.
\",\n \"Urethrogram and Gross Morphology\",\n \"
The safety and functions of the SA/Gel/rGO patches were further proven in clinically relevant rabbit urethral injury models. According to the results of the in vitro mechanical test and biocompatibility of the scaffold, the patches without rGO were selected as the control group, and the rGO concentration of 0.05 and 0.2 mg mL−1 was selected as the experimental group. The patches were sutured to the dorsal urethral defect, and the injury model of the urethra and the surgical process of repair are shown in Figure ##SUPPL##0##S9## (Supporting Information). The recovery of the repaired urethras was detected at 4 and 8 weeks after the operation. The 4‐ and 8‐week urethrogram of the SA/Gel group displayed narrow lumens because of scar formation (Figure\\n##FIG##6##\\n6a,d##). The SA/Gel/rGO0.05 (Figure ##FIG##6##6b,e##) groups showed wide lumens of urethras similar to the normal urethra (Figure ##FIG##6##6g##). In the 4‐week urethrogram, the SA/Gel/rGO0.2 groups had a narrower urethral lumen, the widths developed larger at 8 weeks (Figure ##FIG##6##6c,f##). The reason might be the inflammation‐related swelling of urethral tissue caused by rGO with high concentration. The blockage rate determines whether and how much urine can flow out. The blockage ratio of the urethras treated with the SA/Gel/rGO0.05 patches could approach that of normal lumens (Figure ##FIG##6##6i##). After the incision of the ventral urethral wall, the dorsal wall could be exposed. Gross morphology revealed that urethral tissue in the SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups was significantly better than that in the SA/Gel group. The SA/Gel group showed the growth of severe scar‐like tissue in the urethra (Figure ##FIG##6##6j,m##). At 8 weeks, both of SA/Gel/rGO0.05 (Figure ##FIG##6##6n##) and SA/Gel/rGO0.2 (Figure ##FIG##6##6o##) groups showed smooth epithelial layer similar to the normal urethra of rabbits (Figure ##FIG##6##6h##). However, SA/Gel/rGO0.2 group at 4 weeks (Figure ##FIG##6##6i##) revealed red and swollen mucosa compared with that in the SA/Gel/rGO0.2 group (Figure ##FIG##6##6k##), which is consistent with the lumen width in the urethrogram. Therefore, the treatment of urethral injury could be improved by choosing a low dose of rGO for future application.
\",\n \"Histological Evaluations\",\n \"
The histology analyses of urethral reconstruction at 4‐ and 8‐weeks post‐surgery were performed by hematoxylin and eosin (H&E) and Masson's trichrome staining. The SA/Gel patches with/without rGO showed various outcomes. Additionally, there was no significant infection during the experimental period, suggesting good biocompatibility of the patches. According to the histology, the urethras treated with the SA/Gel patches were associated with thinner/uncomplete urothelium layers, fewer blood vessels, and thicker submucosal tissue (Figure\\n##FIG##7##\\n7a–d##). Due to the lack of regenerated urothelium layers, and over‐deposited ECM, the urethra developed to urethral stricture. In contrast, the reconstructed urethra treated with the SA/Gel/rGO patches had the nearly normal urothelium layers, submucosal tissue, and blood vessels. The continuous and complete urothelium layers were formed on the lumen surfaces for the SA/Gel/rGO0.05 group after 4‐ and 8‐weeks post‐surgery. However, the SA/Gel/rGO0.2 patches could lead to a short period of swelling and inflammation in 4 weeks. These results suggested that rGO at a lower concentration (0.05 mg mL−1) could perform best function in the regeneration of urothelium layers and blood vessels, but not at a higher concentration (0.2 mg mL−1). The mucosal coverage ratios by the SA/Gel/rGO patches were measured (Figure ##FIG##7##7e,f##). The urethras treated with the SA/Gel/rGO0.05 patches had more significant coverages, compared with the other groups. The urethral collagen thicknesses were also measured after the wound healing (Figure ##FIG##7##7g,h##). The SA/Gel/rGO0.05 patch could induce a less collagen deposition under the mucosa, therefore developing a scar‐less wound healing. The histology showed that an optimal concentration of rGO would promote the regeneration of epithelial cells and decrease submucosal ECM deposition to inhibit urethral fibrosis.
\",\n \"Immunofluorescence\",\n \"
The distribution of cytokeratin was investigated based on the expression of epithelial cytokeratin AE1/AE3 (an important membrane surface protein marker of ECs) via immunofluorescence staining of the urethra. The expression level for AE1/AE3 on the SA/Gel/rGO0.05 at 4‐and 8‐weeks post‐surgery (4W: 16.70 ± 2.66 and 8W: 24.72 ± 2.81) is significantly higher than that in the other groups. Although the continuous urothelium layer is formed, the urothelium layer of the SA/Gel/rGO0.2 group is thinner than that of the SA/Gel/rGO0.05 group. The expression of cytokeratin in the SA/Gel group is low (4W: 8.19 ± 1.72 and 8W: 8.29 ± 1.65), there is no complete regeneration of a discontinuous urothelium layer (Figure\\n##FIG##8##\\n8a,e##). It suggests that the introduction of rGO has the potential to promote the regeneration of urothelium layers. Studies have shown that the significant pro‐angiogenic properties of rGO depend on its concentration.[\\n##UREF##5##\\n32\\n##, ##REF##29892387##\\n50\\n##\\n] In the results of fluorescence staining at 4‐weeks post‐surgery, the SA/Gel/rGO0.05 group showed a number of CD 31‐positive cells (the blood vessels labeled by CD 31), which were more than them in the SA/Gel and SA/Gel/rGO0.2 group. At the beginning process of urethral regeneration with inflammation, the vessels in tissue would be much more obvious that is consistent with the results.[\\n##REF##12490959##\\n51\\n##\\n] For SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups, the number of CD 31‐positive cells at 8 weeks (SA/Gel/rGO0.05: 14.10 ± 3.99 and SA/Gel/rGO0.2: 8.63 ± 1.23) are lower than them at 4 weeks (SA/Gel/rGO0.05: 15.35 ± 4.55 and SA/Gel/rGO0.2: 14.77 ± 3.07) (Figure ##FIG##8##8b,f##).
\",\n \"
Different degrees of inflammation response at the injured sites would appear because of the transplantation of external scaffolds. CD 206 is used to label M2 macrophages.[\\n##REF##31903147##\\n52\\n##\\n] The results showed that the number of CD206‐positive cells in the SA/Gel group was the lowest. The number of CD206 positive cells in the SA/Gel/rGO0.05 group in 4‐and 8‐weeks post‐surgery (4W: 36.36 ± 3.54 and 8W: 40.74 ± 2.23) were significantly higher than them in the other groups, indicating that the lower level of local inflammatory response was caused by the rGO treated group (Figure ##FIG##8##8c,g##). This indicated that rGO appeared to promote the transition of M2 macrophages and inhibit the inflammatory response during the urethral repair. Meanwhile, the low inflammatory response might promote the proliferation and activation of fibroblasts. The positive expression of proliferating cell nuclear antigen (PCNA) was obviously increased in the SA/Gel/rGO0.05 group at 4‐and 8‐weeks post‐surgery (4W: 7.77 ± 0.25 and 8W: 8.46 ± 0.94) (Figure ##FIG##8##8d,h##).
\",\n \"Flow Cytometric Analysis of RAW264.7 Surface Markers\",\n \"
To evaluate the macrophages polarization status, the M1 surface markers (CD68, CD86) and M2 surface markers (CD206) in RAW 264.7 macrophages were further analyzed by flow cytometric (Figure\\n##FIG##9##\\n9a##). The results revealed that more than 90% of the cells strongly expressed surface antigens such as CD68. The inflammatory cell density was evaluated by CD86 staining. The CD86 inflammatory cell density in the 0.02rGO and 0.1rGO groups was no significant difference in the control group. However, as the amount of rGO increases, it leads to the increase of CD206‐positive macrophages. The expression of CD86 in M1 type was significantly increased by LPS + INF‐r, and the expression of CD206‐positive macrophages was significantly higher than that in the control group after the introduction of rGO (Figure ##FIG##9##9b##). The results of flow cytometry analysis showed that the addition of rGO promoted the polarization of macrophages to the M2 phenotype in vivo.
\",\n \"qRT‐PCR Analysis of mRNA Levels of the Markers in Tissue and RAW 264.7 Cells\",\n \"
The gene expression of Arg1, Fizz, and Ym1 in the 0.02rGO group and the 0.1rGO group was significantly higher than that in the control group, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the control group (Figure\\n##FIG##10##\\n10a##). At the mRNA level, rGO treatment was associated with significant increases in Arg1, Fizz, and Ym1 expression compared to the control and M1 groups, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the M1 group (Figure ##FIG##10##10b##). These results suggest that rGO may also have an inducing effect on the macrophage anti‐inflammatory phenotype in vivo. The expression of SA/Gel/rGO0.05 α‐SMA, COL1A1, COL3A1, CD206, CD31, TGF‐β2 was significantly higher than that in the control group. The expression of TGFBR1, β‐catenin, and VWF was no significant difference in the control group (Figure ##FIG##10##10c–i##). Overall, the low‐dose rGO effectively promoted epithelization and neovascularization while reducing inflammation. These results suggested that rGO can promote urethral healing and regeneration.
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","rGO Characterizations and Ink Preparation","
Commercially available monolayer rGO powder was used. The rGO was dispersed in water according to the information provided by the manufacturer. The monolayer ratio of rGO powder was up to 80%, the diameters were 0.5–5 µm, and the thicknesses were 0.8–1.2 nm. The morphology of rGO was confirmed under scanning electron microscope (SEM) and transmission electron microscope (TEM) (Figure\n##FIG##1##\n1a,b##). The stock rGO dispersion (1 mg mL−1) was diluted to concentrations in a series of 0.02, 0.05, 0.1, and 0.2 mg mL−1, and all suspensions remained stable and homogeneous for several weeks upon evaluation. With the increasing concentration of rGO, the solution color changed from gray to dark (Figure ##FIG##1##1c##). The solutions were also examined under optical microscopy to show the good dispersion with no obvious aggregation for rGO (Figure ##FIG##1##1d–g##). The area distributions of the rGO were mostly at the 1–50 µm2 range (Figure ##FIG##1##1h–k##). The mean area rate (%) is from 4% to 28% (Figure ##SUPPL##0##S2##, Supporting Information). The results demonstrate that the rGO distributes evenly in the water solution, and most of the rGO particles do not adhere to each other.
","Characterizations of SA/Gel/rGO Hydrogels","
Raman spectroscopic analysis of SA/Gel/rGO shows two characteristic bands of G‐ and D‐bands (Figure\n##FIG##2##\n2a##). The D‐band was at ≈1342 cm−1 and the G‐band was at ≈1584 cm−1. The D‐band reflects the disorder between graphite lamellae due to the interference of sp2\n hybridization of carbon. The G‐band reflects the symmetry and degree of crystallization due to the sp2\n hybrid in‐plane stretching vibration of carbon crystals. ID/IG is the intensity ratio between the D‐ and G‐bands, suggesting the degree of defects in the graphite lamellae[\n##REF##33982723##\n40\n##, ##REF##34576956##\n41\n##\n]; the higher the intensity ratio is, the more defects the C atomic crystal has. Chakraborty reported that the ID/IG ratios of GO and rGO were 0.9 and 1.1, respectively.[\n##REF##35548324##\n42\n##\n] To verify the incorporation of rGO into the SA/Gel hydrogels, the composition of the SA/Gel/rGO composite was confirmed through Fourier‐transform infrared (FTIR) spectroscopy (Figure ##FIG##2##2b##). The SA hydroxyl bond (O─H) stretching was at ≈3445 cm−1, the CH2 group stretching was at ≈2935 cm−1, and the carbonyl bond (C═O) stretching of amide I was at 1632 cm−1. The characteristic bands of SA at ≈1632 and 1401 cm−1 are associated with the carboxyl (─COOH) bond and asymmetric and symmetric stretching peaks of carboxylate salt groups, respectively. The peak at 1542 cm−1 is the ─NH group stretching of amide II and the peak at 1238 cm−1 is the ─NH group stretching of amide III of Gel. The peak at 1177 cm−1 is the C─O─C asymmetric stretching. The peak at 1028 cm−1 is the C─O─H stretching of SA. The characteristic peaks of rGO at ≈2930 and 2854 cm−1 are associated with CH2 and CH3 groups, respectively. Interestingly, the strong absorption peaks at ≈2930 and 2854 cm−1 appeared with the increased concentration of rGO. As such, the FTIR spectra clearly indicated the presence of the rGO in the hybrid hydrogels.
","
The swelling behavior of a hydrogel construct plays a crucial role in its surface properties and mechanical integrity for biomaterial applications.[\n##REF##32310981##\n43\n##, ##REF##32500887##\n44\n##\n] The swelling behaviors of the various hydrogel formulations suggested that the swelling ratio increased when the concentration of SA was elevated (1%, 1.5%, and 2%) and the concentration of rGO was reduced (Figure ##FIG##2##2c##). The higher content of SA enhanced the hydrophilicity of the hydrogel network to improve the equilibrium swelling ratio of SA. For the positive effect of rGO concentration, it was likely due to the ability of rGO to interact with the hydrogen bonds of SA/Gel to act like a multi‐functional cross‐linking agent, therefore increasing the density of the cross‐linked network of the SA/Gel hydrogels at a higher concentration.
","
Overtime, the hydrogels would be subjected to hydrolysis and other forms of degradation after being immersed in the liquid environment. The degree of degradation was evaluated by the mass remaining (%) of the hydrogels over 21 days. The various degrees of degradation showed a significant weight reduction in the first 7 days (Figure ##FIG##2##2d##), which might be due to the hydrolysis of the hydrogels. The remaining mass percentages (%) of the hydrogels are ≈42.62 ± 1.98% to 55.58 ± 1.44% in the different groups. In the next few days, there was a slight decrease in the weights of the hydrogels, which might correspond to the reduced degradation of the cross‐linked structure. The total weights of hydrogels had no significant differences between the SA/Gel hydrogels with and without rGO, implying that the incorporation of rGO did not noticeably affect the degradation of the hydrogel formulations.
","Morphological Evaluations on the SA/Gel/rGO Hydrogels","
The colors of the SA/Gel/rGO hydrogels were observed to be darker with the increased concentration of rGO. The SEM images reveal the porous structures of the SA/Gel/rGO hydrogels with different concentrations of rGO (0, 0.02, 0.05, 0.1, and 0.2 mg mL−1) (Figure\n##FIG##3##\n3a–j##). As the rGO loading increases, the pore size decreases and the pore density increases, further indicating that a high rGO concentration led to a high cross‐linking density, resulting in smaller pore sizes in composite hydrogels (Figure ##FIG##3##3b–j##). The pore size of hydrogels is highly correlated with cell adhesion, growth and proliferation, and nutrient exchange in the matrix. Most importantly, the hydrogels with a porous structure could facilitate efficient biomolecule transport within the environment.[\n##REF##35548324##\n42\n##\n] However, it should be noted that the pore sizes obtained by SEM observations may not indicate the actual pore sizes of the hydrogels under the hydrated states.
","
From the distributions of the pore sizes for the SA/Gel/rGO hydrogels (Figure ##FIG##3##3k–o##), there were network structures with the pore sizes of <100 µm for all the hydrogels. The distributions of the pore sizes of the SA/Gel hydrogel were mainly in the range of 80–200 µm; the pore sizes of the SA/Gel/rGO hydrogels decreased with the increasing concentration of rGO (Figure ##FIG##3##3p##). The pore sizes of the SA/Gel and SA/Gel/rGO0.02 hydrogels were 92.82 ± 12.60 and 91.59 ± 11.6 µm, respectively . There was significant difference between SA/Gel/rGO0.05 (69.39 ± 7.97 µm) and SA/Gel/rGO0.1 (47.20 ± 7.26 µm), compared with the SA/Gel hydrogels. The pore size of SA/Gel/rGO0.2 was the smallest among all the hydrogels (36.29 ± 17.77 µm, ). These results showed that the incorporation of rGO indeed influenced the pore size of the hydrogels in a dose‐dependent pattern.
","Morphological and Mechanical Evaluations on SA/Gel/rGO Patches","
We 3D‐printed various SA/Gel scaffolds with different concentrations of rGO from 0 to 0.2 mg mL−1 (Figure\n##FIG##4##\n4a##). We used two freeze‐drying and one cross‐linking process to obtain the patches post‐printing. The gross morphologies were not significantly different except that the color changed from white to dark at the rGO concentration was increased. The 3D printer was an Organ Printing United system (Figure ##SUPPL##0##S3a##, Supporting Information). The mechanical properties of the SA/Gel/rGO patches are a valuable element for urethral repair and tissue regeneration. The tensile mechanical characteristics of the SA/Gel/rGO patches with different concentrations of rGO were evaluated by the stretching process of the patches (Figure ##SUPPL##0##S3b##, Supporting Information), the length of the patches that stretched between the upper and lower grips of the tensile machine could be adjusted. Then, each sample was stretched at 10 mm per minute until reaching its breaking points. All the samples exhibited the linear elastic behavior (Figure ##FIG##4##4b##). Compared with the patches containing rGO, the SA/Gel patch without rGO showed a lower modulus, 139.68 ± 0.144 KPa (Figure ##FIG##4##4c##). The tensile strengths of the SA/Gel patches at concentrations of 0, 0.02, 0.05, and 0.2 mg mL−1 of rGO were in the range of 139.87 ± 1.02–296.65 ± 1.11 KPa (Figure ##FIG##4##4d##). The tensile strength of the SA/Gel/rGO0.2 patch was higher (296.65 ±1.11 KPa) when the concentration of rGO was increased. The elevation of strain at break in the SA/Gel/rGO0.05 was 211.10 ± 1.34% (Figure ##FIG##4##4e##). Besides, the patches with appropriate elongation are flexible and elastic. Interestingly, the strain at break of SA/Gel/rGO0.2 patch was 137.77 ± 0.86% . These observations might be attributed to the SA/Gel patch to become fragile under the higher concentration of rGO. The SA/Gel/rGO patch was saturable and exhibits excellent mechanical properties to fit the requirement of urethral reconstruction. Interestingly, the rGO hybrid patches appeared to possess higher tensile strengths and Young's modulus. The rGO0.05 was an appropriate concentration to achieve the best mechanical performance among the groups examined.
","Characteristics of Cell Behaviors","
SA/Gel/rGO patches served as the templates for cell adhesion, and proliferation, thus superior cytocompatibility is required. The characteristics of cell survival and proliferation were evaluated through seeding fibroblasts on SA/Gel/rGO hydrogels containing different concentrations of rGO. The live and dead cells were stained with calcein AM and propidium iodides (PI) after 1 day, respectively. The fluorescence of living cells in SA/Gel/rGO hydrogels of 0.1 and 0.2 mg mL−1 of rGO was less (Figure\n##FIG##5##\n5a##). The increased number of dead cells might have been affected in part by the relatively high concentrations of rGO present. The cell morphologies were further observed by SEM, where fibroblasts were generally located within the pores of the 3D‐printed SA/Gel/rGO patches (Figure ##SUPPL##0##S4##, Supporting Information).
","
From the expression patterns of differentially expressed proteins (DEPs) by fibroblasts (cell density: 1 × 107) with the different concentrations of rGO (Figure ##FIG##5##5b##), the cells treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 were compared with the cells with no treatment. The culture time was 72 h. Some biological processes and pathways, which are correlated with tissue regeneration or tissue repair, were observed to be significantly enriched in these DEPs (Figures ##SUPPL##0##S5a,S6a##, and ##SUPPL##0##S7a##, Supporting Information). For example, it is reported that the transmembrane receptor protein tyrosine kinase signaling pathway has a role in promoting sprouting of the angiogenic endothelium.[\n##REF##20445537##\n45\n##\n] The Ras/MAPK signaling cascade plays an important role in zebrafish heart regeneration.[\n##UREF##8##\n46\n##\n] The cell proliferation process is required for cardiomyocytes regeneration.[\n##REF##28185170##\n47\n##\n] In addition, the cellular components related to platelet alpha granule, secretory veside, vesicle lumen, and endomembrane system were enriched in DEPs treated with rGO0.02 (Figure ##SUPPL##0##S5b##, Supporting Information). Similar situations were observed when treated with rGO0.05 and treated with rGO0.1 (Figures ##SUPPL##0##S6b## and ##SUPPL##0##S7b##, Supporting Information). In addition, in molecular functions, the growth factor binding, growth factor activity, receptor‐ligand activity, and signaling receptor activator were enriched (Figures ##SUPPL##0##S5c,S6c##, and ##SUPPL##0##S7c##, Supporting Information). Similarly, the enriched Ras and MAPK signaling pathways were also observed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figures ##SUPPL##0##S5d,S6d##, and ##SUPPL##0##S7d##, Supporting Information). The genes involved in these biological processes and pathways might contribute to angiogenesis mediated by rGO. The bubble graph was also shown for gene ontology enrichment treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 for the biological process, molecular process, and cellular components (Figure ##SUPPL##0##S8a–i##, Supporting Information).
","
Scratch assay is a standard for cell migration study in vitro. The migration of fibroblasts for the wound healing process is of great importance; after the injury, fibroblasts were used to proliferate and migrate from neighboring tissues into the wound area.[\n##REF##20824344##\n48\n##\n] To evaluate the potential of rGO in enhancing the migration of fibroblasts, a time‐dependent experiment was carried out (0–24 h). The results of the scratch assay showed that the migration ability of fibroblasts was greatly improved within 24 h after the treatment with rGO concentrations of 0.02 and 0.05 mg mL−1 (Figure ##FIG##5##5c##). With the increasing concentration of rGO, the proliferation of fibroblasts and wound healing increased first and then decreased, with a maximum migration rate of 61.18 ± 2.61% for rGO at 0.05 mg mL−1 (Figure ##FIG##5##5d##). Of note, when the concentration of rGO was set at 0.2 mg mL−1, the migration and proliferation of fibroblasts were significantly inhibited (26.39 ± 3.93%).
","
Cell counting kit‐8 (CCK‐8) assay was subsequently used to assess the cytotoxic effect of SA/Gel/rGO patches on the fibroblasts (Figure ##FIG##5##5e##). After 1 and 4 days of cell culture, the metabolic activities of cells on the SA/Gel/rGO patches exhibited inhibitory effects. The higher concentration of rGO revealed inhospitable environments to cell metabolic activities during this time point. However, after 7 days of cell culture, the cell metabolic activities on SA/Gel/rGO0.02 and SA/Gel/rGO0.05 became higher than the remaining patches close to that for the control patches without rGO. Consequently, it might be undesirably toxic to cells at the concentration of rGO above 0.05 mg mL−1, in alignment with the cell viability staining results. Some reports have suggested that GO concentration of 50 µg mL−1 on fibroblasts led to significant cytotoxicity, while rGO at a concentration higher than 0.1 µg mL−1 would be toxic to endothelial cells.[\n##UREF##5##\n32\n##, ##REF##21082807##\n49\n##\n] Our results were overall consistent with the previous studies.
","Urethrogram and Gross Morphology","
The safety and functions of the SA/Gel/rGO patches were further proven in clinically relevant rabbit urethral injury models. According to the results of the in vitro mechanical test and biocompatibility of the scaffold, the patches without rGO were selected as the control group, and the rGO concentration of 0.05 and 0.2 mg mL−1 was selected as the experimental group. The patches were sutured to the dorsal urethral defect, and the injury model of the urethra and the surgical process of repair are shown in Figure ##SUPPL##0##S9## (Supporting Information). The recovery of the repaired urethras was detected at 4 and 8 weeks after the operation. The 4‐ and 8‐week urethrogram of the SA/Gel group displayed narrow lumens because of scar formation (Figure\n##FIG##6##\n6a,d##). The SA/Gel/rGO0.05 (Figure ##FIG##6##6b,e##) groups showed wide lumens of urethras similar to the normal urethra (Figure ##FIG##6##6g##). In the 4‐week urethrogram, the SA/Gel/rGO0.2 groups had a narrower urethral lumen, the widths developed larger at 8 weeks (Figure ##FIG##6##6c,f##). The reason might be the inflammation‐related swelling of urethral tissue caused by rGO with high concentration. The blockage rate determines whether and how much urine can flow out. The blockage ratio of the urethras treated with the SA/Gel/rGO0.05 patches could approach that of normal lumens (Figure ##FIG##6##6i##). After the incision of the ventral urethral wall, the dorsal wall could be exposed. Gross morphology revealed that urethral tissue in the SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups was significantly better than that in the SA/Gel group. The SA/Gel group showed the growth of severe scar‐like tissue in the urethra (Figure ##FIG##6##6j,m##). At 8 weeks, both of SA/Gel/rGO0.05 (Figure ##FIG##6##6n##) and SA/Gel/rGO0.2 (Figure ##FIG##6##6o##) groups showed smooth epithelial layer similar to the normal urethra of rabbits (Figure ##FIG##6##6h##). However, SA/Gel/rGO0.2 group at 4 weeks (Figure ##FIG##6##6i##) revealed red and swollen mucosa compared with that in the SA/Gel/rGO0.2 group (Figure ##FIG##6##6k##), which is consistent with the lumen width in the urethrogram. Therefore, the treatment of urethral injury could be improved by choosing a low dose of rGO for future application.
","Histological Evaluations","
The histology analyses of urethral reconstruction at 4‐ and 8‐weeks post‐surgery were performed by hematoxylin and eosin (H&E) and Masson's trichrome staining. The SA/Gel patches with/without rGO showed various outcomes. Additionally, there was no significant infection during the experimental period, suggesting good biocompatibility of the patches. According to the histology, the urethras treated with the SA/Gel patches were associated with thinner/uncomplete urothelium layers, fewer blood vessels, and thicker submucosal tissue (Figure\n##FIG##7##\n7a–d##). Due to the lack of regenerated urothelium layers, and over‐deposited ECM, the urethra developed to urethral stricture. In contrast, the reconstructed urethra treated with the SA/Gel/rGO patches had the nearly normal urothelium layers, submucosal tissue, and blood vessels. The continuous and complete urothelium layers were formed on the lumen surfaces for the SA/Gel/rGO0.05 group after 4‐ and 8‐weeks post‐surgery. However, the SA/Gel/rGO0.2 patches could lead to a short period of swelling and inflammation in 4 weeks. These results suggested that rGO at a lower concentration (0.05 mg mL−1) could perform best function in the regeneration of urothelium layers and blood vessels, but not at a higher concentration (0.2 mg mL−1). The mucosal coverage ratios by the SA/Gel/rGO patches were measured (Figure ##FIG##7##7e,f##). The urethras treated with the SA/Gel/rGO0.05 patches had more significant coverages, compared with the other groups. The urethral collagen thicknesses were also measured after the wound healing (Figure ##FIG##7##7g,h##). The SA/Gel/rGO0.05 patch could induce a less collagen deposition under the mucosa, therefore developing a scar‐less wound healing. The histology showed that an optimal concentration of rGO would promote the regeneration of epithelial cells and decrease submucosal ECM deposition to inhibit urethral fibrosis.
","Immunofluorescence","
The distribution of cytokeratin was investigated based on the expression of epithelial cytokeratin AE1/AE3 (an important membrane surface protein marker of ECs) via immunofluorescence staining of the urethra. The expression level for AE1/AE3 on the SA/Gel/rGO0.05 at 4‐and 8‐weeks post‐surgery (4W: 16.70 ± 2.66 and 8W: 24.72 ± 2.81) is significantly higher than that in the other groups. Although the continuous urothelium layer is formed, the urothelium layer of the SA/Gel/rGO0.2 group is thinner than that of the SA/Gel/rGO0.05 group. The expression of cytokeratin in the SA/Gel group is low (4W: 8.19 ± 1.72 and 8W: 8.29 ± 1.65), there is no complete regeneration of a discontinuous urothelium layer (Figure\n##FIG##8##\n8a,e##). It suggests that the introduction of rGO has the potential to promote the regeneration of urothelium layers. Studies have shown that the significant pro‐angiogenic properties of rGO depend on its concentration.[\n##UREF##5##\n32\n##, ##REF##29892387##\n50\n##\n] In the results of fluorescence staining at 4‐weeks post‐surgery, the SA/Gel/rGO0.05 group showed a number of CD 31‐positive cells (the blood vessels labeled by CD 31), which were more than them in the SA/Gel and SA/Gel/rGO0.2 group. At the beginning process of urethral regeneration with inflammation, the vessels in tissue would be much more obvious that is consistent with the results.[\n##REF##12490959##\n51\n##\n] For SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups, the number of CD 31‐positive cells at 8 weeks (SA/Gel/rGO0.05: 14.10 ± 3.99 and SA/Gel/rGO0.2: 8.63 ± 1.23) are lower than them at 4 weeks (SA/Gel/rGO0.05: 15.35 ± 4.55 and SA/Gel/rGO0.2: 14.77 ± 3.07) (Figure ##FIG##8##8b,f##).
","
Different degrees of inflammation response at the injured sites would appear because of the transplantation of external scaffolds. CD 206 is used to label M2 macrophages.[\n##REF##31903147##\n52\n##\n] The results showed that the number of CD206‐positive cells in the SA/Gel group was the lowest. The number of CD206 positive cells in the SA/Gel/rGO0.05 group in 4‐and 8‐weeks post‐surgery (4W: 36.36 ± 3.54 and 8W: 40.74 ± 2.23) were significantly higher than them in the other groups, indicating that the lower level of local inflammatory response was caused by the rGO treated group (Figure ##FIG##8##8c,g##). This indicated that rGO appeared to promote the transition of M2 macrophages and inhibit the inflammatory response during the urethral repair. Meanwhile, the low inflammatory response might promote the proliferation and activation of fibroblasts. The positive expression of proliferating cell nuclear antigen (PCNA) was obviously increased in the SA/Gel/rGO0.05 group at 4‐and 8‐weeks post‐surgery (4W: 7.77 ± 0.25 and 8W: 8.46 ± 0.94) (Figure ##FIG##8##8d,h##).
","Flow Cytometric Analysis of RAW264.7 Surface Markers","
To evaluate the macrophages polarization status, the M1 surface markers (CD68, CD86) and M2 surface markers (CD206) in RAW 264.7 macrophages were further analyzed by flow cytometric (Figure\n##FIG##9##\n9a##). The results revealed that more than 90% of the cells strongly expressed surface antigens such as CD68. The inflammatory cell density was evaluated by CD86 staining. The CD86 inflammatory cell density in the 0.02rGO and 0.1rGO groups was no significant difference in the control group. However, as the amount of rGO increases, it leads to the increase of CD206‐positive macrophages. The expression of CD86 in M1 type was significantly increased by LPS + INF‐r, and the expression of CD206‐positive macrophages was significantly higher than that in the control group after the introduction of rGO (Figure ##FIG##9##9b##). The results of flow cytometry analysis showed that the addition of rGO promoted the polarization of macrophages to the M2 phenotype in vivo.
","qRT‐PCR Analysis of mRNA Levels of the Markers in Tissue and RAW 264.7 Cells","
The gene expression of Arg1, Fizz, and Ym1 in the 0.02rGO group and the 0.1rGO group was significantly higher than that in the control group, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the control group (Figure\n##FIG##10##\n10a##). At the mRNA level, rGO treatment was associated with significant increases in Arg1, Fizz, and Ym1 expression compared to the control and M1 groups, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the M1 group (Figure ##FIG##10##10b##). These results suggest that rGO may also have an inducing effect on the macrophage anti‐inflammatory phenotype in vivo. The expression of SA/Gel/rGO0.05 α‐SMA, COL1A1, COL3A1, CD206, CD31, TGF‐β2 was significantly higher than that in the control group. The expression of TGFBR1, β‐catenin, and VWF was no significant difference in the control group (Figure ##FIG##10##10c–i##). Overall, the low‐dose rGO effectively promoted epithelization and neovascularization while reducing inflammation. These results suggested that rGO can promote urethral healing and regeneration.
"],"string":"[\n \"Results and Discussion\",\n \"rGO Characterizations and Ink Preparation\",\n \"
Commercially available monolayer rGO powder was used. The rGO was dispersed in water according to the information provided by the manufacturer. The monolayer ratio of rGO powder was up to 80%, the diameters were 0.5–5 µm, and the thicknesses were 0.8–1.2 nm. The morphology of rGO was confirmed under scanning electron microscope (SEM) and transmission electron microscope (TEM) (Figure\\n##FIG##1##\\n1a,b##). The stock rGO dispersion (1 mg mL−1) was diluted to concentrations in a series of 0.02, 0.05, 0.1, and 0.2 mg mL−1, and all suspensions remained stable and homogeneous for several weeks upon evaluation. With the increasing concentration of rGO, the solution color changed from gray to dark (Figure ##FIG##1##1c##). The solutions were also examined under optical microscopy to show the good dispersion with no obvious aggregation for rGO (Figure ##FIG##1##1d–g##). The area distributions of the rGO were mostly at the 1–50 µm2 range (Figure ##FIG##1##1h–k##). The mean area rate (%) is from 4% to 28% (Figure ##SUPPL##0##S2##, Supporting Information). The results demonstrate that the rGO distributes evenly in the water solution, and most of the rGO particles do not adhere to each other.
\",\n \"Characterizations of SA/Gel/rGO Hydrogels\",\n \"
Raman spectroscopic analysis of SA/Gel/rGO shows two characteristic bands of G‐ and D‐bands (Figure\\n##FIG##2##\\n2a##). The D‐band was at ≈1342 cm−1 and the G‐band was at ≈1584 cm−1. The D‐band reflects the disorder between graphite lamellae due to the interference of sp2\\n hybridization of carbon. The G‐band reflects the symmetry and degree of crystallization due to the sp2\\n hybrid in‐plane stretching vibration of carbon crystals. ID/IG is the intensity ratio between the D‐ and G‐bands, suggesting the degree of defects in the graphite lamellae[\\n##REF##33982723##\\n40\\n##, ##REF##34576956##\\n41\\n##\\n]; the higher the intensity ratio is, the more defects the C atomic crystal has. Chakraborty reported that the ID/IG ratios of GO and rGO were 0.9 and 1.1, respectively.[\\n##REF##35548324##\\n42\\n##\\n] To verify the incorporation of rGO into the SA/Gel hydrogels, the composition of the SA/Gel/rGO composite was confirmed through Fourier‐transform infrared (FTIR) spectroscopy (Figure ##FIG##2##2b##). The SA hydroxyl bond (O─H) stretching was at ≈3445 cm−1, the CH2 group stretching was at ≈2935 cm−1, and the carbonyl bond (C═O) stretching of amide I was at 1632 cm−1. The characteristic bands of SA at ≈1632 and 1401 cm−1 are associated with the carboxyl (─COOH) bond and asymmetric and symmetric stretching peaks of carboxylate salt groups, respectively. The peak at 1542 cm−1 is the ─NH group stretching of amide II and the peak at 1238 cm−1 is the ─NH group stretching of amide III of Gel. The peak at 1177 cm−1 is the C─O─C asymmetric stretching. The peak at 1028 cm−1 is the C─O─H stretching of SA. The characteristic peaks of rGO at ≈2930 and 2854 cm−1 are associated with CH2 and CH3 groups, respectively. Interestingly, the strong absorption peaks at ≈2930 and 2854 cm−1 appeared with the increased concentration of rGO. As such, the FTIR spectra clearly indicated the presence of the rGO in the hybrid hydrogels.
\",\n \"
The swelling behavior of a hydrogel construct plays a crucial role in its surface properties and mechanical integrity for biomaterial applications.[\\n##REF##32310981##\\n43\\n##, ##REF##32500887##\\n44\\n##\\n] The swelling behaviors of the various hydrogel formulations suggested that the swelling ratio increased when the concentration of SA was elevated (1%, 1.5%, and 2%) and the concentration of rGO was reduced (Figure ##FIG##2##2c##). The higher content of SA enhanced the hydrophilicity of the hydrogel network to improve the equilibrium swelling ratio of SA. For the positive effect of rGO concentration, it was likely due to the ability of rGO to interact with the hydrogen bonds of SA/Gel to act like a multi‐functional cross‐linking agent, therefore increasing the density of the cross‐linked network of the SA/Gel hydrogels at a higher concentration.
\",\n \"
Overtime, the hydrogels would be subjected to hydrolysis and other forms of degradation after being immersed in the liquid environment. The degree of degradation was evaluated by the mass remaining (%) of the hydrogels over 21 days. The various degrees of degradation showed a significant weight reduction in the first 7 days (Figure ##FIG##2##2d##), which might be due to the hydrolysis of the hydrogels. The remaining mass percentages (%) of the hydrogels are ≈42.62 ± 1.98% to 55.58 ± 1.44% in the different groups. In the next few days, there was a slight decrease in the weights of the hydrogels, which might correspond to the reduced degradation of the cross‐linked structure. The total weights of hydrogels had no significant differences between the SA/Gel hydrogels with and without rGO, implying that the incorporation of rGO did not noticeably affect the degradation of the hydrogel formulations.
\",\n \"Morphological Evaluations on the SA/Gel/rGO Hydrogels\",\n \"
The colors of the SA/Gel/rGO hydrogels were observed to be darker with the increased concentration of rGO. The SEM images reveal the porous structures of the SA/Gel/rGO hydrogels with different concentrations of rGO (0, 0.02, 0.05, 0.1, and 0.2 mg mL−1) (Figure\\n##FIG##3##\\n3a–j##). As the rGO loading increases, the pore size decreases and the pore density increases, further indicating that a high rGO concentration led to a high cross‐linking density, resulting in smaller pore sizes in composite hydrogels (Figure ##FIG##3##3b–j##). The pore size of hydrogels is highly correlated with cell adhesion, growth and proliferation, and nutrient exchange in the matrix. Most importantly, the hydrogels with a porous structure could facilitate efficient biomolecule transport within the environment.[\\n##REF##35548324##\\n42\\n##\\n] However, it should be noted that the pore sizes obtained by SEM observations may not indicate the actual pore sizes of the hydrogels under the hydrated states.
\",\n \"
From the distributions of the pore sizes for the SA/Gel/rGO hydrogels (Figure ##FIG##3##3k–o##), there were network structures with the pore sizes of <100 µm for all the hydrogels. The distributions of the pore sizes of the SA/Gel hydrogel were mainly in the range of 80–200 µm; the pore sizes of the SA/Gel/rGO hydrogels decreased with the increasing concentration of rGO (Figure ##FIG##3##3p##). The pore sizes of the SA/Gel and SA/Gel/rGO0.02 hydrogels were 92.82 ± 12.60 and 91.59 ± 11.6 µm, respectively . There was significant difference between SA/Gel/rGO0.05 (69.39 ± 7.97 µm) and SA/Gel/rGO0.1 (47.20 ± 7.26 µm), compared with the SA/Gel hydrogels. The pore size of SA/Gel/rGO0.2 was the smallest among all the hydrogels (36.29 ± 17.77 µm, ). These results showed that the incorporation of rGO indeed influenced the pore size of the hydrogels in a dose‐dependent pattern.
\",\n \"Morphological and Mechanical Evaluations on SA/Gel/rGO Patches\",\n \"
We 3D‐printed various SA/Gel scaffolds with different concentrations of rGO from 0 to 0.2 mg mL−1 (Figure\\n##FIG##4##\\n4a##). We used two freeze‐drying and one cross‐linking process to obtain the patches post‐printing. The gross morphologies were not significantly different except that the color changed from white to dark at the rGO concentration was increased. The 3D printer was an Organ Printing United system (Figure ##SUPPL##0##S3a##, Supporting Information). The mechanical properties of the SA/Gel/rGO patches are a valuable element for urethral repair and tissue regeneration. The tensile mechanical characteristics of the SA/Gel/rGO patches with different concentrations of rGO were evaluated by the stretching process of the patches (Figure ##SUPPL##0##S3b##, Supporting Information), the length of the patches that stretched between the upper and lower grips of the tensile machine could be adjusted. Then, each sample was stretched at 10 mm per minute until reaching its breaking points. All the samples exhibited the linear elastic behavior (Figure ##FIG##4##4b##). Compared with the patches containing rGO, the SA/Gel patch without rGO showed a lower modulus, 139.68 ± 0.144 KPa (Figure ##FIG##4##4c##). The tensile strengths of the SA/Gel patches at concentrations of 0, 0.02, 0.05, and 0.2 mg mL−1 of rGO were in the range of 139.87 ± 1.02–296.65 ± 1.11 KPa (Figure ##FIG##4##4d##). The tensile strength of the SA/Gel/rGO0.2 patch was higher (296.65 ±1.11 KPa) when the concentration of rGO was increased. The elevation of strain at break in the SA/Gel/rGO0.05 was 211.10 ± 1.34% (Figure ##FIG##4##4e##). Besides, the patches with appropriate elongation are flexible and elastic. Interestingly, the strain at break of SA/Gel/rGO0.2 patch was 137.77 ± 0.86% . These observations might be attributed to the SA/Gel patch to become fragile under the higher concentration of rGO. The SA/Gel/rGO patch was saturable and exhibits excellent mechanical properties to fit the requirement of urethral reconstruction. Interestingly, the rGO hybrid patches appeared to possess higher tensile strengths and Young's modulus. The rGO0.05 was an appropriate concentration to achieve the best mechanical performance among the groups examined.
\",\n \"Characteristics of Cell Behaviors\",\n \"
SA/Gel/rGO patches served as the templates for cell adhesion, and proliferation, thus superior cytocompatibility is required. The characteristics of cell survival and proliferation were evaluated through seeding fibroblasts on SA/Gel/rGO hydrogels containing different concentrations of rGO. The live and dead cells were stained with calcein AM and propidium iodides (PI) after 1 day, respectively. The fluorescence of living cells in SA/Gel/rGO hydrogels of 0.1 and 0.2 mg mL−1 of rGO was less (Figure\\n##FIG##5##\\n5a##). The increased number of dead cells might have been affected in part by the relatively high concentrations of rGO present. The cell morphologies were further observed by SEM, where fibroblasts were generally located within the pores of the 3D‐printed SA/Gel/rGO patches (Figure ##SUPPL##0##S4##, Supporting Information).
\",\n \"
From the expression patterns of differentially expressed proteins (DEPs) by fibroblasts (cell density: 1 × 107) with the different concentrations of rGO (Figure ##FIG##5##5b##), the cells treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 were compared with the cells with no treatment. The culture time was 72 h. Some biological processes and pathways, which are correlated with tissue regeneration or tissue repair, were observed to be significantly enriched in these DEPs (Figures ##SUPPL##0##S5a,S6a##, and ##SUPPL##0##S7a##, Supporting Information). For example, it is reported that the transmembrane receptor protein tyrosine kinase signaling pathway has a role in promoting sprouting of the angiogenic endothelium.[\\n##REF##20445537##\\n45\\n##\\n] The Ras/MAPK signaling cascade plays an important role in zebrafish heart regeneration.[\\n##UREF##8##\\n46\\n##\\n] The cell proliferation process is required for cardiomyocytes regeneration.[\\n##REF##28185170##\\n47\\n##\\n] In addition, the cellular components related to platelet alpha granule, secretory veside, vesicle lumen, and endomembrane system were enriched in DEPs treated with rGO0.02 (Figure ##SUPPL##0##S5b##, Supporting Information). Similar situations were observed when treated with rGO0.05 and treated with rGO0.1 (Figures ##SUPPL##0##S6b## and ##SUPPL##0##S7b##, Supporting Information). In addition, in molecular functions, the growth factor binding, growth factor activity, receptor‐ligand activity, and signaling receptor activator were enriched (Figures ##SUPPL##0##S5c,S6c##, and ##SUPPL##0##S7c##, Supporting Information). Similarly, the enriched Ras and MAPK signaling pathways were also observed via the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Figures ##SUPPL##0##S5d,S6d##, and ##SUPPL##0##S7d##, Supporting Information). The genes involved in these biological processes and pathways might contribute to angiogenesis mediated by rGO. The bubble graph was also shown for gene ontology enrichment treated with rGO of 0.02, 0.05, and 0.1 mg mL−1 for the biological process, molecular process, and cellular components (Figure ##SUPPL##0##S8a–i##, Supporting Information).
\",\n \"
Scratch assay is a standard for cell migration study in vitro. The migration of fibroblasts for the wound healing process is of great importance; after the injury, fibroblasts were used to proliferate and migrate from neighboring tissues into the wound area.[\\n##REF##20824344##\\n48\\n##\\n] To evaluate the potential of rGO in enhancing the migration of fibroblasts, a time‐dependent experiment was carried out (0–24 h). The results of the scratch assay showed that the migration ability of fibroblasts was greatly improved within 24 h after the treatment with rGO concentrations of 0.02 and 0.05 mg mL−1 (Figure ##FIG##5##5c##). With the increasing concentration of rGO, the proliferation of fibroblasts and wound healing increased first and then decreased, with a maximum migration rate of 61.18 ± 2.61% for rGO at 0.05 mg mL−1 (Figure ##FIG##5##5d##). Of note, when the concentration of rGO was set at 0.2 mg mL−1, the migration and proliferation of fibroblasts were significantly inhibited (26.39 ± 3.93%).
\",\n \"
Cell counting kit‐8 (CCK‐8) assay was subsequently used to assess the cytotoxic effect of SA/Gel/rGO patches on the fibroblasts (Figure ##FIG##5##5e##). After 1 and 4 days of cell culture, the metabolic activities of cells on the SA/Gel/rGO patches exhibited inhibitory effects. The higher concentration of rGO revealed inhospitable environments to cell metabolic activities during this time point. However, after 7 days of cell culture, the cell metabolic activities on SA/Gel/rGO0.02 and SA/Gel/rGO0.05 became higher than the remaining patches close to that for the control patches without rGO. Consequently, it might be undesirably toxic to cells at the concentration of rGO above 0.05 mg mL−1, in alignment with the cell viability staining results. Some reports have suggested that GO concentration of 50 µg mL−1 on fibroblasts led to significant cytotoxicity, while rGO at a concentration higher than 0.1 µg mL−1 would be toxic to endothelial cells.[\\n##UREF##5##\\n32\\n##, ##REF##21082807##\\n49\\n##\\n] Our results were overall consistent with the previous studies.
\",\n \"Urethrogram and Gross Morphology\",\n \"
The safety and functions of the SA/Gel/rGO patches were further proven in clinically relevant rabbit urethral injury models. According to the results of the in vitro mechanical test and biocompatibility of the scaffold, the patches without rGO were selected as the control group, and the rGO concentration of 0.05 and 0.2 mg mL−1 was selected as the experimental group. The patches were sutured to the dorsal urethral defect, and the injury model of the urethra and the surgical process of repair are shown in Figure ##SUPPL##0##S9## (Supporting Information). The recovery of the repaired urethras was detected at 4 and 8 weeks after the operation. The 4‐ and 8‐week urethrogram of the SA/Gel group displayed narrow lumens because of scar formation (Figure\\n##FIG##6##\\n6a,d##). The SA/Gel/rGO0.05 (Figure ##FIG##6##6b,e##) groups showed wide lumens of urethras similar to the normal urethra (Figure ##FIG##6##6g##). In the 4‐week urethrogram, the SA/Gel/rGO0.2 groups had a narrower urethral lumen, the widths developed larger at 8 weeks (Figure ##FIG##6##6c,f##). The reason might be the inflammation‐related swelling of urethral tissue caused by rGO with high concentration. The blockage rate determines whether and how much urine can flow out. The blockage ratio of the urethras treated with the SA/Gel/rGO0.05 patches could approach that of normal lumens (Figure ##FIG##6##6i##). After the incision of the ventral urethral wall, the dorsal wall could be exposed. Gross morphology revealed that urethral tissue in the SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups was significantly better than that in the SA/Gel group. The SA/Gel group showed the growth of severe scar‐like tissue in the urethra (Figure ##FIG##6##6j,m##). At 8 weeks, both of SA/Gel/rGO0.05 (Figure ##FIG##6##6n##) and SA/Gel/rGO0.2 (Figure ##FIG##6##6o##) groups showed smooth epithelial layer similar to the normal urethra of rabbits (Figure ##FIG##6##6h##). However, SA/Gel/rGO0.2 group at 4 weeks (Figure ##FIG##6##6i##) revealed red and swollen mucosa compared with that in the SA/Gel/rGO0.2 group (Figure ##FIG##6##6k##), which is consistent with the lumen width in the urethrogram. Therefore, the treatment of urethral injury could be improved by choosing a low dose of rGO for future application.
\",\n \"Histological Evaluations\",\n \"
The histology analyses of urethral reconstruction at 4‐ and 8‐weeks post‐surgery were performed by hematoxylin and eosin (H&E) and Masson's trichrome staining. The SA/Gel patches with/without rGO showed various outcomes. Additionally, there was no significant infection during the experimental period, suggesting good biocompatibility of the patches. According to the histology, the urethras treated with the SA/Gel patches were associated with thinner/uncomplete urothelium layers, fewer blood vessels, and thicker submucosal tissue (Figure\\n##FIG##7##\\n7a–d##). Due to the lack of regenerated urothelium layers, and over‐deposited ECM, the urethra developed to urethral stricture. In contrast, the reconstructed urethra treated with the SA/Gel/rGO patches had the nearly normal urothelium layers, submucosal tissue, and blood vessels. The continuous and complete urothelium layers were formed on the lumen surfaces for the SA/Gel/rGO0.05 group after 4‐ and 8‐weeks post‐surgery. However, the SA/Gel/rGO0.2 patches could lead to a short period of swelling and inflammation in 4 weeks. These results suggested that rGO at a lower concentration (0.05 mg mL−1) could perform best function in the regeneration of urothelium layers and blood vessels, but not at a higher concentration (0.2 mg mL−1). The mucosal coverage ratios by the SA/Gel/rGO patches were measured (Figure ##FIG##7##7e,f##). The urethras treated with the SA/Gel/rGO0.05 patches had more significant coverages, compared with the other groups. The urethral collagen thicknesses were also measured after the wound healing (Figure ##FIG##7##7g,h##). The SA/Gel/rGO0.05 patch could induce a less collagen deposition under the mucosa, therefore developing a scar‐less wound healing. The histology showed that an optimal concentration of rGO would promote the regeneration of epithelial cells and decrease submucosal ECM deposition to inhibit urethral fibrosis.
\",\n \"Immunofluorescence\",\n \"
The distribution of cytokeratin was investigated based on the expression of epithelial cytokeratin AE1/AE3 (an important membrane surface protein marker of ECs) via immunofluorescence staining of the urethra. The expression level for AE1/AE3 on the SA/Gel/rGO0.05 at 4‐and 8‐weeks post‐surgery (4W: 16.70 ± 2.66 and 8W: 24.72 ± 2.81) is significantly higher than that in the other groups. Although the continuous urothelium layer is formed, the urothelium layer of the SA/Gel/rGO0.2 group is thinner than that of the SA/Gel/rGO0.05 group. The expression of cytokeratin in the SA/Gel group is low (4W: 8.19 ± 1.72 and 8W: 8.29 ± 1.65), there is no complete regeneration of a discontinuous urothelium layer (Figure\\n##FIG##8##\\n8a,e##). It suggests that the introduction of rGO has the potential to promote the regeneration of urothelium layers. Studies have shown that the significant pro‐angiogenic properties of rGO depend on its concentration.[\\n##UREF##5##\\n32\\n##, ##REF##29892387##\\n50\\n##\\n] In the results of fluorescence staining at 4‐weeks post‐surgery, the SA/Gel/rGO0.05 group showed a number of CD 31‐positive cells (the blood vessels labeled by CD 31), which were more than them in the SA/Gel and SA/Gel/rGO0.2 group. At the beginning process of urethral regeneration with inflammation, the vessels in tissue would be much more obvious that is consistent with the results.[\\n##REF##12490959##\\n51\\n##\\n] For SA/Gel/rGO0.05 and SA/Gel/rGO0.2 groups, the number of CD 31‐positive cells at 8 weeks (SA/Gel/rGO0.05: 14.10 ± 3.99 and SA/Gel/rGO0.2: 8.63 ± 1.23) are lower than them at 4 weeks (SA/Gel/rGO0.05: 15.35 ± 4.55 and SA/Gel/rGO0.2: 14.77 ± 3.07) (Figure ##FIG##8##8b,f##).
\",\n \"
Different degrees of inflammation response at the injured sites would appear because of the transplantation of external scaffolds. CD 206 is used to label M2 macrophages.[\\n##REF##31903147##\\n52\\n##\\n] The results showed that the number of CD206‐positive cells in the SA/Gel group was the lowest. The number of CD206 positive cells in the SA/Gel/rGO0.05 group in 4‐and 8‐weeks post‐surgery (4W: 36.36 ± 3.54 and 8W: 40.74 ± 2.23) were significantly higher than them in the other groups, indicating that the lower level of local inflammatory response was caused by the rGO treated group (Figure ##FIG##8##8c,g##). This indicated that rGO appeared to promote the transition of M2 macrophages and inhibit the inflammatory response during the urethral repair. Meanwhile, the low inflammatory response might promote the proliferation and activation of fibroblasts. The positive expression of proliferating cell nuclear antigen (PCNA) was obviously increased in the SA/Gel/rGO0.05 group at 4‐and 8‐weeks post‐surgery (4W: 7.77 ± 0.25 and 8W: 8.46 ± 0.94) (Figure ##FIG##8##8d,h##).
\",\n \"Flow Cytometric Analysis of RAW264.7 Surface Markers\",\n \"
To evaluate the macrophages polarization status, the M1 surface markers (CD68, CD86) and M2 surface markers (CD206) in RAW 264.7 macrophages were further analyzed by flow cytometric (Figure\\n##FIG##9##\\n9a##). The results revealed that more than 90% of the cells strongly expressed surface antigens such as CD68. The inflammatory cell density was evaluated by CD86 staining. The CD86 inflammatory cell density in the 0.02rGO and 0.1rGO groups was no significant difference in the control group. However, as the amount of rGO increases, it leads to the increase of CD206‐positive macrophages. The expression of CD86 in M1 type was significantly increased by LPS + INF‐r, and the expression of CD206‐positive macrophages was significantly higher than that in the control group after the introduction of rGO (Figure ##FIG##9##9b##). The results of flow cytometry analysis showed that the addition of rGO promoted the polarization of macrophages to the M2 phenotype in vivo.
\",\n \"qRT‐PCR Analysis of mRNA Levels of the Markers in Tissue and RAW 264.7 Cells\",\n \"
The gene expression of Arg1, Fizz, and Ym1 in the 0.02rGO group and the 0.1rGO group was significantly higher than that in the control group, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the control group (Figure\\n##FIG##10##\\n10a##). At the mRNA level, rGO treatment was associated with significant increases in Arg1, Fizz, and Ym1 expression compared to the control and M1 groups, and the gene expression of iNOS and TGF‐β in the 0.02 rGO group and the 0.1rGO group was significantly lower than that in the M1 group (Figure ##FIG##10##10b##). These results suggest that rGO may also have an inducing effect on the macrophage anti‐inflammatory phenotype in vivo. The expression of SA/Gel/rGO0.05 α‐SMA, COL1A1, COL3A1, CD206, CD31, TGF‐β2 was significantly higher than that in the control group. The expression of TGFBR1, β‐catenin, and VWF was no significant difference in the control group (Figure ##FIG##10##10c–i##). Overall, the low‐dose rGO effectively promoted epithelization and neovascularization while reducing inflammation. These results suggested that rGO can promote urethral healing and regeneration.
In this study, the SA/Gel/rGO patches were synthesized by 3D printing with printable, degradation, and tunable mechanical features for urethral reconstruction. Our results were interesting in that the physical and biological properties of SA/Gel/rGO patches were controllable by adjusting the concentration of rGO. rGO with proper intermediate concentrations turned out to promote cell migration, proliferation, and urothelium layer formation. In addition, the fibrosis of the urethra was inhibited by introducing low‐dose rGO. The urethral reconstruction in rabbits model using the optimized SA/Gel/rGO0.05 patches led to the scar‐less urethral regeneration. Totally, our study provided a systematic evaluation of the application potential of rGO‐based scaffold on the urological and other organ defects’ treatment.
"],"string":"[\n \"Conclusion\",\n \"
In this study, the SA/Gel/rGO patches were synthesized by 3D printing with printable, degradation, and tunable mechanical features for urethral reconstruction. Our results were interesting in that the physical and biological properties of SA/Gel/rGO patches were controllable by adjusting the concentration of rGO. rGO with proper intermediate concentrations turned out to promote cell migration, proliferation, and urothelium layer formation. In addition, the fibrosis of the urethra was inhibited by introducing low‐dose rGO. The urethral reconstruction in rabbits model using the optimized SA/Gel/rGO0.05 patches led to the scar‐less urethral regeneration. Totally, our study provided a systematic evaluation of the application potential of rGO‐based scaffold on the urological and other organ defects’ treatment.
The nasty urine microenvironment (UME) is an inherent obstacle that hinders urethral repair due to fibrosis and swelling of the oftentimes adopted hydrogel‐based biomaterials. Here, using reduced graphene oxide (rGO) along with double‐freeze‐drying to strengthen a 3D‐printed patch is reported to realize scarless urethral repair. The sodium alginate/gelatin/reduced graphene oxide (SA/Gel/rGO) biomaterial features tunable stiffness, degradation profile, and anti‐fibrosis performance. Interestingly, the 3D‐printed alginate‐containing composite scaffold is able to respond to Ca2+ present in the urine, leading to enhanced structural stability and strength as well as inhibiting swelling. The investigations present that the swelling behaviors, mechanical properties, and anti‐fibrosis efficacy of the SA/Gel/rGO patch can be modulated by varying the concentration of rGO. In particular, rGO in optimal concentration shows excellent cell viability, migration, and proliferation. In‐depth mechanistic studies reveal that the activation of cell proliferation and angiogenesis‐related proteins, along with inhibition of fibrosis‐related gene expressions, play an important role in scarless repair by the 3D‐printed SA/Gel/rGO patch via promoting urothelium growth, accelerating angiogenesis, and minimizing fibrosis in vivo. The proposed strategy has the potential of resolving the dilemma of necessary biomaterial stiffness and unwanted fibrosis in urethral repair.
","
A sodium alginate/gelatin/rGO biomimetic patch is created with printable, degradable, and adjustable mechanical properties. The inclusion of rGO with the appropriate concentration can promote cell migration, and proliferation, and inhibit urethral fibrosis and the formation of the urothelium. This strategy demonstrates great potential in addressing the dilemma of necessary biomaterial stiffness and unwanted fibrosis in urethral repair.\n\n
The nasty urine microenvironment (UME) is an inherent obstacle that hinders urethral repair due to fibrosis and swelling of the oftentimes adopted hydrogel‐based biomaterials. Here, using reduced graphene oxide (rGO) along with double‐freeze‐drying to strengthen a 3D‐printed patch is reported to realize scarless urethral repair. The sodium alginate/gelatin/reduced graphene oxide (SA/Gel/rGO) biomaterial features tunable stiffness, degradation profile, and anti‐fibrosis performance. Interestingly, the 3D‐printed alginate‐containing composite scaffold is able to respond to Ca2+ present in the urine, leading to enhanced structural stability and strength as well as inhibiting swelling. The investigations present that the swelling behaviors, mechanical properties, and anti‐fibrosis efficacy of the SA/Gel/rGO patch can be modulated by varying the concentration of rGO. In particular, rGO in optimal concentration shows excellent cell viability, migration, and proliferation. In‐depth mechanistic studies reveal that the activation of cell proliferation and angiogenesis‐related proteins, along with inhibition of fibrosis‐related gene expressions, play an important role in scarless repair by the 3D‐printed SA/Gel/rGO patch via promoting urothelium growth, accelerating angiogenesis, and minimizing fibrosis in vivo. The proposed strategy has the potential of resolving the dilemma of necessary biomaterial stiffness and unwanted fibrosis in urethral repair.
\",\n \"
A sodium alginate/gelatin/rGO biomimetic patch is created with printable, degradable, and adjustable mechanical properties. The inclusion of rGO with the appropriate concentration can promote cell migration, and proliferation, and inhibit urethral fibrosis and the formation of the urothelium. This strategy demonstrates great potential in addressing the dilemma of necessary biomaterial stiffness and unwanted fibrosis in urethral repair.\\n\\n
\"\n]"},"body":{"kind":"list like","value":["Experimental Section","Inks preparation and rGO characterization","
The rGO was dispersed (1 mg mL−1) in water, and the solution was prepared according to the instructions. Briefly, the graphene powder (rGO, 20 mg, XFNANO, Nanjing, China) was dispersed in 20 mL double‐distilled water. And the aqueous suspension was obtained by ultrasonication at 40 KHz for 2 h. Polyvinyl pyrrolidone (PVP, MW = 58 000, 100 mg, Aladdin, Shanghai, China) was added to rGO aqueous suspension for sonication at ambient temperature for 2 h. After that, the rGO suspension was diluted to 0.02, 0.05, 0.1, and 0.2 mg mL−1, respectively.
","
SA powder (2% weight/volume, from brown algae, Sigma–Aldrich, USA) and Gel powder (2% w/v, from porcine skin, Tape A, gel strength ≈300 g Bloom, Sigma–Aldrich, USA) were added to sterile water to prepare the 0 rGO group as a control. Five GA/Gel/rGO hybrid ink solutions containing different rGO concentrations, 0, 0.02, 0.05, 0.1, and 0.2 mg mL−1 rGO, were named as SA/Gel, SA/Gel/rGO0.02, SA/Gel/rGO0.05, SA/Gel/rGO0.1, and SA/Gel/rGO0.2, respectively. To keep the same SA and Gel concentrations, different volumes of sterile water and rGO dispersion were prepared. The amounts of chemicals used for preparing the different SA/Gel/rGO hybrid ink solution are listed (Table ##SUPPL##0##S1##, Supporting Information). As an example, to prepare the SA/Gel/rGO0.05 ink solution, 1.0 mL rGO solution (1 mg mL−1) was mixed with 19 mL sterile water to a homogeneous solution. Then, 400 mg SA and 400 mg Gel were dissolved in the above solution at 37 °C for 24 h. The mixture was kept standing to homogenize, sufficiently dissolve the polymer, and eliminate air bubbles. The rGO morphology was studied by using TEM (FEI, Hillsboro, USA) and SEM (Hitachi TM‐100, Tokyo, Japan). The spatial distribution of rGO was observed under an inverted phase contrast microscope (Olympus, Japan).
","Characterizations of SA/Gel/rGO hydrogel","
\n
\nRaman Shift: The rGO incorporation into the hydrogel was analyzed qualitatively via Raman spectroscopy (DXR 2Xi, ThermoFisher, United States).
\nFourier‐Transform Infrared (FTIR): The functional groups of hydrogels were analyzed by FTIR (iS50, ThermoFisher, United States). The hydrogel was freeze‐dried in a vacuum freeze‐dryer (SCIENTZ‐10N, Ningbo, China) to obtain the powder. The hydrogel powder was finely grounded with KBr and compressed into slices for FTIR measurements, over the range of 500–4000 cm−1.
\nSwelling Ratio: The swelling property was assessed after immersion in double‐distilled water for 5 days by the general gravimetric method, which was referred to by Liu.[\n##UREF##9##\n53\n##\n] Three different concentrations of SA (1%, 1.5%, and 2%) and rGO hybrid hydrogel were investigated (n = 4 per group). The freeze‐dried hydrogels samples were weighed (W\nd) and placed in a 24‐well plate filled with double‐distilled water at room temperature. Then, the samples were taken out and rubbed with filter paper to remove the excess water. Finally, the samples were weighed (W\nw) at the point of swelling equilibrium. The swelling ratio was calculated according to the following formula:\n\n
\n
","
Where W\nd represents the weights of freeze‐dried hydrogel samples, W\nw stands for the weights of hydrogels after absorbing water.\n
\nIn Vitro Degradation: The degradable properties of SA/Gel hydrogel with different concentrations of rGO were examined via quantifying the mass remaining in vitro. These samples were immersed in double‐distilled water and lyophilized for 7, 14, and 21 days. Then, the lyophilized samples were weighed. WI was the initial dry weights of the sample and WT was the weights after T days of immersion in water (T = 7, 14, and 21 days). Finally, the remaining weights were calculated by the following formula. These experiments were performed in triplicates and repeated thrice.\n\n
\n
","Scanning Electron Microscope (SEM)","
The morphology of the hybrid hydrogel samples was observed by SEM. Briefly, the samples were prepared in a 24‐well plate (0.4 cm in height), frozen at 4 °C and −80 °C for 2 h, respectively, and then lyophilized overnight in a vacuum freeze‐dryer (SCIENTZ‐10N, Ningbo, China). Next, the freeze‐dried hydrogel samples were cross‐linked with 5% CaCl2 for 30 min and washed thoroughly with sterile water. Then, they were freeze‐dried and stored in a vacuum container. The distribution of pore size and porosity of hydrogels was counted using Image J visualization software (National Institutes of Health, Bethesda, MD, USA) (n = 10) from SEM images.
","Cell Culture","
Human fibroblasts were obtained from the human foreskin dermal tissue from the National Collection of Authenticated Cell Culture. The cells were cultured in a T‐75 flask (Corning, USA) with high‐glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone, UT), and supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin‐streptomycin in an incubator (37°C, 5% CO2). The complete medium was updated every 2 days. After 85–90% confluence, the passage of 10 to 15 cells was used for subsequent experiments. The growth and proliferation of cells were observed with an inverted phase contrast microscope (CKX53, Olympus, Japan).
","In Vitro Cell Biocompatibility Measurements","
Fibroblasts were used to test cell adhesion and proliferation on the hydrogel. The cells at a density of 5 × 104 cells mL−1 were seeded on the surface of SA/Gel hydrogel. Cell viability was identified by 2 µm Calcein AM (live cell stain, 490 nm) (Life Technologies, USA) and 4 µm propidium iodides (dead cell stain, 535 nm) (PI, Life Technologies, USA). The living and dead cells were marked in green and red, respectively. Samples were washed three times by PBS and stained with Calcein AM and PI for 30 min in the dark. Then, they were washed with PBS solution. A fluorescence microscope with an imaging system (IX73, Olympus, Japan) was used for image acquisition. The cell cytotoxicity of rGO for fibroblasts was evaluated by the CCK‐8 (Dojindo, Japan) at days 1, 4, and 7 after seeding according to the protocol. Briefly, fibroblasts seeded on hydrogel with or without rGO were washed with PBS three times. Then, 100 µL of CCK‐8 and 1 mL of complete culture medium were added to each well, followed by incubation in the dark for 4 h at 37 °C. 200 µL of the medium was transferred into a 96‐well plate after incubation. Each group contained five replicate wells. The CCK‐8 medium was used as the black control. The optical density (OD) was read by the multifunctional enzyme marker (Varioskan Flash, Thermo Fisher Scientific, USA) at 450 nm. five samples were tested for each group.
","
Cell Migration: Wound scratch assay was used for the cell migration study. The fibroblasts at the density were 2 × 105 cells well−1 were seeded on the 24‐well plate. A scratch wound was formed using a pipette tip of 1000 µL when the confluence of cells reached 80–90%. Then, cells were washed with PBS. The distance of scratches was observed and recorded with an inverted phase contrast microscope (W\nd0). The rGO dispersion in a certain volume ratio blended with the complete medium was added to the wells. The cells were cultured for 24 h and observed under the microscope again. And the distance of scratches (W\ndt) was recorded. Wound contraction was quantified from the images by the following equation. Experiments were repeated in triplicates.\n\n
","Printing of Acellular SA/Gel/rGO Patches","
In this study, the SA/Gel/rGO patches were printed using the Organ Printing United system (OPUS, Novaprint Therapeutics Suzhou Co., Ltd, Suzhou, China); it includes multi‐nozzle printing and multi‐material mixing. The patch model was a lattice‐rod structure with 15 mm ×15 mm × 0.75 mm, which was converted into a G‐Code file using Slic3r with the layer height of 0.15 mm (5 layers), the pore size of 1.5 mm. An inner diameter of 200 µm of the printing nozzle was selected. The ink solution was liquid at 37 °C and introduced into a special printing syringe (3 cc), but it cannot be printed in this state. Therefore, the printing syringe with the ink was placed and cooled at 4 °C for 8–10 min. And the temperature of the printer chamber was set to 19 °C. The SA/Gel/rGO patches were printed in a layer‐by‐layer deposition fashion on the printer platform. After printing each patch, they were cooled to −80 °C. Briefly, the above printed acellular patches (15 mm × 15 mm) were frozen at −80 °C, and then lyophilized overnight in a vacuum freeze‐dryer. Next, the freeze‐dried patches were cross‐linked with 5% CaCl2 for 30 min and washed thoroughly with sterile water. The patches were freeze‐dried again and stored in a vacuum container.
","Patch Mechanics","
Multifunctional materials testing machine (HD‐5000B, Yangzhou, China) was used to characterize the tensile behavior of the SA/Gel/rGO patches. The SA/Gel patch was used as the control material. The patch samples of longitudinal strips with a length of 50 mm and a width of 20 mm were prepared. Then, the distance between the clamps was measured. The patches were stretched at a speed of 10 mm per minute at room temperature until reaching their breaking points. Five samples were measured in each group, and the average value and standard deviation were calculated. The final tensile strength was calculated in the following formula.\nwhere δ was the tensile strength, F was the maximum force at the breaking points and S was the cross‐sectional area of patch.
","Cell Morphologies on Patches","
The patches after the final freeze‐drying were sterilized with UV for 8 h and put on a 6‐well plate. Fibroblasts were seeded onto the patches at a density of 2 × 105 cells cm−2 and incubated at 5% CO2 and 37 °C for 3 days. Additionally, the cell culture medium was updated every 2 days. Then, the cells with patches were fixed in 2.5% glutaraldehyde for 2 h at room temperature and then rinsed with PBS. The patches were then vacuum‐dried. The patches were sputter‐coated with gold for 30 sec and examined under a scanning electron microscope at 15 kV (SU8100, Hitachi, Japan).
","Protein Detection","
The total protein of fibroblasts was extracted using ice‐cold Cell & Tissue Protein Extraction Reagent (Kang Chen. Shanghai, China). Then, according to the manufacturer's protocol, the protein concentration was determined using BCA Protein Assay Kit (Kang Chen, Shanghai, China). Briefly, protein array membranes were blocked in blocking buffer for 30 min and incubated with samples at room temperature for 2 h. Then samples were decanted, and membranes were washed. Next, membranes were incubated with diluted biotin‐conjugated antibodies at room temperature for 2 h. The membranes were washed with washing buffer and reacted with HRP‐conjugated streptavidin (1:1000 dilution) at room temperature for 2 h. Membranes were washed thoroughly and exposed to detection buffer in the dark. After acting to detect buffer, the membranes were exposed to X‐ray film. The image was developed using a film scanner. Relative expression levels of cytokines were made by comparing the signal intensities and quantified by densitometry. Positive controls were used to standardize the results from different membranes. And fold changes were calculated in protein expression.
","Gene Ontology and KEGG Enrichment Analysis","
Gene ontology (http://www.geneontology.org) a systematical approach for gene and protein annotation in terms of biological process, molecular process, aisnd cellular component.[\n##REF##10802651##\n54\n##\n] Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) is an online database depositing biological pathways of genes and biochemicals. The enriched GO terms and KEGG pathways were annotated using the R package cluster Profiler.
","Urethroplasty and Postoperative Examinations in Rabbit","
To verify the biocompatibility of the 3D printed SA/Gel/rGO patches in vivo, the patches were placed in the urethral defect of rabbits for in vivo experiments. Nine adult New Zealand white rabbits had an average weight of ≈2.5 kg and were randomly divided into three groups for the urethral defect and subsequent urethroplasty. Rabbits in group 1 (n = 3) were repaired with the SA/Gel patch. Rabbits in group 2 (n = 3) were repaired with the SA/Gel/rGO0.05 patch. Rabbits in group 3 (n = 3) were repaired with the SA/Gel/rGO0.2 patch. These rabbits were first performed by general anesthesia with intravenous injection of pentobarbital, then the rabbit's urethras were disinfected with alcohol. The skins were sectioned at ≈3 cm proximal to the external urethral orifices, and the urethral lumens were exposed. Ventral urethral defects with a mean length × width of 2.0 cm × 0.8 cm were created in the anterior urethra of rabbits. The scaffolds with a length × width of 1.5 cm × 0.5 cm were sutured to the edge of the defect using 6‐0 absorbable polyglactin sutures (Ethicon, USA), and the whole urethral defects were sutured with 6‐0 absorbable polyglactin sutures. The rabbits were observed twice a day.
","
To observe the urethral leakage and stricture of the three groups of rabbits, the contrast solution was injected into the urethral lumens at 4‐ and 8‐weeks post‐surgery. Meanwhile, the rabbits were undertaken contrast‐enhanced urethrogram tests to check the condition of lumen in the urethra at 4‐ and 8‐weeks post‐surgery. The rabbits were euthanized after retrograde urethrograms at 4‐ and 8‐ weeks, and the urethral tissue for the following histology staining was collected. All the animal experiments were in accordance with the guidelines for animal care. The animal protocol (SYXK 2017‐0240) was approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital.
","Histology Assessment and Immunofluorescence","
The urethras were harvested after 4 weeks and 8 weeks postoperatively for histology analysis. Hematoxylin and eosin staining (H&E) and Masson's trichrome staining tests were conducted to identify the epithelial layer and collagen distribution of urethra. The specimens were fixed in 4% paraformaldehyde for 30 min at room temperature. Then, they were dehydrated with different grades of alcohol and embedded in the paraffin blocks. Histological sections were prepared and observed using an optical microscope. To further demonstrate the repair of urethral function, the samples were stained for immunofluorescence for epithelial cytokeratin AE1/AE3 (Santa Cruz Biotechnology, Inc.), CD31 (blood vessels, 1:100, Proteintech Group, Inc), CD206 (1:500, Proteintech Group, Inc), and PCNA (Proliferating Cell Nuclear Antigen 1:500, Abcam plc). Nuclei were stained with DAPI (1:500, Life Technologies). Afterward, the specimens were imaged and observed by an optical microscope.
","Flow cytometry","
After 85–90% confluence, leukemia cells in mouse macrophage (RAW 264.7 cells) were digested with pancreatic enzyme, and centrifuged at 1000 rpm for 5 min. Then the supernatant was removed, and cells were resuspended with a complete medium. RAW264.7 cells were seeded in 6‐well plates (5 × 105 cells/well), and placed in a CO2 incubator at 37 °C overnight. After 1 day, cells were divided into two groups when the cell density reached ≈40–50%. Group 1 (Control; 0.02rGO; 0.1rGO) was temporarily not treated. Group 2 (Control; M1; M1 + 0.02rGO; M1 + 0.1rGO), where M1; M1 + 0.02rGO; M1+ 0.1rGO, first treated with LPS (100 ng/mL) + INF‐r (50 ng mL−1) for 24 h to induce M1 type. On the third day, when the cell density reaches ≈70–80%, the corresponding concentration of rGO was added to the two groups, which were 0.02rGO, 0.1rGO, and treated for 24 h. RAW264.7 cells in the two groups of plates were collected separately for flow cytometry and RT‐PCR detection after 24 h. Flow cytometry was used to determine the effect of graphene on the polarization of Raw264.7 to M1, M2. Fluorescently labeled antibodies CD68, CD86, and CD206 were added to both groups of cells. Before staining, Raw264.7 cells were fixed and broken, then mixed with antibodies and incubated at 4 °C for 30 min in the dark. Then, the cells were resuspended with a cell staining buffer, followed by centrifugation for 5 min at 300 g, and discard the supernatant. Finally, the cells were resuspended by 200 µL cell stained buffer for detection and analysis with flow cytometry (Sysmex, CyFlow Cube8).
","RT‐PCR","
Relative quantification for mRNA gene expression studies. Extraction of mRNA with Invitrogen's trizol. The extracted mRNA was then reverse‐transcribed to become a cDNA template. Finally, a real‐time PCR experiment was performed using cDNA as a template.
","Statistical Analysis","
All the statistical significance for the experiments was analyzed using the one‐way ANOVA and paired‐samples t‐test. The error bar represents the mean ± standard deviation (SD) of measurements performed on each sample group. It was a statistically significant difference based on p <0.05 (*p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001).
","Conflict of Interest","
The authors declare no conflict of interest.
","Supporting information"],"string":"[\n \"Experimental Section\",\n \"Inks preparation and rGO characterization\",\n \"
The rGO was dispersed (1 mg mL−1) in water, and the solution was prepared according to the instructions. Briefly, the graphene powder (rGO, 20 mg, XFNANO, Nanjing, China) was dispersed in 20 mL double‐distilled water. And the aqueous suspension was obtained by ultrasonication at 40 KHz for 2 h. Polyvinyl pyrrolidone (PVP, MW = 58 000, 100 mg, Aladdin, Shanghai, China) was added to rGO aqueous suspension for sonication at ambient temperature for 2 h. After that, the rGO suspension was diluted to 0.02, 0.05, 0.1, and 0.2 mg mL−1, respectively.
\",\n \"
SA powder (2% weight/volume, from brown algae, Sigma–Aldrich, USA) and Gel powder (2% w/v, from porcine skin, Tape A, gel strength ≈300 g Bloom, Sigma–Aldrich, USA) were added to sterile water to prepare the 0 rGO group as a control. Five GA/Gel/rGO hybrid ink solutions containing different rGO concentrations, 0, 0.02, 0.05, 0.1, and 0.2 mg mL−1 rGO, were named as SA/Gel, SA/Gel/rGO0.02, SA/Gel/rGO0.05, SA/Gel/rGO0.1, and SA/Gel/rGO0.2, respectively. To keep the same SA and Gel concentrations, different volumes of sterile water and rGO dispersion were prepared. The amounts of chemicals used for preparing the different SA/Gel/rGO hybrid ink solution are listed (Table ##SUPPL##0##S1##, Supporting Information). As an example, to prepare the SA/Gel/rGO0.05 ink solution, 1.0 mL rGO solution (1 mg mL−1) was mixed with 19 mL sterile water to a homogeneous solution. Then, 400 mg SA and 400 mg Gel were dissolved in the above solution at 37 °C for 24 h. The mixture was kept standing to homogenize, sufficiently dissolve the polymer, and eliminate air bubbles. The rGO morphology was studied by using TEM (FEI, Hillsboro, USA) and SEM (Hitachi TM‐100, Tokyo, Japan). The spatial distribution of rGO was observed under an inverted phase contrast microscope (Olympus, Japan).
\",\n \"Characterizations of SA/Gel/rGO hydrogel\",\n \"
\\n
\\nRaman Shift: The rGO incorporation into the hydrogel was analyzed qualitatively via Raman spectroscopy (DXR 2Xi, ThermoFisher, United States).
\\nFourier‐Transform Infrared (FTIR): The functional groups of hydrogels were analyzed by FTIR (iS50, ThermoFisher, United States). The hydrogel was freeze‐dried in a vacuum freeze‐dryer (SCIENTZ‐10N, Ningbo, China) to obtain the powder. The hydrogel powder was finely grounded with KBr and compressed into slices for FTIR measurements, over the range of 500–4000 cm−1.
\\nSwelling Ratio: The swelling property was assessed after immersion in double‐distilled water for 5 days by the general gravimetric method, which was referred to by Liu.[\\n##UREF##9##\\n53\\n##\\n] Three different concentrations of SA (1%, 1.5%, and 2%) and rGO hybrid hydrogel were investigated (n = 4 per group). The freeze‐dried hydrogels samples were weighed (W\\nd) and placed in a 24‐well plate filled with double‐distilled water at room temperature. Then, the samples were taken out and rubbed with filter paper to remove the excess water. Finally, the samples were weighed (W\\nw) at the point of swelling equilibrium. The swelling ratio was calculated according to the following formula:\\n\\n
\\n
\",\n \"
Where W\\nd represents the weights of freeze‐dried hydrogel samples, W\\nw stands for the weights of hydrogels after absorbing water.\\n
\\nIn Vitro Degradation: The degradable properties of SA/Gel hydrogel with different concentrations of rGO were examined via quantifying the mass remaining in vitro. These samples were immersed in double‐distilled water and lyophilized for 7, 14, and 21 days. Then, the lyophilized samples were weighed. WI was the initial dry weights of the sample and WT was the weights after T days of immersion in water (T = 7, 14, and 21 days). Finally, the remaining weights were calculated by the following formula. These experiments were performed in triplicates and repeated thrice.\\n\\n
\\n
\",\n \"Scanning Electron Microscope (SEM)\",\n \"
The morphology of the hybrid hydrogel samples was observed by SEM. Briefly, the samples were prepared in a 24‐well plate (0.4 cm in height), frozen at 4 °C and −80 °C for 2 h, respectively, and then lyophilized overnight in a vacuum freeze‐dryer (SCIENTZ‐10N, Ningbo, China). Next, the freeze‐dried hydrogel samples were cross‐linked with 5% CaCl2 for 30 min and washed thoroughly with sterile water. Then, they were freeze‐dried and stored in a vacuum container. The distribution of pore size and porosity of hydrogels was counted using Image J visualization software (National Institutes of Health, Bethesda, MD, USA) (n = 10) from SEM images.
\",\n \"Cell Culture\",\n \"
Human fibroblasts were obtained from the human foreskin dermal tissue from the National Collection of Authenticated Cell Culture. The cells were cultured in a T‐75 flask (Corning, USA) with high‐glucose Dulbecco's modified Eagle's medium (DMEM, Hyclone, UT), and supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin‐streptomycin in an incubator (37°C, 5% CO2). The complete medium was updated every 2 days. After 85–90% confluence, the passage of 10 to 15 cells was used for subsequent experiments. The growth and proliferation of cells were observed with an inverted phase contrast microscope (CKX53, Olympus, Japan).
Fibroblasts were used to test cell adhesion and proliferation on the hydrogel. The cells at a density of 5 × 104 cells mL−1 were seeded on the surface of SA/Gel hydrogel. Cell viability was identified by 2 µm Calcein AM (live cell stain, 490 nm) (Life Technologies, USA) and 4 µm propidium iodides (dead cell stain, 535 nm) (PI, Life Technologies, USA). The living and dead cells were marked in green and red, respectively. Samples were washed three times by PBS and stained with Calcein AM and PI for 30 min in the dark. Then, they were washed with PBS solution. A fluorescence microscope with an imaging system (IX73, Olympus, Japan) was used for image acquisition. The cell cytotoxicity of rGO for fibroblasts was evaluated by the CCK‐8 (Dojindo, Japan) at days 1, 4, and 7 after seeding according to the protocol. Briefly, fibroblasts seeded on hydrogel with or without rGO were washed with PBS three times. Then, 100 µL of CCK‐8 and 1 mL of complete culture medium were added to each well, followed by incubation in the dark for 4 h at 37 °C. 200 µL of the medium was transferred into a 96‐well plate after incubation. Each group contained five replicate wells. The CCK‐8 medium was used as the black control. The optical density (OD) was read by the multifunctional enzyme marker (Varioskan Flash, Thermo Fisher Scientific, USA) at 450 nm. five samples were tested for each group.
\",\n \"
Cell Migration: Wound scratch assay was used for the cell migration study. The fibroblasts at the density were 2 × 105 cells well−1 were seeded on the 24‐well plate. A scratch wound was formed using a pipette tip of 1000 µL when the confluence of cells reached 80–90%. Then, cells were washed with PBS. The distance of scratches was observed and recorded with an inverted phase contrast microscope (W\\nd0). The rGO dispersion in a certain volume ratio blended with the complete medium was added to the wells. The cells were cultured for 24 h and observed under the microscope again. And the distance of scratches (W\\ndt) was recorded. Wound contraction was quantified from the images by the following equation. Experiments were repeated in triplicates.\\n\\n
\",\n \"Printing of Acellular SA/Gel/rGO Patches\",\n \"
In this study, the SA/Gel/rGO patches were printed using the Organ Printing United system (OPUS, Novaprint Therapeutics Suzhou Co., Ltd, Suzhou, China); it includes multi‐nozzle printing and multi‐material mixing. The patch model was a lattice‐rod structure with 15 mm ×15 mm × 0.75 mm, which was converted into a G‐Code file using Slic3r with the layer height of 0.15 mm (5 layers), the pore size of 1.5 mm. An inner diameter of 200 µm of the printing nozzle was selected. The ink solution was liquid at 37 °C and introduced into a special printing syringe (3 cc), but it cannot be printed in this state. Therefore, the printing syringe with the ink was placed and cooled at 4 °C for 8–10 min. And the temperature of the printer chamber was set to 19 °C. The SA/Gel/rGO patches were printed in a layer‐by‐layer deposition fashion on the printer platform. After printing each patch, they were cooled to −80 °C. Briefly, the above printed acellular patches (15 mm × 15 mm) were frozen at −80 °C, and then lyophilized overnight in a vacuum freeze‐dryer. Next, the freeze‐dried patches were cross‐linked with 5% CaCl2 for 30 min and washed thoroughly with sterile water. The patches were freeze‐dried again and stored in a vacuum container.
\",\n \"Patch Mechanics\",\n \"
Multifunctional materials testing machine (HD‐5000B, Yangzhou, China) was used to characterize the tensile behavior of the SA/Gel/rGO patches. The SA/Gel patch was used as the control material. The patch samples of longitudinal strips with a length of 50 mm and a width of 20 mm were prepared. Then, the distance between the clamps was measured. The patches were stretched at a speed of 10 mm per minute at room temperature until reaching their breaking points. Five samples were measured in each group, and the average value and standard deviation were calculated. The final tensile strength was calculated in the following formula.\\nwhere δ was the tensile strength, F was the maximum force at the breaking points and S was the cross‐sectional area of patch.
\",\n \"Cell Morphologies on Patches\",\n \"
The patches after the final freeze‐drying were sterilized with UV for 8 h and put on a 6‐well plate. Fibroblasts were seeded onto the patches at a density of 2 × 105 cells cm−2 and incubated at 5% CO2 and 37 °C for 3 days. Additionally, the cell culture medium was updated every 2 days. Then, the cells with patches were fixed in 2.5% glutaraldehyde for 2 h at room temperature and then rinsed with PBS. The patches were then vacuum‐dried. The patches were sputter‐coated with gold for 30 sec and examined under a scanning electron microscope at 15 kV (SU8100, Hitachi, Japan).
\",\n \"Protein Detection\",\n \"
The total protein of fibroblasts was extracted using ice‐cold Cell & Tissue Protein Extraction Reagent (Kang Chen. Shanghai, China). Then, according to the manufacturer's protocol, the protein concentration was determined using BCA Protein Assay Kit (Kang Chen, Shanghai, China). Briefly, protein array membranes were blocked in blocking buffer for 30 min and incubated with samples at room temperature for 2 h. Then samples were decanted, and membranes were washed. Next, membranes were incubated with diluted biotin‐conjugated antibodies at room temperature for 2 h. The membranes were washed with washing buffer and reacted with HRP‐conjugated streptavidin (1:1000 dilution) at room temperature for 2 h. Membranes were washed thoroughly and exposed to detection buffer in the dark. After acting to detect buffer, the membranes were exposed to X‐ray film. The image was developed using a film scanner. Relative expression levels of cytokines were made by comparing the signal intensities and quantified by densitometry. Positive controls were used to standardize the results from different membranes. And fold changes were calculated in protein expression.
\",\n \"Gene Ontology and KEGG Enrichment Analysis\",\n \"
Gene ontology (http://www.geneontology.org) a systematical approach for gene and protein annotation in terms of biological process, molecular process, aisnd cellular component.[\\n##REF##10802651##\\n54\\n##\\n] Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) is an online database depositing biological pathways of genes and biochemicals. The enriched GO terms and KEGG pathways were annotated using the R package cluster Profiler.
\",\n \"Urethroplasty and Postoperative Examinations in Rabbit\",\n \"
To verify the biocompatibility of the 3D printed SA/Gel/rGO patches in vivo, the patches were placed in the urethral defect of rabbits for in vivo experiments. Nine adult New Zealand white rabbits had an average weight of ≈2.5 kg and were randomly divided into three groups for the urethral defect and subsequent urethroplasty. Rabbits in group 1 (n = 3) were repaired with the SA/Gel patch. Rabbits in group 2 (n = 3) were repaired with the SA/Gel/rGO0.05 patch. Rabbits in group 3 (n = 3) were repaired with the SA/Gel/rGO0.2 patch. These rabbits were first performed by general anesthesia with intravenous injection of pentobarbital, then the rabbit's urethras were disinfected with alcohol. The skins were sectioned at ≈3 cm proximal to the external urethral orifices, and the urethral lumens were exposed. Ventral urethral defects with a mean length × width of 2.0 cm × 0.8 cm were created in the anterior urethra of rabbits. The scaffolds with a length × width of 1.5 cm × 0.5 cm were sutured to the edge of the defect using 6‐0 absorbable polyglactin sutures (Ethicon, USA), and the whole urethral defects were sutured with 6‐0 absorbable polyglactin sutures. The rabbits were observed twice a day.
\",\n \"
To observe the urethral leakage and stricture of the three groups of rabbits, the contrast solution was injected into the urethral lumens at 4‐ and 8‐weeks post‐surgery. Meanwhile, the rabbits were undertaken contrast‐enhanced urethrogram tests to check the condition of lumen in the urethra at 4‐ and 8‐weeks post‐surgery. The rabbits were euthanized after retrograde urethrograms at 4‐ and 8‐ weeks, and the urethral tissue for the following histology staining was collected. All the animal experiments were in accordance with the guidelines for animal care. The animal protocol (SYXK 2017‐0240) was approved by the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University Affiliated Sixth People's Hospital.
\",\n \"Histology Assessment and Immunofluorescence\",\n \"
The urethras were harvested after 4 weeks and 8 weeks postoperatively for histology analysis. Hematoxylin and eosin staining (H&E) and Masson's trichrome staining tests were conducted to identify the epithelial layer and collagen distribution of urethra. The specimens were fixed in 4% paraformaldehyde for 30 min at room temperature. Then, they were dehydrated with different grades of alcohol and embedded in the paraffin blocks. Histological sections were prepared and observed using an optical microscope. To further demonstrate the repair of urethral function, the samples were stained for immunofluorescence for epithelial cytokeratin AE1/AE3 (Santa Cruz Biotechnology, Inc.), CD31 (blood vessels, 1:100, Proteintech Group, Inc), CD206 (1:500, Proteintech Group, Inc), and PCNA (Proliferating Cell Nuclear Antigen 1:500, Abcam plc). Nuclei were stained with DAPI (1:500, Life Technologies). Afterward, the specimens were imaged and observed by an optical microscope.
\",\n \"Flow cytometry\",\n \"
After 85–90% confluence, leukemia cells in mouse macrophage (RAW 264.7 cells) were digested with pancreatic enzyme, and centrifuged at 1000 rpm for 5 min. Then the supernatant was removed, and cells were resuspended with a complete medium. RAW264.7 cells were seeded in 6‐well plates (5 × 105 cells/well), and placed in a CO2 incubator at 37 °C overnight. After 1 day, cells were divided into two groups when the cell density reached ≈40–50%. Group 1 (Control; 0.02rGO; 0.1rGO) was temporarily not treated. Group 2 (Control; M1; M1 + 0.02rGO; M1 + 0.1rGO), where M1; M1 + 0.02rGO; M1+ 0.1rGO, first treated with LPS (100 ng/mL) + INF‐r (50 ng mL−1) for 24 h to induce M1 type. On the third day, when the cell density reaches ≈70–80%, the corresponding concentration of rGO was added to the two groups, which were 0.02rGO, 0.1rGO, and treated for 24 h. RAW264.7 cells in the two groups of plates were collected separately for flow cytometry and RT‐PCR detection after 24 h. Flow cytometry was used to determine the effect of graphene on the polarization of Raw264.7 to M1, M2. Fluorescently labeled antibodies CD68, CD86, and CD206 were added to both groups of cells. Before staining, Raw264.7 cells were fixed and broken, then mixed with antibodies and incubated at 4 °C for 30 min in the dark. Then, the cells were resuspended with a cell staining buffer, followed by centrifugation for 5 min at 300 g, and discard the supernatant. Finally, the cells were resuspended by 200 µL cell stained buffer for detection and analysis with flow cytometry (Sysmex, CyFlow Cube8).
\",\n \"RT‐PCR\",\n \"
Relative quantification for mRNA gene expression studies. Extraction of mRNA with Invitrogen's trizol. The extracted mRNA was then reverse‐transcribed to become a cDNA template. Finally, a real‐time PCR experiment was performed using cDNA as a template.
\",\n \"Statistical Analysis\",\n \"
All the statistical significance for the experiments was analyzed using the one‐way ANOVA and paired‐samples t‐test. The error bar represents the mean ± standard deviation (SD) of measurements performed on each sample group. It was a statistically significant difference based on p <0.05 (*p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001).
L.W., K.W., and M.Y contributed equally to this work. This work was supported by the following grants: National Key Research and Development Program of China (2018YFA0703100), Jiangsu Key Technology Research Development Program (BE2017664), Shanghai Jiao Tong University Biomedical Engineering Cross Research Foundation (YG2022ZD022 and YG2017QN15) and the National Natural Science Foundation of China (82072217 and 81772135). Shanghai health committee (20184Y0053), Shanghai “Rising stars of medical talent” Youth development program, Shanghai Jiao Tong University K. C. Wong Medical Fellowship Fund, and Shanghai sixth people's hospital foundational research program. National Natural Science Foundation of China (Grant No. 62374107), The Talent Progrom of Shanghai University of Engineering Science (QNTD202104), Shanghai Local University Capacity Building Project of Science and Technology Innovation Action Program (21010501700), Class III Peak Discipline of Shanghai‐Materials Science and Engineering (High‐Energy Beam Intelligent Processing and Green Manufacturing). Natural Science Foundation of Shanghai(20ZR1442100).
","Data Availability Statement","
Research data are not shared.
"],"string":"[\n \"Acknowledgements\",\n \"
L.W., K.W., and M.Y contributed equally to this work. This work was supported by the following grants: National Key Research and Development Program of China (2018YFA0703100), Jiangsu Key Technology Research Development Program (BE2017664), Shanghai Jiao Tong University Biomedical Engineering Cross Research Foundation (YG2022ZD022 and YG2017QN15) and the National Natural Science Foundation of China (82072217 and 81772135). Shanghai health committee (20184Y0053), Shanghai “Rising stars of medical talent” Youth development program, Shanghai Jiao Tong University K. C. Wong Medical Fellowship Fund, and Shanghai sixth people's hospital foundational research program. National Natural Science Foundation of China (Grant No. 62374107), The Talent Progrom of Shanghai University of Engineering Science (QNTD202104), Shanghai Local University Capacity Building Project of Science and Technology Innovation Action Program (21010501700), Class III Peak Discipline of Shanghai‐Materials Science and Engineering (High‐Energy Beam Intelligent Processing and Green Manufacturing). Natural Science Foundation of Shanghai(20ZR1442100).
\",\n \"Data Availability Statement\",\n \"
Research data are not shared.
\"\n]"},"figure":{"kind":"list like","value":["
Schematics on the development of 3D‐printed SA/Gel/rGO patches for urethral repair. The SA/Gel/rGO patches were prepared by a 3D‐printing system and cross‐linked by Ca2+. The patches were then sutured in the damaged urethra of rabbits, wherein the rGO component facilitated regeneration of mucosa, promoted vascularization, and exhibited anti‐fibrotic activity.
","
Morphologies of rGO and optical images of rGO/hydrogel‐precursor dispersions. a) SEM and b) TEM images of rGO. c) Photographs of different concentrations of rGO/hydrogel‐precursor suspensions at rGO concentrations of 0.02, 0.05, 0.1, and 0.2 mg mL−1. d–g) Corresponding optical microscopy images. h–k) Area counts of the rGO distributed in the corresponding suspensions; *p <0.05, ****p <0.0001, n.s. means not significant.
","
Physicochemical characterizations of the SA/Gel/rGO hydrogels. a) Raman spectra of SA, SA/Gel, and SA/Gel/rGO hydrogels. b) FTIR spectra of i) SA/Gel hydrogel, ii) SA/Gel/rGO0.02 hydrogel, iii) SA/Gel/rGO0.05 hydrogel, and iv) SA/Gel/rGO0.2 hydrogel. c) Swelling ratios of the different concentrations of SA and SA/Gel/rGO composite inks at different concentrations of rGO. d) Degradation profiles of the hydrogels.
","
SEM images and gross appearances of SA/Gel/rGO hydrogels with various concentrations of rGO: 0, 0.02, 0.05, 0.1, and 0.2 mg mL−1. a–e) SEM images at 1000× magnification and optical images of the hydrogels; f–j) SEM images at 300× magnification. k–o) Distributions of pore sizes of the different hydrogels. p) Average pore sizes of the different hydrogels; **p <0.01; ***p <0.001; ****p <0.0001; n.s. means not significant.
","
a) Morphology of the 3D‐printed SA/Gel/rGO patches containing different concentrations of rGO, and the double‐freeze‐drying process. b) Tensile stress‐strain curves; c) Young's moduli; d) tensile strengths at break; and e) strains at the break for the different 3D‐printed patches; **p <0.01; ***p <0.001; and ****p <0.0001.
","
In vitro cytocompatibility of rGO and rGO composite hydrogel. a) Cell viability of fibroblasts seeded on different concentrations of SA/Gel/rGO hydrogels at day 1. Living cells are depicted in green and dead cells in red. b) Heat map of DEPs. Red and green colors indicate high and low expressions, respectively. c) wound healing was observed in fibroblast cells treated with the different concentrations of rGO (0, 0.02, 0.05, 0.1, and 0.2 mg mL−1). fibroblast cells treated without rGO were used as a positive control experiment. The scale bar at the right lower corner is 500 µm. d) The percentage of wound contraction was measured using Image J analysis software. Statistical significance was calculated by using a t‐test. e) Cell proliferation of fibroblasts cultured on hydrogels for 1, 4, and 7 days; n = 6, **p <0.01, ***p <0.001, ****p <0.0001.
","
Urethrography examinations and appearances of urethral repair from the biomaterial with SA/Gel, SA/Gel/rGO0.05, and SA/Gel/rGO0.2. The results of urethras from a,d,g,j) SA/Gel, b,e,h,k) SA/Gel/rGO0.05, and c,f,i,l) SA/Gel/rGO0.2 at 4‐ and 8‐weeks post‐surgery. The red arrows indicate the lumen of the urethra. The red dotted boxes indicate the urethral epithelial layer. *p <0.05, ****p <0.0001, n.s. means not significant.
","
Histological examination of the regenerated tissues at the damaged urethra sites after staining the sections with H&E and Masson's Trichrome. Representative images of urethral cross‐sections of the reconstructed urethra using SA/Gel, SA/Gel/rGO0.05, and SA/Gel/rGO0.2 of a,c) at 4 weeks and b,d) at 8 weeks. The mucosal coverage of urethra at 4 weeks (a) and at 8 weeks (b); The collagen thickness of urethra at 4 weeks (a) and at 8 weeks (b). The green dotted lines indicate the border line of collagen thickness. Data are means ± standard error, n = 3; **p <0.01, ***p <0.001, ****p <0.0001, n.s. means not significant.
","
Immunofluorescence staining of specific biomarkers in the SA/Gel, SA/Gel/rGO0.05, and SA/Gel/rGO0.2 repair after 4‐ and 8‐weeks post‐surgery. Representative immunofluorescence images of a) AE1/AE3, b) CD31, c) CD206, d) PCNA. Scale bar: 50 µm. Blue: Nuclei. e) Quantification of expression levels of epithelial cytokeratin AE1/AE3, f) the blood vessels based on the CD 31, g) the inflammation based on CD206 (M2 macrophages), and h) cell proliferation based on PCNA. Data are mean ± standard error, n = 3; *p <0.05, **p <0.01, ***p <0.001; ****p <0.0001, n.s. means not significant.
","
Flow cytometric analysis of RAW264.7 surface markers (CD68, CD86, and CD206). a) group 1 (Control; 0.02rGO; 0.1rGO). b) group 2 (Control; M1; M1 + 0.02rGO; M1 + 0.1rGO), where, M1; M1 + 0.02rGO; M1+ 0.1rGO, first treated with LPS (100 ng mL−1) + INF‐r (50 ng/mL) for 24 h to induce M1 type.
","
qRT‐PCR analysis of mRNA levels of the indicated macrophage markers in rGO co‐cultured with the RAW 264.7 cells and/or factors. The gene expression levels of a) iNOS, TGF‐α, Arg‐1, Fizz, Ym1, and b) iNOS, TGF‐α, Arg‐1, Fizz, Ym1. qRT‐PCR analysis of mRNA levels of the markers and factors in tissue. The gene expression levels of c) α‐SMA, d) COL1A1, e) COL3A1, f) CD206, g) PCNA, h) TGFBR1, i) CD31, j) β‐catenin, k) TGF‐β2, l) VWF. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, n.s. means not significant.
"],"string":"[\n \"
Schematics on the development of 3D‐printed SA/Gel/rGO patches for urethral repair. The SA/Gel/rGO patches were prepared by a 3D‐printing system and cross‐linked by Ca2+. The patches were then sutured in the damaged urethra of rabbits, wherein the rGO component facilitated regeneration of mucosa, promoted vascularization, and exhibited anti‐fibrotic activity.
\",\n \"
Morphologies of rGO and optical images of rGO/hydrogel‐precursor dispersions. a) SEM and b) TEM images of rGO. c) Photographs of different concentrations of rGO/hydrogel‐precursor suspensions at rGO concentrations of 0.02, 0.05, 0.1, and 0.2 mg mL−1. d–g) Corresponding optical microscopy images. h–k) Area counts of the rGO distributed in the corresponding suspensions; *p <0.05, ****p <0.0001, n.s. means not significant.
\",\n \"
Physicochemical characterizations of the SA/Gel/rGO hydrogels. a) Raman spectra of SA, SA/Gel, and SA/Gel/rGO hydrogels. b) FTIR spectra of i) SA/Gel hydrogel, ii) SA/Gel/rGO0.02 hydrogel, iii) SA/Gel/rGO0.05 hydrogel, and iv) SA/Gel/rGO0.2 hydrogel. c) Swelling ratios of the different concentrations of SA and SA/Gel/rGO composite inks at different concentrations of rGO. d) Degradation profiles of the hydrogels.
\",\n \"
SEM images and gross appearances of SA/Gel/rGO hydrogels with various concentrations of rGO: 0, 0.02, 0.05, 0.1, and 0.2 mg mL−1. a–e) SEM images at 1000× magnification and optical images of the hydrogels; f–j) SEM images at 300× magnification. k–o) Distributions of pore sizes of the different hydrogels. p) Average pore sizes of the different hydrogels; **p <0.01; ***p <0.001; ****p <0.0001; n.s. means not significant.
\",\n \"
a) Morphology of the 3D‐printed SA/Gel/rGO patches containing different concentrations of rGO, and the double‐freeze‐drying process. b) Tensile stress‐strain curves; c) Young's moduli; d) tensile strengths at break; and e) strains at the break for the different 3D‐printed patches; **p <0.01; ***p <0.001; and ****p <0.0001.
\",\n \"
In vitro cytocompatibility of rGO and rGO composite hydrogel. a) Cell viability of fibroblasts seeded on different concentrations of SA/Gel/rGO hydrogels at day 1. Living cells are depicted in green and dead cells in red. b) Heat map of DEPs. Red and green colors indicate high and low expressions, respectively. c) wound healing was observed in fibroblast cells treated with the different concentrations of rGO (0, 0.02, 0.05, 0.1, and 0.2 mg mL−1). fibroblast cells treated without rGO were used as a positive control experiment. The scale bar at the right lower corner is 500 µm. d) The percentage of wound contraction was measured using Image J analysis software. Statistical significance was calculated by using a t‐test. e) Cell proliferation of fibroblasts cultured on hydrogels for 1, 4, and 7 days; n = 6, **p <0.01, ***p <0.001, ****p <0.0001.
\",\n \"
Urethrography examinations and appearances of urethral repair from the biomaterial with SA/Gel, SA/Gel/rGO0.05, and SA/Gel/rGO0.2. The results of urethras from a,d,g,j) SA/Gel, b,e,h,k) SA/Gel/rGO0.05, and c,f,i,l) SA/Gel/rGO0.2 at 4‐ and 8‐weeks post‐surgery. The red arrows indicate the lumen of the urethra. The red dotted boxes indicate the urethral epithelial layer. *p <0.05, ****p <0.0001, n.s. means not significant.
\",\n \"
Histological examination of the regenerated tissues at the damaged urethra sites after staining the sections with H&E and Masson's Trichrome. Representative images of urethral cross‐sections of the reconstructed urethra using SA/Gel, SA/Gel/rGO0.05, and SA/Gel/rGO0.2 of a,c) at 4 weeks and b,d) at 8 weeks. The mucosal coverage of urethra at 4 weeks (a) and at 8 weeks (b); The collagen thickness of urethra at 4 weeks (a) and at 8 weeks (b). The green dotted lines indicate the border line of collagen thickness. Data are means ± standard error, n = 3; **p <0.01, ***p <0.001, ****p <0.0001, n.s. means not significant.
\",\n \"
Immunofluorescence staining of specific biomarkers in the SA/Gel, SA/Gel/rGO0.05, and SA/Gel/rGO0.2 repair after 4‐ and 8‐weeks post‐surgery. Representative immunofluorescence images of a) AE1/AE3, b) CD31, c) CD206, d) PCNA. Scale bar: 50 µm. Blue: Nuclei. e) Quantification of expression levels of epithelial cytokeratin AE1/AE3, f) the blood vessels based on the CD 31, g) the inflammation based on CD206 (M2 macrophages), and h) cell proliferation based on PCNA. Data are mean ± standard error, n = 3; *p <0.05, **p <0.01, ***p <0.001; ****p <0.0001, n.s. means not significant.
\",\n \"
Flow cytometric analysis of RAW264.7 surface markers (CD68, CD86, and CD206). a) group 1 (Control; 0.02rGO; 0.1rGO). b) group 2 (Control; M1; M1 + 0.02rGO; M1 + 0.1rGO), where, M1; M1 + 0.02rGO; M1+ 0.1rGO, first treated with LPS (100 ng mL−1) + INF‐r (50 ng/mL) for 24 h to induce M1 type.
\",\n \"
qRT‐PCR analysis of mRNA levels of the indicated macrophage markers in rGO co‐cultured with the RAW 264.7 cells and/or factors. The gene expression levels of a) iNOS, TGF‐α, Arg‐1, Fizz, Ym1, and b) iNOS, TGF‐α, Arg‐1, Fizz, Ym1. qRT‐PCR analysis of mRNA levels of the markers and factors in tissue. The gene expression levels of c) α‐SMA, d) COL1A1, e) COL3A1, f) CD206, g) PCNA, h) TGFBR1, i) CD31, j) β‐catenin, k) TGF‐β2, l) VWF. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, n.s. means not significant.
Plastic products have become deeply rooted in human society. Globally, more than 100 million tons of plastic waste are annually discarded into the environment, which are landfilled, incinerated, or discharged into the ocean, resulting in serious ecological damages.[\n##REF##32703909##\n1\n##\n,2] Epoxy resins (EPs), derived from petroleum, are cost‐effective thermosetting materials that are ubiquitously used in aerospace, transportation, architecture, and electronic and electrical devices.[\n##UREF##0##\n3\n##, ##UREF##1##\n4\n##\n] However, thermosets have a limited capacity to degrade and recycle than thermoplastics or supramolecular plastics due to their three‐dimensional (3D) crosslinked structure.[\n##REF##36163266##\n5\n##, ##REF##28386008##\n6\n##, ##UREF##2##\n7\n##, ##UREF##3##\n8\n##\n] Once cured, it will cause permanent plastic wastes that require hundreds of years to naturally clean up. To resolve the problem, tremendous efforts are still necessitated toward degradable and closed‐loop cycling epoxies as alternatives to traditional ones,[\n##UREF##4##\n9\n##, ##UREF##5##\n10\n##\n] such as developing bio‐based monomers.[\n##UREF##6##\n11\n##\n] However, there still remain intractable challenges for these structures such as their high cost, and how to make a trade‐off between high structural strength and easy degradability, as well as further realize multifunctional material purposes.[\n##UREF##7##\n12\n##, ##UREF##8##\n13\n##\n]\n
","
Alternatively, dynamic‐covalent and/or non‐covalent crosslinking strategies are particularly attractive for the designs of next‐generation materials with healable and chemical recycling properties.[\n##UREF##9##\n14\n##, ##REF##32999924##\n15\n##, ##UREF##10##\n16\n##, ##REF##33393759##\n17\n##, ##REF##22890548##\n18\n##, ##UREF##11##\n19\n##, ##UREF##12##\n20\n##, ##REF##32699399##\n21\n##, ##UREF##13##\n22\n##\n] This fact derives a new class of polymers named vitrimers, which merge the combinative features of thermoplastics and thermosets.[\n##REF##22096195##\n23\n##, ##UREF##14##\n24\n##, ##REF##28386008##\n25\n##, ##UREF##15##\n26\n##, ##REF##31565382##\n27\n##, ##UREF##16##\n28\n##\n] Epoxy vitrimers are emerging and explored as the time demand for programming degradable and recyclable epoxies and even their thermosets.[\n##REF##36299449##\n29\n##, ##UREF##17##\n30\n##\n] Vitrimers are generally constructed by dynamic‐covalent bonds linked with polymer networks (DCPNs), wherein bond reversion is driven by catalysts or external stimuli.[\n##UREF##18##\n31\n##, ##UREF##19##\n32\n##, ##UREF##20##\n33\n##\n] In many cases, the control exerted over the crosslinked structure by governing dynamic covalent bonds tends to compromise their natural thermosetting virtues (for example, thermal and chemical resistance, and mechanical robustness). Therefore, a holistic perspective involving supramolecular modes and covalent adaptable modes will contribute to an in‐depth understanding of crosslinking behavior from chemical bonds to linear monomers, and even to the whole crosslinked structure of polymer networks.[\n##UREF##21##\n34\n##, ##UREF##22##\n35\n##, ##UREF##23##\n36\n##, ##UREF##24##\n37\n##\n]\n
","
Unlike classical linear polymers, hyperbranched polymers (HBPs) possess spatial molecular configurations with 3D‐branched architecture and abundant external terminals.[\n##UREF##25##\n38\n##, ##REF##25902871##\n39\n##\n] They can be easily functionalized for polymer modification and are a well‐established strategy for achieving high strength and toughness of thermoset polymers.[\n##UREF##26##\n40\n##\n] Considerable progress has been made in developing various hyperbranched structures and prototyping them in relevant functional applications.[\n##REF##25902871##\n41\n##, ##UREF##27##\n42\n##, ##REF##33492940##\n43\n##, ##UREF##28##\n44\n##, ##UREF##29##\n45\n##, ##UREF##30##\n46\n##\n] Benefiting the virtue of their hyperbranched architecture, HBPs with broad intramolecular cavities can reduce network density to accommodate solvent molecules, thus driving dynamic behaviors. Hence, they hold promise for tailoring degradable and in‐processable vitrimers from native thermosets. The interpenetrating networks formed by HBPs and epoxy matrices, characterized by network topologies and dynamic linkages, are poised to bestow upon thermoset polymers unique and exceptional characteristics. Moreover, they offer ease of functionalization, particularly in compensating for the lack of mechanical robustness.
","
Herein, we report hyperbranched dynamic crosslinking networks (HDCNs) to program an industrial thermoset into a degradable and reconfigurable vitrimer. The conceptual basis underlying this approach is depicted in Figure ##FIG##0##\n1\n##. The HDCNs are structured with multiple dynamics via introducing a hyperbranched macromonomer (HBPPB) bearing dynamic units and reactive terminals. Through the integration of boron and phosphorus units, the molecular configuration can be well stretched into a topology, exploiting the planar‐like nature (sp2 hybridization) of boron and the sp3 hybridization of phosphorus esters. This hybridized structure establishes covalent crosslinks with DGEBA monomer (ring‐opening) and anhydride monomer (forming ester bond), as well as supramolecular crosslinking via H‐bonding (Figure ##FIG##0##1c##). Consequently, the installation of HDCNs makes a thermoset network from a permanently 3D structure to a dynamic topologically crosslinked architecture, wherein the branching architecture benefits an expanded crosslinking network (low‐crosslink‐density, Figure ##FIG##3##4c##) that is capable of adapting solvent molecule entrance to drive the dynamic bond exchange. Such structural features render HDCNs susceptible to mild solvent‐dissolved degradation (room temperature, Figure ##FIG##0##1b##) and confer transformative material property[\n##UREF##31##\n47\n##, ##UREF##32##\n48\n##, ##REF##23579959##\n49\n##\n] from a rigid vitrimer to an elastomer upon heating in ethanol (See Movie ##SUPPL##1##S1##, Supporting Information). More attractively, the after‐treated vitrimer showcases a suprahigh modulus (5.45 GPa) at extremely low temperature (−150 °C), posing them and even their composites supranormal uses for astrospace, superconducting energy storage, medical equipment, and beyond. Furthermore, the vitrimer exhibits high flexural modulus and toughness, and excellent thermal stability, among other high added values, demonstrating an extension of existing vitrimers and vitrimer properties. In light, this design principle via HDCNs may hold promise for the breakthrough of conventional concepts toward the next generation of sustainable and advanced thermoset polymers.
"],"string":"[\n \"Introduction\",\n \"
Plastic products have become deeply rooted in human society. Globally, more than 100 million tons of plastic waste are annually discarded into the environment, which are landfilled, incinerated, or discharged into the ocean, resulting in serious ecological damages.[\\n##REF##32703909##\\n1\\n##\\n,2] Epoxy resins (EPs), derived from petroleum, are cost‐effective thermosetting materials that are ubiquitously used in aerospace, transportation, architecture, and electronic and electrical devices.[\\n##UREF##0##\\n3\\n##, ##UREF##1##\\n4\\n##\\n] However, thermosets have a limited capacity to degrade and recycle than thermoplastics or supramolecular plastics due to their three‐dimensional (3D) crosslinked structure.[\\n##REF##36163266##\\n5\\n##, ##REF##28386008##\\n6\\n##, ##UREF##2##\\n7\\n##, ##UREF##3##\\n8\\n##\\n] Once cured, it will cause permanent plastic wastes that require hundreds of years to naturally clean up. To resolve the problem, tremendous efforts are still necessitated toward degradable and closed‐loop cycling epoxies as alternatives to traditional ones,[\\n##UREF##4##\\n9\\n##, ##UREF##5##\\n10\\n##\\n] such as developing bio‐based monomers.[\\n##UREF##6##\\n11\\n##\\n] However, there still remain intractable challenges for these structures such as their high cost, and how to make a trade‐off between high structural strength and easy degradability, as well as further realize multifunctional material purposes.[\\n##UREF##7##\\n12\\n##, ##UREF##8##\\n13\\n##\\n]\\n
\",\n \"
Alternatively, dynamic‐covalent and/or non‐covalent crosslinking strategies are particularly attractive for the designs of next‐generation materials with healable and chemical recycling properties.[\\n##UREF##9##\\n14\\n##, ##REF##32999924##\\n15\\n##, ##UREF##10##\\n16\\n##, ##REF##33393759##\\n17\\n##, ##REF##22890548##\\n18\\n##, ##UREF##11##\\n19\\n##, ##UREF##12##\\n20\\n##, ##REF##32699399##\\n21\\n##, ##UREF##13##\\n22\\n##\\n] This fact derives a new class of polymers named vitrimers, which merge the combinative features of thermoplastics and thermosets.[\\n##REF##22096195##\\n23\\n##, ##UREF##14##\\n24\\n##, ##REF##28386008##\\n25\\n##, ##UREF##15##\\n26\\n##, ##REF##31565382##\\n27\\n##, ##UREF##16##\\n28\\n##\\n] Epoxy vitrimers are emerging and explored as the time demand for programming degradable and recyclable epoxies and even their thermosets.[\\n##REF##36299449##\\n29\\n##, ##UREF##17##\\n30\\n##\\n] Vitrimers are generally constructed by dynamic‐covalent bonds linked with polymer networks (DCPNs), wherein bond reversion is driven by catalysts or external stimuli.[\\n##UREF##18##\\n31\\n##, ##UREF##19##\\n32\\n##, ##UREF##20##\\n33\\n##\\n] In many cases, the control exerted over the crosslinked structure by governing dynamic covalent bonds tends to compromise their natural thermosetting virtues (for example, thermal and chemical resistance, and mechanical robustness). Therefore, a holistic perspective involving supramolecular modes and covalent adaptable modes will contribute to an in‐depth understanding of crosslinking behavior from chemical bonds to linear monomers, and even to the whole crosslinked structure of polymer networks.[\\n##UREF##21##\\n34\\n##, ##UREF##22##\\n35\\n##, ##UREF##23##\\n36\\n##, ##UREF##24##\\n37\\n##\\n]\\n
\",\n \"
Unlike classical linear polymers, hyperbranched polymers (HBPs) possess spatial molecular configurations with 3D‐branched architecture and abundant external terminals.[\\n##UREF##25##\\n38\\n##, ##REF##25902871##\\n39\\n##\\n] They can be easily functionalized for polymer modification and are a well‐established strategy for achieving high strength and toughness of thermoset polymers.[\\n##UREF##26##\\n40\\n##\\n] Considerable progress has been made in developing various hyperbranched structures and prototyping them in relevant functional applications.[\\n##REF##25902871##\\n41\\n##, ##UREF##27##\\n42\\n##, ##REF##33492940##\\n43\\n##, ##UREF##28##\\n44\\n##, ##UREF##29##\\n45\\n##, ##UREF##30##\\n46\\n##\\n] Benefiting the virtue of their hyperbranched architecture, HBPs with broad intramolecular cavities can reduce network density to accommodate solvent molecules, thus driving dynamic behaviors. Hence, they hold promise for tailoring degradable and in‐processable vitrimers from native thermosets. The interpenetrating networks formed by HBPs and epoxy matrices, characterized by network topologies and dynamic linkages, are poised to bestow upon thermoset polymers unique and exceptional characteristics. Moreover, they offer ease of functionalization, particularly in compensating for the lack of mechanical robustness.
\",\n \"
Herein, we report hyperbranched dynamic crosslinking networks (HDCNs) to program an industrial thermoset into a degradable and reconfigurable vitrimer. The conceptual basis underlying this approach is depicted in Figure ##FIG##0##\\n1\\n##. The HDCNs are structured with multiple dynamics via introducing a hyperbranched macromonomer (HBPPB) bearing dynamic units and reactive terminals. Through the integration of boron and phosphorus units, the molecular configuration can be well stretched into a topology, exploiting the planar‐like nature (sp2 hybridization) of boron and the sp3 hybridization of phosphorus esters. This hybridized structure establishes covalent crosslinks with DGEBA monomer (ring‐opening) and anhydride monomer (forming ester bond), as well as supramolecular crosslinking via H‐bonding (Figure ##FIG##0##1c##). Consequently, the installation of HDCNs makes a thermoset network from a permanently 3D structure to a dynamic topologically crosslinked architecture, wherein the branching architecture benefits an expanded crosslinking network (low‐crosslink‐density, Figure ##FIG##3##4c##) that is capable of adapting solvent molecule entrance to drive the dynamic bond exchange. Such structural features render HDCNs susceptible to mild solvent‐dissolved degradation (room temperature, Figure ##FIG##0##1b##) and confer transformative material property[\\n##UREF##31##\\n47\\n##, ##UREF##32##\\n48\\n##, ##REF##23579959##\\n49\\n##\\n] from a rigid vitrimer to an elastomer upon heating in ethanol (See Movie ##SUPPL##1##S1##, Supporting Information). More attractively, the after‐treated vitrimer showcases a suprahigh modulus (5.45 GPa) at extremely low temperature (−150 °C), posing them and even their composites supranormal uses for astrospace, superconducting energy storage, medical equipment, and beyond. Furthermore, the vitrimer exhibits high flexural modulus and toughness, and excellent thermal stability, among other high added values, demonstrating an extension of existing vitrimers and vitrimer properties. In light, this design principle via HDCNs may hold promise for the breakthrough of conventional concepts toward the next generation of sustainable and advanced thermoset polymers.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results and Discussion","Synthesis and Characterization of HDCNs","
The synthesis of HDCNs is straightforward via introducing a hyperbranched dynamic macromonomer into a traditional epoxy thermoset network as a dynamic crosslinker. The hyperbranched macromonomer (HBPPB) was synthesized via A2+B2+C3 polycondensation (Section ##SUPPL##0##S1.3a##, Supporting Information). In parallel, a linear poly‐phosphate/borate structure (LPPB), a hyperbranched polyborate (HBPB), and a hyperbranched polyphosphate (HBPP) were synthesized as controls to compare with HDCNs. Synthesis details are described in Sections ##SUPPL##0##S1.4## and ##SUPPL##0##S1.5## (Supporting Information). The molecular structure of the synthetic polymers was characterized using multiple analytical techniques, including Fourier transform infrared (FT‐IR), 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectroscopies, gel permeation chromatography (GPC) (see Section ##SUPPL##0##S1.3b##, Supporting Information). The concentration of hydroxyl groups (─OH) in HBPPB was quantified via a standard titration experiment, yielding a value of 7.97 × 10−3 mol g−1 (Section ##SUPPL##0##S1.3c## and Table ##SUPPL##0##S2##, Supporting Information), indicating the high abundance of ─OH groups.
","
The polymer matrix comprised of a petroleum‐based epoxy (diglycidyl ether bisphenol A, DGEBA, E51) a commercially used anhydride‐type curing agent (methyl tetrahydrophthalic anhydride, MTHPA), and a certain amount of HBPPB. A facile thermosetting workflow was employed to cast EP‐x samples, where x represents the mass concentration of HBPPB. The curing performance was evaluated using isothermal differential scanning calorimetry (Figure ##SUPPL##0##S21c##, Supporting Information), which showed a homogeneous and single peak among all pre‐polymer systems, indicating well compatibility among the three constituents. The natural EP and EP‐9 underwent full curing as proved in its time‐sweep curves by rheology (Figure ##SUPPL##0##S22##, Supporting Information). For subsequent analysis, the epoxy samples containing HBPPB equal to or greater than 9 wt.% were donated as vitrimers due to their degradable and/or reconfigurable dynamic behavior. The other samples were categorized as thermosets due to their predominant thermoset nature. To facilitate comparative investigations, epoxy vitrimers containing varying mass fractions of LPPB, HBPB, and HBPP were prepared using the same procedure as HDCNs, and a comparison of their material properties is presented in Sections ##SUPPL##0##S2.5## and ##SUPPL##0##S2.6## (Supporting Information).
","
The dynamic nature of HDCNs was characterized extensively through stress relaxation (see Section ##SUPPL##0##S2.3##, Supporting Information) and creep‐recovery test (Section ##SUPPL##0##S2.4##, Supporting Information) in tensile mode.[\n##REF##31433176##\n50\n##, ##REF##35590755##\n51\n##, ##REF##35607118##\n52\n##, ##REF##29733205##\n53\n##, ##REF##28557428##\n54\n##\n] In EP‐12, the time‐ and temperature‐dependent modulus revealed a partial relaxation to a plateau regime that deviates from classical linear‐viscoelastic vitrimers. This unique relaxation signifies that HDCNs not only behave with liquid‐like viscoelasticity due to the activation of dynamic exchange above the topology‐freezing transition temperature (T\nv, described by Arrhenius and Williams−Landel−Ferry law), but also retain the resilience and recovery capabilities like an elastic body subjected to Hooke's law. By fitting the results on an Arrhenius plot, a linear correlation between ln(τ*) (relaxation time) and 1000/T (inverse temperature) was observed, enabling the calculation of an activation energy (E\na) of 78.1 kJ mol−1 and T\nv of 71.7 °C (Figures ##SUPPL##0##S24## and ##SUPPL##0##S25##, Supporting Information). It is noteworthy that several examples of vitrimers with T\ng > T\nv have been reported.[\n##UREF##33##\n55\n##, ##REF##28991493##\n56\n##\n] Consequently, the material exhibits dynamic vitrimer behavior, albeit with reduced dynamic capabilities compared to fully relaxed vitrimers. Nonetheless, it demonstrates exceptional dimensional stability and mechanical strength even above the glass‐transition temperature (T\ng) and T\nv. This behavior can be attributed to the presence of permanently crosslinked sites and potentially relates to the network topologies, thereby underlying further understanding of vitrimers and their dynamic behaviors.
","Cleavable and Topological Crosslinks Control over Degradable HDCNs","
Thermoset polymers are generally considered non‐degradable and hard to reprocess, particularly when in comparison to thermoplastic or weakly crosslinked supramolecular plastics.[\n##REF##36318511##\n57\n##, ##UREF##34##\n58\n##\n] However, inserting topological crosslinks and cleavable bonds into the thermoset network via HDCNs leads to the formation of vitrimers that exhibit distinctive behavior upon exposure to solvents (Figure ##FIG##1##\n2\n##; Section ##SUPPL##0##S1.7##, Supporting Information). In this study, a commonly used aprotic solvent (dimethylacetamide, DMAc) was employed to fully immerse the samples. EP‐12, as the most prominent case for degradation, underwent swelling and fragmentation within a mere 2 days at room temperature (Figure ##FIG##1##2a,b##), in stark contrast to the natural thermoset EP, which retained its intact and rigid shape. Moreover, an increasing concentration of HBPPB can accelerate the process. In fact, a rapid degradation was observed within 6 hours when the system temperature was raised to 95 °C in DMAc (Figure ##SUPPL##0##S13##, Supporting Information). Based on these differences, we infer that the solvent‐assisted degradation was associated with cleavable crosslinks in HDCNs.
","
Unlike classic thermoset networks or degradable covalent networks, most of them are specialized for the degradation of weakly crosslinked materials such as thermoplastics and supramolecular plastics but are often helpless for highly crosslinked thermoset networks.[\n##REF##31011169##\n59\n##, ##UREF##35##\n60\n##\n] Whereas HDCNs benefit the stretching of molecular configurations for topological crosslinking purposes, which not only consists of several cleavable units but, more importantly, enables a topologically expanded network interspace that allows solvent molecular entrance to drive dynamic behaviors. The crosslinking density of HDCNs is relatively lower than their thermoset EP counterpart (Figure ##FIG##3##4c##). To assert the breakages when degrading, a schematic model describes the possible mechanisms in Figure ##FIG##1##2e##. Such a structure model encompasses three types of cleavable bonds. The first involves two kinds of carbonic ester bonds (mark a) that result from the reaction between the anhydride with HBPPB and the epoxy monomer (Figure ##SUPPL##0##S10##, Supporting Information). The second breakage is attributed to the boronic ester group (BO3)[\n##REF##29733205##\n53\n##, ##REF##25945818##\n61\n##, ##REF##27387198##\n62\n##\n] and the phosphate group (PO3)[\n##UREF##36##\n63\n##\n] upon the molecular backbone of the hyperbranched crosslinker. Third, non‐covalent H‐bonds are also susceptible to cleavage when the solvent is diffused.
","
To monitor the aforementioned breakages, both EP‐12 and the solvent were subjected to FT‐IR analysis, as shown in Figure ##FIG##1##2f##. The results reveal distinct spectral features, notably the emergence of a newly detected C═O absorption (mark a) and C─H signals within the substituted benzene moiety in the DMAc spectrum after degradation, indicating the species of anhydride‐contained substrates are partially dissolved in DMAc, primarily indicating the cleavage of these carboxyl ester bonds. Furthermore, the degraded sample exhibits a diminished C─O─B/P absorption (mark b), which corresponds to the depolymerization of HBPPB. Complementary insights from the X‐ray photoelectron spectroscopy (XPS, details in Section ##SUPPL##0##S2.8##, Supporting Information) reveal that the beta‐hydroxy ester (formed through the reaction of the anhydride with ‐OH and epoxy groups) underwent breakage, as evidenced by its heightened and broader O─H/COOH intensity in the O1s spectra, as well as the relatively lower signals of C═O and C─O─C in the C1s spectra. More importantly, the degraded sample shows very weak signals of B and P in its high‐resolution XPS, thus further confirming the depolymerized HBPPB. Consequently, a highly cross‐linked network experienced fragmentation as a result of cleavages of these bonds.
","
The degraded products were subsequently processed into fine powder through grinding and drying (Figure ##FIG##1##2c##). Notably, by mixing 1 g of degraded powder with ≈5 g of our pristine epoxy resin (refer to Section ##SUPPL##0##S1.7##, Supporting Information) and curing following the same procedure, we obtained the post‐cycled EP with impact strength and flexural strength comparable to the original (Figure ##FIG##1##2d##). Despite the cleavable bonds being introduced, we note that in thermosetting materials with only adapting additively crosslinked cleavable units, theoretically, it is difficult in practice to realize entirely or closed‐loop recycling of monomers once all of the crosslinks are formed.[\n##REF##36864144##\n64\n##, ##REF##37023198##\n65\n##, ##REF##37100913##\n66\n##\n] In this system, although the native EP thermoset also contains ester dynamic bonds, it remains non‐degradable or slowly degradable compared to the vitrimer. Thus, both the cleavable units and topological crosslinks control the degradation.
","
Regarding the recovery uses (Figure ##FIG##1##2g–i##), carbon fiber composites have been explored for high‐performance applications, while the costly embedded carbon fibers (CFs) cannot be retrieved from bulk materials.[\n##UREF##37##\n67\n##, ##REF##35549086##\n68\n##\n] In this implementation, when carbon fiber fabrics were embedded into HDCNs to fabricate a composite, we were able to quantitatively recover the CFs while effectively separating the end‐of‐use epoxy powders from the composite. The Raman spectrum of the recovered fibers shows a carbon peak closely resembling those of the native fibers, indicating minimal surface impact on the carbon fibers (Figure ##FIG##1##2j##). These results hint at promising opportunities for the cyclic utilization of end‐of‐use epoxy resins and composites.
Next, by a chance following‐up, we note that when the samples were immersed in ethanol following a heating (95 °C, 6 h), the vitrimer sample (EP‐9) via forming HDCNs displayed elastomeric‐like feature upon cooling to room temperature, which allowed for a casual deformation and recovery as well (see Movie ##SUPPL##1##S1##, Supporting Information; Figure ##FIG##2##\n3a,b##), whereas the native thermoset (EP) still remains its rigid stiffness (Figure ##FIG##2##3c##). In our further study, we denoted EP‐9 as an example with suitable elasticity compared to EP‐12. Such transformation in elastomeric characteristics is exciting considering the inherent highly crosslinked nature of its bulk thermosets, especially for industrial epoxy commodities. This suggests that hyperbranched cross‐linking may alter the crosslinking mode of the thermoset network. We coined this type of vitrimer as “reconfigurable” vitrimers,[\n##UREF##10##\n16\n##, ##REF##23579959##\n49\n##\n] that is, a thermosetting network – whose crosslinked structure should be permanent – but can be reconstructed through solvent‐assisted reconfiguration. As illustrated in Figure ##FIG##2##3d,e##, we reasoned the ester‐exchange driving the reconstruction of HDCNs,[\n##REF##35640074##\n69\n##, ##REF##31469270##\n70\n##\n] whereby the ester bonds and hydrogen bonds can be rearranged since the ethanol serves as a single ─OH blocking molecule to substitute the ─OH site from those of hyperbranched crosslinks. The reorganized structure (Figure ##FIG##2##3j##) is corroborated by FT‐IR before‐ and after‐ethanol, temperature‐dependent IR, and dynamic thermomechanometry (Figure ##FIG##2##3g–i##). In particular, Figure ##FIG##2##3g## demonstrates a blueshift in the hydroxyl stretching vibration at 3400 cm−1, along with an increased width and intensity, indicating the transition of hydroxyl groups from a “free” state to an “associated” state. Furthermore, the reduction in peak intensity of C─O─Csym verifies the occurrence of ester exchange in HDCNs since symmetric ether structures (C─O─Csym, 1200–1150 cm−1) typically exhibit an increase in peak intensity. Additional evidence from model reactions (Section ##SUPPL##0##S1.8##, Supporting Information)[\n##REF##31433176##\n50\n##, ##REF##29733205##\n53\n##\n] and the XPS survey (Section ##SUPPL##0##S2.8##, Supporting Information) further supports the ester exchange should be the main mechanism to drive the reconfigurable behavior of the material. Ultimately, the reconstruction of HDCNs introduces polymer chain mobility and reduces the crosslinked structure, leading to the transformation in material nature.
","
To further investigate the network dynamics of HDCNs, dynamic thermomechanometry was investigated over a wide temperature from −150 to 150 °C (Figure ##FIG##2##3i##; Figure ##SUPPL##0##S23##, Supporting Information) to record flexural modulus at three‐point bending mode. As the temperature decreases, the loss modulus exhibits a double‐relaxation of heat dissipation when the material is deformed. The lower (mark 1) relaxation involves a glass‐to‐elastic transition that unfreezes the side chains and groups, resulting in a rapid increase in flexural storage modulus from 3.2 to 5.4 GPa (Figure ##SUPPL##0##S23a##, Supporting Information). This increase is attributed to the ethyl ester (forming by ethanol exchange) occupying the initial crosslinked site of carbonic ester for network reconstruction (Figure ##FIG##2##3j##). The second relaxation (mark 2) signifies an elastic‐to‐liquid transition as the vitrimer network flows and relaxes due to dynamic bond exchange. Real‐temperature IR analysis confirms the defreezing temperature of H‐bonds in close proximity to this transition, as indicated by a significant increase in the normalized intensity of hydroxyl groups, shifting from the “associated” state to the “free” state upon temperature elevation from 0 °C (Figure ##FIG##2##3h##),[\n##UREF##38##\n71\n##\n] while the intensity remains almost unchanged from −50 to 0 °C.
","
In this term, the reconstructed HDCNs display a stronger H‐bonding crosslinked network compared with that of native EP at lower temperature areas, where the polymer segments are frozen and tightly constrained by H‐bonding.[\n##UREF##39##\n72\n##\n] Consequently, we observe a significant increase in G’ with decreasing temperature (Figure ##SUPPL##0##S23a##, Supporting Information), recording as high as 5.45 GPa at −150 °C. This represents an exceptionally high flexural modulus for polymeric materials and approximately twice that of native EP under the same condition. The exceptional modulus at ultra‐low temperatures empowers this polymeric material with supernormal applications in astrospace material, superconducting energy storage, and medical equipment.
","General Properties and Multifunctional Performance of HDCNs","
\nFigure ##FIG##3##\n4\n## provides a comprehensive analysis of the multifaceted performance of the material in harnessing the advantages of HDCNs. First, the glass transition temperature (T\ng, Figure ##SUPPL##0##S21a## and Table ##SUPPL##0##S6##, Supporting Information) of EP, EP‐3, EP‐6, EP‐9, and EP‐12 are 131, 121, 118, 104, and 94 °C, respectively, indicating they are glassy polymers at room temperature. The slight decrease in T\ng can be attributed to the amorphous structure of HBPPB.[\n##UREF##25##\n38\n##\n] Upon crosslinking a hyperbranched macromolecular, network topologies are physically expanded.[\n##UREF##40##\n73\n##, ##REF##27634530##\n74\n##\n] However, the heat loss of chains motion is intensified below T\ng due to increased friction and constraints imposed by the HBPPB within the network (increased loss modulus (Figure ##FIG##3##4a##). Conversely, above T\ng and T\nv, the friction between epoxy chains with HBPPB is reduced due to dynamic bond exchange, and the network flows more easily thus existing a reduced loss modulus. Note that inserting poorly rigid chains often compromises the modulus and strength. Remarkably, the vitrimer, upon introducing an aliphatic hyperbranched crosslinker, demonstrates an even higher storage modulus (E’) compared to that of native EP (Figure ##FIG##3##4b##). Specifically, the addition of 3% HBPPB elevates E’ from 2.38 GPa of native EP up to 3.15 GPa (Figure ##SUPPL##0##S21b##, Supporting Information), followed by a decline with further increasing HBPPB, which substantiates the reinforcement effect of HDCNs. This enhancement can be attributed to that the hyperbranched structure acts as a “supramolecular rivet” to facilitate a robust supramolecular/epoxy interpenetrating network through H‐bonds and covalent bonds (Figure ##FIG##3##4d##).[\n##UREF##41##\n75\n##, ##UREF##42##\n76\n##\n] The branching molecular topology enables an expanded network interspace, resulting in a reduced network crosslinking density (Figure ##FIG##3##4c##), as elaborated in Section ##SUPPL##0##S2.1## (Supporting Information). Consequently, the hyperbranched crosslinker holds significant promise as a topological crosslinking tool for programming low‐crosslinking yet high‐strength polymers.
","
Measurements of impact toughness, flexural strength, and tensile strength were carried out to facilitate a comprehensive understanding of mechanical performance (Figure ##FIG##3##4e,f##; details in Section ##SUPPL##0##S2.6##, Supporting Information). The high crosslinking density and rigid polymer chains render thermosets susceptible to impact failure, resulting in a microscopic “river‐like” brittle fracture (Figure ##SUPPL##0##S30##, Supporting Information) with 12.94 kJ m−2 of impact strength. However, EP‐3 shows a superior increase (98.5%) from 12.94 up to 25.69 kJ m−2, accompanied by obvious “dimple‐fracture” features that promote energy absorption. Thought‐out relevant works regarding hyperbranched polymer‐reinforced thermosets, a supramolecular network‐induced energy dissipation mechanism could be supposed.[\n##UREF##43##\n77\n##, ##REF##35042887##\n78\n##\n] The network topologies furnish cross‐linked polymers with enhanced properties without altering their chemical composition.[\n##REF##35894294##\n79\n##\n] It is worth noting that the increase in impact strength surpasses that of flexural strength, which can be attributed to the presence of flexible aliphatic chains that mitigate the network stiffness. Both the flexural and tensile strength show a consistent and noteworthy improvement from 106.1 up to 132 MPa, and 52.1 up to 81.2 MPa, respectively (Table ##SUPPL##0##S7##, Supporting Information). Notably, the tensile strain‐stress curves indicate that vitrimers are more stretchable than that of native thermoset epoxy (Figure ##SUPPL##0##S31##, Supporting Information). This finding holds great promise for the fabrication of carbon fiber reinforced polymers (CFRPs), where the stress should primarily reside in the fibers rather than the resins. This attribute ensures the exceptional mechanical strength of CFRPs (Section ##SUPPL##0##S2.6.3##, Supporting Information).
","
In this contribution, both the strength and modulus depend on a tightly crosslinked network. The HBPPB, which terminates abundant ─OH groups, acts as a “rivet” within the network through covalent bonding and non‐covalent H‐bonding. The observed increase in E’ is also in agreement with the flexural results. Unfortunately, the optimal mechanical system does not maintain a consistent HBPPB concentration with the degradable and recyclable sample. Despite overloading HBPPB causing deteriorated mechanical strength, it still outperforms native EP. The reduction in impact and flexural strength can be attributed to the local agglomeration of hyperbranched polymers.[\n##UREF##29##\n45\n##, ##UREF##30##\n46\n##\n] Supplementary thermal and thermomechanical parameters are provided in Table ##SUPPL##0##S6## (Supporting Information). The vitrimers exhibit improved T\nmax values and char yields, indicating superior thermal stability of HDCNs, which is primarily attributed to the presence of the inert boron component in the system.
","
We next sought to probe the functional performance of materials. Good fire safety is a crucial consideration for real‐world applications of polymeric materials.[\n##UREF##44##\n80\n##, ##UREF##45##\n81\n##\n] Overall, our results demonstrate that the vitrimer sample exhibits superior fire safety performance compared to the native EP. Specifically, the vitrimer samples, EP‐9 and EP‐12, exhibit reduced thermal hazards and smoke production, along with increased resistance to ignition (higher limit oxygen index), and self‐extinguishing properties in an air atmosphere (Figure ##FIG##3##4g##). A comprehensive investigation was conducted through a full‐scale study of the fire‐retardant performance (Section ##SUPPL##0##S2.10## and Figure ##SUPPL##0##S41##, Supporting Information). The cone calorimeter test, which simulates a real fire scenario, provided crucial parameters to elucidate the flame‐retardant effect and mechanism.[\n##UREF##46##\n82\n##\n] Additionally, XPS analysis and Raman spectra (Figure ##SUPPL##0##S42##, Supporting Information) revealed the condensed fire‐retardant actions. The HDCNs constructed in our materials accumulate two types of flame‐retardant elements (phosphorus and boron) within the hyperbranched backbone for synergistic fire‐retarding action, ensuring the fire safety of the material.
","
One of the facts is that the materials, specifically EP‐9 and EP‐12, exhibit significantly superior transparency and lighter coloration than their native thermoset. The transmittance of materials was measured using UV–vis spectrophotometer (See Figure ##SUPPL##0##S40##, Supporting Information). The average transmittance in both 280–380 nm (UV region) and 380–780 nm (visible region) was acquired in Figure ##FIG##3##4h##, showing significant increases in transmittance to both UV and visible light. The exceptional transparency of these materials can be attributed to the unique construction of HDCNs and the optical properties of HBPPB. Excitingly, this type of hyperbranched polymer has been reported as a class of non‐traditional aggregation‐induced emission luminogens (AIEgens),[\n##UREF##47##\n83\n##, ##UREF##48##\n84\n##\n] posing a promising pathway toward understanding the polymer crosslinking via fluorescent visualization methods. Besides, the exceptional transparency and lightness impart this polymeric material high added‐values for application in packaging materials, photoelectric materials, and flexible electronics.[\n##UREF##49##\n85\n##, ##REF##22789123##\n86\n##\n]\n
"],"string":"[\n \"Results and Discussion\",\n \"Synthesis and Characterization of HDCNs\",\n \"
The synthesis of HDCNs is straightforward via introducing a hyperbranched dynamic macromonomer into a traditional epoxy thermoset network as a dynamic crosslinker. The hyperbranched macromonomer (HBPPB) was synthesized via A2+B2+C3 polycondensation (Section ##SUPPL##0##S1.3a##, Supporting Information). In parallel, a linear poly‐phosphate/borate structure (LPPB), a hyperbranched polyborate (HBPB), and a hyperbranched polyphosphate (HBPP) were synthesized as controls to compare with HDCNs. Synthesis details are described in Sections ##SUPPL##0##S1.4## and ##SUPPL##0##S1.5## (Supporting Information). The molecular structure of the synthetic polymers was characterized using multiple analytical techniques, including Fourier transform infrared (FT‐IR), 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectroscopies, gel permeation chromatography (GPC) (see Section ##SUPPL##0##S1.3b##, Supporting Information). The concentration of hydroxyl groups (─OH) in HBPPB was quantified via a standard titration experiment, yielding a value of 7.97 × 10−3 mol g−1 (Section ##SUPPL##0##S1.3c## and Table ##SUPPL##0##S2##, Supporting Information), indicating the high abundance of ─OH groups.
\",\n \"
The polymer matrix comprised of a petroleum‐based epoxy (diglycidyl ether bisphenol A, DGEBA, E51) a commercially used anhydride‐type curing agent (methyl tetrahydrophthalic anhydride, MTHPA), and a certain amount of HBPPB. A facile thermosetting workflow was employed to cast EP‐x samples, where x represents the mass concentration of HBPPB. The curing performance was evaluated using isothermal differential scanning calorimetry (Figure ##SUPPL##0##S21c##, Supporting Information), which showed a homogeneous and single peak among all pre‐polymer systems, indicating well compatibility among the three constituents. The natural EP and EP‐9 underwent full curing as proved in its time‐sweep curves by rheology (Figure ##SUPPL##0##S22##, Supporting Information). For subsequent analysis, the epoxy samples containing HBPPB equal to or greater than 9 wt.% were donated as vitrimers due to their degradable and/or reconfigurable dynamic behavior. The other samples were categorized as thermosets due to their predominant thermoset nature. To facilitate comparative investigations, epoxy vitrimers containing varying mass fractions of LPPB, HBPB, and HBPP were prepared using the same procedure as HDCNs, and a comparison of their material properties is presented in Sections ##SUPPL##0##S2.5## and ##SUPPL##0##S2.6## (Supporting Information).
\",\n \"
The dynamic nature of HDCNs was characterized extensively through stress relaxation (see Section ##SUPPL##0##S2.3##, Supporting Information) and creep‐recovery test (Section ##SUPPL##0##S2.4##, Supporting Information) in tensile mode.[\\n##REF##31433176##\\n50\\n##, ##REF##35590755##\\n51\\n##, ##REF##35607118##\\n52\\n##, ##REF##29733205##\\n53\\n##, ##REF##28557428##\\n54\\n##\\n] In EP‐12, the time‐ and temperature‐dependent modulus revealed a partial relaxation to a plateau regime that deviates from classical linear‐viscoelastic vitrimers. This unique relaxation signifies that HDCNs not only behave with liquid‐like viscoelasticity due to the activation of dynamic exchange above the topology‐freezing transition temperature (T\\nv, described by Arrhenius and Williams−Landel−Ferry law), but also retain the resilience and recovery capabilities like an elastic body subjected to Hooke's law. By fitting the results on an Arrhenius plot, a linear correlation between ln(τ*) (relaxation time) and 1000/T (inverse temperature) was observed, enabling the calculation of an activation energy (E\\na) of 78.1 kJ mol−1 and T\\nv of 71.7 °C (Figures ##SUPPL##0##S24## and ##SUPPL##0##S25##, Supporting Information). It is noteworthy that several examples of vitrimers with T\\ng > T\\nv have been reported.[\\n##UREF##33##\\n55\\n##, ##REF##28991493##\\n56\\n##\\n] Consequently, the material exhibits dynamic vitrimer behavior, albeit with reduced dynamic capabilities compared to fully relaxed vitrimers. Nonetheless, it demonstrates exceptional dimensional stability and mechanical strength even above the glass‐transition temperature (T\\ng) and T\\nv. This behavior can be attributed to the presence of permanently crosslinked sites and potentially relates to the network topologies, thereby underlying further understanding of vitrimers and their dynamic behaviors.
\",\n \"Cleavable and Topological Crosslinks Control over Degradable HDCNs\",\n \"
Thermoset polymers are generally considered non‐degradable and hard to reprocess, particularly when in comparison to thermoplastic or weakly crosslinked supramolecular plastics.[\\n##REF##36318511##\\n57\\n##, ##UREF##34##\\n58\\n##\\n] However, inserting topological crosslinks and cleavable bonds into the thermoset network via HDCNs leads to the formation of vitrimers that exhibit distinctive behavior upon exposure to solvents (Figure ##FIG##1##\\n2\\n##; Section ##SUPPL##0##S1.7##, Supporting Information). In this study, a commonly used aprotic solvent (dimethylacetamide, DMAc) was employed to fully immerse the samples. EP‐12, as the most prominent case for degradation, underwent swelling and fragmentation within a mere 2 days at room temperature (Figure ##FIG##1##2a,b##), in stark contrast to the natural thermoset EP, which retained its intact and rigid shape. Moreover, an increasing concentration of HBPPB can accelerate the process. In fact, a rapid degradation was observed within 6 hours when the system temperature was raised to 95 °C in DMAc (Figure ##SUPPL##0##S13##, Supporting Information). Based on these differences, we infer that the solvent‐assisted degradation was associated with cleavable crosslinks in HDCNs.
\",\n \"
Unlike classic thermoset networks or degradable covalent networks, most of them are specialized for the degradation of weakly crosslinked materials such as thermoplastics and supramolecular plastics but are often helpless for highly crosslinked thermoset networks.[\\n##REF##31011169##\\n59\\n##, ##UREF##35##\\n60\\n##\\n] Whereas HDCNs benefit the stretching of molecular configurations for topological crosslinking purposes, which not only consists of several cleavable units but, more importantly, enables a topologically expanded network interspace that allows solvent molecular entrance to drive dynamic behaviors. The crosslinking density of HDCNs is relatively lower than their thermoset EP counterpart (Figure ##FIG##3##4c##). To assert the breakages when degrading, a schematic model describes the possible mechanisms in Figure ##FIG##1##2e##. Such a structure model encompasses three types of cleavable bonds. The first involves two kinds of carbonic ester bonds (mark a) that result from the reaction between the anhydride with HBPPB and the epoxy monomer (Figure ##SUPPL##0##S10##, Supporting Information). The second breakage is attributed to the boronic ester group (BO3)[\\n##REF##29733205##\\n53\\n##, ##REF##25945818##\\n61\\n##, ##REF##27387198##\\n62\\n##\\n] and the phosphate group (PO3)[\\n##UREF##36##\\n63\\n##\\n] upon the molecular backbone of the hyperbranched crosslinker. Third, non‐covalent H‐bonds are also susceptible to cleavage when the solvent is diffused.
\",\n \"
To monitor the aforementioned breakages, both EP‐12 and the solvent were subjected to FT‐IR analysis, as shown in Figure ##FIG##1##2f##. The results reveal distinct spectral features, notably the emergence of a newly detected C═O absorption (mark a) and C─H signals within the substituted benzene moiety in the DMAc spectrum after degradation, indicating the species of anhydride‐contained substrates are partially dissolved in DMAc, primarily indicating the cleavage of these carboxyl ester bonds. Furthermore, the degraded sample exhibits a diminished C─O─B/P absorption (mark b), which corresponds to the depolymerization of HBPPB. Complementary insights from the X‐ray photoelectron spectroscopy (XPS, details in Section ##SUPPL##0##S2.8##, Supporting Information) reveal that the beta‐hydroxy ester (formed through the reaction of the anhydride with ‐OH and epoxy groups) underwent breakage, as evidenced by its heightened and broader O─H/COOH intensity in the O1s spectra, as well as the relatively lower signals of C═O and C─O─C in the C1s spectra. More importantly, the degraded sample shows very weak signals of B and P in its high‐resolution XPS, thus further confirming the depolymerized HBPPB. Consequently, a highly cross‐linked network experienced fragmentation as a result of cleavages of these bonds.
\",\n \"
The degraded products were subsequently processed into fine powder through grinding and drying (Figure ##FIG##1##2c##). Notably, by mixing 1 g of degraded powder with ≈5 g of our pristine epoxy resin (refer to Section ##SUPPL##0##S1.7##, Supporting Information) and curing following the same procedure, we obtained the post‐cycled EP with impact strength and flexural strength comparable to the original (Figure ##FIG##1##2d##). Despite the cleavable bonds being introduced, we note that in thermosetting materials with only adapting additively crosslinked cleavable units, theoretically, it is difficult in practice to realize entirely or closed‐loop recycling of monomers once all of the crosslinks are formed.[\\n##REF##36864144##\\n64\\n##, ##REF##37023198##\\n65\\n##, ##REF##37100913##\\n66\\n##\\n] In this system, although the native EP thermoset also contains ester dynamic bonds, it remains non‐degradable or slowly degradable compared to the vitrimer. Thus, both the cleavable units and topological crosslinks control the degradation.
\",\n \"
Regarding the recovery uses (Figure ##FIG##1##2g–i##), carbon fiber composites have been explored for high‐performance applications, while the costly embedded carbon fibers (CFs) cannot be retrieved from bulk materials.[\\n##UREF##37##\\n67\\n##, ##REF##35549086##\\n68\\n##\\n] In this implementation, when carbon fiber fabrics were embedded into HDCNs to fabricate a composite, we were able to quantitatively recover the CFs while effectively separating the end‐of‐use epoxy powders from the composite. The Raman spectrum of the recovered fibers shows a carbon peak closely resembling those of the native fibers, indicating minimal surface impact on the carbon fibers (Figure ##FIG##1##2j##). These results hint at promising opportunities for the cyclic utilization of end‐of‐use epoxy resins and composites.
Next, by a chance following‐up, we note that when the samples were immersed in ethanol following a heating (95 °C, 6 h), the vitrimer sample (EP‐9) via forming HDCNs displayed elastomeric‐like feature upon cooling to room temperature, which allowed for a casual deformation and recovery as well (see Movie ##SUPPL##1##S1##, Supporting Information; Figure ##FIG##2##\\n3a,b##), whereas the native thermoset (EP) still remains its rigid stiffness (Figure ##FIG##2##3c##). In our further study, we denoted EP‐9 as an example with suitable elasticity compared to EP‐12. Such transformation in elastomeric characteristics is exciting considering the inherent highly crosslinked nature of its bulk thermosets, especially for industrial epoxy commodities. This suggests that hyperbranched cross‐linking may alter the crosslinking mode of the thermoset network. We coined this type of vitrimer as “reconfigurable” vitrimers,[\\n##UREF##10##\\n16\\n##, ##REF##23579959##\\n49\\n##\\n] that is, a thermosetting network – whose crosslinked structure should be permanent – but can be reconstructed through solvent‐assisted reconfiguration. As illustrated in Figure ##FIG##2##3d,e##, we reasoned the ester‐exchange driving the reconstruction of HDCNs,[\\n##REF##35640074##\\n69\\n##, ##REF##31469270##\\n70\\n##\\n] whereby the ester bonds and hydrogen bonds can be rearranged since the ethanol serves as a single ─OH blocking molecule to substitute the ─OH site from those of hyperbranched crosslinks. The reorganized structure (Figure ##FIG##2##3j##) is corroborated by FT‐IR before‐ and after‐ethanol, temperature‐dependent IR, and dynamic thermomechanometry (Figure ##FIG##2##3g–i##). In particular, Figure ##FIG##2##3g## demonstrates a blueshift in the hydroxyl stretching vibration at 3400 cm−1, along with an increased width and intensity, indicating the transition of hydroxyl groups from a “free” state to an “associated” state. Furthermore, the reduction in peak intensity of C─O─Csym verifies the occurrence of ester exchange in HDCNs since symmetric ether structures (C─O─Csym, 1200–1150 cm−1) typically exhibit an increase in peak intensity. Additional evidence from model reactions (Section ##SUPPL##0##S1.8##, Supporting Information)[\\n##REF##31433176##\\n50\\n##, ##REF##29733205##\\n53\\n##\\n] and the XPS survey (Section ##SUPPL##0##S2.8##, Supporting Information) further supports the ester exchange should be the main mechanism to drive the reconfigurable behavior of the material. Ultimately, the reconstruction of HDCNs introduces polymer chain mobility and reduces the crosslinked structure, leading to the transformation in material nature.
\",\n \"
To further investigate the network dynamics of HDCNs, dynamic thermomechanometry was investigated over a wide temperature from −150 to 150 °C (Figure ##FIG##2##3i##; Figure ##SUPPL##0##S23##, Supporting Information) to record flexural modulus at three‐point bending mode. As the temperature decreases, the loss modulus exhibits a double‐relaxation of heat dissipation when the material is deformed. The lower (mark 1) relaxation involves a glass‐to‐elastic transition that unfreezes the side chains and groups, resulting in a rapid increase in flexural storage modulus from 3.2 to 5.4 GPa (Figure ##SUPPL##0##S23a##, Supporting Information). This increase is attributed to the ethyl ester (forming by ethanol exchange) occupying the initial crosslinked site of carbonic ester for network reconstruction (Figure ##FIG##2##3j##). The second relaxation (mark 2) signifies an elastic‐to‐liquid transition as the vitrimer network flows and relaxes due to dynamic bond exchange. Real‐temperature IR analysis confirms the defreezing temperature of H‐bonds in close proximity to this transition, as indicated by a significant increase in the normalized intensity of hydroxyl groups, shifting from the “associated” state to the “free” state upon temperature elevation from 0 °C (Figure ##FIG##2##3h##),[\\n##UREF##38##\\n71\\n##\\n] while the intensity remains almost unchanged from −50 to 0 °C.
\",\n \"
In this term, the reconstructed HDCNs display a stronger H‐bonding crosslinked network compared with that of native EP at lower temperature areas, where the polymer segments are frozen and tightly constrained by H‐bonding.[\\n##UREF##39##\\n72\\n##\\n] Consequently, we observe a significant increase in G’ with decreasing temperature (Figure ##SUPPL##0##S23a##, Supporting Information), recording as high as 5.45 GPa at −150 °C. This represents an exceptionally high flexural modulus for polymeric materials and approximately twice that of native EP under the same condition. The exceptional modulus at ultra‐low temperatures empowers this polymeric material with supernormal applications in astrospace material, superconducting energy storage, and medical equipment.
\",\n \"General Properties and Multifunctional Performance of HDCNs\",\n \"
\\nFigure ##FIG##3##\\n4\\n## provides a comprehensive analysis of the multifaceted performance of the material in harnessing the advantages of HDCNs. First, the glass transition temperature (T\\ng, Figure ##SUPPL##0##S21a## and Table ##SUPPL##0##S6##, Supporting Information) of EP, EP‐3, EP‐6, EP‐9, and EP‐12 are 131, 121, 118, 104, and 94 °C, respectively, indicating they are glassy polymers at room temperature. The slight decrease in T\\ng can be attributed to the amorphous structure of HBPPB.[\\n##UREF##25##\\n38\\n##\\n] Upon crosslinking a hyperbranched macromolecular, network topologies are physically expanded.[\\n##UREF##40##\\n73\\n##, ##REF##27634530##\\n74\\n##\\n] However, the heat loss of chains motion is intensified below T\\ng due to increased friction and constraints imposed by the HBPPB within the network (increased loss modulus (Figure ##FIG##3##4a##). Conversely, above T\\ng and T\\nv, the friction between epoxy chains with HBPPB is reduced due to dynamic bond exchange, and the network flows more easily thus existing a reduced loss modulus. Note that inserting poorly rigid chains often compromises the modulus and strength. Remarkably, the vitrimer, upon introducing an aliphatic hyperbranched crosslinker, demonstrates an even higher storage modulus (E’) compared to that of native EP (Figure ##FIG##3##4b##). Specifically, the addition of 3% HBPPB elevates E’ from 2.38 GPa of native EP up to 3.15 GPa (Figure ##SUPPL##0##S21b##, Supporting Information), followed by a decline with further increasing HBPPB, which substantiates the reinforcement effect of HDCNs. This enhancement can be attributed to that the hyperbranched structure acts as a “supramolecular rivet” to facilitate a robust supramolecular/epoxy interpenetrating network through H‐bonds and covalent bonds (Figure ##FIG##3##4d##).[\\n##UREF##41##\\n75\\n##, ##UREF##42##\\n76\\n##\\n] The branching molecular topology enables an expanded network interspace, resulting in a reduced network crosslinking density (Figure ##FIG##3##4c##), as elaborated in Section ##SUPPL##0##S2.1## (Supporting Information). Consequently, the hyperbranched crosslinker holds significant promise as a topological crosslinking tool for programming low‐crosslinking yet high‐strength polymers.
\",\n \"
Measurements of impact toughness, flexural strength, and tensile strength were carried out to facilitate a comprehensive understanding of mechanical performance (Figure ##FIG##3##4e,f##; details in Section ##SUPPL##0##S2.6##, Supporting Information). The high crosslinking density and rigid polymer chains render thermosets susceptible to impact failure, resulting in a microscopic “river‐like” brittle fracture (Figure ##SUPPL##0##S30##, Supporting Information) with 12.94 kJ m−2 of impact strength. However, EP‐3 shows a superior increase (98.5%) from 12.94 up to 25.69 kJ m−2, accompanied by obvious “dimple‐fracture” features that promote energy absorption. Thought‐out relevant works regarding hyperbranched polymer‐reinforced thermosets, a supramolecular network‐induced energy dissipation mechanism could be supposed.[\\n##UREF##43##\\n77\\n##, ##REF##35042887##\\n78\\n##\\n] The network topologies furnish cross‐linked polymers with enhanced properties without altering their chemical composition.[\\n##REF##35894294##\\n79\\n##\\n] It is worth noting that the increase in impact strength surpasses that of flexural strength, which can be attributed to the presence of flexible aliphatic chains that mitigate the network stiffness. Both the flexural and tensile strength show a consistent and noteworthy improvement from 106.1 up to 132 MPa, and 52.1 up to 81.2 MPa, respectively (Table ##SUPPL##0##S7##, Supporting Information). Notably, the tensile strain‐stress curves indicate that vitrimers are more stretchable than that of native thermoset epoxy (Figure ##SUPPL##0##S31##, Supporting Information). This finding holds great promise for the fabrication of carbon fiber reinforced polymers (CFRPs), where the stress should primarily reside in the fibers rather than the resins. This attribute ensures the exceptional mechanical strength of CFRPs (Section ##SUPPL##0##S2.6.3##, Supporting Information).
\",\n \"
In this contribution, both the strength and modulus depend on a tightly crosslinked network. The HBPPB, which terminates abundant ─OH groups, acts as a “rivet” within the network through covalent bonding and non‐covalent H‐bonding. The observed increase in E’ is also in agreement with the flexural results. Unfortunately, the optimal mechanical system does not maintain a consistent HBPPB concentration with the degradable and recyclable sample. Despite overloading HBPPB causing deteriorated mechanical strength, it still outperforms native EP. The reduction in impact and flexural strength can be attributed to the local agglomeration of hyperbranched polymers.[\\n##UREF##29##\\n45\\n##, ##UREF##30##\\n46\\n##\\n] Supplementary thermal and thermomechanical parameters are provided in Table ##SUPPL##0##S6## (Supporting Information). The vitrimers exhibit improved T\\nmax values and char yields, indicating superior thermal stability of HDCNs, which is primarily attributed to the presence of the inert boron component in the system.
\",\n \"
We next sought to probe the functional performance of materials. Good fire safety is a crucial consideration for real‐world applications of polymeric materials.[\\n##UREF##44##\\n80\\n##, ##UREF##45##\\n81\\n##\\n] Overall, our results demonstrate that the vitrimer sample exhibits superior fire safety performance compared to the native EP. Specifically, the vitrimer samples, EP‐9 and EP‐12, exhibit reduced thermal hazards and smoke production, along with increased resistance to ignition (higher limit oxygen index), and self‐extinguishing properties in an air atmosphere (Figure ##FIG##3##4g##). A comprehensive investigation was conducted through a full‐scale study of the fire‐retardant performance (Section ##SUPPL##0##S2.10## and Figure ##SUPPL##0##S41##, Supporting Information). The cone calorimeter test, which simulates a real fire scenario, provided crucial parameters to elucidate the flame‐retardant effect and mechanism.[\\n##UREF##46##\\n82\\n##\\n] Additionally, XPS analysis and Raman spectra (Figure ##SUPPL##0##S42##, Supporting Information) revealed the condensed fire‐retardant actions. The HDCNs constructed in our materials accumulate two types of flame‐retardant elements (phosphorus and boron) within the hyperbranched backbone for synergistic fire‐retarding action, ensuring the fire safety of the material.
\",\n \"
One of the facts is that the materials, specifically EP‐9 and EP‐12, exhibit significantly superior transparency and lighter coloration than their native thermoset. The transmittance of materials was measured using UV–vis spectrophotometer (See Figure ##SUPPL##0##S40##, Supporting Information). The average transmittance in both 280–380 nm (UV region) and 380–780 nm (visible region) was acquired in Figure ##FIG##3##4h##, showing significant increases in transmittance to both UV and visible light. The exceptional transparency of these materials can be attributed to the unique construction of HDCNs and the optical properties of HBPPB. Excitingly, this type of hyperbranched polymer has been reported as a class of non‐traditional aggregation‐induced emission luminogens (AIEgens),[\\n##UREF##47##\\n83\\n##, ##UREF##48##\\n84\\n##\\n] posing a promising pathway toward understanding the polymer crosslinking via fluorescent visualization methods. Besides, the exceptional transparency and lightness impart this polymeric material high added‐values for application in packaging materials, photoelectric materials, and flexible electronics.[\\n##UREF##49##\\n85\\n##, ##REF##22789123##\\n86\\n##\\n]\\n
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","Synthesis and Characterization of HDCNs","
The synthesis of HDCNs is straightforward via introducing a hyperbranched dynamic macromonomer into a traditional epoxy thermoset network as a dynamic crosslinker. The hyperbranched macromonomer (HBPPB) was synthesized via A2+B2+C3 polycondensation (Section ##SUPPL##0##S1.3a##, Supporting Information). In parallel, a linear poly‐phosphate/borate structure (LPPB), a hyperbranched polyborate (HBPB), and a hyperbranched polyphosphate (HBPP) were synthesized as controls to compare with HDCNs. Synthesis details are described in Sections ##SUPPL##0##S1.4## and ##SUPPL##0##S1.5## (Supporting Information). The molecular structure of the synthetic polymers was characterized using multiple analytical techniques, including Fourier transform infrared (FT‐IR), 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectroscopies, gel permeation chromatography (GPC) (see Section ##SUPPL##0##S1.3b##, Supporting Information). The concentration of hydroxyl groups (─OH) in HBPPB was quantified via a standard titration experiment, yielding a value of 7.97 × 10−3 mol g−1 (Section ##SUPPL##0##S1.3c## and Table ##SUPPL##0##S2##, Supporting Information), indicating the high abundance of ─OH groups.
","
The polymer matrix comprised of a petroleum‐based epoxy (diglycidyl ether bisphenol A, DGEBA, E51) a commercially used anhydride‐type curing agent (methyl tetrahydrophthalic anhydride, MTHPA), and a certain amount of HBPPB. A facile thermosetting workflow was employed to cast EP‐x samples, where x represents the mass concentration of HBPPB. The curing performance was evaluated using isothermal differential scanning calorimetry (Figure ##SUPPL##0##S21c##, Supporting Information), which showed a homogeneous and single peak among all pre‐polymer systems, indicating well compatibility among the three constituents. The natural EP and EP‐9 underwent full curing as proved in its time‐sweep curves by rheology (Figure ##SUPPL##0##S22##, Supporting Information). For subsequent analysis, the epoxy samples containing HBPPB equal to or greater than 9 wt.% were donated as vitrimers due to their degradable and/or reconfigurable dynamic behavior. The other samples were categorized as thermosets due to their predominant thermoset nature. To facilitate comparative investigations, epoxy vitrimers containing varying mass fractions of LPPB, HBPB, and HBPP were prepared using the same procedure as HDCNs, and a comparison of their material properties is presented in Sections ##SUPPL##0##S2.5## and ##SUPPL##0##S2.6## (Supporting Information).
","
The dynamic nature of HDCNs was characterized extensively through stress relaxation (see Section ##SUPPL##0##S2.3##, Supporting Information) and creep‐recovery test (Section ##SUPPL##0##S2.4##, Supporting Information) in tensile mode.[\n##REF##31433176##\n50\n##, ##REF##35590755##\n51\n##, ##REF##35607118##\n52\n##, ##REF##29733205##\n53\n##, ##REF##28557428##\n54\n##\n] In EP‐12, the time‐ and temperature‐dependent modulus revealed a partial relaxation to a plateau regime that deviates from classical linear‐viscoelastic vitrimers. This unique relaxation signifies that HDCNs not only behave with liquid‐like viscoelasticity due to the activation of dynamic exchange above the topology‐freezing transition temperature (T\nv, described by Arrhenius and Williams−Landel−Ferry law), but also retain the resilience and recovery capabilities like an elastic body subjected to Hooke's law. By fitting the results on an Arrhenius plot, a linear correlation between ln(τ*) (relaxation time) and 1000/T (inverse temperature) was observed, enabling the calculation of an activation energy (E\na) of 78.1 kJ mol−1 and T\nv of 71.7 °C (Figures ##SUPPL##0##S24## and ##SUPPL##0##S25##, Supporting Information). It is noteworthy that several examples of vitrimers with T\ng > T\nv have been reported.[\n##UREF##33##\n55\n##, ##REF##28991493##\n56\n##\n] Consequently, the material exhibits dynamic vitrimer behavior, albeit with reduced dynamic capabilities compared to fully relaxed vitrimers. Nonetheless, it demonstrates exceptional dimensional stability and mechanical strength even above the glass‐transition temperature (T\ng) and T\nv. This behavior can be attributed to the presence of permanently crosslinked sites and potentially relates to the network topologies, thereby underlying further understanding of vitrimers and their dynamic behaviors.
","Cleavable and Topological Crosslinks Control over Degradable HDCNs","
Thermoset polymers are generally considered non‐degradable and hard to reprocess, particularly when in comparison to thermoplastic or weakly crosslinked supramolecular plastics.[\n##REF##36318511##\n57\n##, ##UREF##34##\n58\n##\n] However, inserting topological crosslinks and cleavable bonds into the thermoset network via HDCNs leads to the formation of vitrimers that exhibit distinctive behavior upon exposure to solvents (Figure ##FIG##1##\n2\n##; Section ##SUPPL##0##S1.7##, Supporting Information). In this study, a commonly used aprotic solvent (dimethylacetamide, DMAc) was employed to fully immerse the samples. EP‐12, as the most prominent case for degradation, underwent swelling and fragmentation within a mere 2 days at room temperature (Figure ##FIG##1##2a,b##), in stark contrast to the natural thermoset EP, which retained its intact and rigid shape. Moreover, an increasing concentration of HBPPB can accelerate the process. In fact, a rapid degradation was observed within 6 hours when the system temperature was raised to 95 °C in DMAc (Figure ##SUPPL##0##S13##, Supporting Information). Based on these differences, we infer that the solvent‐assisted degradation was associated with cleavable crosslinks in HDCNs.
","
Unlike classic thermoset networks or degradable covalent networks, most of them are specialized for the degradation of weakly crosslinked materials such as thermoplastics and supramolecular plastics but are often helpless for highly crosslinked thermoset networks.[\n##REF##31011169##\n59\n##, ##UREF##35##\n60\n##\n] Whereas HDCNs benefit the stretching of molecular configurations for topological crosslinking purposes, which not only consists of several cleavable units but, more importantly, enables a topologically expanded network interspace that allows solvent molecular entrance to drive dynamic behaviors. The crosslinking density of HDCNs is relatively lower than their thermoset EP counterpart (Figure ##FIG##3##4c##). To assert the breakages when degrading, a schematic model describes the possible mechanisms in Figure ##FIG##1##2e##. Such a structure model encompasses three types of cleavable bonds. The first involves two kinds of carbonic ester bonds (mark a) that result from the reaction between the anhydride with HBPPB and the epoxy monomer (Figure ##SUPPL##0##S10##, Supporting Information). The second breakage is attributed to the boronic ester group (BO3)[\n##REF##29733205##\n53\n##, ##REF##25945818##\n61\n##, ##REF##27387198##\n62\n##\n] and the phosphate group (PO3)[\n##UREF##36##\n63\n##\n] upon the molecular backbone of the hyperbranched crosslinker. Third, non‐covalent H‐bonds are also susceptible to cleavage when the solvent is diffused.
","
To monitor the aforementioned breakages, both EP‐12 and the solvent were subjected to FT‐IR analysis, as shown in Figure ##FIG##1##2f##. The results reveal distinct spectral features, notably the emergence of a newly detected C═O absorption (mark a) and C─H signals within the substituted benzene moiety in the DMAc spectrum after degradation, indicating the species of anhydride‐contained substrates are partially dissolved in DMAc, primarily indicating the cleavage of these carboxyl ester bonds. Furthermore, the degraded sample exhibits a diminished C─O─B/P absorption (mark b), which corresponds to the depolymerization of HBPPB. Complementary insights from the X‐ray photoelectron spectroscopy (XPS, details in Section ##SUPPL##0##S2.8##, Supporting Information) reveal that the beta‐hydroxy ester (formed through the reaction of the anhydride with ‐OH and epoxy groups) underwent breakage, as evidenced by its heightened and broader O─H/COOH intensity in the O1s spectra, as well as the relatively lower signals of C═O and C─O─C in the C1s spectra. More importantly, the degraded sample shows very weak signals of B and P in its high‐resolution XPS, thus further confirming the depolymerized HBPPB. Consequently, a highly cross‐linked network experienced fragmentation as a result of cleavages of these bonds.
","
The degraded products were subsequently processed into fine powder through grinding and drying (Figure ##FIG##1##2c##). Notably, by mixing 1 g of degraded powder with ≈5 g of our pristine epoxy resin (refer to Section ##SUPPL##0##S1.7##, Supporting Information) and curing following the same procedure, we obtained the post‐cycled EP with impact strength and flexural strength comparable to the original (Figure ##FIG##1##2d##). Despite the cleavable bonds being introduced, we note that in thermosetting materials with only adapting additively crosslinked cleavable units, theoretically, it is difficult in practice to realize entirely or closed‐loop recycling of monomers once all of the crosslinks are formed.[\n##REF##36864144##\n64\n##, ##REF##37023198##\n65\n##, ##REF##37100913##\n66\n##\n] In this system, although the native EP thermoset also contains ester dynamic bonds, it remains non‐degradable or slowly degradable compared to the vitrimer. Thus, both the cleavable units and topological crosslinks control the degradation.
","
Regarding the recovery uses (Figure ##FIG##1##2g–i##), carbon fiber composites have been explored for high‐performance applications, while the costly embedded carbon fibers (CFs) cannot be retrieved from bulk materials.[\n##UREF##37##\n67\n##, ##REF##35549086##\n68\n##\n] In this implementation, when carbon fiber fabrics were embedded into HDCNs to fabricate a composite, we were able to quantitatively recover the CFs while effectively separating the end‐of‐use epoxy powders from the composite. The Raman spectrum of the recovered fibers shows a carbon peak closely resembling those of the native fibers, indicating minimal surface impact on the carbon fibers (Figure ##FIG##1##2j##). These results hint at promising opportunities for the cyclic utilization of end‐of‐use epoxy resins and composites.
Next, by a chance following‐up, we note that when the samples were immersed in ethanol following a heating (95 °C, 6 h), the vitrimer sample (EP‐9) via forming HDCNs displayed elastomeric‐like feature upon cooling to room temperature, which allowed for a casual deformation and recovery as well (see Movie ##SUPPL##1##S1##, Supporting Information; Figure ##FIG##2##\n3a,b##), whereas the native thermoset (EP) still remains its rigid stiffness (Figure ##FIG##2##3c##). In our further study, we denoted EP‐9 as an example with suitable elasticity compared to EP‐12. Such transformation in elastomeric characteristics is exciting considering the inherent highly crosslinked nature of its bulk thermosets, especially for industrial epoxy commodities. This suggests that hyperbranched cross‐linking may alter the crosslinking mode of the thermoset network. We coined this type of vitrimer as “reconfigurable” vitrimers,[\n##UREF##10##\n16\n##, ##REF##23579959##\n49\n##\n] that is, a thermosetting network – whose crosslinked structure should be permanent – but can be reconstructed through solvent‐assisted reconfiguration. As illustrated in Figure ##FIG##2##3d,e##, we reasoned the ester‐exchange driving the reconstruction of HDCNs,[\n##REF##35640074##\n69\n##, ##REF##31469270##\n70\n##\n] whereby the ester bonds and hydrogen bonds can be rearranged since the ethanol serves as a single ─OH blocking molecule to substitute the ─OH site from those of hyperbranched crosslinks. The reorganized structure (Figure ##FIG##2##3j##) is corroborated by FT‐IR before‐ and after‐ethanol, temperature‐dependent IR, and dynamic thermomechanometry (Figure ##FIG##2##3g–i##). In particular, Figure ##FIG##2##3g## demonstrates a blueshift in the hydroxyl stretching vibration at 3400 cm−1, along with an increased width and intensity, indicating the transition of hydroxyl groups from a “free” state to an “associated” state. Furthermore, the reduction in peak intensity of C─O─Csym verifies the occurrence of ester exchange in HDCNs since symmetric ether structures (C─O─Csym, 1200–1150 cm−1) typically exhibit an increase in peak intensity. Additional evidence from model reactions (Section ##SUPPL##0##S1.8##, Supporting Information)[\n##REF##31433176##\n50\n##, ##REF##29733205##\n53\n##\n] and the XPS survey (Section ##SUPPL##0##S2.8##, Supporting Information) further supports the ester exchange should be the main mechanism to drive the reconfigurable behavior of the material. Ultimately, the reconstruction of HDCNs introduces polymer chain mobility and reduces the crosslinked structure, leading to the transformation in material nature.
","
To further investigate the network dynamics of HDCNs, dynamic thermomechanometry was investigated over a wide temperature from −150 to 150 °C (Figure ##FIG##2##3i##; Figure ##SUPPL##0##S23##, Supporting Information) to record flexural modulus at three‐point bending mode. As the temperature decreases, the loss modulus exhibits a double‐relaxation of heat dissipation when the material is deformed. The lower (mark 1) relaxation involves a glass‐to‐elastic transition that unfreezes the side chains and groups, resulting in a rapid increase in flexural storage modulus from 3.2 to 5.4 GPa (Figure ##SUPPL##0##S23a##, Supporting Information). This increase is attributed to the ethyl ester (forming by ethanol exchange) occupying the initial crosslinked site of carbonic ester for network reconstruction (Figure ##FIG##2##3j##). The second relaxation (mark 2) signifies an elastic‐to‐liquid transition as the vitrimer network flows and relaxes due to dynamic bond exchange. Real‐temperature IR analysis confirms the defreezing temperature of H‐bonds in close proximity to this transition, as indicated by a significant increase in the normalized intensity of hydroxyl groups, shifting from the “associated” state to the “free” state upon temperature elevation from 0 °C (Figure ##FIG##2##3h##),[\n##UREF##38##\n71\n##\n] while the intensity remains almost unchanged from −50 to 0 °C.
","
In this term, the reconstructed HDCNs display a stronger H‐bonding crosslinked network compared with that of native EP at lower temperature areas, where the polymer segments are frozen and tightly constrained by H‐bonding.[\n##UREF##39##\n72\n##\n] Consequently, we observe a significant increase in G’ with decreasing temperature (Figure ##SUPPL##0##S23a##, Supporting Information), recording as high as 5.45 GPa at −150 °C. This represents an exceptionally high flexural modulus for polymeric materials and approximately twice that of native EP under the same condition. The exceptional modulus at ultra‐low temperatures empowers this polymeric material with supernormal applications in astrospace material, superconducting energy storage, and medical equipment.
","General Properties and Multifunctional Performance of HDCNs","
\nFigure ##FIG##3##\n4\n## provides a comprehensive analysis of the multifaceted performance of the material in harnessing the advantages of HDCNs. First, the glass transition temperature (T\ng, Figure ##SUPPL##0##S21a## and Table ##SUPPL##0##S6##, Supporting Information) of EP, EP‐3, EP‐6, EP‐9, and EP‐12 are 131, 121, 118, 104, and 94 °C, respectively, indicating they are glassy polymers at room temperature. The slight decrease in T\ng can be attributed to the amorphous structure of HBPPB.[\n##UREF##25##\n38\n##\n] Upon crosslinking a hyperbranched macromolecular, network topologies are physically expanded.[\n##UREF##40##\n73\n##, ##REF##27634530##\n74\n##\n] However, the heat loss of chains motion is intensified below T\ng due to increased friction and constraints imposed by the HBPPB within the network (increased loss modulus (Figure ##FIG##3##4a##). Conversely, above T\ng and T\nv, the friction between epoxy chains with HBPPB is reduced due to dynamic bond exchange, and the network flows more easily thus existing a reduced loss modulus. Note that inserting poorly rigid chains often compromises the modulus and strength. Remarkably, the vitrimer, upon introducing an aliphatic hyperbranched crosslinker, demonstrates an even higher storage modulus (E’) compared to that of native EP (Figure ##FIG##3##4b##). Specifically, the addition of 3% HBPPB elevates E’ from 2.38 GPa of native EP up to 3.15 GPa (Figure ##SUPPL##0##S21b##, Supporting Information), followed by a decline with further increasing HBPPB, which substantiates the reinforcement effect of HDCNs. This enhancement can be attributed to that the hyperbranched structure acts as a “supramolecular rivet” to facilitate a robust supramolecular/epoxy interpenetrating network through H‐bonds and covalent bonds (Figure ##FIG##3##4d##).[\n##UREF##41##\n75\n##, ##UREF##42##\n76\n##\n] The branching molecular topology enables an expanded network interspace, resulting in a reduced network crosslinking density (Figure ##FIG##3##4c##), as elaborated in Section ##SUPPL##0##S2.1## (Supporting Information). Consequently, the hyperbranched crosslinker holds significant promise as a topological crosslinking tool for programming low‐crosslinking yet high‐strength polymers.
","
Measurements of impact toughness, flexural strength, and tensile strength were carried out to facilitate a comprehensive understanding of mechanical performance (Figure ##FIG##3##4e,f##; details in Section ##SUPPL##0##S2.6##, Supporting Information). The high crosslinking density and rigid polymer chains render thermosets susceptible to impact failure, resulting in a microscopic “river‐like” brittle fracture (Figure ##SUPPL##0##S30##, Supporting Information) with 12.94 kJ m−2 of impact strength. However, EP‐3 shows a superior increase (98.5%) from 12.94 up to 25.69 kJ m−2, accompanied by obvious “dimple‐fracture” features that promote energy absorption. Thought‐out relevant works regarding hyperbranched polymer‐reinforced thermosets, a supramolecular network‐induced energy dissipation mechanism could be supposed.[\n##UREF##43##\n77\n##, ##REF##35042887##\n78\n##\n] The network topologies furnish cross‐linked polymers with enhanced properties without altering their chemical composition.[\n##REF##35894294##\n79\n##\n] It is worth noting that the increase in impact strength surpasses that of flexural strength, which can be attributed to the presence of flexible aliphatic chains that mitigate the network stiffness. Both the flexural and tensile strength show a consistent and noteworthy improvement from 106.1 up to 132 MPa, and 52.1 up to 81.2 MPa, respectively (Table ##SUPPL##0##S7##, Supporting Information). Notably, the tensile strain‐stress curves indicate that vitrimers are more stretchable than that of native thermoset epoxy (Figure ##SUPPL##0##S31##, Supporting Information). This finding holds great promise for the fabrication of carbon fiber reinforced polymers (CFRPs), where the stress should primarily reside in the fibers rather than the resins. This attribute ensures the exceptional mechanical strength of CFRPs (Section ##SUPPL##0##S2.6.3##, Supporting Information).
","
In this contribution, both the strength and modulus depend on a tightly crosslinked network. The HBPPB, which terminates abundant ─OH groups, acts as a “rivet” within the network through covalent bonding and non‐covalent H‐bonding. The observed increase in E’ is also in agreement with the flexural results. Unfortunately, the optimal mechanical system does not maintain a consistent HBPPB concentration with the degradable and recyclable sample. Despite overloading HBPPB causing deteriorated mechanical strength, it still outperforms native EP. The reduction in impact and flexural strength can be attributed to the local agglomeration of hyperbranched polymers.[\n##UREF##29##\n45\n##, ##UREF##30##\n46\n##\n] Supplementary thermal and thermomechanical parameters are provided in Table ##SUPPL##0##S6## (Supporting Information). The vitrimers exhibit improved T\nmax values and char yields, indicating superior thermal stability of HDCNs, which is primarily attributed to the presence of the inert boron component in the system.
","
We next sought to probe the functional performance of materials. Good fire safety is a crucial consideration for real‐world applications of polymeric materials.[\n##UREF##44##\n80\n##, ##UREF##45##\n81\n##\n] Overall, our results demonstrate that the vitrimer sample exhibits superior fire safety performance compared to the native EP. Specifically, the vitrimer samples, EP‐9 and EP‐12, exhibit reduced thermal hazards and smoke production, along with increased resistance to ignition (higher limit oxygen index), and self‐extinguishing properties in an air atmosphere (Figure ##FIG##3##4g##). A comprehensive investigation was conducted through a full‐scale study of the fire‐retardant performance (Section ##SUPPL##0##S2.10## and Figure ##SUPPL##0##S41##, Supporting Information). The cone calorimeter test, which simulates a real fire scenario, provided crucial parameters to elucidate the flame‐retardant effect and mechanism.[\n##UREF##46##\n82\n##\n] Additionally, XPS analysis and Raman spectra (Figure ##SUPPL##0##S42##, Supporting Information) revealed the condensed fire‐retardant actions. The HDCNs constructed in our materials accumulate two types of flame‐retardant elements (phosphorus and boron) within the hyperbranched backbone for synergistic fire‐retarding action, ensuring the fire safety of the material.
","
One of the facts is that the materials, specifically EP‐9 and EP‐12, exhibit significantly superior transparency and lighter coloration than their native thermoset. The transmittance of materials was measured using UV–vis spectrophotometer (See Figure ##SUPPL##0##S40##, Supporting Information). The average transmittance in both 280–380 nm (UV region) and 380–780 nm (visible region) was acquired in Figure ##FIG##3##4h##, showing significant increases in transmittance to both UV and visible light. The exceptional transparency of these materials can be attributed to the unique construction of HDCNs and the optical properties of HBPPB. Excitingly, this type of hyperbranched polymer has been reported as a class of non‐traditional aggregation‐induced emission luminogens (AIEgens),[\n##UREF##47##\n83\n##, ##UREF##48##\n84\n##\n] posing a promising pathway toward understanding the polymer crosslinking via fluorescent visualization methods. Besides, the exceptional transparency and lightness impart this polymeric material high added‐values for application in packaging materials, photoelectric materials, and flexible electronics.[\n##UREF##49##\n85\n##, ##REF##22789123##\n86\n##\n]\n
"],"string":"[\n \"Results and Discussion\",\n \"Synthesis and Characterization of HDCNs\",\n \"
The synthesis of HDCNs is straightforward via introducing a hyperbranched dynamic macromonomer into a traditional epoxy thermoset network as a dynamic crosslinker. The hyperbranched macromonomer (HBPPB) was synthesized via A2+B2+C3 polycondensation (Section ##SUPPL##0##S1.3a##, Supporting Information). In parallel, a linear poly‐phosphate/borate structure (LPPB), a hyperbranched polyborate (HBPB), and a hyperbranched polyphosphate (HBPP) were synthesized as controls to compare with HDCNs. Synthesis details are described in Sections ##SUPPL##0##S1.4## and ##SUPPL##0##S1.5## (Supporting Information). The molecular structure of the synthetic polymers was characterized using multiple analytical techniques, including Fourier transform infrared (FT‐IR), 1H, 13C, and 31P nuclear magnetic resonance (NMR) spectroscopies, gel permeation chromatography (GPC) (see Section ##SUPPL##0##S1.3b##, Supporting Information). The concentration of hydroxyl groups (─OH) in HBPPB was quantified via a standard titration experiment, yielding a value of 7.97 × 10−3 mol g−1 (Section ##SUPPL##0##S1.3c## and Table ##SUPPL##0##S2##, Supporting Information), indicating the high abundance of ─OH groups.
\",\n \"
The polymer matrix comprised of a petroleum‐based epoxy (diglycidyl ether bisphenol A, DGEBA, E51) a commercially used anhydride‐type curing agent (methyl tetrahydrophthalic anhydride, MTHPA), and a certain amount of HBPPB. A facile thermosetting workflow was employed to cast EP‐x samples, where x represents the mass concentration of HBPPB. The curing performance was evaluated using isothermal differential scanning calorimetry (Figure ##SUPPL##0##S21c##, Supporting Information), which showed a homogeneous and single peak among all pre‐polymer systems, indicating well compatibility among the three constituents. The natural EP and EP‐9 underwent full curing as proved in its time‐sweep curves by rheology (Figure ##SUPPL##0##S22##, Supporting Information). For subsequent analysis, the epoxy samples containing HBPPB equal to or greater than 9 wt.% were donated as vitrimers due to their degradable and/or reconfigurable dynamic behavior. The other samples were categorized as thermosets due to their predominant thermoset nature. To facilitate comparative investigations, epoxy vitrimers containing varying mass fractions of LPPB, HBPB, and HBPP were prepared using the same procedure as HDCNs, and a comparison of their material properties is presented in Sections ##SUPPL##0##S2.5## and ##SUPPL##0##S2.6## (Supporting Information).
\",\n \"
The dynamic nature of HDCNs was characterized extensively through stress relaxation (see Section ##SUPPL##0##S2.3##, Supporting Information) and creep‐recovery test (Section ##SUPPL##0##S2.4##, Supporting Information) in tensile mode.[\\n##REF##31433176##\\n50\\n##, ##REF##35590755##\\n51\\n##, ##REF##35607118##\\n52\\n##, ##REF##29733205##\\n53\\n##, ##REF##28557428##\\n54\\n##\\n] In EP‐12, the time‐ and temperature‐dependent modulus revealed a partial relaxation to a plateau regime that deviates from classical linear‐viscoelastic vitrimers. This unique relaxation signifies that HDCNs not only behave with liquid‐like viscoelasticity due to the activation of dynamic exchange above the topology‐freezing transition temperature (T\\nv, described by Arrhenius and Williams−Landel−Ferry law), but also retain the resilience and recovery capabilities like an elastic body subjected to Hooke's law. By fitting the results on an Arrhenius plot, a linear correlation between ln(τ*) (relaxation time) and 1000/T (inverse temperature) was observed, enabling the calculation of an activation energy (E\\na) of 78.1 kJ mol−1 and T\\nv of 71.7 °C (Figures ##SUPPL##0##S24## and ##SUPPL##0##S25##, Supporting Information). It is noteworthy that several examples of vitrimers with T\\ng > T\\nv have been reported.[\\n##UREF##33##\\n55\\n##, ##REF##28991493##\\n56\\n##\\n] Consequently, the material exhibits dynamic vitrimer behavior, albeit with reduced dynamic capabilities compared to fully relaxed vitrimers. Nonetheless, it demonstrates exceptional dimensional stability and mechanical strength even above the glass‐transition temperature (T\\ng) and T\\nv. This behavior can be attributed to the presence of permanently crosslinked sites and potentially relates to the network topologies, thereby underlying further understanding of vitrimers and their dynamic behaviors.
\",\n \"Cleavable and Topological Crosslinks Control over Degradable HDCNs\",\n \"
Thermoset polymers are generally considered non‐degradable and hard to reprocess, particularly when in comparison to thermoplastic or weakly crosslinked supramolecular plastics.[\\n##REF##36318511##\\n57\\n##, ##UREF##34##\\n58\\n##\\n] However, inserting topological crosslinks and cleavable bonds into the thermoset network via HDCNs leads to the formation of vitrimers that exhibit distinctive behavior upon exposure to solvents (Figure ##FIG##1##\\n2\\n##; Section ##SUPPL##0##S1.7##, Supporting Information). In this study, a commonly used aprotic solvent (dimethylacetamide, DMAc) was employed to fully immerse the samples. EP‐12, as the most prominent case for degradation, underwent swelling and fragmentation within a mere 2 days at room temperature (Figure ##FIG##1##2a,b##), in stark contrast to the natural thermoset EP, which retained its intact and rigid shape. Moreover, an increasing concentration of HBPPB can accelerate the process. In fact, a rapid degradation was observed within 6 hours when the system temperature was raised to 95 °C in DMAc (Figure ##SUPPL##0##S13##, Supporting Information). Based on these differences, we infer that the solvent‐assisted degradation was associated with cleavable crosslinks in HDCNs.
\",\n \"
Unlike classic thermoset networks or degradable covalent networks, most of them are specialized for the degradation of weakly crosslinked materials such as thermoplastics and supramolecular plastics but are often helpless for highly crosslinked thermoset networks.[\\n##REF##31011169##\\n59\\n##, ##UREF##35##\\n60\\n##\\n] Whereas HDCNs benefit the stretching of molecular configurations for topological crosslinking purposes, which not only consists of several cleavable units but, more importantly, enables a topologically expanded network interspace that allows solvent molecular entrance to drive dynamic behaviors. The crosslinking density of HDCNs is relatively lower than their thermoset EP counterpart (Figure ##FIG##3##4c##). To assert the breakages when degrading, a schematic model describes the possible mechanisms in Figure ##FIG##1##2e##. Such a structure model encompasses three types of cleavable bonds. The first involves two kinds of carbonic ester bonds (mark a) that result from the reaction between the anhydride with HBPPB and the epoxy monomer (Figure ##SUPPL##0##S10##, Supporting Information). The second breakage is attributed to the boronic ester group (BO3)[\\n##REF##29733205##\\n53\\n##, ##REF##25945818##\\n61\\n##, ##REF##27387198##\\n62\\n##\\n] and the phosphate group (PO3)[\\n##UREF##36##\\n63\\n##\\n] upon the molecular backbone of the hyperbranched crosslinker. Third, non‐covalent H‐bonds are also susceptible to cleavage when the solvent is diffused.
\",\n \"
To monitor the aforementioned breakages, both EP‐12 and the solvent were subjected to FT‐IR analysis, as shown in Figure ##FIG##1##2f##. The results reveal distinct spectral features, notably the emergence of a newly detected C═O absorption (mark a) and C─H signals within the substituted benzene moiety in the DMAc spectrum after degradation, indicating the species of anhydride‐contained substrates are partially dissolved in DMAc, primarily indicating the cleavage of these carboxyl ester bonds. Furthermore, the degraded sample exhibits a diminished C─O─B/P absorption (mark b), which corresponds to the depolymerization of HBPPB. Complementary insights from the X‐ray photoelectron spectroscopy (XPS, details in Section ##SUPPL##0##S2.8##, Supporting Information) reveal that the beta‐hydroxy ester (formed through the reaction of the anhydride with ‐OH and epoxy groups) underwent breakage, as evidenced by its heightened and broader O─H/COOH intensity in the O1s spectra, as well as the relatively lower signals of C═O and C─O─C in the C1s spectra. More importantly, the degraded sample shows very weak signals of B and P in its high‐resolution XPS, thus further confirming the depolymerized HBPPB. Consequently, a highly cross‐linked network experienced fragmentation as a result of cleavages of these bonds.
\",\n \"
The degraded products were subsequently processed into fine powder through grinding and drying (Figure ##FIG##1##2c##). Notably, by mixing 1 g of degraded powder with ≈5 g of our pristine epoxy resin (refer to Section ##SUPPL##0##S1.7##, Supporting Information) and curing following the same procedure, we obtained the post‐cycled EP with impact strength and flexural strength comparable to the original (Figure ##FIG##1##2d##). Despite the cleavable bonds being introduced, we note that in thermosetting materials with only adapting additively crosslinked cleavable units, theoretically, it is difficult in practice to realize entirely or closed‐loop recycling of monomers once all of the crosslinks are formed.[\\n##REF##36864144##\\n64\\n##, ##REF##37023198##\\n65\\n##, ##REF##37100913##\\n66\\n##\\n] In this system, although the native EP thermoset also contains ester dynamic bonds, it remains non‐degradable or slowly degradable compared to the vitrimer. Thus, both the cleavable units and topological crosslinks control the degradation.
\",\n \"
Regarding the recovery uses (Figure ##FIG##1##2g–i##), carbon fiber composites have been explored for high‐performance applications, while the costly embedded carbon fibers (CFs) cannot be retrieved from bulk materials.[\\n##UREF##37##\\n67\\n##, ##REF##35549086##\\n68\\n##\\n] In this implementation, when carbon fiber fabrics were embedded into HDCNs to fabricate a composite, we were able to quantitatively recover the CFs while effectively separating the end‐of‐use epoxy powders from the composite. The Raman spectrum of the recovered fibers shows a carbon peak closely resembling those of the native fibers, indicating minimal surface impact on the carbon fibers (Figure ##FIG##1##2j##). These results hint at promising opportunities for the cyclic utilization of end‐of‐use epoxy resins and composites.
Next, by a chance following‐up, we note that when the samples were immersed in ethanol following a heating (95 °C, 6 h), the vitrimer sample (EP‐9) via forming HDCNs displayed elastomeric‐like feature upon cooling to room temperature, which allowed for a casual deformation and recovery as well (see Movie ##SUPPL##1##S1##, Supporting Information; Figure ##FIG##2##\\n3a,b##), whereas the native thermoset (EP) still remains its rigid stiffness (Figure ##FIG##2##3c##). In our further study, we denoted EP‐9 as an example with suitable elasticity compared to EP‐12. Such transformation in elastomeric characteristics is exciting considering the inherent highly crosslinked nature of its bulk thermosets, especially for industrial epoxy commodities. This suggests that hyperbranched cross‐linking may alter the crosslinking mode of the thermoset network. We coined this type of vitrimer as “reconfigurable” vitrimers,[\\n##UREF##10##\\n16\\n##, ##REF##23579959##\\n49\\n##\\n] that is, a thermosetting network – whose crosslinked structure should be permanent – but can be reconstructed through solvent‐assisted reconfiguration. As illustrated in Figure ##FIG##2##3d,e##, we reasoned the ester‐exchange driving the reconstruction of HDCNs,[\\n##REF##35640074##\\n69\\n##, ##REF##31469270##\\n70\\n##\\n] whereby the ester bonds and hydrogen bonds can be rearranged since the ethanol serves as a single ─OH blocking molecule to substitute the ─OH site from those of hyperbranched crosslinks. The reorganized structure (Figure ##FIG##2##3j##) is corroborated by FT‐IR before‐ and after‐ethanol, temperature‐dependent IR, and dynamic thermomechanometry (Figure ##FIG##2##3g–i##). In particular, Figure ##FIG##2##3g## demonstrates a blueshift in the hydroxyl stretching vibration at 3400 cm−1, along with an increased width and intensity, indicating the transition of hydroxyl groups from a “free” state to an “associated” state. Furthermore, the reduction in peak intensity of C─O─Csym verifies the occurrence of ester exchange in HDCNs since symmetric ether structures (C─O─Csym, 1200–1150 cm−1) typically exhibit an increase in peak intensity. Additional evidence from model reactions (Section ##SUPPL##0##S1.8##, Supporting Information)[\\n##REF##31433176##\\n50\\n##, ##REF##29733205##\\n53\\n##\\n] and the XPS survey (Section ##SUPPL##0##S2.8##, Supporting Information) further supports the ester exchange should be the main mechanism to drive the reconfigurable behavior of the material. Ultimately, the reconstruction of HDCNs introduces polymer chain mobility and reduces the crosslinked structure, leading to the transformation in material nature.
\",\n \"
To further investigate the network dynamics of HDCNs, dynamic thermomechanometry was investigated over a wide temperature from −150 to 150 °C (Figure ##FIG##2##3i##; Figure ##SUPPL##0##S23##, Supporting Information) to record flexural modulus at three‐point bending mode. As the temperature decreases, the loss modulus exhibits a double‐relaxation of heat dissipation when the material is deformed. The lower (mark 1) relaxation involves a glass‐to‐elastic transition that unfreezes the side chains and groups, resulting in a rapid increase in flexural storage modulus from 3.2 to 5.4 GPa (Figure ##SUPPL##0##S23a##, Supporting Information). This increase is attributed to the ethyl ester (forming by ethanol exchange) occupying the initial crosslinked site of carbonic ester for network reconstruction (Figure ##FIG##2##3j##). The second relaxation (mark 2) signifies an elastic‐to‐liquid transition as the vitrimer network flows and relaxes due to dynamic bond exchange. Real‐temperature IR analysis confirms the defreezing temperature of H‐bonds in close proximity to this transition, as indicated by a significant increase in the normalized intensity of hydroxyl groups, shifting from the “associated” state to the “free” state upon temperature elevation from 0 °C (Figure ##FIG##2##3h##),[\\n##UREF##38##\\n71\\n##\\n] while the intensity remains almost unchanged from −50 to 0 °C.
\",\n \"
In this term, the reconstructed HDCNs display a stronger H‐bonding crosslinked network compared with that of native EP at lower temperature areas, where the polymer segments are frozen and tightly constrained by H‐bonding.[\\n##UREF##39##\\n72\\n##\\n] Consequently, we observe a significant increase in G’ with decreasing temperature (Figure ##SUPPL##0##S23a##, Supporting Information), recording as high as 5.45 GPa at −150 °C. This represents an exceptionally high flexural modulus for polymeric materials and approximately twice that of native EP under the same condition. The exceptional modulus at ultra‐low temperatures empowers this polymeric material with supernormal applications in astrospace material, superconducting energy storage, and medical equipment.
\",\n \"General Properties and Multifunctional Performance of HDCNs\",\n \"
\\nFigure ##FIG##3##\\n4\\n## provides a comprehensive analysis of the multifaceted performance of the material in harnessing the advantages of HDCNs. First, the glass transition temperature (T\\ng, Figure ##SUPPL##0##S21a## and Table ##SUPPL##0##S6##, Supporting Information) of EP, EP‐3, EP‐6, EP‐9, and EP‐12 are 131, 121, 118, 104, and 94 °C, respectively, indicating they are glassy polymers at room temperature. The slight decrease in T\\ng can be attributed to the amorphous structure of HBPPB.[\\n##UREF##25##\\n38\\n##\\n] Upon crosslinking a hyperbranched macromolecular, network topologies are physically expanded.[\\n##UREF##40##\\n73\\n##, ##REF##27634530##\\n74\\n##\\n] However, the heat loss of chains motion is intensified below T\\ng due to increased friction and constraints imposed by the HBPPB within the network (increased loss modulus (Figure ##FIG##3##4a##). Conversely, above T\\ng and T\\nv, the friction between epoxy chains with HBPPB is reduced due to dynamic bond exchange, and the network flows more easily thus existing a reduced loss modulus. Note that inserting poorly rigid chains often compromises the modulus and strength. Remarkably, the vitrimer, upon introducing an aliphatic hyperbranched crosslinker, demonstrates an even higher storage modulus (E’) compared to that of native EP (Figure ##FIG##3##4b##). Specifically, the addition of 3% HBPPB elevates E’ from 2.38 GPa of native EP up to 3.15 GPa (Figure ##SUPPL##0##S21b##, Supporting Information), followed by a decline with further increasing HBPPB, which substantiates the reinforcement effect of HDCNs. This enhancement can be attributed to that the hyperbranched structure acts as a “supramolecular rivet” to facilitate a robust supramolecular/epoxy interpenetrating network through H‐bonds and covalent bonds (Figure ##FIG##3##4d##).[\\n##UREF##41##\\n75\\n##, ##UREF##42##\\n76\\n##\\n] The branching molecular topology enables an expanded network interspace, resulting in a reduced network crosslinking density (Figure ##FIG##3##4c##), as elaborated in Section ##SUPPL##0##S2.1## (Supporting Information). Consequently, the hyperbranched crosslinker holds significant promise as a topological crosslinking tool for programming low‐crosslinking yet high‐strength polymers.
\",\n \"
Measurements of impact toughness, flexural strength, and tensile strength were carried out to facilitate a comprehensive understanding of mechanical performance (Figure ##FIG##3##4e,f##; details in Section ##SUPPL##0##S2.6##, Supporting Information). The high crosslinking density and rigid polymer chains render thermosets susceptible to impact failure, resulting in a microscopic “river‐like” brittle fracture (Figure ##SUPPL##0##S30##, Supporting Information) with 12.94 kJ m−2 of impact strength. However, EP‐3 shows a superior increase (98.5%) from 12.94 up to 25.69 kJ m−2, accompanied by obvious “dimple‐fracture” features that promote energy absorption. Thought‐out relevant works regarding hyperbranched polymer‐reinforced thermosets, a supramolecular network‐induced energy dissipation mechanism could be supposed.[\\n##UREF##43##\\n77\\n##, ##REF##35042887##\\n78\\n##\\n] The network topologies furnish cross‐linked polymers with enhanced properties without altering their chemical composition.[\\n##REF##35894294##\\n79\\n##\\n] It is worth noting that the increase in impact strength surpasses that of flexural strength, which can be attributed to the presence of flexible aliphatic chains that mitigate the network stiffness. Both the flexural and tensile strength show a consistent and noteworthy improvement from 106.1 up to 132 MPa, and 52.1 up to 81.2 MPa, respectively (Table ##SUPPL##0##S7##, Supporting Information). Notably, the tensile strain‐stress curves indicate that vitrimers are more stretchable than that of native thermoset epoxy (Figure ##SUPPL##0##S31##, Supporting Information). This finding holds great promise for the fabrication of carbon fiber reinforced polymers (CFRPs), where the stress should primarily reside in the fibers rather than the resins. This attribute ensures the exceptional mechanical strength of CFRPs (Section ##SUPPL##0##S2.6.3##, Supporting Information).
\",\n \"
In this contribution, both the strength and modulus depend on a tightly crosslinked network. The HBPPB, which terminates abundant ─OH groups, acts as a “rivet” within the network through covalent bonding and non‐covalent H‐bonding. The observed increase in E’ is also in agreement with the flexural results. Unfortunately, the optimal mechanical system does not maintain a consistent HBPPB concentration with the degradable and recyclable sample. Despite overloading HBPPB causing deteriorated mechanical strength, it still outperforms native EP. The reduction in impact and flexural strength can be attributed to the local agglomeration of hyperbranched polymers.[\\n##UREF##29##\\n45\\n##, ##UREF##30##\\n46\\n##\\n] Supplementary thermal and thermomechanical parameters are provided in Table ##SUPPL##0##S6## (Supporting Information). The vitrimers exhibit improved T\\nmax values and char yields, indicating superior thermal stability of HDCNs, which is primarily attributed to the presence of the inert boron component in the system.
\",\n \"
We next sought to probe the functional performance of materials. Good fire safety is a crucial consideration for real‐world applications of polymeric materials.[\\n##UREF##44##\\n80\\n##, ##UREF##45##\\n81\\n##\\n] Overall, our results demonstrate that the vitrimer sample exhibits superior fire safety performance compared to the native EP. Specifically, the vitrimer samples, EP‐9 and EP‐12, exhibit reduced thermal hazards and smoke production, along with increased resistance to ignition (higher limit oxygen index), and self‐extinguishing properties in an air atmosphere (Figure ##FIG##3##4g##). A comprehensive investigation was conducted through a full‐scale study of the fire‐retardant performance (Section ##SUPPL##0##S2.10## and Figure ##SUPPL##0##S41##, Supporting Information). The cone calorimeter test, which simulates a real fire scenario, provided crucial parameters to elucidate the flame‐retardant effect and mechanism.[\\n##UREF##46##\\n82\\n##\\n] Additionally, XPS analysis and Raman spectra (Figure ##SUPPL##0##S42##, Supporting Information) revealed the condensed fire‐retardant actions. The HDCNs constructed in our materials accumulate two types of flame‐retardant elements (phosphorus and boron) within the hyperbranched backbone for synergistic fire‐retarding action, ensuring the fire safety of the material.
\",\n \"
One of the facts is that the materials, specifically EP‐9 and EP‐12, exhibit significantly superior transparency and lighter coloration than their native thermoset. The transmittance of materials was measured using UV–vis spectrophotometer (See Figure ##SUPPL##0##S40##, Supporting Information). The average transmittance in both 280–380 nm (UV region) and 380–780 nm (visible region) was acquired in Figure ##FIG##3##4h##, showing significant increases in transmittance to both UV and visible light. The exceptional transparency of these materials can be attributed to the unique construction of HDCNs and the optical properties of HBPPB. Excitingly, this type of hyperbranched polymer has been reported as a class of non‐traditional aggregation‐induced emission luminogens (AIEgens),[\\n##UREF##47##\\n83\\n##, ##UREF##48##\\n84\\n##\\n] posing a promising pathway toward understanding the polymer crosslinking via fluorescent visualization methods. Besides, the exceptional transparency and lightness impart this polymeric material high added‐values for application in packaging materials, photoelectric materials, and flexible electronics.[\\n##UREF##49##\\n85\\n##, ##REF##22789123##\\n86\\n##\\n]\\n
In summary, we concept a hyperbranched dynamic crosslinked network (HDCNs) featuring multi‐dynamic bonds and topological crosslinking network architecture. Such a unique structure not only allows the penetration of solvent molecules for dynamic responsiveness, but also integrates exceptional mechanical strength, high transparency, and fire‐retardant functions into a single material. Through the construction of HDCNs, this work engineers a commercial petroleum‐based epoxy thermoset into a degradable and reconfigurable vitrimer by the breakages of dynamic units and the reconstruction of dynamic networks. The vitrimer showcases mild solvent degradation at room‐temperature and powder reprocessable performance, enabling the recovery of carbon fiber and resin powder from composite. More remarkably, we have made an exciting discovery that HDCNs display reconfigurable behavior via ester‐exchange triggered by ethanol, resulting in a flexible elastomer at 25 °C and a supra‐high flexural modulus of 5.45 GPa at ultralow temperature (−150 °C), thus offering the potential for extraordinary applications. The utilization of HDCNs as a building strategy opens up many inspirations toward designing and customizing sustainable polymeric materials, while also uncovering their fascinating material nature.
"],"string":"[\n \"Conclusion\",\n \"
In summary, we concept a hyperbranched dynamic crosslinked network (HDCNs) featuring multi‐dynamic bonds and topological crosslinking network architecture. Such a unique structure not only allows the penetration of solvent molecules for dynamic responsiveness, but also integrates exceptional mechanical strength, high transparency, and fire‐retardant functions into a single material. Through the construction of HDCNs, this work engineers a commercial petroleum‐based epoxy thermoset into a degradable and reconfigurable vitrimer by the breakages of dynamic units and the reconstruction of dynamic networks. The vitrimer showcases mild solvent degradation at room‐temperature and powder reprocessable performance, enabling the recovery of carbon fiber and resin powder from composite. More remarkably, we have made an exciting discovery that HDCNs display reconfigurable behavior via ester‐exchange triggered by ethanol, resulting in a flexible elastomer at 25 °C and a supra‐high flexural modulus of 5.45 GPa at ultralow temperature (−150 °C), thus offering the potential for extraordinary applications. The utilization of HDCNs as a building strategy opens up many inspirations toward designing and customizing sustainable polymeric materials, while also uncovering their fascinating material nature.
Degradation and reprocessing of thermoset polymers have long been intractable challenges to meet a sustainable future. Star strategies via dynamic cross‐linking hydrogen bonds and/or covalent bonds can afford reprocessable thermosets, but often at the cost of properties or even their functions. Herein, a simple strategy coined as hyperbranched dynamic crosslinking networks (HDCNs) toward in‐practice engineering a petroleum‐based epoxy thermoset into degradable, reconfigurable, and multifunctional vitrimer is provided. The special characteristics of HDCNs involve spatially topological crosslinks for solvent adaption and multi‐dynamic linkages for reversible behaviors. The resulting vitrimer displays mild room‐temperature degradation to dimethylacetamide and can realize the cycling of carbon fiber and epoxy powder from composite. Besides, they have supra toughness and high flexural modulus, high transparency as well as fire‐retardancy surpassing their original thermoset. Notably, it is noted in a chance‐following that ethanol molecule can induce the reconstruction of vitrimer network by ester‐exchange, converting a stiff vitrimer into elastomeric feature, and such material records an ultrahigh modulus (5.45 GPa) at −150 °C for their ultralow‐temperature condition uses. This is shaping up to be a potentially sustainable advanced material to address the post‐consumer thermoset waste, and also provide a newly crosslinked mode for the designs of high‐performance polymer.
","
Hyperbranched dynamic crosslinking networks (HDCNs) convert a thermoset network from permanently three‐dimensional‐structure to dynamically topological‐crosslinked architecture, programming a petroleum‐based thermoset into degradable, reconfigurable and multifunctional vitrimer. They are capable of room‐temperature degradation to solvent, and exhibit reconfigurability from stiff vitrimer to elastomer. Vitrimers also display enhanced modulus, toughness, flame‐retardancy, and high transparency when compared to native commodity thermosets.\n
Degradation and reprocessing of thermoset polymers have long been intractable challenges to meet a sustainable future. Star strategies via dynamic cross‐linking hydrogen bonds and/or covalent bonds can afford reprocessable thermosets, but often at the cost of properties or even their functions. Herein, a simple strategy coined as hyperbranched dynamic crosslinking networks (HDCNs) toward in‐practice engineering a petroleum‐based epoxy thermoset into degradable, reconfigurable, and multifunctional vitrimer is provided. The special characteristics of HDCNs involve spatially topological crosslinks for solvent adaption and multi‐dynamic linkages for reversible behaviors. The resulting vitrimer displays mild room‐temperature degradation to dimethylacetamide and can realize the cycling of carbon fiber and epoxy powder from composite. Besides, they have supra toughness and high flexural modulus, high transparency as well as fire‐retardancy surpassing their original thermoset. Notably, it is noted in a chance‐following that ethanol molecule can induce the reconstruction of vitrimer network by ester‐exchange, converting a stiff vitrimer into elastomeric feature, and such material records an ultrahigh modulus (5.45 GPa) at −150 °C for their ultralow‐temperature condition uses. This is shaping up to be a potentially sustainable advanced material to address the post‐consumer thermoset waste, and also provide a newly crosslinked mode for the designs of high‐performance polymer.
\",\n \"
Hyperbranched dynamic crosslinking networks (HDCNs) convert a thermoset network from permanently three‐dimensional‐structure to dynamically topological‐crosslinked architecture, programming a petroleum‐based thermoset into degradable, reconfigurable and multifunctional vitrimer. They are capable of room‐temperature degradation to solvent, and exhibit reconfigurability from stiff vitrimer to elastomer. Vitrimers also display enhanced modulus, toughness, flame‐retardancy, and high transparency when compared to native commodity thermosets.\\n
This work was sponsored by National Natural Science Foundation of China (Grants 21875188, 22175143, and 22022107), Key Research and Development Project of Shaanxi (Grants 2022GY‐353), Science Center for Gas Turbine Project (Grants P2022‐DB‐V‐001‐001), and Fundamental Research Funds for the Central Universities (D5000230114). Many thanks to the Analytical & Testing Center of Northwestern Polytechnical University for testing assistance.
","Data Availability Statement","
The data that support the findings of this study are available from the corresponding author upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
This work was sponsored by National Natural Science Foundation of China (Grants 21875188, 22175143, and 22022107), Key Research and Development Project of Shaanxi (Grants 2022GY‐353), Science Center for Gas Turbine Project (Grants P2022‐DB‐V‐001‐001), and Fundamental Research Funds for the Central Universities (D5000230114). Many thanks to the Analytical & Testing Center of Northwestern Polytechnical University for testing assistance.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available from the corresponding author upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
The conceptual basis of this work. a) The HDCNs forming strategy via a hyperbranched macromonomer bearing ─OH terminal and dynamic bonds to co‐crosslink with industrial thermoset epoxy. b) The schematic model describing HDCNs enables a strong and tough epoxy vitrimer with reconfigurable and degradable performance. c) Network features: from double topologically crosslinking modes to reduce network density for adapting solvents, as well as multiple dynamic linkages involving covalent PO3/BO3, ester bonds, and non‐covalent hydrogen bonds.
","
Degradation and cycling case of HDCNs. a,b) Samples immersed in DMAc for the solvent‐degradation varying the concentration of HBPPB. c) degradation product underwent drying and grinding into epoxy powder. d) cycling epoxy powder with virgin epoxy resin at a ratio of ≈1:5 for comparable mechanical strength. e) HDCNs braking modes: multiple cleavable units upon the HDCNs. f) IR spectrum before‐ and after‐ degradation. g–i) cycling case for end‐of‐use carbon fiber and epoxy resin from composite. j) Raman spectra of the recovered carbon fiber.
","
Reconstructed HDCNs enable a reconfigurable yet tough vitrimer. a) Samples immersed in ethanol for the reconstruction of HDCNs. b,c) digital photographs of the elastomeric vitrimer (EP‐9) versus rigid thermoset (EP). d,e) ethanol‐induced reconstruction of HDCNs from ester‐exchange. f) E’ showing an ultrahigh flexural modulus at −150 °C for low‐temperature application. g) FTIR spectra of the vitrimer before‐ and after‐ ethanol treatment. h) 2D temperature‐dependent FTIR upon heating from −50 to 150 °C. i) loss modulus (G’’) corresponds to double‐relaxation behaviors. J) Possible HDCNs reconstruction mechanism: reorganized H‐bonds and dynamic ester bonds.
","
General and multifunctional performance of material. DMA determination for a) temperature‐dependent loss modulus and b) storage modulus of materials. c) Crosslinking density showing the reduction in network density due to network topology. d) Simultaneously strengthening and toughening effect through HDCNs. e,f) Impact toughness and flexural strength improvement. g) Self‐extinguishing performance and reduced smoke production for fire‐retardancy. h) Digital photos of typical samples (EP, EP‐9, EP‐12 from left to right) and average transmittance in visible (220–380 nm) and UV region (380–780 nm).
"],"string":"[\n \"
The conceptual basis of this work. a) The HDCNs forming strategy via a hyperbranched macromonomer bearing ─OH terminal and dynamic bonds to co‐crosslink with industrial thermoset epoxy. b) The schematic model describing HDCNs enables a strong and tough epoxy vitrimer with reconfigurable and degradable performance. c) Network features: from double topologically crosslinking modes to reduce network density for adapting solvents, as well as multiple dynamic linkages involving covalent PO3/BO3, ester bonds, and non‐covalent hydrogen bonds.
\",\n \"
Degradation and cycling case of HDCNs. a,b) Samples immersed in DMAc for the solvent‐degradation varying the concentration of HBPPB. c) degradation product underwent drying and grinding into epoxy powder. d) cycling epoxy powder with virgin epoxy resin at a ratio of ≈1:5 for comparable mechanical strength. e) HDCNs braking modes: multiple cleavable units upon the HDCNs. f) IR spectrum before‐ and after‐ degradation. g–i) cycling case for end‐of‐use carbon fiber and epoxy resin from composite. j) Raman spectra of the recovered carbon fiber.
\",\n \"
Reconstructed HDCNs enable a reconfigurable yet tough vitrimer. a) Samples immersed in ethanol for the reconstruction of HDCNs. b,c) digital photographs of the elastomeric vitrimer (EP‐9) versus rigid thermoset (EP). d,e) ethanol‐induced reconstruction of HDCNs from ester‐exchange. f) E’ showing an ultrahigh flexural modulus at −150 °C for low‐temperature application. g) FTIR spectra of the vitrimer before‐ and after‐ ethanol treatment. h) 2D temperature‐dependent FTIR upon heating from −50 to 150 °C. i) loss modulus (G’’) corresponds to double‐relaxation behaviors. J) Possible HDCNs reconstruction mechanism: reorganized H‐bonds and dynamic ester bonds.
\",\n \"
General and multifunctional performance of material. DMA determination for a) temperature‐dependent loss modulus and b) storage modulus of materials. c) Crosslinking density showing the reduction in network density due to network topology. d) Simultaneously strengthening and toughening effect through HDCNs. e,f) Impact toughness and flexural strength improvement. g) Self‐extinguishing performance and reduced smoke production for fire‐retardancy. h) Digital photos of typical samples (EP, EP‐9, EP‐12 from left to right) and average transmittance in visible (220–380 nm) and UV region (380–780 nm).
Since the seminal work of Bloch in 1929,[\n##UREF##0##\n1\n##\n] electron–phonon coupling has emerged as one of the most important topics in modern condensed matter physics. In conventional superconductors, electron‐phonon coupling underlies Cooper pairing. In semiconductors, it sets an upper bound for electron mobility. Electron–phonon coupling also governs numerous thermal and spin relaxation processes in solids. In ionic crystals with f orbitals, spin‐orbit coupling and the crystal electric field (CEF) associated with the ionic or ligand environment split the electronic wavefunction eigenstates into manifolds. Atomic displacements can change the ligand environment and hence the orbital manifolds. This change is typically treated within the adiabatic Born–Oppenheimer approximation, in which atomic motion is slow relative to the electronic degrees of freedom. However, when phonon‐CEF coupling is nearly resonant, the Born–Oppenheimer approximation is inadequate and a new state—a vibronic bound state (VBS), with both orbital character and phonon character—can form.
","
Phonons can carry pseudo‐angular momentum when the 2D eigenvibration can be represented by a circular basis, as shown in Figure ##FIG##0##\n1a##.[\n##UREF##1##\n2\n##, ##REF##26406841##\n3\n##, ##UREF##2##\n4\n##, ##UREF##3##\n5\n##, ##UREF##4##\n6\n##, ##UREF##5##\n7\n##, ##REF##29420291##\n8\n##, ##UREF##6##\n9\n##\n] The conceptual difference between angular momentum and pseudo angular momentum is similar to that between linear momentum and pseudo linear momentum. The prefix pseudo is unnecessary for elementary particles in a vacuum and distinguishes that from motion in a medium, such as a lattice. Regular angular momentum exhibits rotational invariance under the rotation of the entire system while pseudo angular momentum is invariant under the rotation of the field.[\n##UREF##7##\n10\n##\n]\n
","
The ability to control angular‐momentum transfer between phonons and electronic degrees of freedom may unlock new opportunities in quantum information processing by providing new interfaces with spins in materials. However, the transfer of angular momentum between photons, electronic spin, orbital excitations, and phonons is still not well understood. It has been studied, for instance, in the context of paramagnetic spin relaxation,[\n##UREF##8##\n11\n##, ##UREF##9##\n12\n##\n] ultra‐fast demagnetization processes,[\n##REF##31501328##\n13\n##, ##UREF##10##\n14\n##\n] and recently angulon quasiparticles.[\n##UREF##11##\n15\n##\n] Phononic angular momentum is key to several fundamental effects in physics, including the microscopic explanation of the phonon Hall effect,[\n##REF##16241740##\n16\n##\n] the Einstein‐de Haas effect,[\n##UREF##12##\n17\n##, ##REF##30602792##\n18\n##, ##REF##35110761##\n19\n##\n] and zero‐point energies of chiral phonons.[\n##UREF##12##\n17\n##\n]\n
","
NaYbSe2\n[\n##UREF##13##\n20\n##, ##UREF##14##\n21\n##, ##UREF##15##\n22\n##, ##UREF##16##\n23\n##, ##UREF##17##\n24\n##\n] belongs to the family of the form A1 +Yb3 +X22− that have been identified as quantum spin liquid (QSL) candidates. 14 members of this family have been identified to date.[\n##UREF##14##\n21\n##, ##UREF##15##\n22\n##, ##UREF##16##\n23\n##, ##UREF##18##\n25\n##, ##UREF##19##\n26\n##, ##UREF##20##\n27\n##, ##UREF##21##\n28\n##, ##UREF##22##\n29\n##, ##UREF##23##\n30\n##, ##UREF##24##\n31\n##, ##UREF##25##\n32\n##, ##UREF##26##\n33\n##, ##UREF##27##\n34\n##, ##UREF##28##\n35\n##, ##UREF##29##\n36\n##, ##UREF##30##\n37\n##\n] They all have planar triangular lattices decorated by antiferromagnetically coupled spins making them susceptible to geometric frustration, resulting in a lack of long‐range magnetic order down to the lowest probed temperatures. This family of candidate QSLs is objectively less defect prone than Yb(Mg, Ga)O4,[\n##UREF##16##\n23\n##\n] and the breadth of substitutional composites in the family makes it an ideal platform for the study and control of QSL excitations.
","
The effective spin Seff = 1/2 of the system comes from the Yb3 + ion, which has the [Xe]4f13 electronic configuration. Yb3 +, like all the elements in the 4f block, has weak exchange coupling and strong spin‐orbit coupling compared to the 3d‐block elements. The ground state spin‐orbit manifold of Yb3 + has total spin J = 7/2. The next manifold J = 5/2 is more than an eV above the ground state manifold.[\n##UREF##31##\n38\n##, ##UREF##32##\n39\n##\n] The ground state spin‐orbit manifold J = 7/2 splits into four Kramers pairs |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, |ψ3±⟩, where the z‐component of the total angular momentum in the system can take values of mJ = ±1/2, ±3/2, ±5/2, and ±7/2. CEF1, CEF2, and CEF3 describe the transition between the excited doublets and the ground state |ψ0±⟩, respectively. The CEF splitting within the ground‐state manifold is typically only tens of meV due to the well‐shielded nature of the 4f electrons. Structurally, NaYbSe2 has less distortion in its YbSe6 octahedra than other members in the A1 +Yb3 +X22− family.[\n##UREF##18##\n25\n##\n] Its ground states are reported to be described by spinon excitations[\n##UREF##14##\n21\n##\n] or ferrimagnetic quasistatic and dynamic excitations within a QSL matrix.[\n##UREF##33##\n40\n##\n] While pristine NaYbSe2 is insulating, with increasing pressure it exhibits increased conductivity and eventually demonstrates a dip in resistivity attributed to a possible superconducting state.[\n##UREF##13##\n20\n##, ##UREF##17##\n24\n##\n]\n
","
Elementary excitations in solids like magnons, phonons, and CEFs are usually considered decoupled and determined independently. However, strong coupling between normally decoupled excitations can result in fundamentally new material functionality. Thalmeier and Fulde[\n##UREF##34##\n41\n##\n] first theoretically described how a bound state between a crystal field excitation and phonons may form, but VBSs have only been reported in a handful of intermetallics[\n##UREF##34##\n41\n##, ##REF##30894488##\n42\n##, ##REF##31107079##\n43\n##, ##UREF##35##\n44\n##, ##UREF##36##\n45\n##, ##REF##23003286##\n46\n##, ##UREF##37##\n47\n##\n] and oxides,[\n##REF##10044073##\n48\n##, ##UREF##38##\n49\n##, ##UREF##39##\n50\n##\n] including Tb2Ti2O7\n[\n##UREF##40##\n51\n##\n] another geometrically frustrated magnet whose quantum spin liquid state is not yet fully understood.[\n##REF##24483925##\n52\n##, ##UREF##41##\n53\n##\n] Here we report the presence of a VBS in NaYbSe2 and a change of angular momentum ΔJ\n\nz\n = ±1ℏ in an orbital excitation due to phononic coupling.
"],"string":"[\n \"Introduction\",\n \"
Since the seminal work of Bloch in 1929,[\\n##UREF##0##\\n1\\n##\\n] electron–phonon coupling has emerged as one of the most important topics in modern condensed matter physics. In conventional superconductors, electron‐phonon coupling underlies Cooper pairing. In semiconductors, it sets an upper bound for electron mobility. Electron–phonon coupling also governs numerous thermal and spin relaxation processes in solids. In ionic crystals with f orbitals, spin‐orbit coupling and the crystal electric field (CEF) associated with the ionic or ligand environment split the electronic wavefunction eigenstates into manifolds. Atomic displacements can change the ligand environment and hence the orbital manifolds. This change is typically treated within the adiabatic Born–Oppenheimer approximation, in which atomic motion is slow relative to the electronic degrees of freedom. However, when phonon‐CEF coupling is nearly resonant, the Born–Oppenheimer approximation is inadequate and a new state—a vibronic bound state (VBS), with both orbital character and phonon character—can form.
\",\n \"
Phonons can carry pseudo‐angular momentum when the 2D eigenvibration can be represented by a circular basis, as shown in Figure ##FIG##0##\\n1a##.[\\n##UREF##1##\\n2\\n##, ##REF##26406841##\\n3\\n##, ##UREF##2##\\n4\\n##, ##UREF##3##\\n5\\n##, ##UREF##4##\\n6\\n##, ##UREF##5##\\n7\\n##, ##REF##29420291##\\n8\\n##, ##UREF##6##\\n9\\n##\\n] The conceptual difference between angular momentum and pseudo angular momentum is similar to that between linear momentum and pseudo linear momentum. The prefix pseudo is unnecessary for elementary particles in a vacuum and distinguishes that from motion in a medium, such as a lattice. Regular angular momentum exhibits rotational invariance under the rotation of the entire system while pseudo angular momentum is invariant under the rotation of the field.[\\n##UREF##7##\\n10\\n##\\n]\\n
\",\n \"
The ability to control angular‐momentum transfer between phonons and electronic degrees of freedom may unlock new opportunities in quantum information processing by providing new interfaces with spins in materials. However, the transfer of angular momentum between photons, electronic spin, orbital excitations, and phonons is still not well understood. It has been studied, for instance, in the context of paramagnetic spin relaxation,[\\n##UREF##8##\\n11\\n##, ##UREF##9##\\n12\\n##\\n] ultra‐fast demagnetization processes,[\\n##REF##31501328##\\n13\\n##, ##UREF##10##\\n14\\n##\\n] and recently angulon quasiparticles.[\\n##UREF##11##\\n15\\n##\\n] Phononic angular momentum is key to several fundamental effects in physics, including the microscopic explanation of the phonon Hall effect,[\\n##REF##16241740##\\n16\\n##\\n] the Einstein‐de Haas effect,[\\n##UREF##12##\\n17\\n##, ##REF##30602792##\\n18\\n##, ##REF##35110761##\\n19\\n##\\n] and zero‐point energies of chiral phonons.[\\n##UREF##12##\\n17\\n##\\n]\\n
\",\n \"
NaYbSe2\\n[\\n##UREF##13##\\n20\\n##, ##UREF##14##\\n21\\n##, ##UREF##15##\\n22\\n##, ##UREF##16##\\n23\\n##, ##UREF##17##\\n24\\n##\\n] belongs to the family of the form A1 +Yb3 +X22− that have been identified as quantum spin liquid (QSL) candidates. 14 members of this family have been identified to date.[\\n##UREF##14##\\n21\\n##, ##UREF##15##\\n22\\n##, ##UREF##16##\\n23\\n##, ##UREF##18##\\n25\\n##, ##UREF##19##\\n26\\n##, ##UREF##20##\\n27\\n##, ##UREF##21##\\n28\\n##, ##UREF##22##\\n29\\n##, ##UREF##23##\\n30\\n##, ##UREF##24##\\n31\\n##, ##UREF##25##\\n32\\n##, ##UREF##26##\\n33\\n##, ##UREF##27##\\n34\\n##, ##UREF##28##\\n35\\n##, ##UREF##29##\\n36\\n##, ##UREF##30##\\n37\\n##\\n] They all have planar triangular lattices decorated by antiferromagnetically coupled spins making them susceptible to geometric frustration, resulting in a lack of long‐range magnetic order down to the lowest probed temperatures. This family of candidate QSLs is objectively less defect prone than Yb(Mg, Ga)O4,[\\n##UREF##16##\\n23\\n##\\n] and the breadth of substitutional composites in the family makes it an ideal platform for the study and control of QSL excitations.
\",\n \"
The effective spin Seff = 1/2 of the system comes from the Yb3 + ion, which has the [Xe]4f13 electronic configuration. Yb3 +, like all the elements in the 4f block, has weak exchange coupling and strong spin‐orbit coupling compared to the 3d‐block elements. The ground state spin‐orbit manifold of Yb3 + has total spin J = 7/2. The next manifold J = 5/2 is more than an eV above the ground state manifold.[\\n##UREF##31##\\n38\\n##, ##UREF##32##\\n39\\n##\\n] The ground state spin‐orbit manifold J = 7/2 splits into four Kramers pairs |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, |ψ3±⟩, where the z‐component of the total angular momentum in the system can take values of mJ = ±1/2, ±3/2, ±5/2, and ±7/2. CEF1, CEF2, and CEF3 describe the transition between the excited doublets and the ground state |ψ0±⟩, respectively. The CEF splitting within the ground‐state manifold is typically only tens of meV due to the well‐shielded nature of the 4f electrons. Structurally, NaYbSe2 has less distortion in its YbSe6 octahedra than other members in the A1 +Yb3 +X22− family.[\\n##UREF##18##\\n25\\n##\\n] Its ground states are reported to be described by spinon excitations[\\n##UREF##14##\\n21\\n##\\n] or ferrimagnetic quasistatic and dynamic excitations within a QSL matrix.[\\n##UREF##33##\\n40\\n##\\n] While pristine NaYbSe2 is insulating, with increasing pressure it exhibits increased conductivity and eventually demonstrates a dip in resistivity attributed to a possible superconducting state.[\\n##UREF##13##\\n20\\n##, ##UREF##17##\\n24\\n##\\n]\\n
\",\n \"
Elementary excitations in solids like magnons, phonons, and CEFs are usually considered decoupled and determined independently. However, strong coupling between normally decoupled excitations can result in fundamentally new material functionality. Thalmeier and Fulde[\\n##UREF##34##\\n41\\n##\\n] first theoretically described how a bound state between a crystal field excitation and phonons may form, but VBSs have only been reported in a handful of intermetallics[\\n##UREF##34##\\n41\\n##, ##REF##30894488##\\n42\\n##, ##REF##31107079##\\n43\\n##, ##UREF##35##\\n44\\n##, ##UREF##36##\\n45\\n##, ##REF##23003286##\\n46\\n##, ##UREF##37##\\n47\\n##\\n] and oxides,[\\n##REF##10044073##\\n48\\n##, ##UREF##38##\\n49\\n##, ##UREF##39##\\n50\\n##\\n] including Tb2Ti2O7\\n[\\n##UREF##40##\\n51\\n##\\n] another geometrically frustrated magnet whose quantum spin liquid state is not yet fully understood.[\\n##REF##24483925##\\n52\\n##, ##UREF##41##\\n53\\n##\\n] Here we report the presence of a VBS in NaYbSe2 and a change of angular momentum ΔJ\\n\\nz\\n = ±1ℏ in an orbital excitation due to phononic coupling.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results and Discussion","
We first verify the primary CEF excitations and phonon modes with temperature‐dependent Raman spectroscopy. The temperature dependence of the relevant Raman features is depicted in Figure ##FIG##0##1c## while the full spectra are shown in Figures ##SUPPL##0##S2## and ##SUPPL##0##S3##, Supporting Information. Consistent with earlier neutron scattering and Raman reports,[\n##UREF##16##\n23\n##, ##UREF##28##\n35\n##\n] strong CEF modes are observed at 117.2 cm−1 (CEF1), 197.8 cm−1 (CEF2), and 247.0 cm−1 (CEF3). These become significantly stronger in intensity and soften (shift toward lower energy) as the temperature decreases. NaYbSe2 also has two Raman‐active phonon modes: E\ng at 124.4 cm−1 and A\n1g at 172.8 cm−1 within this frequency space. The intensity and energy of these phonon modes exhibit a substantially weaker temperature dependence. Figure ##FIG##0##1b## shows a classical spinning top representation of the spin‐orbit eigenstates for |ψ0+⟩ as well as the relative weights cmj for |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, |ψ3±⟩ as |ψ〉= ∑mj=−72,…72cmj|72,mj⟩. Empty (filled) circles represent cmj=0 (cmj≠0). The blue (pink) cones represent the + (−) branch. Note that the E\ng and CEF1 modes exist at similar energies. This is the case for NaYbSe2,[\n##UREF##16##\n23\n##\n] CsYbSe2,[\n##UREF##28##\n35\n##\n] and KYbSe2.[\n##UREF##30##\n37\n##\n] Using Bayesian inference with a Hamiltonian Monte Carlo model in PyMC3,[\n##UREF##42##\n54\n##\n] we track the peak parameters from the experimental data shown in Figure ##FIG##0##1c##. The extracted peak positions for CEF1 and E\ng are shown in Figure ##FIG##0##1d##. The symbols represent the median values from the Bayesian inference. The darker shaded bands illustrate the 68% (corresponding to 1σ in the central limit) highest density intervals (HDIs), and the lighter shaded bands illustrate the 95% (corresponding to 2σ) HDIs. Other selected modes are described in the Supporting Information. The model has larger error bars from T = 175 to 255 K, but the modes are well resolved elsewhere.
","
A previously unreported mode is clearly seen in our data at 151.2 cm−1. Labeled ω in Figure ##FIG##0##1c,d##, this mode has characteristics in common with both CEFs and phonons: ω has a significantly stronger structure factor at low temperatures, just as the CEFs do, but it does not exhibit the strong softening at lower temperatures that the CEF modes exhibit. Instead, it hardens in a manner consistent with slight phonon hardening in NaYbSe2 at low temperatures. We therefore assign ω as a VBS with parent states CEF1 and E\ng. Earlier Raman spectra in this temperature range[\n##UREF##16##\n23\n##\n] were only helicity‐resolved at room temperature, where ω is weak.
","
In order to better understand the origin of the ω mode, we calculated the phonon dispersion relationship for NaYbSe2 using density functional theory (DFT) and Perdew–Burke–Ernzerhof (PBE) exchange energy, as shown in Figure ##SUPPL##0##S1##, Supporting Information. These calculations suggest that no optical or acoustic phonons are present near ω. If anything, PBE normally overestimates the in‐plane lattice constants, resulting in softer in‐plane vibrations (E\ng) than those measured. Comparing these calculations to published data (and the data in this manuscript) the calculated E\ng at the gamma point is indeed 15 cm−1 softer than that measured. The A\n1g mode is also calculated to be softer than the measured mode, 135 cm−1 compared to the measured 172.8 cm−1. Between these two energies, there are very few modes at the gamma point, and halfway between them there are no phonon branches.
","
In fact, most models have no phonons throughout the Brillouin zone around 150 cm−1. Additionally, published neutron scattering data suggests there are no phonons in this frequency space. Specifically, figure S3J in ref. [##UREF##14##21##] reveals a decrease in spectral weight as the temperature increases. Additionally, Zhang et al.[\n##UREF##16##\n23\n##\n] could not model extra spectral weight at ≈19 meV (see figure ##FIG##1##2## of that manuscript) when the temperature decreased below 100 K. Notably, these neutron data have momentum transfer dependence like a phonon, but the temperature dependence of a crystal field, consistent with a vibronic bound state.
","
The mode ω is present in both NaYbSe2 and CsYbSe2, although it is far less prominent in CsYbSe2.[\n##UREF##28##\n35\n##\n] This is likely because in NaYbSe2, Na is both smaller and lighter. The lattice is therefore more tightly‐packed. The lighter Na also has larger vibrational displacements. Both effects increase the coupling strength, resulting in a VBS far stronger than that in CsYbSe2. Fitting to the Thalmeier–Fulde description of a magnetoelastic vibronic bound state[\n##UREF##34##\n41\n##\n] yields a coupling strength of 32.0 cm−1 (3.97 meV) for NaYbSe2, which is stronger than the 23.6 cm−1 (2.93 meV) coupling strength reported for CsYbSe2 and comparable to the roughly 34.0 cm−1 (4.22 meV) coupling strength reported for Ce2O3.[\n##REF##31107079##\n43\n##\n]\n
","
To gain a deeper understanding of the CEF and the ω Raman modes, we studied the helicity dependence and magnetic field dependence of Raman spectra of NaYbSe2. During the Raman scattering process, the energy transferred from the photons can be used to excite a variety of (quasi)particles in the system, including electronic orbitals, phonons, and their hybridized states, as indicated by the schematic illustration in Figure ##FIG##1##\n2a##. Figure ##FIG##1##2b## shows helicity‐resolved Raman spectra acquired at T = 4 K, with CEF1, CEF2, CEF3, E\ng, A\n1g, and ω highlighted. Here, we can simultaneously observe electronic orbital excitations (CEF1, CEF2, and CEF3), phononic excitations (E\ng and A\n1g), and the entangled state ω between the orbital and phononic excitations by low‐temperature Raman spectroscopy. Furthermore, we discovered that these (quasi)particles exhibit different responses to the helicity of photons. While A\n1g and ω (along with the Rayleigh scattering; see Figure ##SUPPL##0##S7##, Supporting Information) are stronger in the co‐circular polarization configuration, CEF1–CEF3 and E\ng are stronger in the cross‐circular polarization configuration.
","
We also performed magnetic field‐dependent Raman spectroscopy with B || c, which lifts the degeneracy within each of the Kramers pairs and therefore provides further information on the orbital excitations corresponding to CEF1–3. Magnetization and specific heat measurements have been previously used to show that no field‐induced ordering is present in NaYbSe2 for B || c for B < 9 T at T = 4 K.[\n##UREF##15##\n22\n##\n] Therefore, with the 6 T magnetic fields accessible here, no field‐induced transition is expected. Figure ##FIG##2##\n3\n## shows the magnetic field dependence of the CEF levels for CEF1 (Figure ##FIG##2##3a##), CEF2 (Figure ##FIG##2##3b##), ω (Figure ##FIG##2##3c##), and CEF3 (Figure ##FIG##2##3d##) measurements. Again, the CEF levels only show up in cross‐circular polarization configurations, (σ+, σ−) and (σ−, σ+), while the ω mode only appears in co‐circular polarization configurations, (σ+, σ+) and (σ−, σ−). Note that in the (σ+, σ−) configuration and with increasing positive magnetic field, CEF1 and CEF2 increase in energy while CEF3 decreases. This dependence is inverted if the magnetic field is reversed (B → −B) or the helicity is reversed ((σ+, σ−) → (σ−, σ+)).
","
The observed circular polarization dependence and magnetic field dependence of CEF1–3 and ω in Figures ##FIG##1##2## and ##FIG##2##3## clearly indicate different underlying optical selection rules that can be understood based on angular momentum conservation. Each left (right) circularly polarized σ+ (σ−) photon carries angular momentum +ℏ (−ℏ).[\n##UREF##43##\n55\n##\n] The link between helicity and circular polarization is given by h = σ · k, where k is the momentum of the photon, and h the helicity.[\n##REF##29956970##\n56\n##\n] Absorption of a σ+ photon increases the angular momentum of the system by +ℏ, corresponding to being acted on by operator J\n+, while scattering a σ+ photon corresponds to J\n− acting on the system. Therefore, if one considers Raman spectra acquired in the (σincident, σscatter) = (σ+, σ−) configuration where the change of angular momentum in photons is ΔJ\nphoton = −2ℏ, the system could obtain +2ℏ of angular momentum from the photons (i.e., ΔJ\nsystem = +2ℏ), corresponding to J\n+\nJ\n+ acting on the system (a generalization of the closure condition in Axe et al.,[\n##UREF##44##\n57\n##\n] which contracts ∑virtual||〈ψfinal|M\n\ni\n|ψvirtual〉〈ψvirtual|M\n\nj\n|ψinitial〉|| to ||〈ψfinal|M\n\ni\n\nM\n\nj\n|ψinitial〉|| for any dipolar transition M\n\ni\n, M\n\nj\n). Therefore, a mode that is active in the (σ+, σ−) configuration could have dominant matrix‐element contributions from ||〈ψfinal|J\n+\nJ\n+|ψinitial〉||. However, it is important to note that in an analog to the Umklapp process, the threefold rotational symmetry in NaYbSe2\n[\n##UREF##45##\n58\n##\n] allows for discrete angular momentum conservation as long as |ΔJ\nphoton + ΔJ\nsystem|/ℏ = 0 (modulo 3). In other words, the angular momentum conservation rule is relaxed so that the total change of angular momentum can be either zero or a multiple of 3ℏ due to the threefold rotational symmetry.[\n##UREF##46##\n59\n##, ##UREF##47##\n60\n##, ##UREF##48##\n61\n##\n] This means that in the (σ+, σ−) configuration where ΔJ\nphoton = −2ℏ, ΔJ\nsystem can be +2ℏ or −ℏ to satisfy the relaxed angular momentum conservation rule. For ΔJ\nsystem = +2ℏ, it corresponds to the operation of J\n+\nJ\n+, while for ΔJ\nsystem = −ℏ, it corresponds to J\n−. Therefore, a mode that is active in the (σ+, σ−) configuration could also have dominant matrix‐element contributions from ||〈ψfinal|J\n−|ψinitial〉||. In contrast, in the (σincident, (σ−, σ+) configuration where ΔJ\nphoton = +2ℏ, the system could lose 2ℏ of angular momentum to the photons (ΔJ\nsystem = −2ℏ), corresponding to J\n−\nJ\n− acting on the system, or ΔJ\nsystem = +ℏ, corresponding to J\n+ acting on the system. Hence, a mode that is active in the (σ−, σ+) configuration should have dominant matrix‐element contributions from ||〈ψfinal|J\n−\nJ\n−|ψinitial〉|| or ||〈ψfinal|J\n+|ψinitial〉||.
","
Since all the CEF modes are enhanced in the cross‐circular channel and suppressed in the co‐circular channel, all of them should have a finite change of angular momentum after the orbital transitions. As discussed above, ΔJ\nCEF = +2ℏ or −ℏ in the (σ+, σ−) configuration, while ΔJ\nCEF = −2ℏ or +ℏ in the (σ−, σ+) configuration. Considering that an additional Zeeman dependence H\n\nB\n = −g\n\nJ\nμB\nJ\n\nz\n · B is added when a magnetic field is applied, the change of the sign of ΔJ\nCEF from (σ+, σ−) to (σ−, σ+) explains why the magnetic field dependence of CEF1–3 is inverted when the helicity is reversed in Figure ##FIG##2##3##. Another intriguing finding from Figure ##FIG##2##3## is that the magnetic field dependence of CEF1 and CEF2 is opposite to that of CEF3 regardless of the helicity, suggesting that ΔJ\nCEF1 and ΔJ\nCEF2 share the same sign, whereas ΔJ\nCEF3 has the opposite sign. More specifically, in the (σ+, σ−) configuration, we should have ΔJ\nCEF1 = ΔJ\nCEF2 = +2ℏ while ΔJ\nCEF3 = −ℏ, or ΔJ\nCEF1 = ΔJ\nCEF2 = −ℏ while ΔJ\nCEF3 = +2ℏ. Note that ΔJ = −ℏ is allowed for a CEF level due to the threefold rotational symmetry that relaxes the angular momentum conservation rule, but we expect that ΔJ = +2ℏ that satisfies the strict angular momentum conservation should have a higher probability and stronger Raman signals. Since CEF1 and CEF2 have much stronger Raman intensities than CEF3 (Figure ##FIG##1##2b##), it is natural to conclude that ΔJ\nCEF1 = ΔJ\nCEF2 = +2ℏ while ΔJ\nCEF3 = −ℏ in the (σ+, σ−) configuration. Similarly, ΔJ\nCEF1 = ΔJ\nCEF2 = −2ℏ while ΔJ\nCEF3 = +ℏ in the (σ−, σ+) configuration. In terms of matrix operations, ⟨ψ1±|J+J+|ψ0±⟩, ⟨ψ1±|J−J−|ψ0±⟩, ⟨ψ2±|J+J+|ψ0±⟩, ⟨ψ2±|J−J−|ψ0±⟩, ⟨ψ3±|J−|ψ0±⟩, and ⟨ψ3±|J+|ψ0±⟩ should be significant while ⟨ψ3±|J+J+|ψ0±⟩, ⟨ψ3±|J−J−|ψ0±⟩, ⟨ψ1±|J+J−|ψ0±⟩, ⟨ψ1±|J−J+|ψ0±⟩, ⟨ψ2±|J+J−|ψ0±⟩, ⟨ψ2±|J−J+|ψ0±⟩, ⟨ψ3±|J+J−|ψ0±⟩, and ⟨ψ3±|J−J+|ψ0±⟩ should all be small or zero (the last six matrix elements correspond to the co‐circular polarization).
","
As shown in Figure ##FIG##0##1b##, the eigenstates of CEF levels are linear combinations of multiplets m\n\nJ\n = −7/2…7/2: |ψ±〉 = ∑mj=−72,…72cmj±|72,mj⟩. Typically, CEF parameters are fit to a CEF Hamiltonian with Stevens operators given by the symmetry of the ionic environment of the 4f orbitals, as described in the Experimental Section. The procedure is frequently under‐constrained, and there are enough degrees of freedom to fit experimentally observed CEF energy levels with more than one set of CEF parameters that minimize the error that may exist. The order of the eigenstates may not be the same across those sets. However, once the constraints imposed by the optical selection rules discussed above are taken into account, we are able to find a set of CEF parameters, as indicated by filled and empty circles in Figure ##FIG##0##1b##, that can explain the observed helicity and magnetic field dependence in Figures ##FIG##1##2## and ##FIG##2##3##, and we can make final assignments of CEF1‐3. As illustrated in Figure ##FIG##3##\n4a##, CEF1 comes from |ψ0+⟩→|ψ1+⟩ in the (σ+, σ−) configuration, where ΔJ\nphoton = −2ℏ and ΔJ\nCEF1 = +2ℏ, or |ψ0−⟩→|ψ1−⟩ in the (σ−, σ+) configuration, where ΔJ\nphoton = +2ℏ and ΔJ\nCEF1 = −2ℏ (see the arrows for the transitions). We note that the transition of |ψ0+⟩→|ψ1−⟩ with ΔJ\nCEF1 = −ℏ is allowed in (σ+, σ−) and the transition of |ψ0−⟩→|ψ1+⟩ with ΔJ\nCEF1 = +ℏ is allowed in (σ−, σ+), since ΔJ\ntotal = −3ℏ and +3ℏ, respectively, which satisfies the discrete angular momentum conservation. However, these transitions allowed by the crystal threefold rotational symmetry are of a higher order and should be much weaker. Similar transitions apply for CEF2, although they correspond to |ψ0+⟩→|ψ2+⟩ or |ψ0−⟩→|ψ2−⟩. For CEF3 in Figure ##FIG##3##4b##, however, any transition with ΔJ = −2ℏ or +2ℏ is impossible; hence, the transition of |ψ0−⟩→|ψ3−⟩ with ΔJ\nCEF3 = −ℏ is the only one allowed in (σ+, σ−) where ΔJ\ntotal = −3ℏ, and the transition of |ψ0+⟩→|ψ3+⟩ with ΔJ\nCEF3 = +ℏ is the only one allowed in (σ−, σ+) where ΔJ\ntotal = +3ℏ. This could explain why CEF3 is much weaker than CEF1 and CEF2.
","
As for the phonon Raman modes, in general, a nondegenerate Γ‐point phonon (like the A\n1g Raman mode in NaYbSe2) cannot have angular momentum since the eigenvector is a real number. It is therefore only observed in the co‐circular polarization configuration, as shown in Figure ##FIG##1##2b##. However, when the Γ‐point phonon is doubly degenerate, the two real eigenvectors can be reconstructed by a complex superposition as shown in Figure ##FIG##0##1a##. Therefore, doubly degenerate modes like the E\ng Raman mode in NaYbSe2 can have (pseudo)angular momentum of ±ℏ.[\n##REF##26406841##\n3\n##, ##UREF##6##\n9\n##, ##UREF##47##\n60\n##, ##UREF##49##\n62\n##\n] With nonzero angular momentum, it can change the chirality of incident photons and appear in the cross‐circular polarization configuration. As shown in Figure ##FIG##3##4c##, in the (σ+, σ−) configuration, ΔJ\nphoton = −2ℏ and ΔJEg,−=−ℏ, so it is allowed by the discrete angular momentum conservation as ΔJ\ntotal = −3ℏ; In the (σ−, σ+) configuration, ΔJ\nphoton = +2ℏ and ΔJEg,+=+ℏ, giving rise to ΔJ\ntotal = +3ℏ. Such an optical selection rule is similar to that shown for CEF3 in Figure ##FIG##3##4b##, which explains why both are observed in the cross‐circular polarization. Similar results have been reported for E symmetry Raman modes in TMDs, CrBr3, and quartz, among other materials.[\n##UREF##6##\n9\n##, ##UREF##47##\n60\n##, ##UREF##48##\n61\n##\n]\n
","
Finally, moving on to the VBS ω, it is much stronger in the co‐circular polarization configuration and its magnetic field dependence is weak (Figures ##FIG##1##2## and ##FIG##2##3##), in stark contrast to CEF1‐3. This indicates that the change in angular momentum for ω is zero: ΔJ\n\nz\n = 0. Since it is a coupled state between the CEF1 and the E\ng mode, we consider the 2 × ~2 dimensional space |ψ1±⟩⊗|Eg,±⟩. As the angular momentum change ΔJ\n\nz\n for CEF1 is finite (Figure ##FIG##3##4a##), the phonon subsystem has to carry additional angular momentum (Figure ##FIG##3##4c##) in order to yield zero angular momentum for ω. Therefore, the circular basis {$E$g, +, $E$g, $‐$} is the natural choice for describing the eigenvibration. As discussed previously, CEF1 can arise from four possible transitions while E\ng can be $E$g, + and $E$g, $‐$, which gives rise to a total of eight possible transitions for ω. Based on the angular momentum conservation rule, however, we found that two states, ω+=|ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and ω−=|ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, are the most probable (see more details in the Supporting Information). As shown in Figure ##FIG##3##4d##, due to the angular momentum transfer between the CEF1 and E\ng mode, the excited state of ω, |ψ1+⟩⊗|Eg,+⟩ or |ψ1−⟩⊗|Eg,−⟩, has the z‐component of the angular momentum effectively raised or lowered by ℏ compared to the |ψ1+⟩ or |ψ1−⟩ shown in Figure ##FIG##3##4a##, respectively. As a result, the transitions of |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have ΔJ\n\nz\n = 0 to satisfy the selection rule in the co‐circular polarization configuration and explain the weak magnetic field dependence.
","
\nFigure ##FIG##4##\n5a,b## illustrates the two states, |ψ1+⟩⊗|Eg,+⟩ and |ψ1−⟩⊗|Eg,−⟩ based on the observations described above. In Figure ##FIG##4##5a##, when the ground state is |ψ0+⟩ (shown in light pink), the J\n+\nJ\n− or J\n−\nJ\n+ operator excites the system to the + branch, |ψ1+⟩⊗|Eg,+⟩ (where the CEF excitation corresponds to the + branch and the phononic excitation corresponds to + angular momentum). Figure ##FIG##4##5b## shows the same effect for |ψ0−⟩ to |ψ1−⟩⊗|Eg,−⟩. Because the transition is degenerate for |ψ0∓⟩→ω± with J\n+\nJ\n− or J\n−\nJ\n+, this eigenspace, in principle, can be transduced from a QSL ground state. If the QSL ground state is written as ∑j∏iψ0σij – where i is the site index, j the configuration index, and σ\nij\n = +, − is the branch—then J\n+\nJ\n− or J\n−\nJ\n+ should bring the system into ∑j∏iωσij within the point spread volume of the excitation beam, transducing the possible ground state entanglement to the VBS ω±.
","
It is worth noting that, in the original Thalmeier–Fulde model, two VBS modes are expected to emerge from the hybridization of a CEF mode and a phonon mode.[\n##UREF##34##\n41\n##\n] Besides the two most probable VBS transitions shown in Figure ##FIG##3##4d## that give rise to the observed ω peak in the Raman spectra, there are six other transitions that could contribute to the other VBS mode that is not observed in our Raman measurements (see Equation (##SUPPL##0##S1##), Supporting Information). Since the picture of how orbital and phononic excitations couple is not entirely clear, a definitive answer to why other transitions and the other VBS mode do not appear in Raman scattering is beyond the scope of this work. It is expected that the electron–phonon coupling may be more complicated compared to the conventional coupling between orbital angular momentum and spin angular momentum.[\n##UREF##50##\n63\n##\n] Therefore, we hypothesize that the coupling between the CEF1 and the Eg mode is not the same as the conventional coupling between orbital and spin angular momenta. There is a coupling coefficient before the derived ΔJ\nω (see Equation (##SUPPL##0##S1##), Supporting Information), leading to non‐integer ΔJ\nω unless it is zero to begin with. As a consequence, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩, |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, |ψ0+⟩→|ψ1−⟩⊗|Eg,+⟩, and |ψ0−⟩→|ψ1+⟩⊗|Eg,−⟩ that have zero angular momentum can satisfy the angular momentum conservation rule in the co‐circular polarization configuration, and other transitions are forbidden in any polarization channel. Moreover, as discussed previously, since |ψ0+⟩→|ψ1−⟩ and |ψ0−⟩→|ψ1+⟩ are of a higher order and much less probable, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have detectable signals (see Figure ##FIG##3##4d##). This could explain why other transitions and the other VBS modes are not observed.
"],"string":"[\n \"Results and Discussion\",\n \"
We first verify the primary CEF excitations and phonon modes with temperature‐dependent Raman spectroscopy. The temperature dependence of the relevant Raman features is depicted in Figure ##FIG##0##1c## while the full spectra are shown in Figures ##SUPPL##0##S2## and ##SUPPL##0##S3##, Supporting Information. Consistent with earlier neutron scattering and Raman reports,[\\n##UREF##16##\\n23\\n##, ##UREF##28##\\n35\\n##\\n] strong CEF modes are observed at 117.2 cm−1 (CEF1), 197.8 cm−1 (CEF2), and 247.0 cm−1 (CEF3). These become significantly stronger in intensity and soften (shift toward lower energy) as the temperature decreases. NaYbSe2 also has two Raman‐active phonon modes: E\\ng at 124.4 cm−1 and A\\n1g at 172.8 cm−1 within this frequency space. The intensity and energy of these phonon modes exhibit a substantially weaker temperature dependence. Figure ##FIG##0##1b## shows a classical spinning top representation of the spin‐orbit eigenstates for |ψ0+⟩ as well as the relative weights cmj for |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, |ψ3±⟩ as |ψ〉= ∑mj=−72,…72cmj|72,mj⟩. Empty (filled) circles represent cmj=0 (cmj≠0). The blue (pink) cones represent the + (−) branch. Note that the E\\ng and CEF1 modes exist at similar energies. This is the case for NaYbSe2,[\\n##UREF##16##\\n23\\n##\\n] CsYbSe2,[\\n##UREF##28##\\n35\\n##\\n] and KYbSe2.[\\n##UREF##30##\\n37\\n##\\n] Using Bayesian inference with a Hamiltonian Monte Carlo model in PyMC3,[\\n##UREF##42##\\n54\\n##\\n] we track the peak parameters from the experimental data shown in Figure ##FIG##0##1c##. The extracted peak positions for CEF1 and E\\ng are shown in Figure ##FIG##0##1d##. The symbols represent the median values from the Bayesian inference. The darker shaded bands illustrate the 68% (corresponding to 1σ in the central limit) highest density intervals (HDIs), and the lighter shaded bands illustrate the 95% (corresponding to 2σ) HDIs. Other selected modes are described in the Supporting Information. The model has larger error bars from T = 175 to 255 K, but the modes are well resolved elsewhere.
\",\n \"
A previously unreported mode is clearly seen in our data at 151.2 cm−1. Labeled ω in Figure ##FIG##0##1c,d##, this mode has characteristics in common with both CEFs and phonons: ω has a significantly stronger structure factor at low temperatures, just as the CEFs do, but it does not exhibit the strong softening at lower temperatures that the CEF modes exhibit. Instead, it hardens in a manner consistent with slight phonon hardening in NaYbSe2 at low temperatures. We therefore assign ω as a VBS with parent states CEF1 and E\\ng. Earlier Raman spectra in this temperature range[\\n##UREF##16##\\n23\\n##\\n] were only helicity‐resolved at room temperature, where ω is weak.
\",\n \"
In order to better understand the origin of the ω mode, we calculated the phonon dispersion relationship for NaYbSe2 using density functional theory (DFT) and Perdew–Burke–Ernzerhof (PBE) exchange energy, as shown in Figure ##SUPPL##0##S1##, Supporting Information. These calculations suggest that no optical or acoustic phonons are present near ω. If anything, PBE normally overestimates the in‐plane lattice constants, resulting in softer in‐plane vibrations (E\\ng) than those measured. Comparing these calculations to published data (and the data in this manuscript) the calculated E\\ng at the gamma point is indeed 15 cm−1 softer than that measured. The A\\n1g mode is also calculated to be softer than the measured mode, 135 cm−1 compared to the measured 172.8 cm−1. Between these two energies, there are very few modes at the gamma point, and halfway between them there are no phonon branches.
\",\n \"
In fact, most models have no phonons throughout the Brillouin zone around 150 cm−1. Additionally, published neutron scattering data suggests there are no phonons in this frequency space. Specifically, figure S3J in ref. [##UREF##14##21##] reveals a decrease in spectral weight as the temperature increases. Additionally, Zhang et al.[\\n##UREF##16##\\n23\\n##\\n] could not model extra spectral weight at ≈19 meV (see figure ##FIG##1##2## of that manuscript) when the temperature decreased below 100 K. Notably, these neutron data have momentum transfer dependence like a phonon, but the temperature dependence of a crystal field, consistent with a vibronic bound state.
\",\n \"
The mode ω is present in both NaYbSe2 and CsYbSe2, although it is far less prominent in CsYbSe2.[\\n##UREF##28##\\n35\\n##\\n] This is likely because in NaYbSe2, Na is both smaller and lighter. The lattice is therefore more tightly‐packed. The lighter Na also has larger vibrational displacements. Both effects increase the coupling strength, resulting in a VBS far stronger than that in CsYbSe2. Fitting to the Thalmeier–Fulde description of a magnetoelastic vibronic bound state[\\n##UREF##34##\\n41\\n##\\n] yields a coupling strength of 32.0 cm−1 (3.97 meV) for NaYbSe2, which is stronger than the 23.6 cm−1 (2.93 meV) coupling strength reported for CsYbSe2 and comparable to the roughly 34.0 cm−1 (4.22 meV) coupling strength reported for Ce2O3.[\\n##REF##31107079##\\n43\\n##\\n]\\n
\",\n \"
To gain a deeper understanding of the CEF and the ω Raman modes, we studied the helicity dependence and magnetic field dependence of Raman spectra of NaYbSe2. During the Raman scattering process, the energy transferred from the photons can be used to excite a variety of (quasi)particles in the system, including electronic orbitals, phonons, and their hybridized states, as indicated by the schematic illustration in Figure ##FIG##1##\\n2a##. Figure ##FIG##1##2b## shows helicity‐resolved Raman spectra acquired at T = 4 K, with CEF1, CEF2, CEF3, E\\ng, A\\n1g, and ω highlighted. Here, we can simultaneously observe electronic orbital excitations (CEF1, CEF2, and CEF3), phononic excitations (E\\ng and A\\n1g), and the entangled state ω between the orbital and phononic excitations by low‐temperature Raman spectroscopy. Furthermore, we discovered that these (quasi)particles exhibit different responses to the helicity of photons. While A\\n1g and ω (along with the Rayleigh scattering; see Figure ##SUPPL##0##S7##, Supporting Information) are stronger in the co‐circular polarization configuration, CEF1–CEF3 and E\\ng are stronger in the cross‐circular polarization configuration.
\",\n \"
We also performed magnetic field‐dependent Raman spectroscopy with B || c, which lifts the degeneracy within each of the Kramers pairs and therefore provides further information on the orbital excitations corresponding to CEF1–3. Magnetization and specific heat measurements have been previously used to show that no field‐induced ordering is present in NaYbSe2 for B || c for B < 9 T at T = 4 K.[\\n##UREF##15##\\n22\\n##\\n] Therefore, with the 6 T magnetic fields accessible here, no field‐induced transition is expected. Figure ##FIG##2##\\n3\\n## shows the magnetic field dependence of the CEF levels for CEF1 (Figure ##FIG##2##3a##), CEF2 (Figure ##FIG##2##3b##), ω (Figure ##FIG##2##3c##), and CEF3 (Figure ##FIG##2##3d##) measurements. Again, the CEF levels only show up in cross‐circular polarization configurations, (σ+, σ−) and (σ−, σ+), while the ω mode only appears in co‐circular polarization configurations, (σ+, σ+) and (σ−, σ−). Note that in the (σ+, σ−) configuration and with increasing positive magnetic field, CEF1 and CEF2 increase in energy while CEF3 decreases. This dependence is inverted if the magnetic field is reversed (B → −B) or the helicity is reversed ((σ+, σ−) → (σ−, σ+)).
\",\n \"
The observed circular polarization dependence and magnetic field dependence of CEF1–3 and ω in Figures ##FIG##1##2## and ##FIG##2##3## clearly indicate different underlying optical selection rules that can be understood based on angular momentum conservation. Each left (right) circularly polarized σ+ (σ−) photon carries angular momentum +ℏ (−ℏ).[\\n##UREF##43##\\n55\\n##\\n] The link between helicity and circular polarization is given by h = σ · k, where k is the momentum of the photon, and h the helicity.[\\n##REF##29956970##\\n56\\n##\\n] Absorption of a σ+ photon increases the angular momentum of the system by +ℏ, corresponding to being acted on by operator J\\n+, while scattering a σ+ photon corresponds to J\\n− acting on the system. Therefore, if one considers Raman spectra acquired in the (σincident, σscatter) = (σ+, σ−) configuration where the change of angular momentum in photons is ΔJ\\nphoton = −2ℏ, the system could obtain +2ℏ of angular momentum from the photons (i.e., ΔJ\\nsystem = +2ℏ), corresponding to J\\n+\\nJ\\n+ acting on the system (a generalization of the closure condition in Axe et al.,[\\n##UREF##44##\\n57\\n##\\n] which contracts ∑virtual||〈ψfinal|M\\n\\ni\\n|ψvirtual〉〈ψvirtual|M\\n\\nj\\n|ψinitial〉|| to ||〈ψfinal|M\\n\\ni\\n\\nM\\n\\nj\\n|ψinitial〉|| for any dipolar transition M\\n\\ni\\n, M\\n\\nj\\n). Therefore, a mode that is active in the (σ+, σ−) configuration could have dominant matrix‐element contributions from ||〈ψfinal|J\\n+\\nJ\\n+|ψinitial〉||. However, it is important to note that in an analog to the Umklapp process, the threefold rotational symmetry in NaYbSe2\\n[\\n##UREF##45##\\n58\\n##\\n] allows for discrete angular momentum conservation as long as |ΔJ\\nphoton + ΔJ\\nsystem|/ℏ = 0 (modulo 3). In other words, the angular momentum conservation rule is relaxed so that the total change of angular momentum can be either zero or a multiple of 3ℏ due to the threefold rotational symmetry.[\\n##UREF##46##\\n59\\n##, ##UREF##47##\\n60\\n##, ##UREF##48##\\n61\\n##\\n] This means that in the (σ+, σ−) configuration where ΔJ\\nphoton = −2ℏ, ΔJ\\nsystem can be +2ℏ or −ℏ to satisfy the relaxed angular momentum conservation rule. For ΔJ\\nsystem = +2ℏ, it corresponds to the operation of J\\n+\\nJ\\n+, while for ΔJ\\nsystem = −ℏ, it corresponds to J\\n−. Therefore, a mode that is active in the (σ+, σ−) configuration could also have dominant matrix‐element contributions from ||〈ψfinal|J\\n−|ψinitial〉||. In contrast, in the (σincident, (σ−, σ+) configuration where ΔJ\\nphoton = +2ℏ, the system could lose 2ℏ of angular momentum to the photons (ΔJ\\nsystem = −2ℏ), corresponding to J\\n−\\nJ\\n− acting on the system, or ΔJ\\nsystem = +ℏ, corresponding to J\\n+ acting on the system. Hence, a mode that is active in the (σ−, σ+) configuration should have dominant matrix‐element contributions from ||〈ψfinal|J\\n−\\nJ\\n−|ψinitial〉|| or ||〈ψfinal|J\\n+|ψinitial〉||.
\",\n \"
Since all the CEF modes are enhanced in the cross‐circular channel and suppressed in the co‐circular channel, all of them should have a finite change of angular momentum after the orbital transitions. As discussed above, ΔJ\\nCEF = +2ℏ or −ℏ in the (σ+, σ−) configuration, while ΔJ\\nCEF = −2ℏ or +ℏ in the (σ−, σ+) configuration. Considering that an additional Zeeman dependence H\\n\\nB\\n = −g\\n\\nJ\\nμB\\nJ\\n\\nz\\n · B is added when a magnetic field is applied, the change of the sign of ΔJ\\nCEF from (σ+, σ−) to (σ−, σ+) explains why the magnetic field dependence of CEF1–3 is inverted when the helicity is reversed in Figure ##FIG##2##3##. Another intriguing finding from Figure ##FIG##2##3## is that the magnetic field dependence of CEF1 and CEF2 is opposite to that of CEF3 regardless of the helicity, suggesting that ΔJ\\nCEF1 and ΔJ\\nCEF2 share the same sign, whereas ΔJ\\nCEF3 has the opposite sign. More specifically, in the (σ+, σ−) configuration, we should have ΔJ\\nCEF1 = ΔJ\\nCEF2 = +2ℏ while ΔJ\\nCEF3 = −ℏ, or ΔJ\\nCEF1 = ΔJ\\nCEF2 = −ℏ while ΔJ\\nCEF3 = +2ℏ. Note that ΔJ = −ℏ is allowed for a CEF level due to the threefold rotational symmetry that relaxes the angular momentum conservation rule, but we expect that ΔJ = +2ℏ that satisfies the strict angular momentum conservation should have a higher probability and stronger Raman signals. Since CEF1 and CEF2 have much stronger Raman intensities than CEF3 (Figure ##FIG##1##2b##), it is natural to conclude that ΔJ\\nCEF1 = ΔJ\\nCEF2 = +2ℏ while ΔJ\\nCEF3 = −ℏ in the (σ+, σ−) configuration. Similarly, ΔJ\\nCEF1 = ΔJ\\nCEF2 = −2ℏ while ΔJ\\nCEF3 = +ℏ in the (σ−, σ+) configuration. In terms of matrix operations, ⟨ψ1±|J+J+|ψ0±⟩, ⟨ψ1±|J−J−|ψ0±⟩, ⟨ψ2±|J+J+|ψ0±⟩, ⟨ψ2±|J−J−|ψ0±⟩, ⟨ψ3±|J−|ψ0±⟩, and ⟨ψ3±|J+|ψ0±⟩ should be significant while ⟨ψ3±|J+J+|ψ0±⟩, ⟨ψ3±|J−J−|ψ0±⟩, ⟨ψ1±|J+J−|ψ0±⟩, ⟨ψ1±|J−J+|ψ0±⟩, ⟨ψ2±|J+J−|ψ0±⟩, ⟨ψ2±|J−J+|ψ0±⟩, ⟨ψ3±|J+J−|ψ0±⟩, and ⟨ψ3±|J−J+|ψ0±⟩ should all be small or zero (the last six matrix elements correspond to the co‐circular polarization).
\",\n \"
As shown in Figure ##FIG##0##1b##, the eigenstates of CEF levels are linear combinations of multiplets m\\n\\nJ\\n = −7/2…7/2: |ψ±〉 = ∑mj=−72,…72cmj±|72,mj⟩. Typically, CEF parameters are fit to a CEF Hamiltonian with Stevens operators given by the symmetry of the ionic environment of the 4f orbitals, as described in the Experimental Section. The procedure is frequently under‐constrained, and there are enough degrees of freedom to fit experimentally observed CEF energy levels with more than one set of CEF parameters that minimize the error that may exist. The order of the eigenstates may not be the same across those sets. However, once the constraints imposed by the optical selection rules discussed above are taken into account, we are able to find a set of CEF parameters, as indicated by filled and empty circles in Figure ##FIG##0##1b##, that can explain the observed helicity and magnetic field dependence in Figures ##FIG##1##2## and ##FIG##2##3##, and we can make final assignments of CEF1‐3. As illustrated in Figure ##FIG##3##\\n4a##, CEF1 comes from |ψ0+⟩→|ψ1+⟩ in the (σ+, σ−) configuration, where ΔJ\\nphoton = −2ℏ and ΔJ\\nCEF1 = +2ℏ, or |ψ0−⟩→|ψ1−⟩ in the (σ−, σ+) configuration, where ΔJ\\nphoton = +2ℏ and ΔJ\\nCEF1 = −2ℏ (see the arrows for the transitions). We note that the transition of |ψ0+⟩→|ψ1−⟩ with ΔJ\\nCEF1 = −ℏ is allowed in (σ+, σ−) and the transition of |ψ0−⟩→|ψ1+⟩ with ΔJ\\nCEF1 = +ℏ is allowed in (σ−, σ+), since ΔJ\\ntotal = −3ℏ and +3ℏ, respectively, which satisfies the discrete angular momentum conservation. However, these transitions allowed by the crystal threefold rotational symmetry are of a higher order and should be much weaker. Similar transitions apply for CEF2, although they correspond to |ψ0+⟩→|ψ2+⟩ or |ψ0−⟩→|ψ2−⟩. For CEF3 in Figure ##FIG##3##4b##, however, any transition with ΔJ = −2ℏ or +2ℏ is impossible; hence, the transition of |ψ0−⟩→|ψ3−⟩ with ΔJ\\nCEF3 = −ℏ is the only one allowed in (σ+, σ−) where ΔJ\\ntotal = −3ℏ, and the transition of |ψ0+⟩→|ψ3+⟩ with ΔJ\\nCEF3 = +ℏ is the only one allowed in (σ−, σ+) where ΔJ\\ntotal = +3ℏ. This could explain why CEF3 is much weaker than CEF1 and CEF2.
\",\n \"
As for the phonon Raman modes, in general, a nondegenerate Γ‐point phonon (like the A\\n1g Raman mode in NaYbSe2) cannot have angular momentum since the eigenvector is a real number. It is therefore only observed in the co‐circular polarization configuration, as shown in Figure ##FIG##1##2b##. However, when the Γ‐point phonon is doubly degenerate, the two real eigenvectors can be reconstructed by a complex superposition as shown in Figure ##FIG##0##1a##. Therefore, doubly degenerate modes like the E\\ng Raman mode in NaYbSe2 can have (pseudo)angular momentum of ±ℏ.[\\n##REF##26406841##\\n3\\n##, ##UREF##6##\\n9\\n##, ##UREF##47##\\n60\\n##, ##UREF##49##\\n62\\n##\\n] With nonzero angular momentum, it can change the chirality of incident photons and appear in the cross‐circular polarization configuration. As shown in Figure ##FIG##3##4c##, in the (σ+, σ−) configuration, ΔJ\\nphoton = −2ℏ and ΔJEg,−=−ℏ, so it is allowed by the discrete angular momentum conservation as ΔJ\\ntotal = −3ℏ; In the (σ−, σ+) configuration, ΔJ\\nphoton = +2ℏ and ΔJEg,+=+ℏ, giving rise to ΔJ\\ntotal = +3ℏ. Such an optical selection rule is similar to that shown for CEF3 in Figure ##FIG##3##4b##, which explains why both are observed in the cross‐circular polarization. Similar results have been reported for E symmetry Raman modes in TMDs, CrBr3, and quartz, among other materials.[\\n##UREF##6##\\n9\\n##, ##UREF##47##\\n60\\n##, ##UREF##48##\\n61\\n##\\n]\\n
\",\n \"
Finally, moving on to the VBS ω, it is much stronger in the co‐circular polarization configuration and its magnetic field dependence is weak (Figures ##FIG##1##2## and ##FIG##2##3##), in stark contrast to CEF1‐3. This indicates that the change in angular momentum for ω is zero: ΔJ\\n\\nz\\n = 0. Since it is a coupled state between the CEF1 and the E\\ng mode, we consider the 2 × ~2 dimensional space |ψ1±⟩⊗|Eg,±⟩. As the angular momentum change ΔJ\\n\\nz\\n for CEF1 is finite (Figure ##FIG##3##4a##), the phonon subsystem has to carry additional angular momentum (Figure ##FIG##3##4c##) in order to yield zero angular momentum for ω. Therefore, the circular basis {$E$g, +, $E$g, $‐$} is the natural choice for describing the eigenvibration. As discussed previously, CEF1 can arise from four possible transitions while E\\ng can be $E$g, + and $E$g, $‐$, which gives rise to a total of eight possible transitions for ω. Based on the angular momentum conservation rule, however, we found that two states, ω+=|ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and ω−=|ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, are the most probable (see more details in the Supporting Information). As shown in Figure ##FIG##3##4d##, due to the angular momentum transfer between the CEF1 and E\\ng mode, the excited state of ω, |ψ1+⟩⊗|Eg,+⟩ or |ψ1−⟩⊗|Eg,−⟩, has the z‐component of the angular momentum effectively raised or lowered by ℏ compared to the |ψ1+⟩ or |ψ1−⟩ shown in Figure ##FIG##3##4a##, respectively. As a result, the transitions of |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have ΔJ\\n\\nz\\n = 0 to satisfy the selection rule in the co‐circular polarization configuration and explain the weak magnetic field dependence.
\",\n \"
\\nFigure ##FIG##4##\\n5a,b## illustrates the two states, |ψ1+⟩⊗|Eg,+⟩ and |ψ1−⟩⊗|Eg,−⟩ based on the observations described above. In Figure ##FIG##4##5a##, when the ground state is |ψ0+⟩ (shown in light pink), the J\\n+\\nJ\\n− or J\\n−\\nJ\\n+ operator excites the system to the + branch, |ψ1+⟩⊗|Eg,+⟩ (where the CEF excitation corresponds to the + branch and the phononic excitation corresponds to + angular momentum). Figure ##FIG##4##5b## shows the same effect for |ψ0−⟩ to |ψ1−⟩⊗|Eg,−⟩. Because the transition is degenerate for |ψ0∓⟩→ω± with J\\n+\\nJ\\n− or J\\n−\\nJ\\n+, this eigenspace, in principle, can be transduced from a QSL ground state. If the QSL ground state is written as ∑j∏iψ0σij – where i is the site index, j the configuration index, and σ\\nij\\n = +, − is the branch—then J\\n+\\nJ\\n− or J\\n−\\nJ\\n+ should bring the system into ∑j∏iωσij within the point spread volume of the excitation beam, transducing the possible ground state entanglement to the VBS ω±.
\",\n \"
It is worth noting that, in the original Thalmeier–Fulde model, two VBS modes are expected to emerge from the hybridization of a CEF mode and a phonon mode.[\\n##UREF##34##\\n41\\n##\\n] Besides the two most probable VBS transitions shown in Figure ##FIG##3##4d## that give rise to the observed ω peak in the Raman spectra, there are six other transitions that could contribute to the other VBS mode that is not observed in our Raman measurements (see Equation (##SUPPL##0##S1##), Supporting Information). Since the picture of how orbital and phononic excitations couple is not entirely clear, a definitive answer to why other transitions and the other VBS mode do not appear in Raman scattering is beyond the scope of this work. It is expected that the electron–phonon coupling may be more complicated compared to the conventional coupling between orbital angular momentum and spin angular momentum.[\\n##UREF##50##\\n63\\n##\\n] Therefore, we hypothesize that the coupling between the CEF1 and the Eg mode is not the same as the conventional coupling between orbital and spin angular momenta. There is a coupling coefficient before the derived ΔJ\\nω (see Equation (##SUPPL##0##S1##), Supporting Information), leading to non‐integer ΔJ\\nω unless it is zero to begin with. As a consequence, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩, |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, |ψ0+⟩→|ψ1−⟩⊗|Eg,+⟩, and |ψ0−⟩→|ψ1+⟩⊗|Eg,−⟩ that have zero angular momentum can satisfy the angular momentum conservation rule in the co‐circular polarization configuration, and other transitions are forbidden in any polarization channel. Moreover, as discussed previously, since |ψ0+⟩→|ψ1−⟩ and |ψ0−⟩→|ψ1+⟩ are of a higher order and much less probable, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have detectable signals (see Figure ##FIG##3##4d##). This could explain why other transitions and the other VBS modes are not observed.
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","
We first verify the primary CEF excitations and phonon modes with temperature‐dependent Raman spectroscopy. The temperature dependence of the relevant Raman features is depicted in Figure ##FIG##0##1c## while the full spectra are shown in Figures ##SUPPL##0##S2## and ##SUPPL##0##S3##, Supporting Information. Consistent with earlier neutron scattering and Raman reports,[\n##UREF##16##\n23\n##, ##UREF##28##\n35\n##\n] strong CEF modes are observed at 117.2 cm−1 (CEF1), 197.8 cm−1 (CEF2), and 247.0 cm−1 (CEF3). These become significantly stronger in intensity and soften (shift toward lower energy) as the temperature decreases. NaYbSe2 also has two Raman‐active phonon modes: E\ng at 124.4 cm−1 and A\n1g at 172.8 cm−1 within this frequency space. The intensity and energy of these phonon modes exhibit a substantially weaker temperature dependence. Figure ##FIG##0##1b## shows a classical spinning top representation of the spin‐orbit eigenstates for |ψ0+⟩ as well as the relative weights cmj for |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, |ψ3±⟩ as |ψ〉= ∑mj=−72,…72cmj|72,mj⟩. Empty (filled) circles represent cmj=0 (cmj≠0). The blue (pink) cones represent the + (−) branch. Note that the E\ng and CEF1 modes exist at similar energies. This is the case for NaYbSe2,[\n##UREF##16##\n23\n##\n] CsYbSe2,[\n##UREF##28##\n35\n##\n] and KYbSe2.[\n##UREF##30##\n37\n##\n] Using Bayesian inference with a Hamiltonian Monte Carlo model in PyMC3,[\n##UREF##42##\n54\n##\n] we track the peak parameters from the experimental data shown in Figure ##FIG##0##1c##. The extracted peak positions for CEF1 and E\ng are shown in Figure ##FIG##0##1d##. The symbols represent the median values from the Bayesian inference. The darker shaded bands illustrate the 68% (corresponding to 1σ in the central limit) highest density intervals (HDIs), and the lighter shaded bands illustrate the 95% (corresponding to 2σ) HDIs. Other selected modes are described in the Supporting Information. The model has larger error bars from T = 175 to 255 K, but the modes are well resolved elsewhere.
","
A previously unreported mode is clearly seen in our data at 151.2 cm−1. Labeled ω in Figure ##FIG##0##1c,d##, this mode has characteristics in common with both CEFs and phonons: ω has a significantly stronger structure factor at low temperatures, just as the CEFs do, but it does not exhibit the strong softening at lower temperatures that the CEF modes exhibit. Instead, it hardens in a manner consistent with slight phonon hardening in NaYbSe2 at low temperatures. We therefore assign ω as a VBS with parent states CEF1 and E\ng. Earlier Raman spectra in this temperature range[\n##UREF##16##\n23\n##\n] were only helicity‐resolved at room temperature, where ω is weak.
","
In order to better understand the origin of the ω mode, we calculated the phonon dispersion relationship for NaYbSe2 using density functional theory (DFT) and Perdew–Burke–Ernzerhof (PBE) exchange energy, as shown in Figure ##SUPPL##0##S1##, Supporting Information. These calculations suggest that no optical or acoustic phonons are present near ω. If anything, PBE normally overestimates the in‐plane lattice constants, resulting in softer in‐plane vibrations (E\ng) than those measured. Comparing these calculations to published data (and the data in this manuscript) the calculated E\ng at the gamma point is indeed 15 cm−1 softer than that measured. The A\n1g mode is also calculated to be softer than the measured mode, 135 cm−1 compared to the measured 172.8 cm−1. Between these two energies, there are very few modes at the gamma point, and halfway between them there are no phonon branches.
","
In fact, most models have no phonons throughout the Brillouin zone around 150 cm−1. Additionally, published neutron scattering data suggests there are no phonons in this frequency space. Specifically, figure S3J in ref. [##UREF##14##21##] reveals a decrease in spectral weight as the temperature increases. Additionally, Zhang et al.[\n##UREF##16##\n23\n##\n] could not model extra spectral weight at ≈19 meV (see figure ##FIG##1##2## of that manuscript) when the temperature decreased below 100 K. Notably, these neutron data have momentum transfer dependence like a phonon, but the temperature dependence of a crystal field, consistent with a vibronic bound state.
","
The mode ω is present in both NaYbSe2 and CsYbSe2, although it is far less prominent in CsYbSe2.[\n##UREF##28##\n35\n##\n] This is likely because in NaYbSe2, Na is both smaller and lighter. The lattice is therefore more tightly‐packed. The lighter Na also has larger vibrational displacements. Both effects increase the coupling strength, resulting in a VBS far stronger than that in CsYbSe2. Fitting to the Thalmeier–Fulde description of a magnetoelastic vibronic bound state[\n##UREF##34##\n41\n##\n] yields a coupling strength of 32.0 cm−1 (3.97 meV) for NaYbSe2, which is stronger than the 23.6 cm−1 (2.93 meV) coupling strength reported for CsYbSe2 and comparable to the roughly 34.0 cm−1 (4.22 meV) coupling strength reported for Ce2O3.[\n##REF##31107079##\n43\n##\n]\n
","
To gain a deeper understanding of the CEF and the ω Raman modes, we studied the helicity dependence and magnetic field dependence of Raman spectra of NaYbSe2. During the Raman scattering process, the energy transferred from the photons can be used to excite a variety of (quasi)particles in the system, including electronic orbitals, phonons, and their hybridized states, as indicated by the schematic illustration in Figure ##FIG##1##\n2a##. Figure ##FIG##1##2b## shows helicity‐resolved Raman spectra acquired at T = 4 K, with CEF1, CEF2, CEF3, E\ng, A\n1g, and ω highlighted. Here, we can simultaneously observe electronic orbital excitations (CEF1, CEF2, and CEF3), phononic excitations (E\ng and A\n1g), and the entangled state ω between the orbital and phononic excitations by low‐temperature Raman spectroscopy. Furthermore, we discovered that these (quasi)particles exhibit different responses to the helicity of photons. While A\n1g and ω (along with the Rayleigh scattering; see Figure ##SUPPL##0##S7##, Supporting Information) are stronger in the co‐circular polarization configuration, CEF1–CEF3 and E\ng are stronger in the cross‐circular polarization configuration.
","
We also performed magnetic field‐dependent Raman spectroscopy with B || c, which lifts the degeneracy within each of the Kramers pairs and therefore provides further information on the orbital excitations corresponding to CEF1–3. Magnetization and specific heat measurements have been previously used to show that no field‐induced ordering is present in NaYbSe2 for B || c for B < 9 T at T = 4 K.[\n##UREF##15##\n22\n##\n] Therefore, with the 6 T magnetic fields accessible here, no field‐induced transition is expected. Figure ##FIG##2##\n3\n## shows the magnetic field dependence of the CEF levels for CEF1 (Figure ##FIG##2##3a##), CEF2 (Figure ##FIG##2##3b##), ω (Figure ##FIG##2##3c##), and CEF3 (Figure ##FIG##2##3d##) measurements. Again, the CEF levels only show up in cross‐circular polarization configurations, (σ+, σ−) and (σ−, σ+), while the ω mode only appears in co‐circular polarization configurations, (σ+, σ+) and (σ−, σ−). Note that in the (σ+, σ−) configuration and with increasing positive magnetic field, CEF1 and CEF2 increase in energy while CEF3 decreases. This dependence is inverted if the magnetic field is reversed (B → −B) or the helicity is reversed ((σ+, σ−) → (σ−, σ+)).
","
The observed circular polarization dependence and magnetic field dependence of CEF1–3 and ω in Figures ##FIG##1##2## and ##FIG##2##3## clearly indicate different underlying optical selection rules that can be understood based on angular momentum conservation. Each left (right) circularly polarized σ+ (σ−) photon carries angular momentum +ℏ (−ℏ).[\n##UREF##43##\n55\n##\n] The link between helicity and circular polarization is given by h = σ · k, where k is the momentum of the photon, and h the helicity.[\n##REF##29956970##\n56\n##\n] Absorption of a σ+ photon increases the angular momentum of the system by +ℏ, corresponding to being acted on by operator J\n+, while scattering a σ+ photon corresponds to J\n− acting on the system. Therefore, if one considers Raman spectra acquired in the (σincident, σscatter) = (σ+, σ−) configuration where the change of angular momentum in photons is ΔJ\nphoton = −2ℏ, the system could obtain +2ℏ of angular momentum from the photons (i.e., ΔJ\nsystem = +2ℏ), corresponding to J\n+\nJ\n+ acting on the system (a generalization of the closure condition in Axe et al.,[\n##UREF##44##\n57\n##\n] which contracts ∑virtual||〈ψfinal|M\n\ni\n|ψvirtual〉〈ψvirtual|M\n\nj\n|ψinitial〉|| to ||〈ψfinal|M\n\ni\n\nM\n\nj\n|ψinitial〉|| for any dipolar transition M\n\ni\n, M\n\nj\n). Therefore, a mode that is active in the (σ+, σ−) configuration could have dominant matrix‐element contributions from ||〈ψfinal|J\n+\nJ\n+|ψinitial〉||. However, it is important to note that in an analog to the Umklapp process, the threefold rotational symmetry in NaYbSe2\n[\n##UREF##45##\n58\n##\n] allows for discrete angular momentum conservation as long as |ΔJ\nphoton + ΔJ\nsystem|/ℏ = 0 (modulo 3). In other words, the angular momentum conservation rule is relaxed so that the total change of angular momentum can be either zero or a multiple of 3ℏ due to the threefold rotational symmetry.[\n##UREF##46##\n59\n##, ##UREF##47##\n60\n##, ##UREF##48##\n61\n##\n] This means that in the (σ+, σ−) configuration where ΔJ\nphoton = −2ℏ, ΔJ\nsystem can be +2ℏ or −ℏ to satisfy the relaxed angular momentum conservation rule. For ΔJ\nsystem = +2ℏ, it corresponds to the operation of J\n+\nJ\n+, while for ΔJ\nsystem = −ℏ, it corresponds to J\n−. Therefore, a mode that is active in the (σ+, σ−) configuration could also have dominant matrix‐element contributions from ||〈ψfinal|J\n−|ψinitial〉||. In contrast, in the (σincident, (σ−, σ+) configuration where ΔJ\nphoton = +2ℏ, the system could lose 2ℏ of angular momentum to the photons (ΔJ\nsystem = −2ℏ), corresponding to J\n−\nJ\n− acting on the system, or ΔJ\nsystem = +ℏ, corresponding to J\n+ acting on the system. Hence, a mode that is active in the (σ−, σ+) configuration should have dominant matrix‐element contributions from ||〈ψfinal|J\n−\nJ\n−|ψinitial〉|| or ||〈ψfinal|J\n+|ψinitial〉||.
","
Since all the CEF modes are enhanced in the cross‐circular channel and suppressed in the co‐circular channel, all of them should have a finite change of angular momentum after the orbital transitions. As discussed above, ΔJ\nCEF = +2ℏ or −ℏ in the (σ+, σ−) configuration, while ΔJ\nCEF = −2ℏ or +ℏ in the (σ−, σ+) configuration. Considering that an additional Zeeman dependence H\n\nB\n = −g\n\nJ\nμB\nJ\n\nz\n · B is added when a magnetic field is applied, the change of the sign of ΔJ\nCEF from (σ+, σ−) to (σ−, σ+) explains why the magnetic field dependence of CEF1–3 is inverted when the helicity is reversed in Figure ##FIG##2##3##. Another intriguing finding from Figure ##FIG##2##3## is that the magnetic field dependence of CEF1 and CEF2 is opposite to that of CEF3 regardless of the helicity, suggesting that ΔJ\nCEF1 and ΔJ\nCEF2 share the same sign, whereas ΔJ\nCEF3 has the opposite sign. More specifically, in the (σ+, σ−) configuration, we should have ΔJ\nCEF1 = ΔJ\nCEF2 = +2ℏ while ΔJ\nCEF3 = −ℏ, or ΔJ\nCEF1 = ΔJ\nCEF2 = −ℏ while ΔJ\nCEF3 = +2ℏ. Note that ΔJ = −ℏ is allowed for a CEF level due to the threefold rotational symmetry that relaxes the angular momentum conservation rule, but we expect that ΔJ = +2ℏ that satisfies the strict angular momentum conservation should have a higher probability and stronger Raman signals. Since CEF1 and CEF2 have much stronger Raman intensities than CEF3 (Figure ##FIG##1##2b##), it is natural to conclude that ΔJ\nCEF1 = ΔJ\nCEF2 = +2ℏ while ΔJ\nCEF3 = −ℏ in the (σ+, σ−) configuration. Similarly, ΔJ\nCEF1 = ΔJ\nCEF2 = −2ℏ while ΔJ\nCEF3 = +ℏ in the (σ−, σ+) configuration. In terms of matrix operations, ⟨ψ1±|J+J+|ψ0±⟩, ⟨ψ1±|J−J−|ψ0±⟩, ⟨ψ2±|J+J+|ψ0±⟩, ⟨ψ2±|J−J−|ψ0±⟩, ⟨ψ3±|J−|ψ0±⟩, and ⟨ψ3±|J+|ψ0±⟩ should be significant while ⟨ψ3±|J+J+|ψ0±⟩, ⟨ψ3±|J−J−|ψ0±⟩, ⟨ψ1±|J+J−|ψ0±⟩, ⟨ψ1±|J−J+|ψ0±⟩, ⟨ψ2±|J+J−|ψ0±⟩, ⟨ψ2±|J−J+|ψ0±⟩, ⟨ψ3±|J+J−|ψ0±⟩, and ⟨ψ3±|J−J+|ψ0±⟩ should all be small or zero (the last six matrix elements correspond to the co‐circular polarization).
","
As shown in Figure ##FIG##0##1b##, the eigenstates of CEF levels are linear combinations of multiplets m\n\nJ\n = −7/2…7/2: |ψ±〉 = ∑mj=−72,…72cmj±|72,mj⟩. Typically, CEF parameters are fit to a CEF Hamiltonian with Stevens operators given by the symmetry of the ionic environment of the 4f orbitals, as described in the Experimental Section. The procedure is frequently under‐constrained, and there are enough degrees of freedom to fit experimentally observed CEF energy levels with more than one set of CEF parameters that minimize the error that may exist. The order of the eigenstates may not be the same across those sets. However, once the constraints imposed by the optical selection rules discussed above are taken into account, we are able to find a set of CEF parameters, as indicated by filled and empty circles in Figure ##FIG##0##1b##, that can explain the observed helicity and magnetic field dependence in Figures ##FIG##1##2## and ##FIG##2##3##, and we can make final assignments of CEF1‐3. As illustrated in Figure ##FIG##3##\n4a##, CEF1 comes from |ψ0+⟩→|ψ1+⟩ in the (σ+, σ−) configuration, where ΔJ\nphoton = −2ℏ and ΔJ\nCEF1 = +2ℏ, or |ψ0−⟩→|ψ1−⟩ in the (σ−, σ+) configuration, where ΔJ\nphoton = +2ℏ and ΔJ\nCEF1 = −2ℏ (see the arrows for the transitions). We note that the transition of |ψ0+⟩→|ψ1−⟩ with ΔJ\nCEF1 = −ℏ is allowed in (σ+, σ−) and the transition of |ψ0−⟩→|ψ1+⟩ with ΔJ\nCEF1 = +ℏ is allowed in (σ−, σ+), since ΔJ\ntotal = −3ℏ and +3ℏ, respectively, which satisfies the discrete angular momentum conservation. However, these transitions allowed by the crystal threefold rotational symmetry are of a higher order and should be much weaker. Similar transitions apply for CEF2, although they correspond to |ψ0+⟩→|ψ2+⟩ or |ψ0−⟩→|ψ2−⟩. For CEF3 in Figure ##FIG##3##4b##, however, any transition with ΔJ = −2ℏ or +2ℏ is impossible; hence, the transition of |ψ0−⟩→|ψ3−⟩ with ΔJ\nCEF3 = −ℏ is the only one allowed in (σ+, σ−) where ΔJ\ntotal = −3ℏ, and the transition of |ψ0+⟩→|ψ3+⟩ with ΔJ\nCEF3 = +ℏ is the only one allowed in (σ−, σ+) where ΔJ\ntotal = +3ℏ. This could explain why CEF3 is much weaker than CEF1 and CEF2.
","
As for the phonon Raman modes, in general, a nondegenerate Γ‐point phonon (like the A\n1g Raman mode in NaYbSe2) cannot have angular momentum since the eigenvector is a real number. It is therefore only observed in the co‐circular polarization configuration, as shown in Figure ##FIG##1##2b##. However, when the Γ‐point phonon is doubly degenerate, the two real eigenvectors can be reconstructed by a complex superposition as shown in Figure ##FIG##0##1a##. Therefore, doubly degenerate modes like the E\ng Raman mode in NaYbSe2 can have (pseudo)angular momentum of ±ℏ.[\n##REF##26406841##\n3\n##, ##UREF##6##\n9\n##, ##UREF##47##\n60\n##, ##UREF##49##\n62\n##\n] With nonzero angular momentum, it can change the chirality of incident photons and appear in the cross‐circular polarization configuration. As shown in Figure ##FIG##3##4c##, in the (σ+, σ−) configuration, ΔJ\nphoton = −2ℏ and ΔJEg,−=−ℏ, so it is allowed by the discrete angular momentum conservation as ΔJ\ntotal = −3ℏ; In the (σ−, σ+) configuration, ΔJ\nphoton = +2ℏ and ΔJEg,+=+ℏ, giving rise to ΔJ\ntotal = +3ℏ. Such an optical selection rule is similar to that shown for CEF3 in Figure ##FIG##3##4b##, which explains why both are observed in the cross‐circular polarization. Similar results have been reported for E symmetry Raman modes in TMDs, CrBr3, and quartz, among other materials.[\n##UREF##6##\n9\n##, ##UREF##47##\n60\n##, ##UREF##48##\n61\n##\n]\n
","
Finally, moving on to the VBS ω, it is much stronger in the co‐circular polarization configuration and its magnetic field dependence is weak (Figures ##FIG##1##2## and ##FIG##2##3##), in stark contrast to CEF1‐3. This indicates that the change in angular momentum for ω is zero: ΔJ\n\nz\n = 0. Since it is a coupled state between the CEF1 and the E\ng mode, we consider the 2 × ~2 dimensional space |ψ1±⟩⊗|Eg,±⟩. As the angular momentum change ΔJ\n\nz\n for CEF1 is finite (Figure ##FIG##3##4a##), the phonon subsystem has to carry additional angular momentum (Figure ##FIG##3##4c##) in order to yield zero angular momentum for ω. Therefore, the circular basis {$E$g, +, $E$g, $‐$} is the natural choice for describing the eigenvibration. As discussed previously, CEF1 can arise from four possible transitions while E\ng can be $E$g, + and $E$g, $‐$, which gives rise to a total of eight possible transitions for ω. Based on the angular momentum conservation rule, however, we found that two states, ω+=|ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and ω−=|ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, are the most probable (see more details in the Supporting Information). As shown in Figure ##FIG##3##4d##, due to the angular momentum transfer between the CEF1 and E\ng mode, the excited state of ω, |ψ1+⟩⊗|Eg,+⟩ or |ψ1−⟩⊗|Eg,−⟩, has the z‐component of the angular momentum effectively raised or lowered by ℏ compared to the |ψ1+⟩ or |ψ1−⟩ shown in Figure ##FIG##3##4a##, respectively. As a result, the transitions of |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have ΔJ\n\nz\n = 0 to satisfy the selection rule in the co‐circular polarization configuration and explain the weak magnetic field dependence.
","
\nFigure ##FIG##4##\n5a,b## illustrates the two states, |ψ1+⟩⊗|Eg,+⟩ and |ψ1−⟩⊗|Eg,−⟩ based on the observations described above. In Figure ##FIG##4##5a##, when the ground state is |ψ0+⟩ (shown in light pink), the J\n+\nJ\n− or J\n−\nJ\n+ operator excites the system to the + branch, |ψ1+⟩⊗|Eg,+⟩ (where the CEF excitation corresponds to the + branch and the phononic excitation corresponds to + angular momentum). Figure ##FIG##4##5b## shows the same effect for |ψ0−⟩ to |ψ1−⟩⊗|Eg,−⟩. Because the transition is degenerate for |ψ0∓⟩→ω± with J\n+\nJ\n− or J\n−\nJ\n+, this eigenspace, in principle, can be transduced from a QSL ground state. If the QSL ground state is written as ∑j∏iψ0σij – where i is the site index, j the configuration index, and σ\nij\n = +, − is the branch—then J\n+\nJ\n− or J\n−\nJ\n+ should bring the system into ∑j∏iωσij within the point spread volume of the excitation beam, transducing the possible ground state entanglement to the VBS ω±.
","
It is worth noting that, in the original Thalmeier–Fulde model, two VBS modes are expected to emerge from the hybridization of a CEF mode and a phonon mode.[\n##UREF##34##\n41\n##\n] Besides the two most probable VBS transitions shown in Figure ##FIG##3##4d## that give rise to the observed ω peak in the Raman spectra, there are six other transitions that could contribute to the other VBS mode that is not observed in our Raman measurements (see Equation (##SUPPL##0##S1##), Supporting Information). Since the picture of how orbital and phononic excitations couple is not entirely clear, a definitive answer to why other transitions and the other VBS mode do not appear in Raman scattering is beyond the scope of this work. It is expected that the electron–phonon coupling may be more complicated compared to the conventional coupling between orbital angular momentum and spin angular momentum.[\n##UREF##50##\n63\n##\n] Therefore, we hypothesize that the coupling between the CEF1 and the Eg mode is not the same as the conventional coupling between orbital and spin angular momenta. There is a coupling coefficient before the derived ΔJ\nω (see Equation (##SUPPL##0##S1##), Supporting Information), leading to non‐integer ΔJ\nω unless it is zero to begin with. As a consequence, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩, |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, |ψ0+⟩→|ψ1−⟩⊗|Eg,+⟩, and |ψ0−⟩→|ψ1+⟩⊗|Eg,−⟩ that have zero angular momentum can satisfy the angular momentum conservation rule in the co‐circular polarization configuration, and other transitions are forbidden in any polarization channel. Moreover, as discussed previously, since |ψ0+⟩→|ψ1−⟩ and |ψ0−⟩→|ψ1+⟩ are of a higher order and much less probable, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have detectable signals (see Figure ##FIG##3##4d##). This could explain why other transitions and the other VBS modes are not observed.
"],"string":"[\n \"Results and Discussion\",\n \"
We first verify the primary CEF excitations and phonon modes with temperature‐dependent Raman spectroscopy. The temperature dependence of the relevant Raman features is depicted in Figure ##FIG##0##1c## while the full spectra are shown in Figures ##SUPPL##0##S2## and ##SUPPL##0##S3##, Supporting Information. Consistent with earlier neutron scattering and Raman reports,[\\n##UREF##16##\\n23\\n##, ##UREF##28##\\n35\\n##\\n] strong CEF modes are observed at 117.2 cm−1 (CEF1), 197.8 cm−1 (CEF2), and 247.0 cm−1 (CEF3). These become significantly stronger in intensity and soften (shift toward lower energy) as the temperature decreases. NaYbSe2 also has two Raman‐active phonon modes: E\\ng at 124.4 cm−1 and A\\n1g at 172.8 cm−1 within this frequency space. The intensity and energy of these phonon modes exhibit a substantially weaker temperature dependence. Figure ##FIG##0##1b## shows a classical spinning top representation of the spin‐orbit eigenstates for |ψ0+⟩ as well as the relative weights cmj for |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, |ψ3±⟩ as |ψ〉= ∑mj=−72,…72cmj|72,mj⟩. Empty (filled) circles represent cmj=0 (cmj≠0). The blue (pink) cones represent the + (−) branch. Note that the E\\ng and CEF1 modes exist at similar energies. This is the case for NaYbSe2,[\\n##UREF##16##\\n23\\n##\\n] CsYbSe2,[\\n##UREF##28##\\n35\\n##\\n] and KYbSe2.[\\n##UREF##30##\\n37\\n##\\n] Using Bayesian inference with a Hamiltonian Monte Carlo model in PyMC3,[\\n##UREF##42##\\n54\\n##\\n] we track the peak parameters from the experimental data shown in Figure ##FIG##0##1c##. The extracted peak positions for CEF1 and E\\ng are shown in Figure ##FIG##0##1d##. The symbols represent the median values from the Bayesian inference. The darker shaded bands illustrate the 68% (corresponding to 1σ in the central limit) highest density intervals (HDIs), and the lighter shaded bands illustrate the 95% (corresponding to 2σ) HDIs. Other selected modes are described in the Supporting Information. The model has larger error bars from T = 175 to 255 K, but the modes are well resolved elsewhere.
\",\n \"
A previously unreported mode is clearly seen in our data at 151.2 cm−1. Labeled ω in Figure ##FIG##0##1c,d##, this mode has characteristics in common with both CEFs and phonons: ω has a significantly stronger structure factor at low temperatures, just as the CEFs do, but it does not exhibit the strong softening at lower temperatures that the CEF modes exhibit. Instead, it hardens in a manner consistent with slight phonon hardening in NaYbSe2 at low temperatures. We therefore assign ω as a VBS with parent states CEF1 and E\\ng. Earlier Raman spectra in this temperature range[\\n##UREF##16##\\n23\\n##\\n] were only helicity‐resolved at room temperature, where ω is weak.
\",\n \"
In order to better understand the origin of the ω mode, we calculated the phonon dispersion relationship for NaYbSe2 using density functional theory (DFT) and Perdew–Burke–Ernzerhof (PBE) exchange energy, as shown in Figure ##SUPPL##0##S1##, Supporting Information. These calculations suggest that no optical or acoustic phonons are present near ω. If anything, PBE normally overestimates the in‐plane lattice constants, resulting in softer in‐plane vibrations (E\\ng) than those measured. Comparing these calculations to published data (and the data in this manuscript) the calculated E\\ng at the gamma point is indeed 15 cm−1 softer than that measured. The A\\n1g mode is also calculated to be softer than the measured mode, 135 cm−1 compared to the measured 172.8 cm−1. Between these two energies, there are very few modes at the gamma point, and halfway between them there are no phonon branches.
\",\n \"
In fact, most models have no phonons throughout the Brillouin zone around 150 cm−1. Additionally, published neutron scattering data suggests there are no phonons in this frequency space. Specifically, figure S3J in ref. [##UREF##14##21##] reveals a decrease in spectral weight as the temperature increases. Additionally, Zhang et al.[\\n##UREF##16##\\n23\\n##\\n] could not model extra spectral weight at ≈19 meV (see figure ##FIG##1##2## of that manuscript) when the temperature decreased below 100 K. Notably, these neutron data have momentum transfer dependence like a phonon, but the temperature dependence of a crystal field, consistent with a vibronic bound state.
\",\n \"
The mode ω is present in both NaYbSe2 and CsYbSe2, although it is far less prominent in CsYbSe2.[\\n##UREF##28##\\n35\\n##\\n] This is likely because in NaYbSe2, Na is both smaller and lighter. The lattice is therefore more tightly‐packed. The lighter Na also has larger vibrational displacements. Both effects increase the coupling strength, resulting in a VBS far stronger than that in CsYbSe2. Fitting to the Thalmeier–Fulde description of a magnetoelastic vibronic bound state[\\n##UREF##34##\\n41\\n##\\n] yields a coupling strength of 32.0 cm−1 (3.97 meV) for NaYbSe2, which is stronger than the 23.6 cm−1 (2.93 meV) coupling strength reported for CsYbSe2 and comparable to the roughly 34.0 cm−1 (4.22 meV) coupling strength reported for Ce2O3.[\\n##REF##31107079##\\n43\\n##\\n]\\n
\",\n \"
To gain a deeper understanding of the CEF and the ω Raman modes, we studied the helicity dependence and magnetic field dependence of Raman spectra of NaYbSe2. During the Raman scattering process, the energy transferred from the photons can be used to excite a variety of (quasi)particles in the system, including electronic orbitals, phonons, and their hybridized states, as indicated by the schematic illustration in Figure ##FIG##1##\\n2a##. Figure ##FIG##1##2b## shows helicity‐resolved Raman spectra acquired at T = 4 K, with CEF1, CEF2, CEF3, E\\ng, A\\n1g, and ω highlighted. Here, we can simultaneously observe electronic orbital excitations (CEF1, CEF2, and CEF3), phononic excitations (E\\ng and A\\n1g), and the entangled state ω between the orbital and phononic excitations by low‐temperature Raman spectroscopy. Furthermore, we discovered that these (quasi)particles exhibit different responses to the helicity of photons. While A\\n1g and ω (along with the Rayleigh scattering; see Figure ##SUPPL##0##S7##, Supporting Information) are stronger in the co‐circular polarization configuration, CEF1–CEF3 and E\\ng are stronger in the cross‐circular polarization configuration.
\",\n \"
We also performed magnetic field‐dependent Raman spectroscopy with B || c, which lifts the degeneracy within each of the Kramers pairs and therefore provides further information on the orbital excitations corresponding to CEF1–3. Magnetization and specific heat measurements have been previously used to show that no field‐induced ordering is present in NaYbSe2 for B || c for B < 9 T at T = 4 K.[\\n##UREF##15##\\n22\\n##\\n] Therefore, with the 6 T magnetic fields accessible here, no field‐induced transition is expected. Figure ##FIG##2##\\n3\\n## shows the magnetic field dependence of the CEF levels for CEF1 (Figure ##FIG##2##3a##), CEF2 (Figure ##FIG##2##3b##), ω (Figure ##FIG##2##3c##), and CEF3 (Figure ##FIG##2##3d##) measurements. Again, the CEF levels only show up in cross‐circular polarization configurations, (σ+, σ−) and (σ−, σ+), while the ω mode only appears in co‐circular polarization configurations, (σ+, σ+) and (σ−, σ−). Note that in the (σ+, σ−) configuration and with increasing positive magnetic field, CEF1 and CEF2 increase in energy while CEF3 decreases. This dependence is inverted if the magnetic field is reversed (B → −B) or the helicity is reversed ((σ+, σ−) → (σ−, σ+)).
\",\n \"
The observed circular polarization dependence and magnetic field dependence of CEF1–3 and ω in Figures ##FIG##1##2## and ##FIG##2##3## clearly indicate different underlying optical selection rules that can be understood based on angular momentum conservation. Each left (right) circularly polarized σ+ (σ−) photon carries angular momentum +ℏ (−ℏ).[\\n##UREF##43##\\n55\\n##\\n] The link between helicity and circular polarization is given by h = σ · k, where k is the momentum of the photon, and h the helicity.[\\n##REF##29956970##\\n56\\n##\\n] Absorption of a σ+ photon increases the angular momentum of the system by +ℏ, corresponding to being acted on by operator J\\n+, while scattering a σ+ photon corresponds to J\\n− acting on the system. Therefore, if one considers Raman spectra acquired in the (σincident, σscatter) = (σ+, σ−) configuration where the change of angular momentum in photons is ΔJ\\nphoton = −2ℏ, the system could obtain +2ℏ of angular momentum from the photons (i.e., ΔJ\\nsystem = +2ℏ), corresponding to J\\n+\\nJ\\n+ acting on the system (a generalization of the closure condition in Axe et al.,[\\n##UREF##44##\\n57\\n##\\n] which contracts ∑virtual||〈ψfinal|M\\n\\ni\\n|ψvirtual〉〈ψvirtual|M\\n\\nj\\n|ψinitial〉|| to ||〈ψfinal|M\\n\\ni\\n\\nM\\n\\nj\\n|ψinitial〉|| for any dipolar transition M\\n\\ni\\n, M\\n\\nj\\n). Therefore, a mode that is active in the (σ+, σ−) configuration could have dominant matrix‐element contributions from ||〈ψfinal|J\\n+\\nJ\\n+|ψinitial〉||. However, it is important to note that in an analog to the Umklapp process, the threefold rotational symmetry in NaYbSe2\\n[\\n##UREF##45##\\n58\\n##\\n] allows for discrete angular momentum conservation as long as |ΔJ\\nphoton + ΔJ\\nsystem|/ℏ = 0 (modulo 3). In other words, the angular momentum conservation rule is relaxed so that the total change of angular momentum can be either zero or a multiple of 3ℏ due to the threefold rotational symmetry.[\\n##UREF##46##\\n59\\n##, ##UREF##47##\\n60\\n##, ##UREF##48##\\n61\\n##\\n] This means that in the (σ+, σ−) configuration where ΔJ\\nphoton = −2ℏ, ΔJ\\nsystem can be +2ℏ or −ℏ to satisfy the relaxed angular momentum conservation rule. For ΔJ\\nsystem = +2ℏ, it corresponds to the operation of J\\n+\\nJ\\n+, while for ΔJ\\nsystem = −ℏ, it corresponds to J\\n−. Therefore, a mode that is active in the (σ+, σ−) configuration could also have dominant matrix‐element contributions from ||〈ψfinal|J\\n−|ψinitial〉||. In contrast, in the (σincident, (σ−, σ+) configuration where ΔJ\\nphoton = +2ℏ, the system could lose 2ℏ of angular momentum to the photons (ΔJ\\nsystem = −2ℏ), corresponding to J\\n−\\nJ\\n− acting on the system, or ΔJ\\nsystem = +ℏ, corresponding to J\\n+ acting on the system. Hence, a mode that is active in the (σ−, σ+) configuration should have dominant matrix‐element contributions from ||〈ψfinal|J\\n−\\nJ\\n−|ψinitial〉|| or ||〈ψfinal|J\\n+|ψinitial〉||.
\",\n \"
Since all the CEF modes are enhanced in the cross‐circular channel and suppressed in the co‐circular channel, all of them should have a finite change of angular momentum after the orbital transitions. As discussed above, ΔJ\\nCEF = +2ℏ or −ℏ in the (σ+, σ−) configuration, while ΔJ\\nCEF = −2ℏ or +ℏ in the (σ−, σ+) configuration. Considering that an additional Zeeman dependence H\\n\\nB\\n = −g\\n\\nJ\\nμB\\nJ\\n\\nz\\n · B is added when a magnetic field is applied, the change of the sign of ΔJ\\nCEF from (σ+, σ−) to (σ−, σ+) explains why the magnetic field dependence of CEF1–3 is inverted when the helicity is reversed in Figure ##FIG##2##3##. Another intriguing finding from Figure ##FIG##2##3## is that the magnetic field dependence of CEF1 and CEF2 is opposite to that of CEF3 regardless of the helicity, suggesting that ΔJ\\nCEF1 and ΔJ\\nCEF2 share the same sign, whereas ΔJ\\nCEF3 has the opposite sign. More specifically, in the (σ+, σ−) configuration, we should have ΔJ\\nCEF1 = ΔJ\\nCEF2 = +2ℏ while ΔJ\\nCEF3 = −ℏ, or ΔJ\\nCEF1 = ΔJ\\nCEF2 = −ℏ while ΔJ\\nCEF3 = +2ℏ. Note that ΔJ = −ℏ is allowed for a CEF level due to the threefold rotational symmetry that relaxes the angular momentum conservation rule, but we expect that ΔJ = +2ℏ that satisfies the strict angular momentum conservation should have a higher probability and stronger Raman signals. Since CEF1 and CEF2 have much stronger Raman intensities than CEF3 (Figure ##FIG##1##2b##), it is natural to conclude that ΔJ\\nCEF1 = ΔJ\\nCEF2 = +2ℏ while ΔJ\\nCEF3 = −ℏ in the (σ+, σ−) configuration. Similarly, ΔJ\\nCEF1 = ΔJ\\nCEF2 = −2ℏ while ΔJ\\nCEF3 = +ℏ in the (σ−, σ+) configuration. In terms of matrix operations, ⟨ψ1±|J+J+|ψ0±⟩, ⟨ψ1±|J−J−|ψ0±⟩, ⟨ψ2±|J+J+|ψ0±⟩, ⟨ψ2±|J−J−|ψ0±⟩, ⟨ψ3±|J−|ψ0±⟩, and ⟨ψ3±|J+|ψ0±⟩ should be significant while ⟨ψ3±|J+J+|ψ0±⟩, ⟨ψ3±|J−J−|ψ0±⟩, ⟨ψ1±|J+J−|ψ0±⟩, ⟨ψ1±|J−J+|ψ0±⟩, ⟨ψ2±|J+J−|ψ0±⟩, ⟨ψ2±|J−J+|ψ0±⟩, ⟨ψ3±|J+J−|ψ0±⟩, and ⟨ψ3±|J−J+|ψ0±⟩ should all be small or zero (the last six matrix elements correspond to the co‐circular polarization).
\",\n \"
As shown in Figure ##FIG##0##1b##, the eigenstates of CEF levels are linear combinations of multiplets m\\n\\nJ\\n = −7/2…7/2: |ψ±〉 = ∑mj=−72,…72cmj±|72,mj⟩. Typically, CEF parameters are fit to a CEF Hamiltonian with Stevens operators given by the symmetry of the ionic environment of the 4f orbitals, as described in the Experimental Section. The procedure is frequently under‐constrained, and there are enough degrees of freedom to fit experimentally observed CEF energy levels with more than one set of CEF parameters that minimize the error that may exist. The order of the eigenstates may not be the same across those sets. However, once the constraints imposed by the optical selection rules discussed above are taken into account, we are able to find a set of CEF parameters, as indicated by filled and empty circles in Figure ##FIG##0##1b##, that can explain the observed helicity and magnetic field dependence in Figures ##FIG##1##2## and ##FIG##2##3##, and we can make final assignments of CEF1‐3. As illustrated in Figure ##FIG##3##\\n4a##, CEF1 comes from |ψ0+⟩→|ψ1+⟩ in the (σ+, σ−) configuration, where ΔJ\\nphoton = −2ℏ and ΔJ\\nCEF1 = +2ℏ, or |ψ0−⟩→|ψ1−⟩ in the (σ−, σ+) configuration, where ΔJ\\nphoton = +2ℏ and ΔJ\\nCEF1 = −2ℏ (see the arrows for the transitions). We note that the transition of |ψ0+⟩→|ψ1−⟩ with ΔJ\\nCEF1 = −ℏ is allowed in (σ+, σ−) and the transition of |ψ0−⟩→|ψ1+⟩ with ΔJ\\nCEF1 = +ℏ is allowed in (σ−, σ+), since ΔJ\\ntotal = −3ℏ and +3ℏ, respectively, which satisfies the discrete angular momentum conservation. However, these transitions allowed by the crystal threefold rotational symmetry are of a higher order and should be much weaker. Similar transitions apply for CEF2, although they correspond to |ψ0+⟩→|ψ2+⟩ or |ψ0−⟩→|ψ2−⟩. For CEF3 in Figure ##FIG##3##4b##, however, any transition with ΔJ = −2ℏ or +2ℏ is impossible; hence, the transition of |ψ0−⟩→|ψ3−⟩ with ΔJ\\nCEF3 = −ℏ is the only one allowed in (σ+, σ−) where ΔJ\\ntotal = −3ℏ, and the transition of |ψ0+⟩→|ψ3+⟩ with ΔJ\\nCEF3 = +ℏ is the only one allowed in (σ−, σ+) where ΔJ\\ntotal = +3ℏ. This could explain why CEF3 is much weaker than CEF1 and CEF2.
\",\n \"
As for the phonon Raman modes, in general, a nondegenerate Γ‐point phonon (like the A\\n1g Raman mode in NaYbSe2) cannot have angular momentum since the eigenvector is a real number. It is therefore only observed in the co‐circular polarization configuration, as shown in Figure ##FIG##1##2b##. However, when the Γ‐point phonon is doubly degenerate, the two real eigenvectors can be reconstructed by a complex superposition as shown in Figure ##FIG##0##1a##. Therefore, doubly degenerate modes like the E\\ng Raman mode in NaYbSe2 can have (pseudo)angular momentum of ±ℏ.[\\n##REF##26406841##\\n3\\n##, ##UREF##6##\\n9\\n##, ##UREF##47##\\n60\\n##, ##UREF##49##\\n62\\n##\\n] With nonzero angular momentum, it can change the chirality of incident photons and appear in the cross‐circular polarization configuration. As shown in Figure ##FIG##3##4c##, in the (σ+, σ−) configuration, ΔJ\\nphoton = −2ℏ and ΔJEg,−=−ℏ, so it is allowed by the discrete angular momentum conservation as ΔJ\\ntotal = −3ℏ; In the (σ−, σ+) configuration, ΔJ\\nphoton = +2ℏ and ΔJEg,+=+ℏ, giving rise to ΔJ\\ntotal = +3ℏ. Such an optical selection rule is similar to that shown for CEF3 in Figure ##FIG##3##4b##, which explains why both are observed in the cross‐circular polarization. Similar results have been reported for E symmetry Raman modes in TMDs, CrBr3, and quartz, among other materials.[\\n##UREF##6##\\n9\\n##, ##UREF##47##\\n60\\n##, ##UREF##48##\\n61\\n##\\n]\\n
\",\n \"
Finally, moving on to the VBS ω, it is much stronger in the co‐circular polarization configuration and its magnetic field dependence is weak (Figures ##FIG##1##2## and ##FIG##2##3##), in stark contrast to CEF1‐3. This indicates that the change in angular momentum for ω is zero: ΔJ\\n\\nz\\n = 0. Since it is a coupled state between the CEF1 and the E\\ng mode, we consider the 2 × ~2 dimensional space |ψ1±⟩⊗|Eg,±⟩. As the angular momentum change ΔJ\\n\\nz\\n for CEF1 is finite (Figure ##FIG##3##4a##), the phonon subsystem has to carry additional angular momentum (Figure ##FIG##3##4c##) in order to yield zero angular momentum for ω. Therefore, the circular basis {$E$g, +, $E$g, $‐$} is the natural choice for describing the eigenvibration. As discussed previously, CEF1 can arise from four possible transitions while E\\ng can be $E$g, + and $E$g, $‐$, which gives rise to a total of eight possible transitions for ω. Based on the angular momentum conservation rule, however, we found that two states, ω+=|ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and ω−=|ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, are the most probable (see more details in the Supporting Information). As shown in Figure ##FIG##3##4d##, due to the angular momentum transfer between the CEF1 and E\\ng mode, the excited state of ω, |ψ1+⟩⊗|Eg,+⟩ or |ψ1−⟩⊗|Eg,−⟩, has the z‐component of the angular momentum effectively raised or lowered by ℏ compared to the |ψ1+⟩ or |ψ1−⟩ shown in Figure ##FIG##3##4a##, respectively. As a result, the transitions of |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have ΔJ\\n\\nz\\n = 0 to satisfy the selection rule in the co‐circular polarization configuration and explain the weak magnetic field dependence.
\",\n \"
\\nFigure ##FIG##4##\\n5a,b## illustrates the two states, |ψ1+⟩⊗|Eg,+⟩ and |ψ1−⟩⊗|Eg,−⟩ based on the observations described above. In Figure ##FIG##4##5a##, when the ground state is |ψ0+⟩ (shown in light pink), the J\\n+\\nJ\\n− or J\\n−\\nJ\\n+ operator excites the system to the + branch, |ψ1+⟩⊗|Eg,+⟩ (where the CEF excitation corresponds to the + branch and the phononic excitation corresponds to + angular momentum). Figure ##FIG##4##5b## shows the same effect for |ψ0−⟩ to |ψ1−⟩⊗|Eg,−⟩. Because the transition is degenerate for |ψ0∓⟩→ω± with J\\n+\\nJ\\n− or J\\n−\\nJ\\n+, this eigenspace, in principle, can be transduced from a QSL ground state. If the QSL ground state is written as ∑j∏iψ0σij – where i is the site index, j the configuration index, and σ\\nij\\n = +, − is the branch—then J\\n+\\nJ\\n− or J\\n−\\nJ\\n+ should bring the system into ∑j∏iωσij within the point spread volume of the excitation beam, transducing the possible ground state entanglement to the VBS ω±.
\",\n \"
It is worth noting that, in the original Thalmeier–Fulde model, two VBS modes are expected to emerge from the hybridization of a CEF mode and a phonon mode.[\\n##UREF##34##\\n41\\n##\\n] Besides the two most probable VBS transitions shown in Figure ##FIG##3##4d## that give rise to the observed ω peak in the Raman spectra, there are six other transitions that could contribute to the other VBS mode that is not observed in our Raman measurements (see Equation (##SUPPL##0##S1##), Supporting Information). Since the picture of how orbital and phononic excitations couple is not entirely clear, a definitive answer to why other transitions and the other VBS mode do not appear in Raman scattering is beyond the scope of this work. It is expected that the electron–phonon coupling may be more complicated compared to the conventional coupling between orbital angular momentum and spin angular momentum.[\\n##UREF##50##\\n63\\n##\\n] Therefore, we hypothesize that the coupling between the CEF1 and the Eg mode is not the same as the conventional coupling between orbital and spin angular momenta. There is a coupling coefficient before the derived ΔJ\\nω (see Equation (##SUPPL##0##S1##), Supporting Information), leading to non‐integer ΔJ\\nω unless it is zero to begin with. As a consequence, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩, |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩, |ψ0+⟩→|ψ1−⟩⊗|Eg,+⟩, and |ψ0−⟩→|ψ1+⟩⊗|Eg,−⟩ that have zero angular momentum can satisfy the angular momentum conservation rule in the co‐circular polarization configuration, and other transitions are forbidden in any polarization channel. Moreover, as discussed previously, since |ψ0+⟩→|ψ1−⟩ and |ψ0−⟩→|ψ1+⟩ are of a higher order and much less probable, only |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩ have detectable signals (see Figure ##FIG##3##4d##). This could explain why other transitions and the other VBS modes are not observed.
We have used polarized field‐dependent Raman spectroscopy to elucidate a vibronic bound state in the quantum spin liquid, NaYbSe2. Of course, future direct infrared excitation of the modes explored in this manuscript could prove to be a more efficient method for direct control of angular momentum transfer. We have measured quantized angular momentum transfer ΔJ\n\nz\n = ±ℏ between an orbital and a phonon subsystem due to strong coupling between orbitals and E\ng phonons. This was verified by observing clean selection rules between the ground and excited‐state orbital levels as well as the vibronic bound state. This transfer of angular momentum may enable the on‐demand creation of phonon condensates carrying macroscopic angular momentum, though the quantum many‐body physics of the orbital‐phonon interactions does require further study. If the phonon band structure is modified by this coupling, it may also be possible to access the resulting Berry curvature and probe the robustness of potential topological invariants. Additionally, the observation of photon‐ and CEF‐mediated angular‐momentum transfer suggests that there may be many more possible routes to the creation and control of phononic angular momentum by coupling to electronic, spin, and orbital degrees of freedom in a given system. This finding thus creates a framework that may ultimately enable the transduction of possible QSL ground states to experimentally accessible VBSs.
"],"string":"[\n \"Conclusion\",\n \"
We have used polarized field‐dependent Raman spectroscopy to elucidate a vibronic bound state in the quantum spin liquid, NaYbSe2. Of course, future direct infrared excitation of the modes explored in this manuscript could prove to be a more efficient method for direct control of angular momentum transfer. We have measured quantized angular momentum transfer ΔJ\\n\\nz\\n = ±ℏ between an orbital and a phonon subsystem due to strong coupling between orbitals and E\\ng phonons. This was verified by observing clean selection rules between the ground and excited‐state orbital levels as well as the vibronic bound state. This transfer of angular momentum may enable the on‐demand creation of phonon condensates carrying macroscopic angular momentum, though the quantum many‐body physics of the orbital‐phonon interactions does require further study. If the phonon band structure is modified by this coupling, it may also be possible to access the resulting Berry curvature and probe the robustness of potential topological invariants. Additionally, the observation of photon‐ and CEF‐mediated angular‐momentum transfer suggests that there may be many more possible routes to the creation and control of phononic angular momentum by coupling to electronic, spin, and orbital degrees of freedom in a given system. This finding thus creates a framework that may ultimately enable the transduction of possible QSL ground states to experimentally accessible VBSs.
The notion that phonons can carry pseudo‐angular momentum has many major consequences, including topologically protected phonon chirality, Berry curvature of phonon band structure, and the phonon Hall effect. When a phonon is resonantly coupled to an orbital state split by its crystal field environment, a so‐called vibronic bound state forms. Here, a vibronic bound state is observed in NaYbSe2, a quantum spin liquid candidate. In addition, field and polarization dependent Raman microscopy is used to probe an angular momentum transfer of ΔJ\n\nz\n = ±ℏ between phonons and the crystalline electric field mediated by the vibronic bound stat. This angular momentum transfer between electronic and lattice subsystems provides new pathways for selective optical addressability of phononic angular momentum via electronic ancillary states.
","
Angular momentum transfer between phonons and the crystal electric field in a candidate quantum spin liquid is observed with polarization‐ and magnetic‐field‐dependent Raman microscopy. These results provide a new framework for selective optical addressability of phononic angular momentum via electronic, spin, and orbital degrees of freedom in emerging quantum materials.\n\n
The notion that phonons can carry pseudo‐angular momentum has many major consequences, including topologically protected phonon chirality, Berry curvature of phonon band structure, and the phonon Hall effect. When a phonon is resonantly coupled to an orbital state split by its crystal field environment, a so‐called vibronic bound state forms. Here, a vibronic bound state is observed in NaYbSe2, a quantum spin liquid candidate. In addition, field and polarization dependent Raman microscopy is used to probe an angular momentum transfer of ΔJ\\n\\nz\\n = ±ℏ between phonons and the crystalline electric field mediated by the vibronic bound stat. This angular momentum transfer between electronic and lattice subsystems provides new pathways for selective optical addressability of phononic angular momentum via electronic ancillary states.
\",\n \"
Angular momentum transfer between phonons and the crystal electric field in a candidate quantum spin liquid is observed with polarization‐ and magnetic‐field‐dependent Raman microscopy. These results provide a new framework for selective optical addressability of phononic angular momentum via electronic, spin, and orbital degrees of freedom in emerging quantum materials.\\n\\n
Single crystals of NaYbSe2 were synthesized from NaCl (crystalline powder, 99+%), Yb (ingot, 99.9%), and Se (powder, 99.999%) via the flux method. The finely ground flux mixtures of NaCl, Yb, and Se with a molar ratio of 20:1:2.4 were heated slowly to 850 °C in a vacuum. After 2 weeks, the furnace was cooled to room temperature at a rate of 40 °C h−1. The laboratory‐grown single crystals of NaYbSe2 were separated from excess alkali halide flux by washing with deionized water and isopropyl alcohol inside a fume‐hood. CsYbSe2 single crystals were grown using a related flux method that is described in more detail in previous work.[\n##UREF##27##\n34\n##\n]\n
","Raman Spectroscopy","
Variable temperature Raman spectra were acquired in a Montana Instruments closed‐cycle cryostat using an in‐vacuum objective with a numerical aperture of 0.85. A 1.5 mW, 532.03 nm continuous wave laser excited the sample in an out‐of‐plane back scattering geometry (beam path ‖ c). Rayleigh scattering was minimized with either a set of three Optigrate volume Bragg gratings or a set of Semrock RazorEdge ultrasteep dichroic and long‐pass edge filters with cutoff at 90 cm−1, and spectra were acquired with a 30 s exposure time.
","
Magnetic‐field‐dependent Raman spectra were acquired at a fixed temperature of T = 4 K for H ‖ c in a customized Leiden dilution refrigerator with free space optical access to the sample at the mixing chamber stage.[\n##UREF##51##\n64\n##\n] The spectra were taken with an Andor Kymera 193 spectrograph (2400 line mm−1 grating) and a Newton EMCCD DU970P‐BV camera. The same laser and filters were used as with the variable temperature measurements, with the laser power set to 1.0 mW and a typical exposure time of 300 s per spectrum. Achromatic half‐wave plates and quarter‐wave plates were mounted on rotators for automated polarization control in both microscopes.
","Crystal Field Hamiltonian","
For a review of the approaches used here, see, for example, Baqrtolomé et al.[\n##UREF##52##\n65\n##\n] The CEF Hamiltonian for NaYbSe2 within the point charge approximation[\n##UREF##16##\n23\n##, ##UREF##18##\n25\n##, ##UREF##25##\n32\n##, ##UREF##53##\n66\n##\n] that described the ground state manifold J = 7/2 is\n\n
","
The expected helicity dependence of the CEF excitations follows from the selection rule bridging the two relevant states. The eigenstates for the energy levels described in Equation (##FORMU##0##1##) are:\nwhere the relative weights cmj± are determined by B20, B40, B43, B60, B63, and B66. The eigenstates are shown in Figure ##FIG##0##1b##. Due to the threefold symmetry of the Yb3 + environment, a threefold periodicity and hence angular momentum folding, analogous to the the Umklapp process for linear momentum[\n##UREF##49##\n62\n##\n] was expected. Without further constraints, the six parameters have enough degrees of freedom to fit experimentally observed energy levels. More than one set of CEF parameters that minimize the error might exist and the order of the eigenstates might not be the same across those sets. For example, the little group spanned by the special pair with single angular momentum eigenstate |72,32⟩ or |72,−32⟩ was assigned to CEF1 in Zhang et al.[\n##UREF##16##\n23\n##\n] and CEF2 in Scheie et al.[\n##UREF##30##\n37\n##\n] Schimidt et al. pointed out that they cannot be the ground state due to observed in‐plane field dependence at low temperatures.[\n##UREF##18##\n25\n##\n] In addition to the Zeeman dependence H\n\nB\n = −g\n\nJ\nμ\nB\n\nJ\n\nz\n · B, several additional corrections have been considered in the literature. For example, Pocs et al.[\n##UREF##25##\n32\n##\n] considered an H\nXXZ term. Zhang et al. considered anisotropic spin–spin interactions.[\n##UREF##54##\n67\n##\n]\n
","
In the context of magnetostrictive coupling with CEF modes, Callan et al. considered an additional term Hme=−∑Γνζ(Γν)u(Γν)Q(Γν) where ζ is the coupling strength, u(Γν) are phonon operators, and Q(Γν) is the transformed phonon mode octupolar operator on the CEF manifold. The Callen–Callen[\n##UREF##55##\n68\n##, ##UREF##56##\n69\n##\n] magnetoelastic interaction is quadruplar. The quadruple operator (l = 2) is given by A\n1g + 2E\ng. Hence,\n\n
","
For Q(EgII), since Jx2−Jy2=(J+J++J−J−)/2, it induced ΔJ\n\nz\n = ±2ℏ, while for J\n\nx\n\nJ\n\ny\n + J\n\ny\n\nJ\n\nx\n = iJ\n\nz\n, ΔJ\n\nz\n = 0. For Q(EgI), J\n\nz\n\nJ\n\nx\n + J\n\nx\n\nJ\n\nz\n and J\n\nz\n\nJ\n\nx\n + J\n\nx\n\nJ\n\nz\n both induces ΔJ\n\nz\n = ±1ℏ.
","Fitting Experimental Helicitiy‐Resolved Data to a CEF Hamiltonian","
To fit the experimental data, a general non‐linear model normalization procedure in Mathematica was used. To start, a general set of eigenvalues and eigenvectors as a function of the CEF parameters was obtained by direct diagonalization of the CEF Hamiltonian. The eigenvectors were sorted based on their eigenvalues for each test parameter space. Then the cost function was defined by the sum of the squared errors between transitions and the data, with both inter‐branch and intra‐branch transitions considered. The selection rules were added as a term in the cost function when necessary. The final CEF parameters were the set that minimized the cost function. However, it was noted that the field dependence of the model was only qualitatively accurate not quantitatively: the experimentally observed levels were only 30% to 60% of the level shift in magnetic field. It was reported that there was non‐negligible magnetoelastic coupling and the field dependence needed to include corresponding terms in the Hamiltonian.
","Bayesian Inference","
Bayesian inference was employed to extract spectral parameters such as peak positions and widths. This approach is based on Bayes' rule: P(θ|y)=P(y|θ)P(θ)P(y). In the context of spectral data, the priors P(θ) were the true distribution of the parameters such as peak position, peak height, and peak width. The likelihood P(y|θ) was the experimental data. The posterior P(θ|y) was the conditional distribution of the experiment parameters given the experimental data. P(y) was a normalization factor. The distribution of the priors of the parameters was assumed to be Gaussian or uniform. Additional noise, offset, and slope were added to capture backgrounds unrelated to the peak parameters. To carry out the inference, the Hamiltonian Monte Carlo Python package PyMC3\n[\n##UREF##42##\n54\n##\n] was used. A hierarchical model was constructed to concurrently extract peak parameters from a family of spectra, such as the temperature dependence or magnetic field dependence. A no U‐Turns (NUTS) sampler was used with four chains with 3000 samples per chain. It takes between 20 min to 3 h for a peak over a dataset to converge.
","Conflict of Interest","
The authors declare no conflict of interest.
","Author Contributions","
All authors discussed the results thoroughly. Y.‐Y.P., C.E.M., and B.J.L. performed magneto‐Raman measurements. L.L. performed Raman tensor analysis. G.P. and J.X. grew the samples. Y.‐Y.P and L.L. did the data analysis with inputs from L.L., M.C., J.S.G., and B.J.L. X.L. and L.L. performed DFT calculations. A.S.S., D.P., S.W., and B.J.L initiated and oversaw the project. Y.‐Y.P., L.L. and B.J.L wrote most of the manuscript with contributions from all authors.
Single crystals of NaYbSe2 were synthesized from NaCl (crystalline powder, 99+%), Yb (ingot, 99.9%), and Se (powder, 99.999%) via the flux method. The finely ground flux mixtures of NaCl, Yb, and Se with a molar ratio of 20:1:2.4 were heated slowly to 850 °C in a vacuum. After 2 weeks, the furnace was cooled to room temperature at a rate of 40 °C h−1. The laboratory‐grown single crystals of NaYbSe2 were separated from excess alkali halide flux by washing with deionized water and isopropyl alcohol inside a fume‐hood. CsYbSe2 single crystals were grown using a related flux method that is described in more detail in previous work.[\\n##UREF##27##\\n34\\n##\\n]\\n
\",\n \"Raman Spectroscopy\",\n \"
Variable temperature Raman spectra were acquired in a Montana Instruments closed‐cycle cryostat using an in‐vacuum objective with a numerical aperture of 0.85. A 1.5 mW, 532.03 nm continuous wave laser excited the sample in an out‐of‐plane back scattering geometry (beam path ‖ c). Rayleigh scattering was minimized with either a set of three Optigrate volume Bragg gratings or a set of Semrock RazorEdge ultrasteep dichroic and long‐pass edge filters with cutoff at 90 cm−1, and spectra were acquired with a 30 s exposure time.
\",\n \"
Magnetic‐field‐dependent Raman spectra were acquired at a fixed temperature of T = 4 K for H ‖ c in a customized Leiden dilution refrigerator with free space optical access to the sample at the mixing chamber stage.[\\n##UREF##51##\\n64\\n##\\n] The spectra were taken with an Andor Kymera 193 spectrograph (2400 line mm−1 grating) and a Newton EMCCD DU970P‐BV camera. The same laser and filters were used as with the variable temperature measurements, with the laser power set to 1.0 mW and a typical exposure time of 300 s per spectrum. Achromatic half‐wave plates and quarter‐wave plates were mounted on rotators for automated polarization control in both microscopes.
\",\n \"Crystal Field Hamiltonian\",\n \"
For a review of the approaches used here, see, for example, Baqrtolomé et al.[\\n##UREF##52##\\n65\\n##\\n] The CEF Hamiltonian for NaYbSe2 within the point charge approximation[\\n##UREF##16##\\n23\\n##, ##UREF##18##\\n25\\n##, ##UREF##25##\\n32\\n##, ##UREF##53##\\n66\\n##\\n] that described the ground state manifold J = 7/2 is\\n\\n
\",\n \"
The expected helicity dependence of the CEF excitations follows from the selection rule bridging the two relevant states. The eigenstates for the energy levels described in Equation (##FORMU##0##1##) are:\\nwhere the relative weights cmj± are determined by B20, B40, B43, B60, B63, and B66. The eigenstates are shown in Figure ##FIG##0##1b##. Due to the threefold symmetry of the Yb3 + environment, a threefold periodicity and hence angular momentum folding, analogous to the the Umklapp process for linear momentum[\\n##UREF##49##\\n62\\n##\\n] was expected. Without further constraints, the six parameters have enough degrees of freedom to fit experimentally observed energy levels. More than one set of CEF parameters that minimize the error might exist and the order of the eigenstates might not be the same across those sets. For example, the little group spanned by the special pair with single angular momentum eigenstate |72,32⟩ or |72,−32⟩ was assigned to CEF1 in Zhang et al.[\\n##UREF##16##\\n23\\n##\\n] and CEF2 in Scheie et al.[\\n##UREF##30##\\n37\\n##\\n] Schimidt et al. pointed out that they cannot be the ground state due to observed in‐plane field dependence at low temperatures.[\\n##UREF##18##\\n25\\n##\\n] In addition to the Zeeman dependence H\\n\\nB\\n = −g\\n\\nJ\\nμ\\nB\\n\\nJ\\n\\nz\\n · B, several additional corrections have been considered in the literature. For example, Pocs et al.[\\n##UREF##25##\\n32\\n##\\n] considered an H\\nXXZ term. Zhang et al. considered anisotropic spin–spin interactions.[\\n##UREF##54##\\n67\\n##\\n]\\n
\",\n \"
In the context of magnetostrictive coupling with CEF modes, Callan et al. considered an additional term Hme=−∑Γνζ(Γν)u(Γν)Q(Γν) where ζ is the coupling strength, u(Γν) are phonon operators, and Q(Γν) is the transformed phonon mode octupolar operator on the CEF manifold. The Callen–Callen[\\n##UREF##55##\\n68\\n##, ##UREF##56##\\n69\\n##\\n] magnetoelastic interaction is quadruplar. The quadruple operator (l = 2) is given by A\\n1g + 2E\\ng. Hence,\\n\\n
\",\n \"
For Q(EgII), since Jx2−Jy2=(J+J++J−J−)/2, it induced ΔJ\\n\\nz\\n = ±2ℏ, while for J\\n\\nx\\n\\nJ\\n\\ny\\n + J\\n\\ny\\n\\nJ\\n\\nx\\n = iJ\\n\\nz\\n, ΔJ\\n\\nz\\n = 0. For Q(EgI), J\\n\\nz\\n\\nJ\\n\\nx\\n + J\\n\\nx\\n\\nJ\\n\\nz\\n and J\\n\\nz\\n\\nJ\\n\\nx\\n + J\\n\\nx\\n\\nJ\\n\\nz\\n both induces ΔJ\\n\\nz\\n = ±1ℏ.
\",\n \"Fitting Experimental Helicitiy‐Resolved Data to a CEF Hamiltonian\",\n \"
To fit the experimental data, a general non‐linear model normalization procedure in Mathematica was used. To start, a general set of eigenvalues and eigenvectors as a function of the CEF parameters was obtained by direct diagonalization of the CEF Hamiltonian. The eigenvectors were sorted based on their eigenvalues for each test parameter space. Then the cost function was defined by the sum of the squared errors between transitions and the data, with both inter‐branch and intra‐branch transitions considered. The selection rules were added as a term in the cost function when necessary. The final CEF parameters were the set that minimized the cost function. However, it was noted that the field dependence of the model was only qualitatively accurate not quantitatively: the experimentally observed levels were only 30% to 60% of the level shift in magnetic field. It was reported that there was non‐negligible magnetoelastic coupling and the field dependence needed to include corresponding terms in the Hamiltonian.
\",\n \"Bayesian Inference\",\n \"
Bayesian inference was employed to extract spectral parameters such as peak positions and widths. This approach is based on Bayes' rule: P(θ|y)=P(y|θ)P(θ)P(y). In the context of spectral data, the priors P(θ) were the true distribution of the parameters such as peak position, peak height, and peak width. The likelihood P(y|θ) was the experimental data. The posterior P(θ|y) was the conditional distribution of the experiment parameters given the experimental data. P(y) was a normalization factor. The distribution of the priors of the parameters was assumed to be Gaussian or uniform. Additional noise, offset, and slope were added to capture backgrounds unrelated to the peak parameters. To carry out the inference, the Hamiltonian Monte Carlo Python package PyMC3\\n[\\n##UREF##42##\\n54\\n##\\n] was used. A hierarchical model was constructed to concurrently extract peak parameters from a family of spectra, such as the temperature dependence or magnetic field dependence. A no U‐Turns (NUTS) sampler was used with four chains with 3000 samples per chain. It takes between 20 min to 3 h for a peak over a dataset to converge.
\",\n \"Conflict of Interest\",\n \"
The authors declare no conflict of interest.
\",\n \"Author Contributions\",\n \"
All authors discussed the results thoroughly. Y.‐Y.P., C.E.M., and B.J.L. performed magneto‐Raman measurements. L.L. performed Raman tensor analysis. G.P. and J.X. grew the samples. Y.‐Y.P and L.L. did the data analysis with inputs from L.L., M.C., J.S.G., and B.J.L. X.L. and L.L. performed DFT calculations. A.S.S., D.P., S.W., and B.J.L initiated and oversaw the project. Y.‐Y.P., L.L. and B.J.L wrote most of the manuscript with contributions from all authors.
The authors would like to acknowledge insightful discussion with Michael A. McGuire, Allen Scheie, Xinshu Zhang, Yi Luo, Cristian Batista, Alan Tennant, and Vyacheslav Bryantsev. This research was sponsored by the U. S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division. Some of the first‐principles phonon calculations and all of the variable‐temperature, zero‐field Raman microscopy were performed at the Center for Nanophase Materials Sciences, which is a U.S. Department of Energy Office of Science User Facility. S.D.W. and G.P. acknowledge support by the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under award DE‐SC0017752. Postdoctoral research support was provided by the Intelligence Community Postdoctoral Research Fellowship Program at the Oak Ridge National Laboratory, administered by Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Office of the Director of National Intelligence.
","Data Availability Statement","
The data that support the findings of this study are available from the corresponding author upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
The authors would like to acknowledge insightful discussion with Michael A. McGuire, Allen Scheie, Xinshu Zhang, Yi Luo, Cristian Batista, Alan Tennant, and Vyacheslav Bryantsev. This research was sponsored by the U. S. Department of Energy, Office of Science, Basic Energy Sciences, Materials Sciences and Engineering Division. Some of the first‐principles phonon calculations and all of the variable‐temperature, zero‐field Raman microscopy were performed at the Center for Nanophase Materials Sciences, which is a U.S. Department of Energy Office of Science User Facility. S.D.W. and G.P. acknowledge support by the US Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under award DE‐SC0017752. Postdoctoral research support was provided by the Intelligence Community Postdoctoral Research Fellowship Program at the Oak Ridge National Laboratory, administered by Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the Office of the Director of National Intelligence.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available from the corresponding author upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
a) The E\ng eigenspace is spanned by the basis {$E$g, $x$, $E$g, $y$}, which can also be spanned by {$E$g, +, $E$g, $‐$} = {$E$g, $x$ + i$E$g, $y$, $E$g, $x$ − i$E$g, $y$}. b) Representations of the CEF eigenstates |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, and |ψ3±⟩. The eigenstates are linear combinations of multiplets m\n\nJ\n = −7/2…7/2. For example, |ψ1+⟩=|72,32⟩ is represented by precessing cones with c32≠0 for |ψ1+⟩=c32|J,32⟩. Hence, all but the mJ=32 cones are transparent (and all but the mJ=32 circles are empty). The same follows for |ψ1−⟩ and |ψ0,2,3±⟩. c) Temperature dependent Raman spectra for CEF1, $E$g, and the VBS, ω, from T = 3.3 K to T = 260 K. d) Peak positions for CEF1, E\ng and ω extracted from Bayesian inference. The symbols are medians of the posterior distribution, and the 68% and 95% HDIs are represented by darker and lighter shading, respectively.
","
a) Schematic illustration of Raman scattering where CEF1‐3, phonon modes, and ω can be excited in the process. b) Helicity‐resolved Raman spectra at T = 4 K, B = 0 T. Four prominent modes are CEF1 (with an E\ng phonon mode at its shoulder), ω, CEF2, and CEF3. The Rayleigh scattering and ω are of (σ+, σ+) and (σ−, σ−) co‐circular scattering channels, while CEF1‐3 and E\ng are of (σ+, σ−) and (σ−, σ+) cross‐circular channels.
","
Helicity‐resolved magnetic field dependence of CEFs and ω for NaYbSe2 at T = 4 K. a) CEF1, b) CEF2, c) ω, and d) CEF3. The 68% and 95% HDIs are represented by darker and lighter shading, respectively. See Supporting Information for the raw spectra that the peak positions were extracted from.
","
a) Selection rule for CEF1 corresponding to |ψ0+⟩→|ψ1+⟩ or |ψ0−⟩→|ψ1−⟩. A similar rule applies for CEF2, although the transition corresponds to |ψ0+⟩→|ψ2+⟩ or |ψ0−⟩→|ψ2−⟩. b) Selection rule for CEF3 corresponding to |ψ0−⟩→|ψ3−⟩ or |ψ0+⟩→|ψ3+⟩. Note that the threefold rotational symmetry in NaYbSe2 allows for discrete angular momentum conservation: |ΔJ\nphoton + ΔJ\nparticle|/ℏ = 0 (modulo 3). Here ΔJ\ntotal = −3ℏ or ΔJ\ntotal = 3ℏ. c) Selection rule for the doubly degenerate E\ng mode that can have (pseudo)angular momentum of ±ℏ. d) Selection rule for the vibronic bound state ω. Due to the angular momentum transfer between the CEF1 and E\ng mode, the excited state of ω, |ψ1+⟩⊗|Eg,+⟩ or |ψ1−⟩⊗|Eg,−⟩, has a completely different helicity selection rule and magnetic field dependence than CEF1.
","
Schematic illustrations of the vibronic bound state ω: a) |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and b) |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩.
"],"string":"[\n \"
a) The E\\ng eigenspace is spanned by the basis {$E$g, $x$, $E$g, $y$}, which can also be spanned by {$E$g, +, $E$g, $‐$} = {$E$g, $x$ + i$E$g, $y$, $E$g, $x$ − i$E$g, $y$}. b) Representations of the CEF eigenstates |ψ0±⟩, |ψ1±⟩, |ψ2±⟩, and |ψ3±⟩. The eigenstates are linear combinations of multiplets m\\n\\nJ\\n = −7/2…7/2. For example, |ψ1+⟩=|72,32⟩ is represented by precessing cones with c32≠0 for |ψ1+⟩=c32|J,32⟩. Hence, all but the mJ=32 cones are transparent (and all but the mJ=32 circles are empty). The same follows for |ψ1−⟩ and |ψ0,2,3±⟩. c) Temperature dependent Raman spectra for CEF1, $E$g, and the VBS, ω, from T = 3.3 K to T = 260 K. d) Peak positions for CEF1, E\\ng and ω extracted from Bayesian inference. The symbols are medians of the posterior distribution, and the 68% and 95% HDIs are represented by darker and lighter shading, respectively.
\",\n \"
a) Schematic illustration of Raman scattering where CEF1‐3, phonon modes, and ω can be excited in the process. b) Helicity‐resolved Raman spectra at T = 4 K, B = 0 T. Four prominent modes are CEF1 (with an E\\ng phonon mode at its shoulder), ω, CEF2, and CEF3. The Rayleigh scattering and ω are of (σ+, σ+) and (σ−, σ−) co‐circular scattering channels, while CEF1‐3 and E\\ng are of (σ+, σ−) and (σ−, σ+) cross‐circular channels.
\",\n \"
Helicity‐resolved magnetic field dependence of CEFs and ω for NaYbSe2 at T = 4 K. a) CEF1, b) CEF2, c) ω, and d) CEF3. The 68% and 95% HDIs are represented by darker and lighter shading, respectively. See Supporting Information for the raw spectra that the peak positions were extracted from.
\",\n \"
a) Selection rule for CEF1 corresponding to |ψ0+⟩→|ψ1+⟩ or |ψ0−⟩→|ψ1−⟩. A similar rule applies for CEF2, although the transition corresponds to |ψ0+⟩→|ψ2+⟩ or |ψ0−⟩→|ψ2−⟩. b) Selection rule for CEF3 corresponding to |ψ0−⟩→|ψ3−⟩ or |ψ0+⟩→|ψ3+⟩. Note that the threefold rotational symmetry in NaYbSe2 allows for discrete angular momentum conservation: |ΔJ\\nphoton + ΔJ\\nparticle|/ℏ = 0 (modulo 3). Here ΔJ\\ntotal = −3ℏ or ΔJ\\ntotal = 3ℏ. c) Selection rule for the doubly degenerate E\\ng mode that can have (pseudo)angular momentum of ±ℏ. d) Selection rule for the vibronic bound state ω. Due to the angular momentum transfer between the CEF1 and E\\ng mode, the excited state of ω, |ψ1+⟩⊗|Eg,+⟩ or |ψ1−⟩⊗|Eg,−⟩, has a completely different helicity selection rule and magnetic field dependence than CEF1.
\",\n \"
Schematic illustrations of the vibronic bound state ω: a) |ψ0+⟩→|ψ1+⟩⊗|Eg,+⟩ and b) |ψ0−⟩→|ψ1−⟩⊗|Eg,−⟩.
Electron–phonon coupling (EPC) is essential in condensed matter physics, leading to pivotal phenomena such as superconductivity,[\n##UREF##0##\n1\n##, ##REF##27015504##\n3\n##\n‐3] ultrahigh thermal conductivity,[\n##UREF##2##\n4\n##\n] charge density wave,[\n##REF##11328180##\n5\n##\n] colossal magneto‐resistant,[\n##UREF##3##\n6\n##\n] etc. To date, there has been rarely reports on experimental determination of the EPC strength in transition metal perovskites, except for superconducting oxides.[\n##UREF##4##\n7\n##\n] Needless to say, understanding the EPC is also important for such non‐superconducting oxides to reveal their underlying microscopic mechanisms.[\n##UREF##5##\n8\n##, ##UREF##6##\n9\n##\n]\n
","
Transition‐metal perovskite LaCoO3 exhibits a diamagnetic insulating low‐spin phase (t2g6eg0,S=0) at low temperatures, whereas it becomes metallic at higher temperature with the Co ions assuming a higher spin state (t2g4eg2,S=2 or t2g5eg1,S=1).[\n##UREF##7##\n10\n##, ##UREF##8##\n11\n##, ##UREF##9##\n12\n##, ##REF##17280319##\n13\n##\n] Recently, it was found that epitaxial tensile‐strained LaCoO3 thin films become ferromagnetic below 85 K,[\n##UREF##10##\n15\n##, ##REF##33929867##\n16\n##, ##UREF##11##\n17\n##, ##UREF##12##\n18\n##\n] exhibiting application potentials in magnetic devices. To date, the EPC strength in LaCoO3 has rarely been measured, especially for thin film samples, although the EPC is one of the primary concerns.[\n##UREF##9##\n12\n##, ##UREF##10##\n15\n##\n] Moreover, the phonon–phonon scattering (PPS) in LaCoO3 has rarely been investigated either.
","
It has rarely been reported whether the EPC and PPS are spatially separated in such oxides or other solids, although it is well known that these two processes are largely detached temporally by exhibiting different characteristic lifetimes.[\n##UREF##13##\n19\n##, ##REF##12618781##\n20\n##\n] Ultrafast time‐resolved pump‐probe spectroscopy is the most viable experimental tool to detect both the EPC and PPS in quantum materials.[\n##UREF##1##\n2\n##, ##REF##27015504##\n3\n##, ##UREF##14##\n21\n##, ##REF##34852544##\n22\n##\n] In parallel, coherent phonon can also be generated and detected by ultrafast pump‐probe spectroscopy.[\n##UREF##15##\n23\n##, ##REF##25031087##\n24\n##, ##UREF##16##\n25\n##\n]\n
","
In this work, we investigate the fluence‐dependent photo‐carriers dynamics in a 40 nm thick LaCoO3 film on a SrTiO3 substrate. Two distinct relaxation processes with lifetimes τ\nfast = 0.2 ps and τ\nslow = 0.9 ps are experimentally observed. The nominal EPC strength λE\n\ng is experimentally determined to be 0.30. We also detected a coherent acoustic phonon in our experiment. Significantly, we identify that the EPC and PPS are basically spatially separated by the interface of the sample.
"],"string":"[\n \"Introduction\",\n \"
Electron–phonon coupling (EPC) is essential in condensed matter physics, leading to pivotal phenomena such as superconductivity,[\\n##UREF##0##\\n1\\n##, ##REF##27015504##\\n3\\n##\\n‐3] ultrahigh thermal conductivity,[\\n##UREF##2##\\n4\\n##\\n] charge density wave,[\\n##REF##11328180##\\n5\\n##\\n] colossal magneto‐resistant,[\\n##UREF##3##\\n6\\n##\\n] etc. To date, there has been rarely reports on experimental determination of the EPC strength in transition metal perovskites, except for superconducting oxides.[\\n##UREF##4##\\n7\\n##\\n] Needless to say, understanding the EPC is also important for such non‐superconducting oxides to reveal their underlying microscopic mechanisms.[\\n##UREF##5##\\n8\\n##, ##UREF##6##\\n9\\n##\\n]\\n
\",\n \"
Transition‐metal perovskite LaCoO3 exhibits a diamagnetic insulating low‐spin phase (t2g6eg0,S=0) at low temperatures, whereas it becomes metallic at higher temperature with the Co ions assuming a higher spin state (t2g4eg2,S=2 or t2g5eg1,S=1).[\\n##UREF##7##\\n10\\n##, ##UREF##8##\\n11\\n##, ##UREF##9##\\n12\\n##, ##REF##17280319##\\n13\\n##\\n] Recently, it was found that epitaxial tensile‐strained LaCoO3 thin films become ferromagnetic below 85 K,[\\n##UREF##10##\\n15\\n##, ##REF##33929867##\\n16\\n##, ##UREF##11##\\n17\\n##, ##UREF##12##\\n18\\n##\\n] exhibiting application potentials in magnetic devices. To date, the EPC strength in LaCoO3 has rarely been measured, especially for thin film samples, although the EPC is one of the primary concerns.[\\n##UREF##9##\\n12\\n##, ##UREF##10##\\n15\\n##\\n] Moreover, the phonon–phonon scattering (PPS) in LaCoO3 has rarely been investigated either.
\",\n \"
It has rarely been reported whether the EPC and PPS are spatially separated in such oxides or other solids, although it is well known that these two processes are largely detached temporally by exhibiting different characteristic lifetimes.[\\n##UREF##13##\\n19\\n##, ##REF##12618781##\\n20\\n##\\n] Ultrafast time‐resolved pump‐probe spectroscopy is the most viable experimental tool to detect both the EPC and PPS in quantum materials.[\\n##UREF##1##\\n2\\n##, ##REF##27015504##\\n3\\n##, ##UREF##14##\\n21\\n##, ##REF##34852544##\\n22\\n##\\n] In parallel, coherent phonon can also be generated and detected by ultrafast pump‐probe spectroscopy.[\\n##UREF##15##\\n23\\n##, ##REF##25031087##\\n24\\n##, ##UREF##16##\\n25\\n##\\n]\\n
\",\n \"
In this work, we investigate the fluence‐dependent photo‐carriers dynamics in a 40 nm thick LaCoO3 film on a SrTiO3 substrate. Two distinct relaxation processes with lifetimes τ\\nfast = 0.2 ps and τ\\nslow = 0.9 ps are experimentally observed. The nominal EPC strength λE\\n\\ng is experimentally determined to be 0.30. We also detected a coherent acoustic phonon in our experiment. Significantly, we identify that the EPC and PPS are basically spatially separated by the interface of the sample.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results","Photo‐Carrier Relaxation Dynamics in LaCoO<sub>3</sub>/SrTiO<sub>3</sub>\n","
We detect the relative transient differential reflectivity ΔR/R\n0 of LaCoO3/SrTiO3 as a function of delay time, for which the data recorded at various pump fluences are presented in Figure\n##FIG##0##\n1\n##. In Figure ##FIG##0##1a##, the dots are the experimental results and the solid curves are fitting results (see a latter paragraph for a quantitative description). The signal we measure is proportional to the density of the photo‐excited carriers (abbreviated as photo‐carriers), which is intrinsically due to the Fermi transition and obeys the Fermi golden rule.[\n##UREF##1##\n2\n##, ##UREF##17##\n26\n##\n] Upon the pump pulse excitation, the density of the photo carriers reaches a maximum value at time t = 0 fs, then decays through various relaxation channels, including the EPC, PPS, electron‐hole recombination, etc.[\n##UREF##14##\n21\n##\n]\n
","
In the inset of Figure ##FIG##0##1a## we summarize the max value of |ΔR/R\n0| as a function of the excitation fluence. The value |ΔR/R\n0|max increases with fluence and a linear fit (solid line) to the signal |ΔR/R\n0|max can cover most of the range of the experimental condition. At above 3.2 mJ cm−2 an off‐linear behavior arises, which indicates the occurrence of a thermal effect in the experiment. Note that the off‐linear (i.e., saturation) behavior in |ΔR/R\n0|max is a strict criterion for identifying whether there is a thermal effect (for details, see the Supporting Information of ref. [##UREF##1##2##] and references therein). The thermal (pink) and non‐thermal (blue) effect regimes are depicted by different colors as a guide for the identification.
","
To better reveal the fluence dependence of the photo‐carriers ultrafast dynamics, the normalized ΔR/R\n0 is shown in Figure ##FIG##0##1b##. The data are normalized to the |ΔR/R\n0|max value at the highest fluence excitation. To see the initial dynamics clearly, we illustrate a higher temporal resolution view of the scanning trace in the inset of Figure ##FIG##0##1b##. With increasing fluence, the normalized ΔR/R\n0 exhibits prominent changes, whereby the relaxation becomes more gradual. This reveals that the ultrafast photo‐carriers relaxation in LaCoO3/SrTiO3 is clearly dependent on the pump fluence. Note that all the data in this work are one identical set of experimental results, although they are presented in different ways to emphasize different aspects. The transient reflectivity of the SrTiO3 substrate is also measured, which is significantly different from that of the LaCoO3/SrTiO3 sample (Figure ##SUPPL##0##S2##, Supporting Information). The control experiment demonstrates that the photo‐carriers relaxation dynamics in Figure ##FIG##0##1a## is mainly attributed to the LaCoO3 thin film.
","
We quantitatively analyze the experimental results. Because the electron–phonon interaction has a characteristic lifetime at the order of 1 ps,[\n##UREF##1##\n2\n##, ##UREF##2##\n4\n##, ##UREF##13##\n19\n##, ##REF##12618781##\n20\n##\n] and the presence of a hump centered at 10 ps in the dynamics[\n##UREF##18##\n27\n##\n] may affect the assignment of the relaxation components, we choose the data fitting range to be from −0.7 to 3.6 ps to minimize the interference of the hump. Before we do the quantitative data fitting, we use a log‐scale coordinate to show ΔR/R\n0 (Figure ##SUPPL##0##S4##, Supporting Information), which more clearly reveals how many components are present in the photo‐carriers relaxation dynamics. As seen, there are three components, and the slowest one is very flat, which can be reasonably represented by a constant term. A convoluted exponential‐decay function is employed to fit the photo‐carriers relaxation dynamics, along with a decaying cosine function to fit the coherent phonon, as:\nwhere A and τ represent the amplitude and lifetime, respectively, the subscript fast, slow, and 0 mark the three components, 12πpet2/2p2is the Gaussian response function,[\n28] Ω is the angular frequency of the coherent phonon, and φ is the initial phase of the coherent phonon. The fitting results compare well with our data [Figure ##FIG##0##1a##].
","
The quantitative analysis of the fluence‐dependent results yields fluence‐dependent amplitudes and lifetimes, which are summarized in Figure\n##FIG##1##\n2\n##. The A\nfast and A\nslow exhibit positive correlations with the fluence in the non‐thermal effect regime [Figures ##FIG##1##2a,c##], which is in line with the results shown in the inset of Figure ##FIG##0##1##. The value of τ\nfast slightly increases with fluence (Figure ##FIG##1##2b##), and τ\nslow is nearly a constant (Figure ##FIG##1##2d##). Following the convention, the fast component is dominated by the EPC, and the slow component is mainly connected to the PPS. [\n##UREF##1##\n2\n##, ##REF##27015504##\n3\n##, ##UREF##2##\n4\n##\n] Such assignment is based on the characteristic interaction times for different processes (Figure ##SUPPL##0##S5##, Supporting Information),[\n##UREF##13##\n19\n##\n] which is experimentally tested true and consistent in previous investigations. From the fast component, we can obtain the explicit value of the EPC strength λ.
","Obtaining the EPC Strength <italic toggle=\"yes\">λ</italic>\n","
In a recent work,[\n4] we developed a method to obtain λ by the fluence dependence of the fast component, for which the advantages are two‐folded: 1) one can obtain the EPC strength λ at room temperature, which usually does not vary much with temperature, and 2) one can circumvent the frequently encountered dilemma that the light penetration depth and the heat capacity coefficient of a material are usually unavailable. The initial work[\n##UREF##19##\n28\n##\n] is developed for low temperature and medium‐high fluence regime. Later on, we have developed a more comprehensive version, which is extended to room temperature (thus easier to implement) and can also be applied to medium‐high fluence regime.[\n##UREF##2##\n4\n##\n] We name this model as fluence‐dependence model (FDM).[\n##UREF##2##\n4\n##, ##UREF##19##\n28\n##\n] The FDM is indeed derived from the Allen model.[\n##UREF##20##\n29\n##\n] There have also been several other sophisticated discussions and models along this direction.[\n##UREF##21##\n30\n##, ##REF##21231613##\n31\n##, ##UREF##22##\n32\n##, ##UREF##23##\n33\n##\n] In our FDM approach, the value of λ is related to the fast component lifetime τ\nfast by \nwhere kB\n is the Boltzmann constant, TL\n is the lattice temperature, Ω is the angular frequency of the phonon (usually optical phonon[\n##UREF##24##\n34\n##\n]), and Θ is an effective absorption coefficient of fluence (when a laser beam of fluence F is incident on the sample, the electron temperature increases from the equilibrium temperature TL\n to TL2+ΘF). At medium‐high fluence regime whereby the sample assumes the non‐thermal regime (i.e., the density of photocarriers is proportional to the fluence, see the inset of Figure ##FIG##0##1a##), Θ is a constant.
","
Using the equation of FDM, the fitting curve compares well with the experimental results at various fluences [Figure ##FIG##1##2b##]. We obtain that λ˂Ω3>/<Ω> = 294.9 ± 25.4 ps−2 (i.e., 129.3 ± 11.1 meV2). Usually, we take the lowest energy optical phonon mode as an example to obtain the nominal EPC strength for a quantum material. For our LaCoO3/SrTiO3 sample, it has four E\ng modes, where the lowest energy E\ng mode is the lowest energy optical phonon mode (E\ng) for the material. The second highest energy E\ng mode is related to the orbital‐phonon coupling and the Jahn–Teller distortion.[\n##REF##15524742##\n35\n##\n] Still, in the Raman result, the second lowest E\ng mode is much more prominent than the lowest energy E\ng mode, and it is the second lowest energy optical phonon mode (another A\ngg mode is silent).[\n##UREF##25##\n36\n##\n] We take the second lowest E\ng mode (with a frequency of 175.3 cm−1 [\n##REF##15524742##\n35\n##, ##UREF##25##\n36\n##, ##UREF##26##\n37\n##\n]) to be the characteristic phonon mode to obtain the nominal EPC strength. In such a way, we obtain λE\n\ng = 0.30. Note that the EPC strength of LaCoO3/SrTiO3 we obtain here is close to those of superconducting oxides YBa2Cu3O6.5 (λ ≥ 0.25) and La1.85Sr0.15CuO4 (λ ≥ 0.5).[\n##REF##21231613##\n31\n##\n] The λ = 0.30 is a regular EPC value, in the middle of the various EPC values reported. Such an EPC strength is enough to cause significant Jahn–Teller distortion.[\n##UREF##9##\n12\n##, ##UREF##10##\n15\n##\n]\n
","Coherent Phonon in Thin Film LaCoO<sub>3</sub>/SrTiO<sub>3</sub>\n","
Furthermore, as seen in Figure ##FIG##0##1b##, there is a coherent oscillation that is unambiguously observed in our fluence‐dependent ΔR/R\n0 signal. We re‐plot one of the signals (with the fluence of 1.41 mJ cm−2) in Figure\n##FIG##2##\n3a##. Here, Figure ##FIG##2##3a## shows the data with a longer temporal range than that of Figure ##FIG##0##1##. The data at a relatively longer time scale are fitted by a red wavy curve using a decaying cosine function, which corresponds to the phonon term in Equation (##FORMU##0##1##). A regular periodic oscillation is clearly seen. The inset of Figure ##FIG##2##3a## provides a closer zoom‐in view of the oscillation and the fitting curve compares well with the data. A broad hump is observed at the 4–30 ps range, which is not a part of the coherent phonon oscillation, due to the opposite phase. Previous investigations in cobalt perovskite had assigned it to the propagation of the photo‐induced metallic domain.[\n##REF##19659241##\n38\n##, ##UREF##27##\n39\n##\n] In this work we mainly focus on the coherent phonon, rather than this hump.
","
In Figures ##FIG##2##3b‐d##, we present the quantitative analysis of the coherent phonon. To better reveal the coherent oscillation, we subtract the photo‐carriers relaxation from the ΔR/R\n0 signal. The coherent oscillations at different fluences are illustrated in Figure ##FIG##2##3b##, which are offset for clarity. Each experimental oscillation trace is fitted by the decaying cosine function in Equation (##FORMU##0##1##). All the values of A\nphonon, τ\nphonon, φ, and Ω (see Equation (##FORMU##0##1##)) are obtained through the data fitting in Figure ##FIG##2##3b##. The phonon frequency Ω is shown in Figure ##FIG##2##3c##, whose average value is 45.3 GHz, nearly unchanged even up to the high fluence regime whereby the thermal effect inaugurates. The value of the phonon frequency is much smaller than a regular optical phonon frequency. We attribute it to be a coherent acoustic phonon, which is generated by the transient thermal strain induced by the ultrafast light pulses.[\n##REF##25031087##\n24\n##, ##UREF##28##\n40\n##, ##UREF##29##\n41\n##\n] Here the phonon frequency is independent of the film thickness (Figure ##SUPPL##0##S8##, Supporting Information).[\n##REF##25031087##\n24\n##, ##UREF##30##\n42\n##\n] The phonon amplitudes A\nphonon are summarized in Figure ##FIG##2##3d##, which increase linearly with fluence. Thus, unlike that for the photo‐carriers dynamics (Figure ##FIG##0##1a## and Figure ##FIG##1##2a##), no prominent saturation on coherent phonons is observed in the thermal effect regime. This indicates the lattice does not experience irreversible damage by shining the ultrafast light pulses on. Similar property has also been found in other materials (e.g., in Cd3As2\n[\n##UREF##16##\n25\n##\n]).
","
Significantly, we investigate the effect of the substrate on the photo‐carriers relaxation dynamics by comparing the ultrafast dynamics of two different samples. In the control experiment, the second sample is a 40 nm thickness LaCoO3 on a (100) LaAlO3 substrate. The pump and probe beam fluences are 0.86 and 0.14 mJ cm−2, respectively. The dynamics we obtain is shown in Figure\n##FIG##3##\n4a##, which is normalized to compare with that of the LaCoO3/SrTiO3 sample. The data for LaCoO3/SrTiO3 are obtained under a pump fluence of 0.91 mJ cm−2 and a probe fluence of 0.13 mJ cm−2, which are nearly identical to those for the LaCoO3/LaAlO3 sample. In the longer temporal range, the dynamics of these two samples exhibit an apparent difference. However, for the shorter temporal range, the dynamics for the two samples nearly overlap in the initial range (see the inset of Figure ##FIG##3##4a## for better revealed the ultrafast relaxation at initial stage). This indicates that the fast and slow components behave in a different way. To better illaustrate the data, following the aforementioned procedures (Equation (##FORMU##0##1##)), we display the fast and slow components for both samples in Figure ##FIG##3##4b##, along with a normalized version in its inset. The solid curves are fast components and the dashed curves are slow components. The fast components for the two samples are nearly overlapped; as a contrast, the slow components for the two samples are clearly different. While the lifetime τ\nfast = 0.17 ± 0.03 ps is nearly identical for both samples (τ\nfast for the LaCoO3/SrTiO3 sample is 0.18 ps (Figure ##FIG##1##2b##), the lifetime τ\nslow = 0.67 ± 0.18 ps is different from that for the other sample (τ\nslow = 0.90 ± 0.17 ps (Figure ##FIG##1##2d##) for the LaCoO3/SrTiO3 sample). These results indicate that the EPC strength for the two samples is very close; however, the PPS rate in LaCoO3/SrTiO3 is noticeably lower than in LaCoO3/LaAlO3.
","
In the control experiment, we follow the same procedure to analyze the coherent acoustic phonon in the LaCoO3/LaAlO3 sample. The oscillations were obtained by subtracting the photo‐carriers relaxation in Figure ##FIG##3##4a##, and are presented in Figure ##FIG##3##4c##, both fitted with cosine decaying functions. The frequency domain results are obtained through Fast Fourier Transformation (FFT) (see Figure ##FIG##3##4d##). Interestingly, the two frequency domain peaks are located at 33.3 (for LaCoO3/LaAlO3) and 45.6 GHz (for LaCoO3/SrTiO3), respectively. While these two values are different, we find that the value for LaCoO3/LaAlO3 is in well agreement with that in Fe2O3/LaAlO3,[\n##UREF##31##\n43\n##\n] and that for LaCoO3/SrTiO3 is in agreement with that in Fe2O3/SrTiO3 and LaRhO3/SrTiO3.[\n##UREF##30##\n42\n##, ##UREF##31##\n43\n##\n]\n
"],"string":"[\n \"Results\",\n \"Photo‐Carrier Relaxation Dynamics in LaCoO<sub>3</sub>/SrTiO<sub>3</sub>\\n\",\n \"
We detect the relative transient differential reflectivity ΔR/R\\n0 of LaCoO3/SrTiO3 as a function of delay time, for which the data recorded at various pump fluences are presented in Figure\\n##FIG##0##\\n1\\n##. In Figure ##FIG##0##1a##, the dots are the experimental results and the solid curves are fitting results (see a latter paragraph for a quantitative description). The signal we measure is proportional to the density of the photo‐excited carriers (abbreviated as photo‐carriers), which is intrinsically due to the Fermi transition and obeys the Fermi golden rule.[\\n##UREF##1##\\n2\\n##, ##UREF##17##\\n26\\n##\\n] Upon the pump pulse excitation, the density of the photo carriers reaches a maximum value at time t = 0 fs, then decays through various relaxation channels, including the EPC, PPS, electron‐hole recombination, etc.[\\n##UREF##14##\\n21\\n##\\n]\\n
\",\n \"
In the inset of Figure ##FIG##0##1a## we summarize the max value of |ΔR/R\\n0| as a function of the excitation fluence. The value |ΔR/R\\n0|max increases with fluence and a linear fit (solid line) to the signal |ΔR/R\\n0|max can cover most of the range of the experimental condition. At above 3.2 mJ cm−2 an off‐linear behavior arises, which indicates the occurrence of a thermal effect in the experiment. Note that the off‐linear (i.e., saturation) behavior in |ΔR/R\\n0|max is a strict criterion for identifying whether there is a thermal effect (for details, see the Supporting Information of ref. [##UREF##1##2##] and references therein). The thermal (pink) and non‐thermal (blue) effect regimes are depicted by different colors as a guide for the identification.
\",\n \"
To better reveal the fluence dependence of the photo‐carriers ultrafast dynamics, the normalized ΔR/R\\n0 is shown in Figure ##FIG##0##1b##. The data are normalized to the |ΔR/R\\n0|max value at the highest fluence excitation. To see the initial dynamics clearly, we illustrate a higher temporal resolution view of the scanning trace in the inset of Figure ##FIG##0##1b##. With increasing fluence, the normalized ΔR/R\\n0 exhibits prominent changes, whereby the relaxation becomes more gradual. This reveals that the ultrafast photo‐carriers relaxation in LaCoO3/SrTiO3 is clearly dependent on the pump fluence. Note that all the data in this work are one identical set of experimental results, although they are presented in different ways to emphasize different aspects. The transient reflectivity of the SrTiO3 substrate is also measured, which is significantly different from that of the LaCoO3/SrTiO3 sample (Figure ##SUPPL##0##S2##, Supporting Information). The control experiment demonstrates that the photo‐carriers relaxation dynamics in Figure ##FIG##0##1a## is mainly attributed to the LaCoO3 thin film.
\",\n \"
We quantitatively analyze the experimental results. Because the electron–phonon interaction has a characteristic lifetime at the order of 1 ps,[\\n##UREF##1##\\n2\\n##, ##UREF##2##\\n4\\n##, ##UREF##13##\\n19\\n##, ##REF##12618781##\\n20\\n##\\n] and the presence of a hump centered at 10 ps in the dynamics[\\n##UREF##18##\\n27\\n##\\n] may affect the assignment of the relaxation components, we choose the data fitting range to be from −0.7 to 3.6 ps to minimize the interference of the hump. Before we do the quantitative data fitting, we use a log‐scale coordinate to show ΔR/R\\n0 (Figure ##SUPPL##0##S4##, Supporting Information), which more clearly reveals how many components are present in the photo‐carriers relaxation dynamics. As seen, there are three components, and the slowest one is very flat, which can be reasonably represented by a constant term. A convoluted exponential‐decay function is employed to fit the photo‐carriers relaxation dynamics, along with a decaying cosine function to fit the coherent phonon, as:\\nwhere A and τ represent the amplitude and lifetime, respectively, the subscript fast, slow, and 0 mark the three components, 12πpet2/2p2is the Gaussian response function,[\\n28] Ω is the angular frequency of the coherent phonon, and φ is the initial phase of the coherent phonon. The fitting results compare well with our data [Figure ##FIG##0##1a##].
\",\n \"
The quantitative analysis of the fluence‐dependent results yields fluence‐dependent amplitudes and lifetimes, which are summarized in Figure\\n##FIG##1##\\n2\\n##. The A\\nfast and A\\nslow exhibit positive correlations with the fluence in the non‐thermal effect regime [Figures ##FIG##1##2a,c##], which is in line with the results shown in the inset of Figure ##FIG##0##1##. The value of τ\\nfast slightly increases with fluence (Figure ##FIG##1##2b##), and τ\\nslow is nearly a constant (Figure ##FIG##1##2d##). Following the convention, the fast component is dominated by the EPC, and the slow component is mainly connected to the PPS. [\\n##UREF##1##\\n2\\n##, ##REF##27015504##\\n3\\n##, ##UREF##2##\\n4\\n##\\n] Such assignment is based on the characteristic interaction times for different processes (Figure ##SUPPL##0##S5##, Supporting Information),[\\n##UREF##13##\\n19\\n##\\n] which is experimentally tested true and consistent in previous investigations. From the fast component, we can obtain the explicit value of the EPC strength λ.
\",\n \"Obtaining the EPC Strength <italic toggle=\\\"yes\\\">λ</italic>\\n\",\n \"
In a recent work,[\\n4] we developed a method to obtain λ by the fluence dependence of the fast component, for which the advantages are two‐folded: 1) one can obtain the EPC strength λ at room temperature, which usually does not vary much with temperature, and 2) one can circumvent the frequently encountered dilemma that the light penetration depth and the heat capacity coefficient of a material are usually unavailable. The initial work[\\n##UREF##19##\\n28\\n##\\n] is developed for low temperature and medium‐high fluence regime. Later on, we have developed a more comprehensive version, which is extended to room temperature (thus easier to implement) and can also be applied to medium‐high fluence regime.[\\n##UREF##2##\\n4\\n##\\n] We name this model as fluence‐dependence model (FDM).[\\n##UREF##2##\\n4\\n##, ##UREF##19##\\n28\\n##\\n] The FDM is indeed derived from the Allen model.[\\n##UREF##20##\\n29\\n##\\n] There have also been several other sophisticated discussions and models along this direction.[\\n##UREF##21##\\n30\\n##, ##REF##21231613##\\n31\\n##, ##UREF##22##\\n32\\n##, ##UREF##23##\\n33\\n##\\n] In our FDM approach, the value of λ is related to the fast component lifetime τ\\nfast by \\nwhere kB\\n is the Boltzmann constant, TL\\n is the lattice temperature, Ω is the angular frequency of the phonon (usually optical phonon[\\n##UREF##24##\\n34\\n##\\n]), and Θ is an effective absorption coefficient of fluence (when a laser beam of fluence F is incident on the sample, the electron temperature increases from the equilibrium temperature TL\\n to TL2+ΘF). At medium‐high fluence regime whereby the sample assumes the non‐thermal regime (i.e., the density of photocarriers is proportional to the fluence, see the inset of Figure ##FIG##0##1a##), Θ is a constant.
\",\n \"
Using the equation of FDM, the fitting curve compares well with the experimental results at various fluences [Figure ##FIG##1##2b##]. We obtain that λ˂Ω3>/<Ω> = 294.9 ± 25.4 ps−2 (i.e., 129.3 ± 11.1 meV2). Usually, we take the lowest energy optical phonon mode as an example to obtain the nominal EPC strength for a quantum material. For our LaCoO3/SrTiO3 sample, it has four E\\ng modes, where the lowest energy E\\ng mode is the lowest energy optical phonon mode (E\\ng) for the material. The second highest energy E\\ng mode is related to the orbital‐phonon coupling and the Jahn–Teller distortion.[\\n##REF##15524742##\\n35\\n##\\n] Still, in the Raman result, the second lowest E\\ng mode is much more prominent than the lowest energy E\\ng mode, and it is the second lowest energy optical phonon mode (another A\\ngg mode is silent).[\\n##UREF##25##\\n36\\n##\\n] We take the second lowest E\\ng mode (with a frequency of 175.3 cm−1 [\\n##REF##15524742##\\n35\\n##, ##UREF##25##\\n36\\n##, ##UREF##26##\\n37\\n##\\n]) to be the characteristic phonon mode to obtain the nominal EPC strength. In such a way, we obtain λE\\n\\ng = 0.30. Note that the EPC strength of LaCoO3/SrTiO3 we obtain here is close to those of superconducting oxides YBa2Cu3O6.5 (λ ≥ 0.25) and La1.85Sr0.15CuO4 (λ ≥ 0.5).[\\n##REF##21231613##\\n31\\n##\\n] The λ = 0.30 is a regular EPC value, in the middle of the various EPC values reported. Such an EPC strength is enough to cause significant Jahn–Teller distortion.[\\n##UREF##9##\\n12\\n##, ##UREF##10##\\n15\\n##\\n]\\n
\",\n \"Coherent Phonon in Thin Film LaCoO<sub>3</sub>/SrTiO<sub>3</sub>\\n\",\n \"
Furthermore, as seen in Figure ##FIG##0##1b##, there is a coherent oscillation that is unambiguously observed in our fluence‐dependent ΔR/R\\n0 signal. We re‐plot one of the signals (with the fluence of 1.41 mJ cm−2) in Figure\\n##FIG##2##\\n3a##. Here, Figure ##FIG##2##3a## shows the data with a longer temporal range than that of Figure ##FIG##0##1##. The data at a relatively longer time scale are fitted by a red wavy curve using a decaying cosine function, which corresponds to the phonon term in Equation (##FORMU##0##1##). A regular periodic oscillation is clearly seen. The inset of Figure ##FIG##2##3a## provides a closer zoom‐in view of the oscillation and the fitting curve compares well with the data. A broad hump is observed at the 4–30 ps range, which is not a part of the coherent phonon oscillation, due to the opposite phase. Previous investigations in cobalt perovskite had assigned it to the propagation of the photo‐induced metallic domain.[\\n##REF##19659241##\\n38\\n##, ##UREF##27##\\n39\\n##\\n] In this work we mainly focus on the coherent phonon, rather than this hump.
\",\n \"
In Figures ##FIG##2##3b‐d##, we present the quantitative analysis of the coherent phonon. To better reveal the coherent oscillation, we subtract the photo‐carriers relaxation from the ΔR/R\\n0 signal. The coherent oscillations at different fluences are illustrated in Figure ##FIG##2##3b##, which are offset for clarity. Each experimental oscillation trace is fitted by the decaying cosine function in Equation (##FORMU##0##1##). All the values of A\\nphonon, τ\\nphonon, φ, and Ω (see Equation (##FORMU##0##1##)) are obtained through the data fitting in Figure ##FIG##2##3b##. The phonon frequency Ω is shown in Figure ##FIG##2##3c##, whose average value is 45.3 GHz, nearly unchanged even up to the high fluence regime whereby the thermal effect inaugurates. The value of the phonon frequency is much smaller than a regular optical phonon frequency. We attribute it to be a coherent acoustic phonon, which is generated by the transient thermal strain induced by the ultrafast light pulses.[\\n##REF##25031087##\\n24\\n##, ##UREF##28##\\n40\\n##, ##UREF##29##\\n41\\n##\\n] Here the phonon frequency is independent of the film thickness (Figure ##SUPPL##0##S8##, Supporting Information).[\\n##REF##25031087##\\n24\\n##, ##UREF##30##\\n42\\n##\\n] The phonon amplitudes A\\nphonon are summarized in Figure ##FIG##2##3d##, which increase linearly with fluence. Thus, unlike that for the photo‐carriers dynamics (Figure ##FIG##0##1a## and Figure ##FIG##1##2a##), no prominent saturation on coherent phonons is observed in the thermal effect regime. This indicates the lattice does not experience irreversible damage by shining the ultrafast light pulses on. Similar property has also been found in other materials (e.g., in Cd3As2\\n[\\n##UREF##16##\\n25\\n##\\n]).
\",\n \"
Significantly, we investigate the effect of the substrate on the photo‐carriers relaxation dynamics by comparing the ultrafast dynamics of two different samples. In the control experiment, the second sample is a 40 nm thickness LaCoO3 on a (100) LaAlO3 substrate. The pump and probe beam fluences are 0.86 and 0.14 mJ cm−2, respectively. The dynamics we obtain is shown in Figure\\n##FIG##3##\\n4a##, which is normalized to compare with that of the LaCoO3/SrTiO3 sample. The data for LaCoO3/SrTiO3 are obtained under a pump fluence of 0.91 mJ cm−2 and a probe fluence of 0.13 mJ cm−2, which are nearly identical to those for the LaCoO3/LaAlO3 sample. In the longer temporal range, the dynamics of these two samples exhibit an apparent difference. However, for the shorter temporal range, the dynamics for the two samples nearly overlap in the initial range (see the inset of Figure ##FIG##3##4a## for better revealed the ultrafast relaxation at initial stage). This indicates that the fast and slow components behave in a different way. To better illaustrate the data, following the aforementioned procedures (Equation (##FORMU##0##1##)), we display the fast and slow components for both samples in Figure ##FIG##3##4b##, along with a normalized version in its inset. The solid curves are fast components and the dashed curves are slow components. The fast components for the two samples are nearly overlapped; as a contrast, the slow components for the two samples are clearly different. While the lifetime τ\\nfast = 0.17 ± 0.03 ps is nearly identical for both samples (τ\\nfast for the LaCoO3/SrTiO3 sample is 0.18 ps (Figure ##FIG##1##2b##), the lifetime τ\\nslow = 0.67 ± 0.18 ps is different from that for the other sample (τ\\nslow = 0.90 ± 0.17 ps (Figure ##FIG##1##2d##) for the LaCoO3/SrTiO3 sample). These results indicate that the EPC strength for the two samples is very close; however, the PPS rate in LaCoO3/SrTiO3 is noticeably lower than in LaCoO3/LaAlO3.
\",\n \"
In the control experiment, we follow the same procedure to analyze the coherent acoustic phonon in the LaCoO3/LaAlO3 sample. The oscillations were obtained by subtracting the photo‐carriers relaxation in Figure ##FIG##3##4a##, and are presented in Figure ##FIG##3##4c##, both fitted with cosine decaying functions. The frequency domain results are obtained through Fast Fourier Transformation (FFT) (see Figure ##FIG##3##4d##). Interestingly, the two frequency domain peaks are located at 33.3 (for LaCoO3/LaAlO3) and 45.6 GHz (for LaCoO3/SrTiO3), respectively. While these two values are different, we find that the value for LaCoO3/LaAlO3 is in well agreement with that in Fe2O3/LaAlO3,[\\n##UREF##31##\\n43\\n##\\n] and that for LaCoO3/SrTiO3 is in agreement with that in Fe2O3/SrTiO3 and LaRhO3/SrTiO3.[\\n##UREF##30##\\n42\\n##, ##UREF##31##\\n43\\n##\\n]\\n
We summarize a few typical reported coherent acoustic phonon frequencies in oxides thin films, as well as in some semiconductor thin films, in Table\n##TAB##0##\n1\n##. All these values are obtained at room temperature and probed with 800 nm light pulses. From the table, the coherent acoustic phonon frequency is nearly identical for samples with identical substrates, regardless of the material of the thin films. This indicates that the coherent acoustic phonons are all mainly generated in the substrate. Note that for Figure ##FIG##2##3b## by sample we mean the whole heterostructure including the film and substrate. All these results are in consensus with each other, indicating that the coherent acoustic phonons are mainly determined by the substrate. It is very plausible that the coherent acoustic phonons are generated (especially at a few atomic layers in the substrate nearby the interface) and detected in the substrates (Figure ##SUPPL##0##S9##, Supporting Information). The penetration depth of LaCoO3 is reported to be 110 nm,[\n##UREF##32##\n44\n##\n] which allows for the prominent transmission through a 40 nm thick thin film. In such a scenario, the pump pulse generates a transient thermal strain, producing longitudinal temperature gradients that spread at the interface between the film and substrate to generate the coherent acoustic phonon.[\n##UREF##28##\n40\n##, ##UREF##33##\n45\n##\n] Note that the coherent acoustic phonon in a bare SrTiO3 substrate is not easy to observe.[\n##UREF##30##\n42\n##\n] Usually, one needs the interference between the reflections from the surface and the strain wave to detect the coherent acoustic phonon (Figure ##SUPPL##0##S9##, Supporting Information).[\n##UREF##30##\n42\n##\n]\n
","
We schematically illustrate the scenario underlying the whole process in Figure\n##FIG##4##\n5\n##. The photo‐carriers are excited to the excited states and then relax through the coupling with optical phonons in the thin films (not mainly in the substrate).[\n##UREF##2##\n4\n##, ##UREF##24##\n34\n##\n] Energy is exchanged between the photo‐carriers and the thin film crystal lattice. Consequently, the thin film lattice absorbs the energy from the photo‐carriers, generating a vast quantity of non‐equilibrium optical phonons, which relax mainly through PPS, decaying into lower energy acoustic phonons. The deviation in the PPS rate in LaCoO3/SrTiO3 and LaCoO3/LaAlO3 suggests that this OP→AP relaxation process mainly occurs in the substrate, especially at the several atomic layers in the substrate nearby the interface, instead of the thin film. Note that the inset of Figure ##FIG##3##4b## shows a very similar EPC relaxation rate, indicating that strain[\n##REF##30778061##\n48\n##\n] does not affect EPC (Figure ##SUPPL##0##S6##, Supporting Information); also, possible effects caused by interfacial structural configuration or band alignment[\n##UREF##36##\n49\n##, ##UREF##37##\n50\n##\n] will be masked by our LaCoO3 40 nm thick film, thus becoming negligible (Figure ##SUPPL##0##S7##, Supporting Information).
","
Assuming that it is other than the above scenario—supposing the EPC and PPS occur both in the thin film or substrate, we should observe nearly identical τ\nfast and τ\nslow in the control experiment, which is apparently not the fact. Hence, we conclude that the EPC and PPS are spatially separated by the interface. This scenario is also confirmed by the distinctive coherent acoustic phonons observed in samples with distinctive substrates. Owing to such a scenario, the OP→AP decaying process inevitably penetrates the interface, which is in line with the longitudinal nature of the acoustic phonons.[\n##REF##25031087##\n24\n##, ##UREF##16##\n25\n##\n] As a result, the penetration and annihilation/creation of phonons perpendicular to the interface[\n##REF##27015504##\n3\n##, ##REF##30822064##\n51\n##, ##UREF##38##\n52\n##, ##REF##30770805##\n53\n##\n] naturally occur. A temporal evolution for the ultrafast processes is depicted in the caption.
"],"string":"[\n \"Discussion\",\n \"
We summarize a few typical reported coherent acoustic phonon frequencies in oxides thin films, as well as in some semiconductor thin films, in Table\\n##TAB##0##\\n1\\n##. All these values are obtained at room temperature and probed with 800 nm light pulses. From the table, the coherent acoustic phonon frequency is nearly identical for samples with identical substrates, regardless of the material of the thin films. This indicates that the coherent acoustic phonons are all mainly generated in the substrate. Note that for Figure ##FIG##2##3b## by sample we mean the whole heterostructure including the film and substrate. All these results are in consensus with each other, indicating that the coherent acoustic phonons are mainly determined by the substrate. It is very plausible that the coherent acoustic phonons are generated (especially at a few atomic layers in the substrate nearby the interface) and detected in the substrates (Figure ##SUPPL##0##S9##, Supporting Information). The penetration depth of LaCoO3 is reported to be 110 nm,[\\n##UREF##32##\\n44\\n##\\n] which allows for the prominent transmission through a 40 nm thick thin film. In such a scenario, the pump pulse generates a transient thermal strain, producing longitudinal temperature gradients that spread at the interface between the film and substrate to generate the coherent acoustic phonon.[\\n##UREF##28##\\n40\\n##, ##UREF##33##\\n45\\n##\\n] Note that the coherent acoustic phonon in a bare SrTiO3 substrate is not easy to observe.[\\n##UREF##30##\\n42\\n##\\n] Usually, one needs the interference between the reflections from the surface and the strain wave to detect the coherent acoustic phonon (Figure ##SUPPL##0##S9##, Supporting Information).[\\n##UREF##30##\\n42\\n##\\n]\\n
\",\n \"
We schematically illustrate the scenario underlying the whole process in Figure\\n##FIG##4##\\n5\\n##. The photo‐carriers are excited to the excited states and then relax through the coupling with optical phonons in the thin films (not mainly in the substrate).[\\n##UREF##2##\\n4\\n##, ##UREF##24##\\n34\\n##\\n] Energy is exchanged between the photo‐carriers and the thin film crystal lattice. Consequently, the thin film lattice absorbs the energy from the photo‐carriers, generating a vast quantity of non‐equilibrium optical phonons, which relax mainly through PPS, decaying into lower energy acoustic phonons. The deviation in the PPS rate in LaCoO3/SrTiO3 and LaCoO3/LaAlO3 suggests that this OP→AP relaxation process mainly occurs in the substrate, especially at the several atomic layers in the substrate nearby the interface, instead of the thin film. Note that the inset of Figure ##FIG##3##4b## shows a very similar EPC relaxation rate, indicating that strain[\\n##REF##30778061##\\n48\\n##\\n] does not affect EPC (Figure ##SUPPL##0##S6##, Supporting Information); also, possible effects caused by interfacial structural configuration or band alignment[\\n##UREF##36##\\n49\\n##, ##UREF##37##\\n50\\n##\\n] will be masked by our LaCoO3 40 nm thick film, thus becoming negligible (Figure ##SUPPL##0##S7##, Supporting Information).
\",\n \"
Assuming that it is other than the above scenario—supposing the EPC and PPS occur both in the thin film or substrate, we should observe nearly identical τ\\nfast and τ\\nslow in the control experiment, which is apparently not the fact. Hence, we conclude that the EPC and PPS are spatially separated by the interface. This scenario is also confirmed by the distinctive coherent acoustic phonons observed in samples with distinctive substrates. Owing to such a scenario, the OP→AP decaying process inevitably penetrates the interface, which is in line with the longitudinal nature of the acoustic phonons.[\\n##REF##25031087##\\n24\\n##, ##UREF##16##\\n25\\n##\\n] As a result, the penetration and annihilation/creation of phonons perpendicular to the interface[\\n##REF##27015504##\\n3\\n##, ##REF##30822064##\\n51\\n##, ##UREF##38##\\n52\\n##, ##REF##30770805##\\n53\\n##\\n] naturally occur. A temporal evolution for the ultrafast processes is depicted in the caption.
Electron–phonon coupling (EPC) and phonon–phonon scattering (PPS) are at the core of the microscopic physics mechanisms of vast quantum materials. However, to date, there are rarely reports that these two processes can be spatially separated, although they are usually temporally detached with different characteristic lifetimes. Here, by employing ultrafast spectroscopy to investigate the photo‐carrier ultrafast dynamics in a LaCoO3 thin film on a (100) SrTiO3 substrate, intriguing evidence is found that the two interactions are indeed spatially separated. The EPC mainly occurs in the thin film, whereas PPS is largely in the substrate, especially at the several atomic layers near the interface. Across‐interface penetration and decay of optical phonons into acoustic phonons thus naturally occur. An EPC strength λEg\n = 0.30 is also obtained and an acoustic phonon mode at 45.3 GHz is observed. The finding lays out a cornerstone for future quantum nano device designs.
","
Electron–phonon coupling (EPC) and phonon‐phonon scattering (PPS) are at the core of the microscopic physics mechanisms of vast quantum materials. Here, evidence of spatially separated EPC and PPS is unveiled by using ultrafast spectroscopy on a 40 nm LaCoO3 thin films grown on SrTiO3 substrate. The EPC mainly occurs in thin film and the PPS occurs largely in the substrate, especially at the several atomic layers in the substrate nearby the interface.\n\n
","
\n\n\nW.\nHao\n, \nM.\nGu\n, \nZ.\nTian\n, \nS.\nFu\n, \nM.\nMeng\n, \nH.\nZhang\n, \nJ.\nGuo\n, \nJ.\nZhao\n, Separated Electron–Phonon and Phonon–Phonon Scatterings Across Interface in Thin Film LaCoO3/SrTiO3\n. Adv. Sci.\n2024, 11, 2305900. 10.1002/advs.202305900\n\n
"],"string":"[\n \"Abstract\",\n \"
Electron–phonon coupling (EPC) and phonon–phonon scattering (PPS) are at the core of the microscopic physics mechanisms of vast quantum materials. However, to date, there are rarely reports that these two processes can be spatially separated, although they are usually temporally detached with different characteristic lifetimes. Here, by employing ultrafast spectroscopy to investigate the photo‐carrier ultrafast dynamics in a LaCoO3 thin film on a (100) SrTiO3 substrate, intriguing evidence is found that the two interactions are indeed spatially separated. The EPC mainly occurs in the thin film, whereas PPS is largely in the substrate, especially at the several atomic layers near the interface. Across‐interface penetration and decay of optical phonons into acoustic phonons thus naturally occur. An EPC strength λEg\\n = 0.30 is also obtained and an acoustic phonon mode at 45.3 GHz is observed. The finding lays out a cornerstone for future quantum nano device designs.
\",\n \"
Electron–phonon coupling (EPC) and phonon‐phonon scattering (PPS) are at the core of the microscopic physics mechanisms of vast quantum materials. Here, evidence of spatially separated EPC and PPS is unveiled by using ultrafast spectroscopy on a 40 nm LaCoO3 thin films grown on SrTiO3 substrate. The EPC mainly occurs in thin film and the PPS occurs largely in the substrate, especially at the several atomic layers in the substrate nearby the interface.\\n\\n
\",\n \"
\\n\\n\\nW.\\nHao\\n, \\nM.\\nGu\\n, \\nZ.\\nTian\\n, \\nS.\\nFu\\n, \\nM.\\nMeng\\n, \\nH.\\nZhang\\n, \\nJ.\\nGuo\\n, \\nJ.\\nZhao\\n, Separated Electron–Phonon and Phonon–Phonon Scatterings Across Interface in Thin Film LaCoO3/SrTiO3\\n. Adv. Sci.\\n2024, 11, 2305900. 10.1002/advs.202305900\\n\\n
In summary, we investigate the ultrafast dynamics of LaCoO3/SrTiO3 and perform the control experiment with a LaCoO3/LaAlO3 sample. By the lifetime of the fast photo‐carriers relaxation component, we obtain the EPC strength in the LaCoO3 thin film to be λ˂Ω3>/<Ω> = 129.3 ± 11.1 meV2, which corresponds to a nominal EPC strength of λE\n\ng = 0.30. A coherent acoustic phonon mode with a frequency of 45.3 GHz is also generated and detected. We attribute it to light pulse‐induced thermal strain. Intriguingly, through the control experiment, we discover that the EPC mainly occurs in the thin film and the PPS is dominated by the substrate, especially at the several atomic layers in the substrate nearby the interface, whereby the optical phonons penetrate across the interface to decay into acoustic phonons. Our findings reveal a rarely observed/reported phenomena that can hardly be detected by any other experimental means, and lays down an important physics mechanism foundation for the relevant future designs of quantum nano devices.
","Experimental Section","
Ultrafast laser pulses with 800 nm central wavelength, 70 fs pulse duration, and 250 kHz repetition rate were used. The spot diameters of the pump and probe beams were 60 and 55 µm, respectively, on the sample surface. The pump fluence ranges from 0.16 to 4.81 mJ cm−2, while the probe fluence was kept at 0.13 mJ cm−2. The fluences F were obtained through F= 4W/Rr\nπd\n2, where W was the laser beam power, Rr\n was the repetition rate of the laser, and d was the beam spot diameter. Cross‐polarization detection was implemented in order to reduce noise. We conducted all the experiments at room temperature (i.e., 298 K).
","
Our samples were grown by using the pulsed laser deposition method, where two ≈100 unit‐cell‐thick (approximately 40 nm) LaCoO3 films were grown on twin‐polished SrTiO3(100) and LaAlO3(100) substrates, respectively. The oxygen partial pressure was optimized at 15 Pa and the growth temperature was 670 °C. After in‐situ annealing for 1 h, the samples were cooled down to room temperature under the same oxygen pressure (15 Pa). The substrates were 5 × 5 × 0.5 mm3 in volume. The general grown method, X‐ray diffraction, and reflection high‐energy electron diffraction characterizations were presented in Supporting Information, Figure ##SUPPL##0##S1##. Our magnetic and electrical characterizations shown that there was no prominent oxygen vacancy (or in‐gap state) in the SrTiO3(100) and LaAlO3(100) substrates (Figure ##SUPPL##0##S3## Supporting Information ##SUPPL##1##S1##).
","Conflict of Interest","
The authors declare no conflict of interest.
","Author Contributions","
J.Z. conceived the idea and supervised the project. W.H. and J.Z. conducted the ultrafast spectroscopy experiment. M.G., M.M., and J.G. fabricated the sample and did the characterization. W.H., Z.T., S.F., and J.Z. analyzed the data. With constructive inputs from H.Z. and J.G., W.H. prepared the draft and J.Z. wrote the paper.
In summary, we investigate the ultrafast dynamics of LaCoO3/SrTiO3 and perform the control experiment with a LaCoO3/LaAlO3 sample. By the lifetime of the fast photo‐carriers relaxation component, we obtain the EPC strength in the LaCoO3 thin film to be λ˂Ω3>/<Ω> = 129.3 ± 11.1 meV2, which corresponds to a nominal EPC strength of λE\\n\\ng = 0.30. A coherent acoustic phonon mode with a frequency of 45.3 GHz is also generated and detected. We attribute it to light pulse‐induced thermal strain. Intriguingly, through the control experiment, we discover that the EPC mainly occurs in the thin film and the PPS is dominated by the substrate, especially at the several atomic layers in the substrate nearby the interface, whereby the optical phonons penetrate across the interface to decay into acoustic phonons. Our findings reveal a rarely observed/reported phenomena that can hardly be detected by any other experimental means, and lays down an important physics mechanism foundation for the relevant future designs of quantum nano devices.
\",\n \"Experimental Section\",\n \"
Ultrafast laser pulses with 800 nm central wavelength, 70 fs pulse duration, and 250 kHz repetition rate were used. The spot diameters of the pump and probe beams were 60 and 55 µm, respectively, on the sample surface. The pump fluence ranges from 0.16 to 4.81 mJ cm−2, while the probe fluence was kept at 0.13 mJ cm−2. The fluences F were obtained through F= 4W/Rr\\nπd\\n2, where W was the laser beam power, Rr\\n was the repetition rate of the laser, and d was the beam spot diameter. Cross‐polarization detection was implemented in order to reduce noise. We conducted all the experiments at room temperature (i.e., 298 K).
\",\n \"
Our samples were grown by using the pulsed laser deposition method, where two ≈100 unit‐cell‐thick (approximately 40 nm) LaCoO3 films were grown on twin‐polished SrTiO3(100) and LaAlO3(100) substrates, respectively. The oxygen partial pressure was optimized at 15 Pa and the growth temperature was 670 °C. After in‐situ annealing for 1 h, the samples were cooled down to room temperature under the same oxygen pressure (15 Pa). The substrates were 5 × 5 × 0.5 mm3 in volume. The general grown method, X‐ray diffraction, and reflection high‐energy electron diffraction characterizations were presented in Supporting Information, Figure ##SUPPL##0##S1##. Our magnetic and electrical characterizations shown that there was no prominent oxygen vacancy (or in‐gap state) in the SrTiO3(100) and LaAlO3(100) substrates (Figure ##SUPPL##0##S3## Supporting Information ##SUPPL##1##S1##).
\",\n \"Conflict of Interest\",\n \"
The authors declare no conflict of interest.
\",\n \"Author Contributions\",\n \"
J.Z. conceived the idea and supervised the project. W.H. and J.Z. conducted the ultrafast spectroscopy experiment. M.G., M.M., and J.G. fabricated the sample and did the characterization. W.H., Z.T., S.F., and J.Z. analyzed the data. With constructive inputs from H.Z. and J.G., W.H. prepared the draft and J.Z. wrote the paper.
This work was supported by the National Key Research and Development Program of China (Grant Nos. 2021YFA1400201 and 2017YFA0303600), the CAS Project for Young Scientists in Basic Research (Grant No. YSBR‐059), the Beijing Natural Science Foundation (Grant No. 4191003), the National Natural Science Foundation of China (Grant Nos. 11774408 and 11974253), the Strategic Priority Research Program of CAS (Grant No. XDB30000000), the International Partnership Program of Chinese Academy of Sciences (Grant No. GJHZ1826), and CAS Interdisciplinary Innovation Team.
","Data Availability Statement","
The data that support the findings of this study are available from the corresponding author upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
This work was supported by the National Key Research and Development Program of China (Grant Nos. 2021YFA1400201 and 2017YFA0303600), the CAS Project for Young Scientists in Basic Research (Grant No. YSBR‐059), the Beijing Natural Science Foundation (Grant No. 4191003), the National Natural Science Foundation of China (Grant Nos. 11774408 and 11974253), the Strategic Priority Research Program of CAS (Grant No. XDB30000000), the International Partnership Program of Chinese Academy of Sciences (Grant No. GJHZ1826), and CAS Interdisciplinary Innovation Team.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available from the corresponding author upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
Relative transient differential reflectivity of LaCoO3/SrTiO3. a) Fluence‐dependent relative differential reflectivity ΔR/R\n0. Solid curves: fitting results using Equation (##FORMU##0##1##). The arrow marks the increase in pump fluence. Inset: fluence‐dependence of |ΔR/R\n0|max. Blue and pink colors: non‐thermal and thermal effect regimes. Solid line: linear fit. b) Normalized ΔR/R\n0. Inset: zoom‐in view of the normalized signal ΔR/R\n0.
","
Fluence‐dependence of the amplitudes and lifetimes. Quantitatively obtained fitting results: a) A\nfast, b) τ\nfast, c) A\nslow, and d) τ\nslow. Solid curve in b): Fitted curve using a FDM[\n##UREF##2##\n4\n##\n] of obtaining the EPC strength at room temperature.
","
Pump fluence dependence of the coherent phonon oscillation. a) Ultrafast photo‐carriers relaxation dynamics under 1.41 mJ cm−2 pump fluence. Red wave: fitting curve of the coherent phonon oscillation with relaxation. b) Coherent phonon oscillation at different fluences (data are offset for clarity). Solid curves: decaying cosine functions. c,d) Fluence dependence of the phonon frequency and amplitude. Dark red lines: constant and linear fits to the data.
","
Photo‐carriers relaxation dynamics of LaCoO3/SrTiO3 and LaCoO3/LaAlO3. a) Normalized differential reflectivity ΔR/R\n0 under close pump and probe fluences. Inset: zoom‐in view for initial temporal range. b) The fitting components for fast and slow components in the inset of 5a. Inset: Normalized components. c) Coherent phonon oscillation with cosine decay fitting (offset for clarity). d) Fourier transformation of the oscillations. Note, for LaCoO3/SrTiO3, the pump fluence is 0.91 mJ cm−2 and the probe fluence is 0.13 mJ cm−2.
","
Schematic illustration of the separated EPC and PPS processes across the interface. The vibrational balls depict the real‐space atomic position fluctuations. OP: optical phonons; AP: acoustic phonons. The pump laser pulses instantly generate excited state photo‐carriers (within femtoseconds), which then excite optical phonons mainly within the thin film (with a typical interaction lifetime of 0.15–0.25 ps, see Figure ##FIG##1##2##). Concomitantly, these optical phonons annihilate to create acoustic phonons mainly within the substrate, especially at the several atomic layers in the substrate nearby the interface (with a characteristic interaction lifetime of 0.8–1.2 ps, see Figure ##FIG##1##2##). Such a process naturally involves the penetration of phonons across the interface.
"],"string":"[\n \"
Relative transient differential reflectivity of LaCoO3/SrTiO3. a) Fluence‐dependent relative differential reflectivity ΔR/R\\n0. Solid curves: fitting results using Equation (##FORMU##0##1##). The arrow marks the increase in pump fluence. Inset: fluence‐dependence of |ΔR/R\\n0|max. Blue and pink colors: non‐thermal and thermal effect regimes. Solid line: linear fit. b) Normalized ΔR/R\\n0. Inset: zoom‐in view of the normalized signal ΔR/R\\n0.
\",\n \"
Fluence‐dependence of the amplitudes and lifetimes. Quantitatively obtained fitting results: a) A\\nfast, b) τ\\nfast, c) A\\nslow, and d) τ\\nslow. Solid curve in b): Fitted curve using a FDM[\\n##UREF##2##\\n4\\n##\\n] of obtaining the EPC strength at room temperature.
\",\n \"
Pump fluence dependence of the coherent phonon oscillation. a) Ultrafast photo‐carriers relaxation dynamics under 1.41 mJ cm−2 pump fluence. Red wave: fitting curve of the coherent phonon oscillation with relaxation. b) Coherent phonon oscillation at different fluences (data are offset for clarity). Solid curves: decaying cosine functions. c,d) Fluence dependence of the phonon frequency and amplitude. Dark red lines: constant and linear fits to the data.
\",\n \"
Photo‐carriers relaxation dynamics of LaCoO3/SrTiO3 and LaCoO3/LaAlO3. a) Normalized differential reflectivity ΔR/R\\n0 under close pump and probe fluences. Inset: zoom‐in view for initial temporal range. b) The fitting components for fast and slow components in the inset of 5a. Inset: Normalized components. c) Coherent phonon oscillation with cosine decay fitting (offset for clarity). d) Fourier transformation of the oscillations. Note, for LaCoO3/SrTiO3, the pump fluence is 0.91 mJ cm−2 and the probe fluence is 0.13 mJ cm−2.
\",\n \"
Schematic illustration of the separated EPC and PPS processes across the interface. The vibrational balls depict the real‐space atomic position fluctuations. OP: optical phonons; AP: acoustic phonons. The pump laser pulses instantly generate excited state photo‐carriers (within femtoseconds), which then excite optical phonons mainly within the thin film (with a typical interaction lifetime of 0.15–0.25 ps, see Figure ##FIG##1##2##). Concomitantly, these optical phonons annihilate to create acoustic phonons mainly within the substrate, especially at the several atomic layers in the substrate nearby the interface (with a characteristic interaction lifetime of 0.8–1.2 ps, see Figure ##FIG##1##2##). Such a process naturally involves the penetration of phonons across the interface.
\"\n]"},"table":{"kind":"list like","value":["
Coherent acoustic phonon frequencies in various oxide and semiconductor thin film samples (from this work and adapted from[\n##UREF##30##\n42\n##, ##UREF##31##\n43\n##, ##UREF##34##\n46\n##, ##UREF##35##\n47\n##\n]).
Sample
Phonon frequency [GHz]
LaRhO3/SrTiO3(110)
45[\n##UREF##30##\n42\n##\n]\n
LaCoO3/SrTiO3(100)
45.3[this work]\n
Fe2O3/SrTiO3(100)
44.7[\n##UREF##31##\n43\n##\n]\n
Fe2O3/BaTiO3(100)
30.9[\n##UREF##31##\n43\n##\n]\n
LaCoO3/LaAlO3(100)
33.3[this work]\n
Fe2O3/LaAlO3(100)
33.9[\n##UREF##31##\n43\n##\n]\n
LaRhO3/(LaAlO3)0.3(Sr2TaAlO6)0.7(110)
35.05[\n##UREF##30##\n42\n##\n]\n
GaSb/GaAs
43[\n##UREF##34##\n46\n##\n]\n
Co2MnAl/GaAs
43.5[\n##UREF##35##\n47\n##\n]\n
John Wiley & Sons, Ltd."],"string":"[\n \"
Coherent acoustic phonon frequencies in various oxide and semiconductor thin film samples (from this work and adapted from[\\n##UREF##30##\\n42\\n##, ##UREF##31##\\n43\\n##, ##UREF##34##\\n46\\n##, ##UREF##35##\\n47\\n##\\n]).
Sample
Phonon frequency [GHz]
LaRhO3/SrTiO3(110)
45[\\n##UREF##30##\\n42\\n##\\n]\\n
LaCoO3/SrTiO3(100)
45.3[this work]\\n
Fe2O3/SrTiO3(100)
44.7[\\n##UREF##31##\\n43\\n##\\n]\\n
Fe2O3/BaTiO3(100)
30.9[\\n##UREF##31##\\n43\\n##\\n]\\n
LaCoO3/LaAlO3(100)
33.3[this work]\\n
Fe2O3/LaAlO3(100)
33.9[\\n##UREF##31##\\n43\\n##\\n]\\n
LaRhO3/(LaAlO3)0.3(Sr2TaAlO6)0.7(110)
35.05[\\n##UREF##30##\\n42\\n##\\n]\\n
GaSb/GaAs
43[\\n##UREF##34##\\n46\\n##\\n]\\n
Co2MnAl/GaAs
43.5[\\n##UREF##35##\\n47\\n##\\n]\\n
John Wiley & Sons, Ltd.\"\n]"},"formula":{"kind":"list like","value":["\n\nΔR/R0=(Afaste−t/τfast+Aslowe−t/τslow+A0)⊗(12πpet2/2p2)+Aphonone−t/τphononcos(Ωt+φ)\n","\n\nτfast=πkB2TL2+ΘF(TL2+ΘF−TL)3λℏ2Ω31eℏΩ/(kBTL2+ΘF)−1−1eℏΩ/(kBTL)−1\n"],"string":"[\n \"\\n\\nΔR/R0=(Afaste−t/τfast+Aslowe−t/τslow+A0)⊗(12πpet2/2p2)+Aphonone−t/τphononcos(Ωt+φ)\\n\",\n \"\\n\\nτfast=πkB2TL2+ΘF(TL2+ΘF−TL)3λℏ2Ω31eℏΩ/(kBTL2+ΘF)−1−1eℏΩ/(kBTL)−1\\n\"\n]"},"box":{"kind":"list like","value":[""],"string":"[\n \"\"\n]"},"code":{"kind":"list like","value":[],"string":"[]"},"quote":{"kind":"list like","value":[],"string":"[]"},"chemical":{"kind":"list like","value":[],"string":"[]"},"supplementary":{"kind":"list like","value":["","
Supporting Information
"],"string":"[\n \"\",\n \"
Supporting Information
\"\n]"},"footnote":{"kind":"list like","value":["
The phonon frequencies in samples with identical substrates exhibit close values, not depending on the thin film material. Identical thin films on different substrates exhibit different frequency values. These facts indicate that the acoustic phonons are dominated by the substrates.
"],"string":"[\n \"
The phonon frequencies in samples with identical substrates exhibit close values, not depending on the thin film material. Identical thin films on different substrates exhibit different frequency values. These facts indicate that the acoustic phonons are dominated by the substrates.
Liver fibrosis is a common feature of chronic liver diseases, associated with advanced disease progression and poor prognosis. Unless effectively treated, fibrosis tends to develop into cirrhosis and subsequently into liver failure, requiring transplantation.[\n##REF##31590120##\n1\n##\n] The underlying etiologies of liver fibrosis include hepatitis B and C infections, alcohol abuse, non‐alcoholic fatty liver disease (NAFLD), and cholestatic injury.[\n##REF##34923653##\n2\n##\n] Hepatic stellate cells (HSCs) play a crucial role in the development of fibrotic processes as they possess the ability to undergo activation and trans‐differentiation into myofibroblast‐like cells. These cells are a primary source of the extracellular matrix (ECM).[\n##REF##28487545##\n3\n##\n] The activation of HSCs involves a complex interaction with other resident cells and their secreted profibrotic mediators, including chemokines, growth factors, reactive oxygen species, and metabolic products.[\n##UREF##0##\n4\n##\n] Although numerous studies have contributed to our understanding of the pathogenesis of liver fibrosis, the intercellular crosstalk between dysregulated hepatocyte metabolism and HSC activation remains unclear.
","
Bile acids (BAs) are the end products of cholesterol catabolism in hepatocytes. They are known for liver cholesterol secretion and intestine lipid absorption.[\n##REF##35165436##\n5\n##\n] However, dysregulated BAs homeostasis may result in chronic liver diseases.[\n##REF##34555862##\n6\n##\n] Recent studies have reported increased BAs levels with increasing fibrosis stage.[\n##REF##30506692##\n7\n##\n] The composition of BAs in patients with cirrhosis has been previously reported to be abnormal.[\n##REF##25964117##\n8\n##, ##REF##33754726##\n9\n##\n] The accumulation of BAs in the liver may lead to hepatocyte mitochondrial injury, cholangiocyte proliferation, or macrophage activation, which result in cholestatic liver injuries and inflammatory responses.[\n##REF##33987435##\n10\n##, ##REF##28120434##\n11\n##, ##REF##33724957##\n12\n##\n] Defects in specific genes involved in BAs metabolism contribute to chronic cholestatic liver fibrosis.[\n##REF##35229330##\n13\n##\n]\n
","
Solute carrier family 27 member 5 (SLC27A5), also known as long‐chain fatty acid transport protein 5 (FATP5), is mainly expressed in the liver, specifically in the basement membrane of hepatocytes, where it participates in fatty acid transport.[\n##REF##16618416##\n14\n##\n] SLC27A5 exhibits bile acid‐CoA ligase (BAL) activity, which converts unconjugated BAs to their CoA thioester derivatives and catalyzes the conjugation of BAs with amino acids.[\n##REF##12454267##\n15\n##, ##REF##16618417##\n16\n##\n] Mice lacking SLC27A5 have shown to exhibit higher levels of unconjugated BAs and lower levels of conjugated BAs, as compared to normal mice.[\n##REF##16618417##\n16\n##, ##REF##21826528##\n17\n##\n] This is consistent with elevated unconjugated BAs observed in patients with genetic defects in SLC27A5.[\n##REF##22089923##\n18\n##, ##REF##23415802##\n19\n##\n] Silencing of SLC27A5 in mice corresponds to the high proportion of unconjugated BAs reported in these patients. Notably, a neonate with a homozygotic missense mutation in SLC27A5 was found to develop extensive liver fibrosis.[\n##REF##22089923##\n18\n##\n] Additionally, Enooku et al. demonstrated that lower SLC27A5 expression is associated with the progression of ballooning and fibrosis in patients with NAFLD.[\n##REF##31602526##\n20\n##\n] However, the specific mechanisms underlying SLC27A5 deficiency in liver fibrosis remain unclear.
","
Therefore, we aimed to elucidate the role of SLC27A5 in regulating BAs conjugation during the progression of liver fibrosis. Furthermore, we monitored the effect of the lack of SLC27A5 in mice on the activation of HSCs and liver fibrosis and investigated the associated molecular mechanisms. We believe that our findings can provide mechanistic insights into the role of SLC27A5 in regulating liver fibrosis.
"],"string":"[\n \"Introduction\",\n \"
Liver fibrosis is a common feature of chronic liver diseases, associated with advanced disease progression and poor prognosis. Unless effectively treated, fibrosis tends to develop into cirrhosis and subsequently into liver failure, requiring transplantation.[\\n##REF##31590120##\\n1\\n##\\n] The underlying etiologies of liver fibrosis include hepatitis B and C infections, alcohol abuse, non‐alcoholic fatty liver disease (NAFLD), and cholestatic injury.[\\n##REF##34923653##\\n2\\n##\\n] Hepatic stellate cells (HSCs) play a crucial role in the development of fibrotic processes as they possess the ability to undergo activation and trans‐differentiation into myofibroblast‐like cells. These cells are a primary source of the extracellular matrix (ECM).[\\n##REF##28487545##\\n3\\n##\\n] The activation of HSCs involves a complex interaction with other resident cells and their secreted profibrotic mediators, including chemokines, growth factors, reactive oxygen species, and metabolic products.[\\n##UREF##0##\\n4\\n##\\n] Although numerous studies have contributed to our understanding of the pathogenesis of liver fibrosis, the intercellular crosstalk between dysregulated hepatocyte metabolism and HSC activation remains unclear.
\",\n \"
Bile acids (BAs) are the end products of cholesterol catabolism in hepatocytes. They are known for liver cholesterol secretion and intestine lipid absorption.[\\n##REF##35165436##\\n5\\n##\\n] However, dysregulated BAs homeostasis may result in chronic liver diseases.[\\n##REF##34555862##\\n6\\n##\\n] Recent studies have reported increased BAs levels with increasing fibrosis stage.[\\n##REF##30506692##\\n7\\n##\\n] The composition of BAs in patients with cirrhosis has been previously reported to be abnormal.[\\n##REF##25964117##\\n8\\n##, ##REF##33754726##\\n9\\n##\\n] The accumulation of BAs in the liver may lead to hepatocyte mitochondrial injury, cholangiocyte proliferation, or macrophage activation, which result in cholestatic liver injuries and inflammatory responses.[\\n##REF##33987435##\\n10\\n##, ##REF##28120434##\\n11\\n##, ##REF##33724957##\\n12\\n##\\n] Defects in specific genes involved in BAs metabolism contribute to chronic cholestatic liver fibrosis.[\\n##REF##35229330##\\n13\\n##\\n]\\n
\",\n \"
Solute carrier family 27 member 5 (SLC27A5), also known as long‐chain fatty acid transport protein 5 (FATP5), is mainly expressed in the liver, specifically in the basement membrane of hepatocytes, where it participates in fatty acid transport.[\\n##REF##16618416##\\n14\\n##\\n] SLC27A5 exhibits bile acid‐CoA ligase (BAL) activity, which converts unconjugated BAs to their CoA thioester derivatives and catalyzes the conjugation of BAs with amino acids.[\\n##REF##12454267##\\n15\\n##, ##REF##16618417##\\n16\\n##\\n] Mice lacking SLC27A5 have shown to exhibit higher levels of unconjugated BAs and lower levels of conjugated BAs, as compared to normal mice.[\\n##REF##16618417##\\n16\\n##, ##REF##21826528##\\n17\\n##\\n] This is consistent with elevated unconjugated BAs observed in patients with genetic defects in SLC27A5.[\\n##REF##22089923##\\n18\\n##, ##REF##23415802##\\n19\\n##\\n] Silencing of SLC27A5 in mice corresponds to the high proportion of unconjugated BAs reported in these patients. Notably, a neonate with a homozygotic missense mutation in SLC27A5 was found to develop extensive liver fibrosis.[\\n##REF##22089923##\\n18\\n##\\n] Additionally, Enooku et al. demonstrated that lower SLC27A5 expression is associated with the progression of ballooning and fibrosis in patients with NAFLD.[\\n##REF##31602526##\\n20\\n##\\n] However, the specific mechanisms underlying SLC27A5 deficiency in liver fibrosis remain unclear.
\",\n \"
Therefore, we aimed to elucidate the role of SLC27A5 in regulating BAs conjugation during the progression of liver fibrosis. Furthermore, we monitored the effect of the lack of SLC27A5 in mice on the activation of HSCs and liver fibrosis and investigated the associated molecular mechanisms. We believe that our findings can provide mechanistic insights into the role of SLC27A5 in regulating liver fibrosis.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results","SLC27A5 Expression is Downregulated in Human and Murine Liver Fibrosis","
To evaluate the role of SLC27A5 in the development of liver fibrosis, we analyzed SLC27A5 mRNA expression levels in normal and fibrotic liver biopsy samples from the Gene Expression Omnibus (GEO) database. We observed downregulation of hepatic SLC27A5 mRNA expression in patients with NAFLD coupled with fibrosis, non‐alcoholic steatohepatitis (NASH) with fibrosis, and cirrhosis, as displayed in Figure\n##FIG##0##\n1A##. We also observed a stepwise decrease in the level of SLC27A5 transcript from fibrosis stages 0 to 4 in a cohort of patients with hepatitis B virus (HBV)‐related liver fibrosis (Figure ##FIG##0##1A##). Analyzing the cirrhosis dataset (GSE25097) revealed that the expression of SLC27A5 was negatively correlated with that of fibrosis‐related genes such as ACTA2, COL1A1, and COL3A1 (Figure ##SUPPL##0##S1A##, Supporting Information). Consistently, human cirrhotic tissue samples (Figure ##FIG##0##1B##; Figure ##SUPPL##0##S1B,C##, Supporting Information) and mouse models of liver fibrosis induced by carbon tetrachloride (CCI4) and thioacetamide (TAA) (Figure ##SUPPL##0##S1D–I##, Supporting Information) also revealed reduced SLC27A5 expression compared with that in the control trials.
","
To identify the putative transcription factors (TFs) responsible for the downregulation of SLC27A5 in liver fibrosis, we analyzed the potential promoter region of SLC27A5 using the JASPAR database considering the 2‐kb upstream sequence of the transcription start site of SLC27A5.[\n##REF##34850907##\n21\n##\n] Further analysis helped identify five TFs (NR2C2, REST, RXRA, HNF4α, and RUNX2) that may bind to the SLC27A5 promoter region (Figure ##SUPPL##0##S2A,B##, Supporting Information). Notably, qRT‐PCR demonstrated a significant upregulation of Runx2 mRNA levels in CCI4‐induced fibrotic liver tissues of mice, whereas Hnf4a and Rest indicated only minor changes (Figure ##FIG##0##1C##). Additionally, substantially elevated RUNX2 expression was observed in the liver tissues of patients with cirrhosis and fibrosis model mice (Figure ##FIG##0##1D##; Figure ##SUPPL##0##S2C–F##, Supporting Information).
","
Subsequently, we investigated whether RUNX2 was involved in the transcriptional regulation of SLC27A5. According to the prediction made using JASPAR, RUNX2 may bind the promoter of SLC27A5 at several possible sites (Figure ##FIG##0##1E##). To determine regulatory regions of RUNX2, the SLC27A5 promoter regions containing different RUNX2 binding sites were cloned into the pGL3 basic vector to perform luciferase reporter assay. We truncated the full‐length region of the SLC27A5 promoter into two fragments, −2023/+366 (pGL3‐P1) and −1001/+182 (pGL3‐P2) (Figure ##FIG##0##1E##). Notably, RUNX2 substantially decreased the activity of both SLC27A5 promoter fragments in the normal human liver cell line MIHA, which further indicates that SLC27A5 is transcriptionally inhibited by RUNX2 (Figure ##FIG##0##1F##). Chromatin immunoprecipitation assay confirmed the binding of RUNX2 to the SLC27A5 promoter (Figure ##FIG##0##1G##). RUNX2 overexpression significantly downregulated SLC27A5 expression in MIHA cells (Figure ##FIG##0##1H##), whereas the inhibition of RUNX2 by shRNA led to elevated expression of SLC27A5 (Figure ##FIG##0##1I##). These findings suggest that RUNX2 is responsible for the downregulation of SLC27A5.
","
The upregulation of RUNX2 was verified in the cirrhosis, NAFLD, NASH and HBV‐related fibrosis datasets (Figure ##FIG##0##1J##; Figure ##SUPPL##0##S2G–I##, Supporting Information). RUNX2 expression was negatively correlated with SLC27A5 mRNA levels in cirrhotic tissues (GSE25097) (Figure ##FIG##0##1K##). These results indicate that the elevated expression of the repressor RUNX2 impairs SLC27A5 transcription in patients with liver fibrosis and mouse models.
","SLC27A5 Deficiency Induces Spontaneous Hepatic Fibrosis in 24‐Month‐Old Mice","
We used the CRISPR‐Cas9 system to generate whole‐body SLC27A5 knockout (Slc27a5\n−/−) mice by deleting the protein‐coding exons of Slc27a5 (Figure ##SUPPL##0##S3A##, Supporting Information) to investigate the loss‐of‐function effect of SLC27A5 in vivo. The genotypes of the mice were determined through PCR analysis of tail DNA (Figure ##SUPPL##0##S3B##, Supporting Information). The serum ALT, AST and ALP levels showed a mild increase in Slc27a5\n−/− mice at 12 months compared to WT littermates (Figure ##SUPPL##0##S3C##, Supporting Information). The mRNA levels of inflammatory genes (Tnfa, Il6, Il1b, Ccl2, Adgre1) were substantially upregulated in the livers of 12‐month‐old Slc27a5\n−/− mice (Figure ##SUPPL##0##S3D##, Supporting Information). These results indicated that Slc27a5\n−/− mice developed liver injury and inflammatory response at 12 months of age.
","
We then maintained wild‐type (WT) and Slc27a5\n−/− mice for 24 months and observed an increase in the size of gallbladders in Slc27a5\n−/− mice (Figure\n##FIG##1##\n2A##). SLC27A5\n−/− mice exhibited a significant increase in the liver‐to‐body weight ratio and elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) compared with that in WT littermates (Figure ##FIG##1##2B##; Figure ##SUPPL##0##S3E,F##, Supporting Information). Histological analysis using H&E and Sirius red staining revealed mild collagen deposition in Slc27a5\n−/− mice at 12 months old (Figure ##FIG##1##2C,D##). With aging, Slc27a5\n−/− mice displayed a massive diffuse ECM and pseudo‐lobular nodule formation at 18 and 24 months, respectively (Figure ##FIG##1##2C,D##). We observed that mRNA levels of profibrotic genes (Acta2, Col1a1, Col3a1, Timp1, and Vim) and inflammatory genes were substantially upregulated in the livers of 24‐month‐old Slc27a5\n−/− mice (Figure ##FIG##1##2E,F##). Liver hydroxyproline (HYP) levels were also increased in Slc27a5\n−/− mice (Figure ##SUPPL##0##S3G##, Supporting Information). Western blotting analysis demonstrated a significant increase in the expression of activated HSCs marker α‐SMA and collagen deposition markers COL1A1 and COL3A1 in Slc27a5\n−/− mice liver tissues (Figure ##SUPPL##0##S3H##, Supporting Information). Furthermore, the expression levels of hepatic genes involved in bile acid synthesis, including Cyp7a1, Cyp8b1, and Cyp27a1, were significantly increased in Slc27a5\n−/− mice (Figure ##FIG##1##2G##). These findings demonstrate that Slc27a5 deficiency in mice induces spontaneous liver fibrosis at 24 months of age.
","SLC27A5 Deficiency Promotes Liver Fibrosis in Chemical‐Induced Fibrosis Murine Models","
To further examine the role of SLC27A5 in liver fibrosis, we investigated fibrogenesis in Slc27a5\n−/− mice subjected to CCl4 and TAA treatment. We observed an increase in the liver‐to‐body weight ratio in Slc27a5\n\n−/−\n mice after six weeks of CCl4 treatment (Figure\n##FIG##2##\n3A##; Figure ##SUPPL##0##S4A##, Supporting Information). Furthermore, H&E, Sirius red staining, α‐SMA, and F4/80 immunostaining revealed elevated collagen deposition, fibrogenesis, and macrophage infiltration in Slc27a5\n\n−/−\n mice (Figure ##FIG##2##3B,C##). Hydroxyproline measurements confirmed increased collagen deposition in Slc27a5\n\n−/−\n mice (Figure ##SUPPL##0##S4B##, Supporting Information). Compared with WT mice, Slc27a5\n\n−/−\n mice displayed significantly higher levels of serum ALT, AST, ALP, and total bilirubin, indicating increased liver injury (Figure ##FIG##2##3D##; Figure ##SUPPL##0##S4C##, Supporting Information). The upregulation of profibrotic and inflammatory genes were also observed in the livers of Slc27a5\n\n−/−\n mice (Figure ##SUPPL##0##S4D–F##, Supporting Information). Eight weeks after TAA injection (Figure ##FIG##2##3E##), Slc27a5\n\n−/−\n mice demonstrated increased collagen deposition, α‐SMA expression, and liver injury (Figure ##FIG##2##3F–H##; Figure ##SUPPL##0##S4G–I##, Supporting Information). Similar to the CCl4 treatment, the hepatic expression of profibrotic and inflammatory genes increased in Slc27a5\n\n−/−\n mice compared with that in WT controls (Figure S##FIG##3##4J–L##, Supporting Information). These findings collectively suggest that SLC27A5 deficiency exacerbates liver fibrosis in mouse models.
","SLC27A5 Loss in Hepatocytes Promotes HSC Activation","
We compared the primary HSCs isolated from Slc27a5\n−/− and WT mice to assess their culture‐induced activation. The HSCs from Slc27a5\n−/− mice exhibited enhanced activation, as evidenced by changes in cell morphology, loss of lipid droplets, and a myofibroblast‐like appearance (Figure\n##FIG##3##\n4A##). The expression of fibrogenic marker genes was induced in these cells (Figure ##FIG##3##4B–D##). We further determined the sensitivity of Slc27a5\n−/− HSCs to the transforming growth factor β1 (TGFβ1), which is regarded as a critical factor in the HSCs activation. The primary HSCs derived from Slc27a5\n−/− mice displayed increased sensitivity to TGFβ1‐stimulated activation compared with that of WT HSCs (Figure ##SUPPL##0##S5A–C##, Supporting Information). These findings suggest that primary HSCs from Slc27a5\n−/− mice exhibit enhanced culture‐induced activation and increased sensitivity to TGFβ1 stimulation in vitro.
","
The crosstalk between hepatocytes and HSCs generates a permissive fibrotic microenvironment that contributes to fibrosis.[\n##UREF##0##\n4\n##\n] Given that SLC27A5 mainly functions in hepatocytes,[\n##REF##16618416##\n14\n##\n] we first examined the expression of SLC27A5 in primary mouse hepatocytes (PMHs) and primary HSCs of WT mice, and observed that SLC27A5 was only expressed in PMHs but not in primary HSCs (Figure ##SUPPL##0##S5D##, Supporting Information). We next investigated the contribution of Slc27a5\n−/− PMHs to fibrotic progression. The WT or Slc27a5\n−/− PMH supernatant was added to a primary culture of WT mice HSCs for 48 h (Figure ##FIG##3##4E##). Our results demonstrated a significant up‐regulation of fibrogenic markers in WT HSCs exposed to Slc27a5\n−/− PMH supernatant (Figure ##FIG##3##4F–H##). Next, we performed a co‐culture experiment using a combination of the human HSC cell line LX‐2 and MIHA cells in a contact‐independent manner (Figure ##FIG##3##4I##). The increased expression of fibrogenic genes in LX‐2 cells co‐cultured with SLC27A5‐KO MIHA cells was similar to those observed earlier in this study (Figure ##FIG##3##4J,K##; Figure ##SUPPL##0##S5E,F##, Supporting Information). These findings indicate that the loss of SLC27A5 in hepatocytes contributes to HSC activation and increased sensitivity to fibrosis.
SLC27A5 is required for bile acid conjugation and plays an essential role in regulating BAs homeostasis in the liver.[\n##REF##32760200##\n22\n##\n] In this study, we observed an increase in unconjugated BAs, including cholic acid (CA), deoxycholic acid (DCA), and muricholic acid (MCA) in 6‐month‐old Slc27a5\n−/− mice liver tissues and serum (Figure ##SUPPL##0##S6A–D##, Supporting Information). Furthermore, analysis of the bile acid composition of 24‐month‐old Slc27a5\n−/− mice revealed a significant increase in CA and DCA (Figure\n##FIG##4##\n5A,B##), two major bile acids present in both mice and humans. The reported range of BA levels in human portal venous plasma or serum is 9—43 µм.[\n##REF##31616073##\n23\n##\n] To determine whether the elevated BAs in Slc27a5\n−/− mice contribute to the activation of HSCs, we treated human HSC cell line LX‐2 with CA or DCA at concentrations of 25, 50, and 100 µм. Interestingly, CA treatment upregulated the expression of HSCs activation markers in a dose‐dependent manner (Figure ##FIG##4##5C##; Figure ##SUPPL##0##S6E,F##, Supporting Information). This was further confirmed in primary HSCs from WT mice treated with CA at 50 µм. (Figure ##FIG##4##5D–F##). However, DCA supplementation did not strongly affect LX‐2 activation (Figure ##SUPPL##0##S6G–H##, Supporting Information). The mRNA and protein expression of fibrogenic genes were also increased in primary HSCs from Slc27a5\n−/− mice after CA treatment (Figure ##SUPPL##0##S6I,J##, Supporting Information). Furthermore, the serum levels of CA, but not DCA, were significantly increased in patients with cirrhosis (Figure ##FIG##4##5G##). Consistent with these, CA levels were also higher in SLC27A5‐KO MIHA cells and primary mouse hepatocytes (PMHs) compared with that in controls (Figure ##FIG##4##5H,I##). Based on these observations, we concluded that the abnormal elevation of unconjugated CA in mouse models and patients with cirrhosis promotes HSC activation.
","
To determine whether the inhibition of CA levels could diminish the activation of HSCs induced by the supernatant of Slc27a5\n−/− PMHs, WT and Slc27a5\n−/− mice were treated with BSH‐IN‐1, a covalent pan‐inhibitor of gut bacterial bile salt hydrolases (BSHs),[\n##REF##32042200##\n24\n##\n] which decreased unconjugated bile acids (especially CA in Slc27a5−/− mice) in vivo (Figure ##SUPPL##0##S7A##, Supporting Information). Then, the supernatant was added to HSCs from normal WT mice (Figure ##SUPPL##0##S7B##, Supporting Information). The results suggested that the expression of profibrotic genes in primary HSCs was decreased by the treatment of conditional medium from primary PMHs of Slc27a5\n−/− mice with BSH‐IN‐1 gavage (Figure ##SUPPL##0##S7C##, Supporting Information). The CA levels were also decreased in the conditional medium of BSH‐IN‐1 group, whereas the TCA levels were increased in the same medium (Figure ##SUPPL##0##S7D,E##, Supporting Information). Taken together, these results indicate that inhibition of unconjugated CA levels could decrease the expression of profibrotic genes in primary HSCs co‐cultured with the supernatant of Slc27a5\n−/− PMHs.
","Cholic Acid‐Triggered Upregulation of Early Growth Response (EGR3) Promotes HSC Activation","
To investigate how CA induced the activation of HSCs, we performed RNA‐seq analysis on LX‐2 cells treated with CA at a concentration of 50 µм or vehicle controls. The treatment with CA effectively increased the expression of regulatory genes associated with fibrosis in LX‐2 cells (Figure\n##FIG##5##\n6A##). Among the upregulated genes in LX‐2 cells, we observed the most significant upregulation in the expression of pro‐fibrotic transcription factor EGR3 following CA treatment (Figure ##FIG##5##6B##). However, taurodeoxycholate (TDCA), DCA, or taurocholic acid (TCA) supplementation did not significantly affect EGR3 expression (Figure ##SUPPL##0##S8A##, Supporting Information). EGR3 is upregulated in the fibrotic dermis of mice with scleroderma, and increased EGR3 expression contributes to the upregulation of fibrotic genes such as ACTA2 and COL1A1.[\n##REF##23906810##\n25\n##\n] To determine the role of EGR3 in CA‐induced activation of HSCs, we performed ChIP assay to confirm the binding of EGR3 on the promoter of ACTA2 and COL1A1 gene in LX‐2 cells. The data indicated that EGR3 could bind the promoter of ACTA2 and COL1A1 directly, and the recruitment of EGR3 on ACTA2 and COL1A1 promoter was increased by CA stimulation (Figure ##SUPPL##0##S8B##, Supporting Information). Based on these results, we investigated whether CA triggers HSC activation in an EGR3‐dependent manner. Treatment of LX‐2 cells with CA induced the expression of fibrotic genes. However, the induction of these genes was significantly compromised by EGR3 silencing (Figure ##FIG##5##6C,D##). Furthermore, immunofluorescence assays demonstrated that shEGR3 treatment attenuated the CA‐induced expression of α‐SMA (Figure ##FIG##5##6E##). Notably, the upregulation of α‐SMA, COL1A1, and COL3A1 was similarly compromised by EGR3 silencing in primary mouse HSCs (Figure ##FIG##5##6F–H##). We also utilized the MIHA and LX‐2 cells to explore the role of EGR3. As displayed in Figure ##SUPPL##0##S8B–D## (Supporting Information), EGR3 silencing inhibited LX‐2 cell activation induced by SLC27A5‐KO MIHA cell co‐culture. Our findings thus indicate that CA‐induced HSCs activation depends on the transcription factor EGR3.
","Overexpression of SLC27A5 or Inhibition of Intestinal Bile Acid Absorption Ameliorates CCl<sub>4</sub>‐Induced Liver Fibrosis","
Based on our observations, we observed that SLC2A75 expression was downregulated in human and mouse liver fibrosis tissues (Figure ##FIG##0##1##). Furthermore, we noted that SLC27A5 deficiency promoted liver fibrosis (Figure ##FIG##2##3##). We thus hypothesize that SLC27A5 overexpression might protect against liver fibrosis. To test this hypothesis, specifically overexpressed SLC27A5 in the livers of WT and Slc27a5\n−/− (KO) mice through tail vein injection of adeno‐associated virus (AAV) harboring Slc27a5 (AAV‐Slc27a5) or AAV‐Control (AAV‐Con) in a CCl4‐induced liver fibrosis model (Figure\n##FIG##6##\n7A##). Concordantly, the re‐expression of SLC27A5 in Slc27a5\n\n−/−\n mice alleviated collagen deposition, fibrosis, and liver injury (Figure ##FIG##6##7B–F## and Figure ##SUPPL##0##S9A–E##, Supporting Information). Both serum and liver CA accumulation was reduced in Slc27a5\n\n−/−\n mice injected with AAV‐Slc27a5 (Figure ##FIG##6##7G,H##). Notably, WT mice with SLC27A5 overexpression exhibited a mild alleviation of liver fibrosis compared with those injected with AAV‐Control (Figure ##FIG##6##7B–E##). These results suggest that the AAV‐mediated restoration of hepatic SLC27A5 protects against CCl4‐induced liver fibrosis in mice.
","
To further investigate the potential therapeutic strategies for liver fibrosis, we examined the effect of A4250, a specific ASBT inhibitor that decreases hepatic bile acid levels by inhibiting intestinal bile acid absorption,[\n##REF##35780808##\n26\n##\n] on CCl4‐induced liver fibrosis. We subjected WT and Slc27a5\n\n−/−\n mice to CCl4 injection and treated them with A4250 through gavage (Figure ##FIG##6##7I##). After administering the A4250, we observed an evident improvement in liver fibrosis in Slc27a5\n\n−/−\n mice (Figure ##SUPPL##0##S9F##, Supporting Information). Furthermore, the attenuation of hepatic collagen deposition and α‐SMA expression, along with the improvement of liver function, were evident in Slc27a5\n\n−/−\n mice with A4250 administration (Figure ##FIG##6##7J–N##; Figure ##SUPPL##0##S9G–##J, Supporting Information). Moreover, both serum and liver CA levels were markedly decreased in Slc27a5\n\n−/−\n mice following the A4250 treatment (Figure ##FIG##6##7O,P##). Overall, these findings suggest that the re‐expression of SLC27A5 or inhibition of CA accumulation ameliorates the pathogenesis of liver fibrosis, providing a potential strategy for treating this condition.
"],"string":"[\n \"Results\",\n \"SLC27A5 Expression is Downregulated in Human and Murine Liver Fibrosis\",\n \"
To evaluate the role of SLC27A5 in the development of liver fibrosis, we analyzed SLC27A5 mRNA expression levels in normal and fibrotic liver biopsy samples from the Gene Expression Omnibus (GEO) database. We observed downregulation of hepatic SLC27A5 mRNA expression in patients with NAFLD coupled with fibrosis, non‐alcoholic steatohepatitis (NASH) with fibrosis, and cirrhosis, as displayed in Figure\\n##FIG##0##\\n1A##. We also observed a stepwise decrease in the level of SLC27A5 transcript from fibrosis stages 0 to 4 in a cohort of patients with hepatitis B virus (HBV)‐related liver fibrosis (Figure ##FIG##0##1A##). Analyzing the cirrhosis dataset (GSE25097) revealed that the expression of SLC27A5 was negatively correlated with that of fibrosis‐related genes such as ACTA2, COL1A1, and COL3A1 (Figure ##SUPPL##0##S1A##, Supporting Information). Consistently, human cirrhotic tissue samples (Figure ##FIG##0##1B##; Figure ##SUPPL##0##S1B,C##, Supporting Information) and mouse models of liver fibrosis induced by carbon tetrachloride (CCI4) and thioacetamide (TAA) (Figure ##SUPPL##0##S1D–I##, Supporting Information) also revealed reduced SLC27A5 expression compared with that in the control trials.
\",\n \"
To identify the putative transcription factors (TFs) responsible for the downregulation of SLC27A5 in liver fibrosis, we analyzed the potential promoter region of SLC27A5 using the JASPAR database considering the 2‐kb upstream sequence of the transcription start site of SLC27A5.[\\n##REF##34850907##\\n21\\n##\\n] Further analysis helped identify five TFs (NR2C2, REST, RXRA, HNF4α, and RUNX2) that may bind to the SLC27A5 promoter region (Figure ##SUPPL##0##S2A,B##, Supporting Information). Notably, qRT‐PCR demonstrated a significant upregulation of Runx2 mRNA levels in CCI4‐induced fibrotic liver tissues of mice, whereas Hnf4a and Rest indicated only minor changes (Figure ##FIG##0##1C##). Additionally, substantially elevated RUNX2 expression was observed in the liver tissues of patients with cirrhosis and fibrosis model mice (Figure ##FIG##0##1D##; Figure ##SUPPL##0##S2C–F##, Supporting Information).
\",\n \"
Subsequently, we investigated whether RUNX2 was involved in the transcriptional regulation of SLC27A5. According to the prediction made using JASPAR, RUNX2 may bind the promoter of SLC27A5 at several possible sites (Figure ##FIG##0##1E##). To determine regulatory regions of RUNX2, the SLC27A5 promoter regions containing different RUNX2 binding sites were cloned into the pGL3 basic vector to perform luciferase reporter assay. We truncated the full‐length region of the SLC27A5 promoter into two fragments, −2023/+366 (pGL3‐P1) and −1001/+182 (pGL3‐P2) (Figure ##FIG##0##1E##). Notably, RUNX2 substantially decreased the activity of both SLC27A5 promoter fragments in the normal human liver cell line MIHA, which further indicates that SLC27A5 is transcriptionally inhibited by RUNX2 (Figure ##FIG##0##1F##). Chromatin immunoprecipitation assay confirmed the binding of RUNX2 to the SLC27A5 promoter (Figure ##FIG##0##1G##). RUNX2 overexpression significantly downregulated SLC27A5 expression in MIHA cells (Figure ##FIG##0##1H##), whereas the inhibition of RUNX2 by shRNA led to elevated expression of SLC27A5 (Figure ##FIG##0##1I##). These findings suggest that RUNX2 is responsible for the downregulation of SLC27A5.
\",\n \"
The upregulation of RUNX2 was verified in the cirrhosis, NAFLD, NASH and HBV‐related fibrosis datasets (Figure ##FIG##0##1J##; Figure ##SUPPL##0##S2G–I##, Supporting Information). RUNX2 expression was negatively correlated with SLC27A5 mRNA levels in cirrhotic tissues (GSE25097) (Figure ##FIG##0##1K##). These results indicate that the elevated expression of the repressor RUNX2 impairs SLC27A5 transcription in patients with liver fibrosis and mouse models.
We used the CRISPR‐Cas9 system to generate whole‐body SLC27A5 knockout (Slc27a5\\n−/−) mice by deleting the protein‐coding exons of Slc27a5 (Figure ##SUPPL##0##S3A##, Supporting Information) to investigate the loss‐of‐function effect of SLC27A5 in vivo. The genotypes of the mice were determined through PCR analysis of tail DNA (Figure ##SUPPL##0##S3B##, Supporting Information). The serum ALT, AST and ALP levels showed a mild increase in Slc27a5\\n−/− mice at 12 months compared to WT littermates (Figure ##SUPPL##0##S3C##, Supporting Information). The mRNA levels of inflammatory genes (Tnfa, Il6, Il1b, Ccl2, Adgre1) were substantially upregulated in the livers of 12‐month‐old Slc27a5\\n−/− mice (Figure ##SUPPL##0##S3D##, Supporting Information). These results indicated that Slc27a5\\n−/− mice developed liver injury and inflammatory response at 12 months of age.
\",\n \"
We then maintained wild‐type (WT) and Slc27a5\\n−/− mice for 24 months and observed an increase in the size of gallbladders in Slc27a5\\n−/− mice (Figure\\n##FIG##1##\\n2A##). SLC27A5\\n−/− mice exhibited a significant increase in the liver‐to‐body weight ratio and elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil) compared with that in WT littermates (Figure ##FIG##1##2B##; Figure ##SUPPL##0##S3E,F##, Supporting Information). Histological analysis using H&E and Sirius red staining revealed mild collagen deposition in Slc27a5\\n−/− mice at 12 months old (Figure ##FIG##1##2C,D##). With aging, Slc27a5\\n−/− mice displayed a massive diffuse ECM and pseudo‐lobular nodule formation at 18 and 24 months, respectively (Figure ##FIG##1##2C,D##). We observed that mRNA levels of profibrotic genes (Acta2, Col1a1, Col3a1, Timp1, and Vim) and inflammatory genes were substantially upregulated in the livers of 24‐month‐old Slc27a5\\n−/− mice (Figure ##FIG##1##2E,F##). Liver hydroxyproline (HYP) levels were also increased in Slc27a5\\n−/− mice (Figure ##SUPPL##0##S3G##, Supporting Information). Western blotting analysis demonstrated a significant increase in the expression of activated HSCs marker α‐SMA and collagen deposition markers COL1A1 and COL3A1 in Slc27a5\\n−/− mice liver tissues (Figure ##SUPPL##0##S3H##, Supporting Information). Furthermore, the expression levels of hepatic genes involved in bile acid synthesis, including Cyp7a1, Cyp8b1, and Cyp27a1, were significantly increased in Slc27a5\\n−/− mice (Figure ##FIG##1##2G##). These findings demonstrate that Slc27a5 deficiency in mice induces spontaneous liver fibrosis at 24 months of age.
To further examine the role of SLC27A5 in liver fibrosis, we investigated fibrogenesis in Slc27a5\\n−/− mice subjected to CCl4 and TAA treatment. We observed an increase in the liver‐to‐body weight ratio in Slc27a5\\n\\n−/−\\n mice after six weeks of CCl4 treatment (Figure\\n##FIG##2##\\n3A##; Figure ##SUPPL##0##S4A##, Supporting Information). Furthermore, H&E, Sirius red staining, α‐SMA, and F4/80 immunostaining revealed elevated collagen deposition, fibrogenesis, and macrophage infiltration in Slc27a5\\n\\n−/−\\n mice (Figure ##FIG##2##3B,C##). Hydroxyproline measurements confirmed increased collagen deposition in Slc27a5\\n\\n−/−\\n mice (Figure ##SUPPL##0##S4B##, Supporting Information). Compared with WT mice, Slc27a5\\n\\n−/−\\n mice displayed significantly higher levels of serum ALT, AST, ALP, and total bilirubin, indicating increased liver injury (Figure ##FIG##2##3D##; Figure ##SUPPL##0##S4C##, Supporting Information). The upregulation of profibrotic and inflammatory genes were also observed in the livers of Slc27a5\\n\\n−/−\\n mice (Figure ##SUPPL##0##S4D–F##, Supporting Information). Eight weeks after TAA injection (Figure ##FIG##2##3E##), Slc27a5\\n\\n−/−\\n mice demonstrated increased collagen deposition, α‐SMA expression, and liver injury (Figure ##FIG##2##3F–H##; Figure ##SUPPL##0##S4G–I##, Supporting Information). Similar to the CCl4 treatment, the hepatic expression of profibrotic and inflammatory genes increased in Slc27a5\\n\\n−/−\\n mice compared with that in WT controls (Figure S##FIG##3##4J–L##, Supporting Information). These findings collectively suggest that SLC27A5 deficiency exacerbates liver fibrosis in mouse models.
\",\n \"SLC27A5 Loss in Hepatocytes Promotes HSC Activation\",\n \"
We compared the primary HSCs isolated from Slc27a5\\n−/− and WT mice to assess their culture‐induced activation. The HSCs from Slc27a5\\n−/− mice exhibited enhanced activation, as evidenced by changes in cell morphology, loss of lipid droplets, and a myofibroblast‐like appearance (Figure\\n##FIG##3##\\n4A##). The expression of fibrogenic marker genes was induced in these cells (Figure ##FIG##3##4B–D##). We further determined the sensitivity of Slc27a5\\n−/− HSCs to the transforming growth factor β1 (TGFβ1), which is regarded as a critical factor in the HSCs activation. The primary HSCs derived from Slc27a5\\n−/− mice displayed increased sensitivity to TGFβ1‐stimulated activation compared with that of WT HSCs (Figure ##SUPPL##0##S5A–C##, Supporting Information). These findings suggest that primary HSCs from Slc27a5\\n−/− mice exhibit enhanced culture‐induced activation and increased sensitivity to TGFβ1 stimulation in vitro.
\",\n \"
The crosstalk between hepatocytes and HSCs generates a permissive fibrotic microenvironment that contributes to fibrosis.[\\n##UREF##0##\\n4\\n##\\n] Given that SLC27A5 mainly functions in hepatocytes,[\\n##REF##16618416##\\n14\\n##\\n] we first examined the expression of SLC27A5 in primary mouse hepatocytes (PMHs) and primary HSCs of WT mice, and observed that SLC27A5 was only expressed in PMHs but not in primary HSCs (Figure ##SUPPL##0##S5D##, Supporting Information). We next investigated the contribution of Slc27a5\\n−/− PMHs to fibrotic progression. The WT or Slc27a5\\n−/− PMH supernatant was added to a primary culture of WT mice HSCs for 48 h (Figure ##FIG##3##4E##). Our results demonstrated a significant up‐regulation of fibrogenic markers in WT HSCs exposed to Slc27a5\\n−/− PMH supernatant (Figure ##FIG##3##4F–H##). Next, we performed a co‐culture experiment using a combination of the human HSC cell line LX‐2 and MIHA cells in a contact‐independent manner (Figure ##FIG##3##4I##). The increased expression of fibrogenic genes in LX‐2 cells co‐cultured with SLC27A5‐KO MIHA cells was similar to those observed earlier in this study (Figure ##FIG##3##4J,K##; Figure ##SUPPL##0##S5E,F##, Supporting Information). These findings indicate that the loss of SLC27A5 in hepatocytes contributes to HSC activation and increased sensitivity to fibrosis.
SLC27A5 is required for bile acid conjugation and plays an essential role in regulating BAs homeostasis in the liver.[\\n##REF##32760200##\\n22\\n##\\n] In this study, we observed an increase in unconjugated BAs, including cholic acid (CA), deoxycholic acid (DCA), and muricholic acid (MCA) in 6‐month‐old Slc27a5\\n−/− mice liver tissues and serum (Figure ##SUPPL##0##S6A–D##, Supporting Information). Furthermore, analysis of the bile acid composition of 24‐month‐old Slc27a5\\n−/− mice revealed a significant increase in CA and DCA (Figure\\n##FIG##4##\\n5A,B##), two major bile acids present in both mice and humans. The reported range of BA levels in human portal venous plasma or serum is 9—43 µм.[\\n##REF##31616073##\\n23\\n##\\n] To determine whether the elevated BAs in Slc27a5\\n−/− mice contribute to the activation of HSCs, we treated human HSC cell line LX‐2 with CA or DCA at concentrations of 25, 50, and 100 µм. Interestingly, CA treatment upregulated the expression of HSCs activation markers in a dose‐dependent manner (Figure ##FIG##4##5C##; Figure ##SUPPL##0##S6E,F##, Supporting Information). This was further confirmed in primary HSCs from WT mice treated with CA at 50 µм. (Figure ##FIG##4##5D–F##). However, DCA supplementation did not strongly affect LX‐2 activation (Figure ##SUPPL##0##S6G–H##, Supporting Information). The mRNA and protein expression of fibrogenic genes were also increased in primary HSCs from Slc27a5\\n−/− mice after CA treatment (Figure ##SUPPL##0##S6I,J##, Supporting Information). Furthermore, the serum levels of CA, but not DCA, were significantly increased in patients with cirrhosis (Figure ##FIG##4##5G##). Consistent with these, CA levels were also higher in SLC27A5‐KO MIHA cells and primary mouse hepatocytes (PMHs) compared with that in controls (Figure ##FIG##4##5H,I##). Based on these observations, we concluded that the abnormal elevation of unconjugated CA in mouse models and patients with cirrhosis promotes HSC activation.
\",\n \"
To determine whether the inhibition of CA levels could diminish the activation of HSCs induced by the supernatant of Slc27a5\\n−/− PMHs, WT and Slc27a5\\n−/− mice were treated with BSH‐IN‐1, a covalent pan‐inhibitor of gut bacterial bile salt hydrolases (BSHs),[\\n##REF##32042200##\\n24\\n##\\n] which decreased unconjugated bile acids (especially CA in Slc27a5−/− mice) in vivo (Figure ##SUPPL##0##S7A##, Supporting Information). Then, the supernatant was added to HSCs from normal WT mice (Figure ##SUPPL##0##S7B##, Supporting Information). The results suggested that the expression of profibrotic genes in primary HSCs was decreased by the treatment of conditional medium from primary PMHs of Slc27a5\\n−/− mice with BSH‐IN‐1 gavage (Figure ##SUPPL##0##S7C##, Supporting Information). The CA levels were also decreased in the conditional medium of BSH‐IN‐1 group, whereas the TCA levels were increased in the same medium (Figure ##SUPPL##0##S7D,E##, Supporting Information). Taken together, these results indicate that inhibition of unconjugated CA levels could decrease the expression of profibrotic genes in primary HSCs co‐cultured with the supernatant of Slc27a5\\n−/− PMHs.
\",\n \"Cholic Acid‐Triggered Upregulation of Early Growth Response (EGR3) Promotes HSC Activation\",\n \"
To investigate how CA induced the activation of HSCs, we performed RNA‐seq analysis on LX‐2 cells treated with CA at a concentration of 50 µм or vehicle controls. The treatment with CA effectively increased the expression of regulatory genes associated with fibrosis in LX‐2 cells (Figure\\n##FIG##5##\\n6A##). Among the upregulated genes in LX‐2 cells, we observed the most significant upregulation in the expression of pro‐fibrotic transcription factor EGR3 following CA treatment (Figure ##FIG##5##6B##). However, taurodeoxycholate (TDCA), DCA, or taurocholic acid (TCA) supplementation did not significantly affect EGR3 expression (Figure ##SUPPL##0##S8A##, Supporting Information). EGR3 is upregulated in the fibrotic dermis of mice with scleroderma, and increased EGR3 expression contributes to the upregulation of fibrotic genes such as ACTA2 and COL1A1.[\\n##REF##23906810##\\n25\\n##\\n] To determine the role of EGR3 in CA‐induced activation of HSCs, we performed ChIP assay to confirm the binding of EGR3 on the promoter of ACTA2 and COL1A1 gene in LX‐2 cells. The data indicated that EGR3 could bind the promoter of ACTA2 and COL1A1 directly, and the recruitment of EGR3 on ACTA2 and COL1A1 promoter was increased by CA stimulation (Figure ##SUPPL##0##S8B##, Supporting Information). Based on these results, we investigated whether CA triggers HSC activation in an EGR3‐dependent manner. Treatment of LX‐2 cells with CA induced the expression of fibrotic genes. However, the induction of these genes was significantly compromised by EGR3 silencing (Figure ##FIG##5##6C,D##). Furthermore, immunofluorescence assays demonstrated that shEGR3 treatment attenuated the CA‐induced expression of α‐SMA (Figure ##FIG##5##6E##). Notably, the upregulation of α‐SMA, COL1A1, and COL3A1 was similarly compromised by EGR3 silencing in primary mouse HSCs (Figure ##FIG##5##6F–H##). We also utilized the MIHA and LX‐2 cells to explore the role of EGR3. As displayed in Figure ##SUPPL##0##S8B–D## (Supporting Information), EGR3 silencing inhibited LX‐2 cell activation induced by SLC27A5‐KO MIHA cell co‐culture. Our findings thus indicate that CA‐induced HSCs activation depends on the transcription factor EGR3.
\",\n \"Overexpression of SLC27A5 or Inhibition of Intestinal Bile Acid Absorption Ameliorates CCl<sub>4</sub>‐Induced Liver Fibrosis\",\n \"
Based on our observations, we observed that SLC2A75 expression was downregulated in human and mouse liver fibrosis tissues (Figure ##FIG##0##1##). Furthermore, we noted that SLC27A5 deficiency promoted liver fibrosis (Figure ##FIG##2##3##). We thus hypothesize that SLC27A5 overexpression might protect against liver fibrosis. To test this hypothesis, specifically overexpressed SLC27A5 in the livers of WT and Slc27a5\\n−/− (KO) mice through tail vein injection of adeno‐associated virus (AAV) harboring Slc27a5 (AAV‐Slc27a5) or AAV‐Control (AAV‐Con) in a CCl4‐induced liver fibrosis model (Figure\\n##FIG##6##\\n7A##). Concordantly, the re‐expression of SLC27A5 in Slc27a5\\n\\n−/−\\n mice alleviated collagen deposition, fibrosis, and liver injury (Figure ##FIG##6##7B–F## and Figure ##SUPPL##0##S9A–E##, Supporting Information). Both serum and liver CA accumulation was reduced in Slc27a5\\n\\n−/−\\n mice injected with AAV‐Slc27a5 (Figure ##FIG##6##7G,H##). Notably, WT mice with SLC27A5 overexpression exhibited a mild alleviation of liver fibrosis compared with those injected with AAV‐Control (Figure ##FIG##6##7B–E##). These results suggest that the AAV‐mediated restoration of hepatic SLC27A5 protects against CCl4‐induced liver fibrosis in mice.
\",\n \"
To further investigate the potential therapeutic strategies for liver fibrosis, we examined the effect of A4250, a specific ASBT inhibitor that decreases hepatic bile acid levels by inhibiting intestinal bile acid absorption,[\\n##REF##35780808##\\n26\\n##\\n] on CCl4‐induced liver fibrosis. We subjected WT and Slc27a5\\n\\n−/−\\n mice to CCl4 injection and treated them with A4250 through gavage (Figure ##FIG##6##7I##). After administering the A4250, we observed an evident improvement in liver fibrosis in Slc27a5\\n\\n−/−\\n mice (Figure ##SUPPL##0##S9F##, Supporting Information). Furthermore, the attenuation of hepatic collagen deposition and α‐SMA expression, along with the improvement of liver function, were evident in Slc27a5\\n\\n−/−\\n mice with A4250 administration (Figure ##FIG##6##7J–N##; Figure ##SUPPL##0##S9G–##J, Supporting Information). Moreover, both serum and liver CA levels were markedly decreased in Slc27a5\\n\\n−/−\\n mice following the A4250 treatment (Figure ##FIG##6##7O,P##). Overall, these findings suggest that the re‐expression of SLC27A5 or inhibition of CA accumulation ameliorates the pathogenesis of liver fibrosis, providing a potential strategy for treating this condition.
In the present study, we identified SLC27A5 as a novel regulator of liver fibrosis progression. Our findings demonstrate that the expression of SLC27A5 is diminished in the livers of cirrhotic patients and mice with liver fibrosis. Moreover, SLC27A5 deficiency aggravates the progression of liver fibrosis by activating hepatic stellate cells (HSCs) via the regulation of bile acid (BA) conjugation (Figure ##FIG##7##\n8\n##). These results provide important insights into the role of unconjugated BAs in liver fibrosis.
","
The abnormal metabolism of BAs has been associated with various causes of liver fibrosis.[\n##REF##32498677##\n27\n##, ##REF##32243703##\n28\n##, ##UREF##1##\n29\n##\n] However, the specific roles of BAs metabolic enzymes in liver fibrosis have not been systematically explored. In our study, we observed that the expression of the bile acid‐CoA ligase SLC27A5 was decreased in patients with cirrhosis and fibrosis mouse models. A recent study found that SLC27A5 mRNA levels were upregulated in patients with NAFLD and a NAFLD rat model;[\n##REF##36851897##\n30\n##\n] however, SLC27A5 expression decreased as fibrosis progressed.[\n##REF##31602526##\n20\n##\n] Lower expression of Slc27a5 was related to chronic CCl4‐induced liver fibrosis.[\n##UREF##2##\n31\n##\n] Similarly, we previously suggested that SLC27A5 expression was downregulated in HCC tissues owing to DNA hypermethylation.[\n##REF##31367013##\n32\n##\n] This lower expression may be useful for predicting HCC prognosis.[\n##REF##33777129##\n33\n##\n] These findings suggest that the downregulation of SLC27A5 plays a vital role in disease progression, particularly in the development of liver fibrosis, cirrhosis, and HCC.[\n##REF##35124283##\n34\n##\n]\n
","
The maintenance of BAs homeostasis is regulated by the farnesoid X receptor (FXR) via transcriptional repression of key enzymes involved in BAs synthesis, such as cholesterol 7αhydroxylase (CYP7A1) and sterol 12α‐hydroxylase (CYP8B1).[\n##REF##34555862##\n6\n##\n]\nSLC27A5 mRNA expression was upregulated after FXR agonist GW4064 treatment.[\n##REF##30520052##\n35\n##\n] However, the underlying mechanisms of SLC27A5 downregulation in liver fibrosis remain unclear. In this study, we identified RUNX2 as a transcriptional repressor of SLC27A5. The RUNX2 is a master transcription factor in regulating osteoblast differentiation, angiogenesis, cancer metastasis, and, in particular, the fibrosis response.[\n##REF##32788656##\n36\n##, ##REF##37108164##\n37\n##, ##REF##27242197##\n38\n##\n] The upregulation of RUNX2 promotes aortic and pulmonary fibrosis.[\n##REF##26208651##\n39\n##, ##REF##28986417##\n40\n##\n] Several fibrosis‐related genes, such as COL1A1 and TIMP1, are regulated by RUNX2.[\n##UREF##3##\n41\n##, ##REF##15051730##\n42\n##\n] Notably, putative RUNX2‐binding sites are localized in the SLC27A5 promoter, and SLC27A5 expression is repressed by RUNX2, which offers at least one underlying reason for SLC27A5 downregulation in patients with cirrhosis and mice with liver fibrosis. These findings suggest SLC27A5 as a potential diagnostic marker of liver fibrosis.
","
Another important finding of this study was the physiological role of SLC27A5 in liver fibrosis. This finding is supported by several lines of evidence. The experimental Slc27a5\n\n−/−\n mice exhibited significantly increased collagen deposition and α‐SMA expression, with an abundance of unconjugated BAs in the serum and liver. This is consistent with a previous report of a child with a homozygous mutation in SLC2A75 who developed extensive fibrosis.[\n##REF##22089923##\n18\n##\n] Although SLC27A5 expression was not reduced in the liver biopsy, over 85% of the unconjugated BAs were present in the plasma of this child, leading us to speculate that the deficiency of BAs conjugation may result in liver fibrosis.
","
Patients with disrupted BAs conjugation exhibited fat‐soluble vitamin deficiency, whereas some developed liver disease.[\n##REF##23415802##\n19\n##\n] Meanwhile, increased levels of DCA, an unconjugated bile acid from the gut microbiota, promote liver cancer through the senescence of HSCs.[\n##REF##23803760##\n43\n##\n] Elevated BAs levels induce cholestatic disease and fibrosis, which may be attributed to hepatocyte injury or cholangiopathy.[\n##REF##33987435##\n10\n##, ##REF##36899928##\n44\n##\n] However, the role of BAs in HSC activation has not been fully explored.
","
TCA is significantly increased in patients with cirrhosis and promotes HSC activation.[\n##REF##29996772##\n45\n##, ##REF##36800698##\n46\n##\n] Increased levels of conjugated BAs such as TDCA and glycodeoxycholate (GDCA) in patients with NASH and fibrosis mouse models could also increase the protein expression of fibrosis‐related markers in LX‐2 cells.[\n##REF##33752128##\n47\n##\n] However, elevated serum BAs of normal composition may not lead to liver injury or fibrosis, which is supported by the fact that patients with Na+‐taurocholate co‐transporting polypeptide (NTCP) deficiency present with significantly elevated levels of conjugated BAs in the plasma without distinct clinical symptoms.[\n##REF##24867799##\n48\n##\n] In this study, we demonstrated that SLC27A5 knockout in mice substantially increased hepatic and serum‐unconjugated CA levels; this subsequently promoted HSC activation and liver fibrosis. Meanwhile, male C57BL/6J mice fed a chow diet supplemented with 0.5% (w/w) CA reportedly exhibit significant liver fibrosis.[\n##REF##35326188##\n49\n##\n] Although unconjugated DCA was also increased in Slc27a5\n\n−/−\n mice, the DCA may not promote HSCs activation, which is consistent with the unchanged levels of DCA in cirrhotic patients as previously reported.[\n##REF##29996772##\n45\n##\n]\n
","
Furthermore, attenuation of BA levels alleviated CCI4‐induced liver fibrosis in Slc27a5\n\n−/−\n mice. Bile acids are synthesized in the liver and secreted into the intestine to facilitate the absorption of lipophilic nutrients, with 95% of BAs being reabsorbed via the ASBT transporter.[\n##REF##35165436##\n5\n##\n] Thus, the interruption of ASBT has been suggested as a promising strategy to attenuate bile acid‐related diseases.[\n##REF##32201314##\n50\n##\n] Recently, the novel ASBT inhibitor A4250 has demonstrated promise in patients with progressive familial intrahepatic cholestasis.[\n##REF##34499340##\n51\n##, ##REF##35780807##\n52\n##\n] This study suggests that A4250 reduced CA levels and liver fibrosis progression in CCI4‐treated Slc27a5\n\n−/−\n mice. Although A4250 did not specifically reduce CA levels in mice, the presence of abundant CA in the liver and serum of Slc27a5\n\n−/−\n mice suggests that it can be an effective therapy. Hence, we conclude that SLC27A5 deficiency promotes liver fibrosis progression by upregulating unconjugated CA levels.
","
Various BAs can induce the proliferation of activated rat HSCs via the epidermal growth factor receptor (EGFR) pathway. Notably, these bile acids did not affect the expression of collagen I.[\n##REF##15825085##\n53\n##\n] Furthermore, BAs stimulate the expression of early growth response (EGR) genes and protein kinase C signal transduction pathways in HSCs.[\n##REF##8670150##\n54\n##\n] However, the precise details and functions of this signaling mechanism are yet to be determined. The EGR family of transcription factors comprises four members that play a role in immune regulation.[\n##REF##20035903##\n55\n##\n] Notably, a specific member of this family, EGR3, reportedly modulates TGF‐β1 transcription in T cells.[\n##REF##33011290##\n56\n##\n] This member also exerts a profibrotic role in skin and cardiac fibroblasts by stimulating the expression of ACTA2 and COL1A1 genes.[\n##REF##23906810##\n25\n##, ##REF##36182775##\n57\n##\n] Our RNA‐seq data revealed a significant upregulation of EGR3 in CA‐treated LX‐2 cells. To further determine the specific role of EGR3, we conducted an experiment to knock down EGR3 using shRNA. The results suggested that the knockdown of EGR3 attenuated the activation of HSCs induced by CA treatment, indicating the importance of unconjugated CA in HSC activation and fibrosis. However, the specific mechanism underlying the upregulation of EGR3 in CA‐treated HSCs requires further investigation.
","
In the present study, we have mainly focused on the role of SLC27A5 in regulation of BAs conjugation during the progression of liver fibrosis. However, other functions of SLC27A5, such as transportation of LCFA [\n##REF##16618416##\n14\n##\n] in liver fibrosis, need further investigation. Additionally, the injection of AAV‐Slc27a5 at an earlier time point may be a better choice to investigate the effects of SLC27A5 overexpression on the treatment of liver fibrosis.
","
In summary, these results suggest that SLC27A5 deficiency in mice promotes liver fibrosis by inhibiting BAs conjugation. This leads to the accumulation of unconjugated CA in the liver, which activates HSCs via EGR3. These findings provide novel insights into the role of SLC27A5 in the progression of fibrosis. The dysregulation of BAs composition is thus suggested to be closely linked to HSC activation. Therefore, the restoration of SLC27A5 expression in hepatocytes and repression of profibrotic CA levels represent potential applications in the treatment of fibrosis.
"],"string":"[\n \"Discussion\",\n \"
In the present study, we identified SLC27A5 as a novel regulator of liver fibrosis progression. Our findings demonstrate that the expression of SLC27A5 is diminished in the livers of cirrhotic patients and mice with liver fibrosis. Moreover, SLC27A5 deficiency aggravates the progression of liver fibrosis by activating hepatic stellate cells (HSCs) via the regulation of bile acid (BA) conjugation (Figure ##FIG##7##\\n8\\n##). These results provide important insights into the role of unconjugated BAs in liver fibrosis.
\",\n \"
The abnormal metabolism of BAs has been associated with various causes of liver fibrosis.[\\n##REF##32498677##\\n27\\n##, ##REF##32243703##\\n28\\n##, ##UREF##1##\\n29\\n##\\n] However, the specific roles of BAs metabolic enzymes in liver fibrosis have not been systematically explored. In our study, we observed that the expression of the bile acid‐CoA ligase SLC27A5 was decreased in patients with cirrhosis and fibrosis mouse models. A recent study found that SLC27A5 mRNA levels were upregulated in patients with NAFLD and a NAFLD rat model;[\\n##REF##36851897##\\n30\\n##\\n] however, SLC27A5 expression decreased as fibrosis progressed.[\\n##REF##31602526##\\n20\\n##\\n] Lower expression of Slc27a5 was related to chronic CCl4‐induced liver fibrosis.[\\n##UREF##2##\\n31\\n##\\n] Similarly, we previously suggested that SLC27A5 expression was downregulated in HCC tissues owing to DNA hypermethylation.[\\n##REF##31367013##\\n32\\n##\\n] This lower expression may be useful for predicting HCC prognosis.[\\n##REF##33777129##\\n33\\n##\\n] These findings suggest that the downregulation of SLC27A5 plays a vital role in disease progression, particularly in the development of liver fibrosis, cirrhosis, and HCC.[\\n##REF##35124283##\\n34\\n##\\n]\\n
\",\n \"
The maintenance of BAs homeostasis is regulated by the farnesoid X receptor (FXR) via transcriptional repression of key enzymes involved in BAs synthesis, such as cholesterol 7αhydroxylase (CYP7A1) and sterol 12α‐hydroxylase (CYP8B1).[\\n##REF##34555862##\\n6\\n##\\n]\\nSLC27A5 mRNA expression was upregulated after FXR agonist GW4064 treatment.[\\n##REF##30520052##\\n35\\n##\\n] However, the underlying mechanisms of SLC27A5 downregulation in liver fibrosis remain unclear. In this study, we identified RUNX2 as a transcriptional repressor of SLC27A5. The RUNX2 is a master transcription factor in regulating osteoblast differentiation, angiogenesis, cancer metastasis, and, in particular, the fibrosis response.[\\n##REF##32788656##\\n36\\n##, ##REF##37108164##\\n37\\n##, ##REF##27242197##\\n38\\n##\\n] The upregulation of RUNX2 promotes aortic and pulmonary fibrosis.[\\n##REF##26208651##\\n39\\n##, ##REF##28986417##\\n40\\n##\\n] Several fibrosis‐related genes, such as COL1A1 and TIMP1, are regulated by RUNX2.[\\n##UREF##3##\\n41\\n##, ##REF##15051730##\\n42\\n##\\n] Notably, putative RUNX2‐binding sites are localized in the SLC27A5 promoter, and SLC27A5 expression is repressed by RUNX2, which offers at least one underlying reason for SLC27A5 downregulation in patients with cirrhosis and mice with liver fibrosis. These findings suggest SLC27A5 as a potential diagnostic marker of liver fibrosis.
\",\n \"
Another important finding of this study was the physiological role of SLC27A5 in liver fibrosis. This finding is supported by several lines of evidence. The experimental Slc27a5\\n\\n−/−\\n mice exhibited significantly increased collagen deposition and α‐SMA expression, with an abundance of unconjugated BAs in the serum and liver. This is consistent with a previous report of a child with a homozygous mutation in SLC2A75 who developed extensive fibrosis.[\\n##REF##22089923##\\n18\\n##\\n] Although SLC27A5 expression was not reduced in the liver biopsy, over 85% of the unconjugated BAs were present in the plasma of this child, leading us to speculate that the deficiency of BAs conjugation may result in liver fibrosis.
\",\n \"
Patients with disrupted BAs conjugation exhibited fat‐soluble vitamin deficiency, whereas some developed liver disease.[\\n##REF##23415802##\\n19\\n##\\n] Meanwhile, increased levels of DCA, an unconjugated bile acid from the gut microbiota, promote liver cancer through the senescence of HSCs.[\\n##REF##23803760##\\n43\\n##\\n] Elevated BAs levels induce cholestatic disease and fibrosis, which may be attributed to hepatocyte injury or cholangiopathy.[\\n##REF##33987435##\\n10\\n##, ##REF##36899928##\\n44\\n##\\n] However, the role of BAs in HSC activation has not been fully explored.
\",\n \"
TCA is significantly increased in patients with cirrhosis and promotes HSC activation.[\\n##REF##29996772##\\n45\\n##, ##REF##36800698##\\n46\\n##\\n] Increased levels of conjugated BAs such as TDCA and glycodeoxycholate (GDCA) in patients with NASH and fibrosis mouse models could also increase the protein expression of fibrosis‐related markers in LX‐2 cells.[\\n##REF##33752128##\\n47\\n##\\n] However, elevated serum BAs of normal composition may not lead to liver injury or fibrosis, which is supported by the fact that patients with Na+‐taurocholate co‐transporting polypeptide (NTCP) deficiency present with significantly elevated levels of conjugated BAs in the plasma without distinct clinical symptoms.[\\n##REF##24867799##\\n48\\n##\\n] In this study, we demonstrated that SLC27A5 knockout in mice substantially increased hepatic and serum‐unconjugated CA levels; this subsequently promoted HSC activation and liver fibrosis. Meanwhile, male C57BL/6J mice fed a chow diet supplemented with 0.5% (w/w) CA reportedly exhibit significant liver fibrosis.[\\n##REF##35326188##\\n49\\n##\\n] Although unconjugated DCA was also increased in Slc27a5\\n\\n−/−\\n mice, the DCA may not promote HSCs activation, which is consistent with the unchanged levels of DCA in cirrhotic patients as previously reported.[\\n##REF##29996772##\\n45\\n##\\n]\\n
\",\n \"
Furthermore, attenuation of BA levels alleviated CCI4‐induced liver fibrosis in Slc27a5\\n\\n−/−\\n mice. Bile acids are synthesized in the liver and secreted into the intestine to facilitate the absorption of lipophilic nutrients, with 95% of BAs being reabsorbed via the ASBT transporter.[\\n##REF##35165436##\\n5\\n##\\n] Thus, the interruption of ASBT has been suggested as a promising strategy to attenuate bile acid‐related diseases.[\\n##REF##32201314##\\n50\\n##\\n] Recently, the novel ASBT inhibitor A4250 has demonstrated promise in patients with progressive familial intrahepatic cholestasis.[\\n##REF##34499340##\\n51\\n##, ##REF##35780807##\\n52\\n##\\n] This study suggests that A4250 reduced CA levels and liver fibrosis progression in CCI4‐treated Slc27a5\\n\\n−/−\\n mice. Although A4250 did not specifically reduce CA levels in mice, the presence of abundant CA in the liver and serum of Slc27a5\\n\\n−/−\\n mice suggests that it can be an effective therapy. Hence, we conclude that SLC27A5 deficiency promotes liver fibrosis progression by upregulating unconjugated CA levels.
\",\n \"
Various BAs can induce the proliferation of activated rat HSCs via the epidermal growth factor receptor (EGFR) pathway. Notably, these bile acids did not affect the expression of collagen I.[\\n##REF##15825085##\\n53\\n##\\n] Furthermore, BAs stimulate the expression of early growth response (EGR) genes and protein kinase C signal transduction pathways in HSCs.[\\n##REF##8670150##\\n54\\n##\\n] However, the precise details and functions of this signaling mechanism are yet to be determined. The EGR family of transcription factors comprises four members that play a role in immune regulation.[\\n##REF##20035903##\\n55\\n##\\n] Notably, a specific member of this family, EGR3, reportedly modulates TGF‐β1 transcription in T cells.[\\n##REF##33011290##\\n56\\n##\\n] This member also exerts a profibrotic role in skin and cardiac fibroblasts by stimulating the expression of ACTA2 and COL1A1 genes.[\\n##REF##23906810##\\n25\\n##, ##REF##36182775##\\n57\\n##\\n] Our RNA‐seq data revealed a significant upregulation of EGR3 in CA‐treated LX‐2 cells. To further determine the specific role of EGR3, we conducted an experiment to knock down EGR3 using shRNA. The results suggested that the knockdown of EGR3 attenuated the activation of HSCs induced by CA treatment, indicating the importance of unconjugated CA in HSC activation and fibrosis. However, the specific mechanism underlying the upregulation of EGR3 in CA‐treated HSCs requires further investigation.
\",\n \"
In the present study, we have mainly focused on the role of SLC27A5 in regulation of BAs conjugation during the progression of liver fibrosis. However, other functions of SLC27A5, such as transportation of LCFA [\\n##REF##16618416##\\n14\\n##\\n] in liver fibrosis, need further investigation. Additionally, the injection of AAV‐Slc27a5 at an earlier time point may be a better choice to investigate the effects of SLC27A5 overexpression on the treatment of liver fibrosis.
\",\n \"
In summary, these results suggest that SLC27A5 deficiency in mice promotes liver fibrosis by inhibiting BAs conjugation. This leads to the accumulation of unconjugated CA in the liver, which activates HSCs via EGR3. These findings provide novel insights into the role of SLC27A5 in the progression of fibrosis. The dysregulation of BAs composition is thus suggested to be closely linked to HSC activation. Therefore, the restoration of SLC27A5 expression in hepatocytes and repression of profibrotic CA levels represent potential applications in the treatment of fibrosis.
Although the dysregulation of bile acid (BA) composition has been associated with fibrosis progression, its precise roles in liver fibrosis is poorly understood. This study demonstrates that solute carrier family 27 member 5 (SLC27A5), an enzyme involved in BAs metabolism, is substantially downregulated in the liver tissues of patients with cirrhosis and fibrosis mouse models. The downregulation of SLC27A5 depends on RUNX family transcription factor 2 (RUNX2), which serves as a transcriptional repressor. The findings reveal that experimental SLC27A5 knockout (Slc27a5\n\n−/−\n) mice display spontaneous liver fibrosis after 24 months. The loss of SLC27A5 aggravates liver fibrosis induced by carbon tetrachloride (CCI4) and thioacetamide (TAA). Mechanistically, SLC27A5 deficiency results in the accumulation of unconjugated BA, particularly cholic acid (CA), in the liver. This accumulation leads to the activation of hepatic stellate cells (HSCs) by upregulated expression of early growth response protein 3 (EGR3). The re‐expression of hepatic SLC27A5 by an adeno‐associated virus or the reduction of CA levels in the liver using A4250, an apical sodium‐dependent bile acid transporter (ASBT) inhibitor, ameliorates liver fibrosis in Slc27a5\n−/−\n mice. In conclusion, SLC27A5 deficiency in mice drives hepatic fibrosis through CA‐induced activation of HSCs, highlighting its significant implications for liver fibrosis treatment.
","
The authors propose a novel mechanism of aberrant bile acid metabolism in liver fibrosis. Loss of solute carrier family 27 member 5 (SLC27A5) accumulates cholic acids (CA) in the liver and promotes hepatic stellate cells activation via early growth response protein 3 (EGR3). Re‐expression of SLC27A5 or inhibition of CA levels could be a promising strategy for liver fibrosis treatment.\n\n
","
\n\n\nK.\nWu\n, \nY.\nLiu\n, \nJ.\nXia\n, \nJ.\nLiu\n, \nK.\nWang\n, \nH.\nLiang\n, \nF.\nXu\n, \nD.\nLiu\n, \nD.\nNie\n, \nX.\nTang\n, \nA.\nHuang\n, \nC.\nChen\n, \nN.\nTang\n, Loss of SLC27A5 Activates Hepatic Stellate Cells and Promotes Liver Fibrosis via Unconjugated Cholic Acid. Adv. Sci.\n2024, 11, 2304408. 10.1002/advs.202304408\n\n
"],"string":"[\n \"Abstract\",\n \"
Although the dysregulation of bile acid (BA) composition has been associated with fibrosis progression, its precise roles in liver fibrosis is poorly understood. This study demonstrates that solute carrier family 27 member 5 (SLC27A5), an enzyme involved in BAs metabolism, is substantially downregulated in the liver tissues of patients with cirrhosis and fibrosis mouse models. The downregulation of SLC27A5 depends on RUNX family transcription factor 2 (RUNX2), which serves as a transcriptional repressor. The findings reveal that experimental SLC27A5 knockout (Slc27a5\\n\\n−/−\\n) mice display spontaneous liver fibrosis after 24 months. The loss of SLC27A5 aggravates liver fibrosis induced by carbon tetrachloride (CCI4) and thioacetamide (TAA). Mechanistically, SLC27A5 deficiency results in the accumulation of unconjugated BA, particularly cholic acid (CA), in the liver. This accumulation leads to the activation of hepatic stellate cells (HSCs) by upregulated expression of early growth response protein 3 (EGR3). The re‐expression of hepatic SLC27A5 by an adeno‐associated virus or the reduction of CA levels in the liver using A4250, an apical sodium‐dependent bile acid transporter (ASBT) inhibitor, ameliorates liver fibrosis in Slc27a5\\n−/−\\n mice. In conclusion, SLC27A5 deficiency in mice drives hepatic fibrosis through CA‐induced activation of HSCs, highlighting its significant implications for liver fibrosis treatment.
\",\n \"
The authors propose a novel mechanism of aberrant bile acid metabolism in liver fibrosis. Loss of solute carrier family 27 member 5 (SLC27A5) accumulates cholic acids (CA) in the liver and promotes hepatic stellate cells activation via early growth response protein 3 (EGR3). Re‐expression of SLC27A5 or inhibition of CA levels could be a promising strategy for liver fibrosis treatment.\\n\\n
\",\n \"
\\n\\n\\nK.\\nWu\\n, \\nY.\\nLiu\\n, \\nJ.\\nXia\\n, \\nJ.\\nLiu\\n, \\nK.\\nWang\\n, \\nH.\\nLiang\\n, \\nF.\\nXu\\n, \\nD.\\nLiu\\n, \\nD.\\nNie\\n, \\nX.\\nTang\\n, \\nA.\\nHuang\\n, \\nC.\\nChen\\n, \\nN.\\nTang\\n, Loss of SLC27A5 Activates Hepatic Stellate Cells and Promotes Liver Fibrosis via Unconjugated Cholic Acid. Adv. Sci.\\n2024, 11, 2304408. 10.1002/advs.202304408\\n\\n
Liver tissue samples from healthy controls (n = 14) and cirrhotic patients (n = 20) were obtained from the Second Affiliated Hospital of Chongqing Medical University. Fasting serum samples were collected from healthy controls (n = 18) and cirrhotic patients (n = 18) recruited from the Second Affiliated Hospital of Chongqing Medical University. Fasting blood specimens were collected from all volunteers and centrifuged at 2000 g for 10 min at 4 °C. The resulting sera were collected and stored at −80 °C until analysis. Clinical characteristics of participants were summarized in Table ##SUPPL##0##S1## (Supporting Information). Informed consents were obtained from all involved participants. This study was approved by the Research Ethics Committee of Chongqing Medical University (Approval number: 2022054).
","Cell Cultures and Reagents","
Human hepatic stellate cell line LX‐2 was obtained from the China Center for Type Culture Collection (CCTC, Wuhan, China). An immortalized human liver cell line MIHA was kindly provided by Prof. Ben C. B. Ko (The Hong Kong Polytechnic University, Hong Kong, China).[\n##REF##25708728##\n58\n##\n] Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Corning, NY, USA), and 1% penicillin‐streptomycin (MedChemExpress, NJ, USA) at 37 °C containing 5% CO2.
","
For the bile acid treatment experiments, LX‐2 cells were seeded in 6‐well plates, and cells were stimulated by fresh 2% FBS medium with or without individual BAs (Sigma‐Aldrich, St Louis, MO, USA) at 25, 50, 75, and 100 µм for 48 h, and DMSO were used as a vehicle control.
","
For co‐culture experiments, LX‐2 cells were seeded in 12‐well plates at densities of 50000 cells cm−2. MIHA cells were seeded and adhered to the porous polyester (PET) membrane surface of trans‐well inserts (0.4 µm pores, BIOFIL, Guangzhou, China) at a density of 100000 cells cm−2. Inserts containing MIHA cells were incubated overnight to adapt to the new conditions before being placed in 12‐well plates containing HSCs. The co‐culture system was incubated for 48 h in DMEM medium with 2% FBS. Trans‐well inserts were then carefully removed and the LX‐2 cells in 12‐well plates were immediately collected for analysis.
","Animals","
Heterozygous C57BL/6N‐Slc27a5em1cyagen mice were created using the CRISPR‐Cas9 technology by Cyagen Biosciences (Suzhou, China) and were crossed to breed wild‐type (WT) and Slc27a5\n−/− mice. The genotype of WT and Slc27a5\n−/− mice was confirmed by PCR amplification of tail DNA (PCR primers were listed in Table ##SUPPL##0##S2##, Supporting Information). All mice were maintained under specific pathogen‐free conditions in the laboratory animal center of Chongqing Medical University. All animal experiments were performed under the guidelines of the institutional Animal Care and Use Committee at Chongqing Medical University. All animal procedures were also approved by the Animal Experimentation Ethics Committees of Chongqing Medical University (Approval number: 2022054).
","
For the carbon tetrachloride (CCl4) model of liver fibrosis, 6‐ to 8‐week‐old male WT and Slc27a5\n−/− mice were given intraperitoneal (i.p.) injections of CCl4 (1.0 mL kg−1 body weight, dissolved in corn oil at a ratio of 1:9) (Macklin, Shanghai, China) or vehicle (corn oil) twice a week for 6 weeks (n = 5 per group). For the thioacetamide (TAA) model of liver fibrosis, 6‐ to 8‐week‐old male WT and Slc27a5\n−/− mice were intraperitoneally injected with phosphate‐buffered saline (PBS) or TAA (100 mg kg−1 body weight) (Macklin, Shanghai, China) three times a week for 8 weeks (n = 5 per group). The mice were starved overnight and sacrificed 2 days after the final injection. The mouse livers and serum were collected for subsequent experiments. Mouse serum ALT, AST, ALP. and TBil were detected using an automatic biochemical analyzer (Catalyst One, IDEXX, USA).
","
For overexpression of SLC27A5, AAV8‐TBG‐control or AAV8‐TBG‐Slc27a5 (OBiO Technology, Corp., Ltd. Shanghai, China) was injected via tail vein of WT and Slc27a5\n−/− male mice at 8 weeks of age (2 × 1011 genome copies per mouse). After 8 weeks of CCI4 injection, mice were starved overnight and sacrificed for analysis.
","
For inhibition of the hepatic CA accumulation, 8‐week‐old male WT and Slc27a5\n\n−/−\n mice were subjected to the CCl4 injection for 6 weeks and treated with vehicle or bile acid transporter inhibitor A4250 (10 mg kg−1 body weight) (HY‐109120, MedChemExpress) by daily gavages in the last 4 weeks. After 4 weeks of A4250 treatment, mice were starved overnight and sacrificed for analysis.
","
For treatment of BSH‐IN‐1, Adult male WT or Slc27a5\n\n−/−\n mice were gavaged with a single dose of BSH‐IN‐1 (10 mg kg−1, TargetMol, USA) or vehicle control (n = 3 per group). The primary mice hepatocytes were isolated after 24 h of the gavage to perform the co‐culture experiment.
","Extraction and Profiling of Bile Acids","
Serum bile acids extraction was performed by mixing 50 µL of serum with 500 µL of methanol containing 20 µL of 1 µg mL−1 of Cholic acid‐d4 (CDN Isotopes Inc, Pointe‐Claire, Canada) used as the internal standard. For liver bile acid extraction, ≈60 mg of frozen liver samples was weighed and homogenized in 600 mL of methanol. An amount of 500 µL of liver homogenate was spiked with 20 µL of internal standards. The serum or liver mixture was then vortexed at 12000 g at 4 °C for 30 seconds and incubated for 30 min at 65 °C. The samples were then heated on boiling water bath for 3 min and cooled to room temperature. The mixture was centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was collected. The pellet was resuspended in 500 µL of methanol, vortexed for 2 min, and centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was combined with that collected earlier, and dried under vacuum. The residue was redissolved with 50% methanol to a final volume of 100 µL. After centrifugation at 12000 g at for 10 min at 4 °C, an aliquot of 70 µL of supernatant was used for liquid chromatography‐tandem mass spectrometry (LC‐MS) analysis as previously described.[\n##REF##36379100##\n59\n##\n] Briefly, the separation and detection were performed on a Waters ACQUITY UPLC CSH C18 column (2.1 ×100 mm, 1.7 µm) with an Agilent 1290—6495 C ultra performance liquid chromatography‐triple quadrupole tandem mass spectrometry system. Ammonium formate solution of 5 mм with 0.1% formic acid was used as mobile phase A, and methanol was chosen as mobile phase B. The dynamic multiple reaction monitoring (DMRM) was used for MS analysis.
","
For bile acid quantitation, calibration curves of standard samples containing various bile acids were used according to recently published methods.[\n##REF##36379100##\n59\n##\n] Serial standard mixtures were obtained by gradient dilution (4 ×). The standard mixtures were then mixed with internal standard and underwent the same sample preparation as the test samples. Bile acid quantitation was based on the peak area ratio of the targeted bile acids to Cholic acid‐d4. The bile acids concentrations of test samples were quantified using slopes, but not intercepts, of the calibration curves to deduct background signals.
","Primary Mouse Liver Cell Isolation and Culture","
Primary murine hepatocytes were isolated from the livers of male WT and Slc27a5\n−/− mice aged 8–12 weeks according to a reported protocol [\n##UREF##4##\n60\n##\n] with some modification that includes the following steps: in situ pronase (P5147, Sigma‐Aldrich) /collagenase (V900893, Sigma‐Aldrich) perfusion of mouse liver, perfused livers were minced, filtered through 70 µm cell strainer (BS‐70‐XBS, Biosharp, Beijing, China), and centrifuged at 40 g for 5 min at 4 °C to separate hepatocytes. Hepatocytes were plated in Dulbecco's modified Eagle's medium with 1 g L−1 glucose (DMEM‐low glucose, SH30021.01, HyClone) supplemented with 5% FBS, 15 mmol L−1 HEPES (H1095, Solarbio, Beijing, China), and 1% penicillin‐streptomycin. Cells were maintained overnight in serum‐free DMEM containing 1 g L−1 glucose. After 48 h of cell seeding in coated plates, medium was collected and concentrated by a speed‐vacuum to measure bile acids by LC‐MS.
","
HSCs were isolated according to a previously published method,[\n##REF##25612230##\n61\n##\n] and the supernatant was further centrifuged at 580 g for 10 min at 4 °C, resuspended in density gradient‐based Nycodenz (1002424‐1, Alere Technologios AS), and centrifuged at 1400 g for 17 min at 4 °C. HSCs were collected from the interface and cultured in DMEM with 10% FBS and 1% penicillin‐streptomycin.
","
The 48 h culture supernatant from primary hepatocytes of WT or Slc27a5\n\n−/−\n mice was collected and centrifuged at 2000 g for 5 min to prepare it for the conditional culture. The supernatant was collected and labelled as conditioned medium. Twenty‐four hours post isolation, the medium of the primary HSCs was replaced by the conditioned medium from WT or Slc27a5\n\n−/−\n mice primary hepatocytes and incubated for another 48 h. For TGFβ1 treatment, the primary HSCs after 24 h isolation were treated with and without TGFβ1 (4 ng mL−1, Novoprotein, Beijing, China) for another 48 h. For CA treatment, the primary HSCs after 24 h isolation were treated with CA (50 µм) for another 48 h.
","Western Blotting","
Proteins were extracted from cells or liver tissues in the lysis buffer (Beyotime, Shanghai, China) consisting of protease inhibitor cocktail (1:100, TargetMol, MA, USA). The concentration of proteins was determined using BCA Protein Assay kit (Beyotime). The extracted proteins were separated by 10% SDS/PAGE and electro‐transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking in 5% milk for 1 h, the membranes were probed with primary antibodies against SLC27A5 (1:1000, NBP2‐37412, Novus Biologicals, CO, USA), α‐SMA (1:1000, ER1003, Huabio, Hangzhou, China), COL1A1 (1:1000, WL0088, Wanleibio, Shenyang, China), COL3A1 (1:1000, 22734‐1‐AP, Proteintech, IL, USA), RUNX2 (1:1000, 20700‐1‐AP, Proteintech, IL, USA), EGR3 (1:500, sc‐390967, Santa Cruz Biotechnology, USA), β‐actin (1:3000, BL005B, Biosharp), or GAPDH (1:3000, AG019‐1, Beyotime, Shanghai, China) at 4 °C overnight. Membranes were then incubated with horseradish peroxidase‐conjugated secondary antibody (Abcam, Cambridge, UK). The staining was visualized using ClarityTM Western ECL Substrate (Bio‐Rad, CA, USA).
","Liver histological and Immunohistological (IHC) Staining","
Liver specimens were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 µm sections. Then, the specimens were deparaffinized, hydrated and stained by standard methods. To examine hepatic morphology and assess liver fibrosis, H&E and Sirius Red staining were performed. Sections were immune‐stained for SLC27A5 (1:500, NBP2‐37412, Novus Biologicals, CO, USA), α‐SMA (1:300, 19245T, CST, MA, USA), F4/80 (1:300, 70076T, CST), or RUNX2 (1:300, 20700‐1‐AP, Proteintech, IL, USA) overnight at 4 °C. Sections were then incubated with a secondary anti‐rabbit or anti‐mouse IgG (ZSGB‐BIO, Beijing, China) and stained using 3,3′‐diaminobenzidine (ZSGB‐BIO). Stained slides were scanned with a Pannoramic Scan 250 Flash and images were acquired using Pannoramic Viewer 1.15.2 (3DHistech, Budapest, Hungary). Immunostaining and Sirius Red staining were quantified by threshold analysis using the NIH ImageJ software. The immunohistochemical staining of SLC27A5 and RUNX2 was semi‐quantitatively analyzed using the immunoreactive scoring system.[\n##REF##34921145##\n62\n##\n]\n
","Hepatic Hydroxyproline (HYP) Measurement","
The HYP levels of fresh liver samples of mice were quantified. Concentration was calculated by a standard curve using the HYP Content Assay Kit (BC0255, Solarbio, China) according to the manufacturer protocol.
","Immunofluorescence","
HSCs were seeded and treated with CA (50 µм) in slide chambers. After being fixed in 4% paraformaldehyde, cells were permeabilized in 0.5% Triton X‐100 for 10 min. After being blocked in 5% goat serum for 1 h, slides were incubated with primary antibody against α‐SMA (1:300, 19245T, CST) overnight at 4 °C. The slides were then washed 5 times in PBS and incubated with goat anti‐rabbit conjugated with Alexa Fluor 488 or 552 secondary antibody (1:100, ZF‐0311, ZSGB‐bio) for 1 h at 37 °C. Nucleus was stained using 1 µg mL−1 4′,6‐diamidino‐2‐phenylindole (DAPI,1:2000, Roche, Basel, Switzerland). Stained sections were observed by a laser‐scanning confocal microscope (Leica TCS SP8, Solms, Germany).
Total RNA of tissue or cells was isolated with Trizol reagent (Invitrogen, Rockville, MD). Reverse transcription (RT) reactions were performed using PrimeScript RT Reagent Kit (RR047A, TaKaRa, TKY, Japan). Real‐time PCR was carried out using Bio‐Rad CFX96 machine (Bio‐Rad, Hercules, CA, USA). The level of GAPDH or β‐actin RNA expression was used to normalize the data. The sequences of qRT‐PCR primers were listed in Table ##SUPPL##0##S2## (Supporting Information).
","Construction of Plasmids","
The expression vector of human RUNX2 was inserted into the Sal I and Xba I sites of the shuttle vector pAdTrack‐TO4 (from Dr T‐C He, University of Chicago, USA). The 5′‐flanking region (from −2023 to +366 nt) of SLC27A5 gene was inserted into the Hind III and Xho I sites of the pGL3‐Basic vector (Promega, Madison, WI, USA), named pGL3‐P1. To construct another length of luciferase reporter plasmid of SLC27A5, the region from −1001 to +182 nt of SLC27A5 gene was inserted into the pGL3‐Basic vector, named pGL3‐P2. Oligonucleotide sequences were listed in Table ##SUPPL##0##S2## (Supporting Information).
","Lentivirus‐Meditated RNA Interference","
The small double‐strand hairpin shRNA expressing constructs (shRUNX2, shEGR3, and shEgr3) were designed and annealed into the Hpa I and Xho I sites of pLL3.7 lentivirus vector (kindly provided by Prof. Bing Sun, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China). A negative control construct (shCon) was also generated. The lentiviral supernatants were produced in HEK293T cells as previously described.[\n##REF##34650217##\n63\n##\n] Oligonucleotide sequences were listed in Table ##SUPPL##0##S2## (Supporting Information).
","CRISPR/Cas9‐Mediated Knockout Cells","
SLC27A5‐knockout cells were constructed by the CRISPR‐Cas9 system provided by Prof. Ding Xue (Tsinghua University, Beijing, China), as previously described.[\n##REF##31367013##\n32\n##\n] The Knockout of SLC27A5 in MIHA cells were validated by immunoblotting. The sgRNA target sequences were listed in Table ##SUPPL##0##S2## (Supporting Information).
","Luciferase Reporter Assay","
MIHA cells were transfected in twelve‐well plates containing 4 µL of Lipofectamine 8000, 0.5 µg of pADTrack‐TO4 control or pADTrack‐RUNX2 overexpression vectors, 0.5 µg of each luciferase reporter plasmids pGL3‐basic (Promega, Madison, WI, USA), pGL3‐P1 (from −2023 to +366 nt of the promoter region of the SLC27A5 gene), or pGL3‐P2 (from −1001 to +182 nt of the promoter region of the SLC27A5 gene), and 10 ng of pRL‐TK‐Renilla (as transfection control) for 48 h. Cells were harvested and assayed for luciferase activity using the Dual Luciferase Assay Kit (Promega, Madison, WI, USA). All experiments were performed at least three times and expressed as mean ± SEM.
","Chromatin Immunoprecipitation (ChIP) Assay","
MIHA cells of 6 × 106 were cross‐linked using 1% paraformaldehyde for 8 min at 37 °C. Cell lysates were sonicated at 30% power for 10 cycles (15 seconds ON and 15 seconds OFF). Supernatants were separated and incubated with anti‐RUNX2 (20700‐1‐AP, Proteintech, IL, USA), anti‐EGR3 (sc‐390967, Santa Cruz Biotechnology, USA), or control IgG overnight at 4 °C. Chromatin‐antibody complexes were collected by protein A/G agarose beads (Millipore), washed and then eluted. Real‐time PCR was used to analyze the RUNX2‐binding DNA fragments from ChIP assays. The ChIP and ChIP‐qPCR assays were performed as previously described.[\n##REF##34650217##\n63\n##\n] Primers were listed in Table ##SUPPL##0##S2## (Supporting Information).
","RNA‐Seq Analysis","
Total RNA was extracted in LX‐2 cells treated with CA (50 µм) or vehicle for 48 h using Trizol reagent and subjected to library preparation. RNA‐seq was performed by Majorbio Bio‐pharm Technology Co.,Ltd (Shanghai, China). Differential expression genes (DEGs) were analyzed using the DESeq2. Heatmap of DEGs was performed using the “ggplot2” packages in R (v4.2.1, The R Project for Statistical Computing, Vienna, Austria).
","Online Database Analysis","
UCSC (https://genome.ucsc.edu/) was used to predict transcriptional factors (TFs) that could affect SLC27A5.[\n##REF##36420891##\n64\n##\n] JASPAR (http://jaspar.genereg.net/) was used to predict the potential binding sites of TFs on SLC27A5 promoter.[\n##REF##34850907##\n21\n##\n]\n
","Gene Expression Omnibus Database Mining","
Four data sets from GEO database (https://www.ncbi.nlm.nih.gov/geo/) were analyzed with GEO2R to profile gene expression between different samples, such as mild fibrosis versus advanced fibrosis in NAFLD patients (GSE31803), control versus NASH patients (GSE48452), normal versus cirrhosis patients (GSE25097), and different stage of fibrosis in HBV infected patients (GSE84044).
","Statistical Analysis","
All data were presented as the means ± SEM. Tests used to examine the differences between groups include Student's t test, one‐way ANOVA and with the Tukey's post hoc test, and two‐way ANOVA with Tukey's multiple comparisons test. Pearson correlation coefficient (r) was used to test the linear correlation. P‐values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were conducted using GraphPad Prism 8.0 software (La Jolla, CA, USA).
","Ethics Approval Statement","
This study was approved by the Research Ethics Committee of Chongqing Medical University (approval number: 2022054). All animal experiments were performed under the guidelines of the institutional Animal Care and Use Committee at Chongqing Medical University.
","Patient Consent Statement","
Informed consents were obtained from all involved participants.
","Conflict of Interest","
The authors declare no conflict of interest.
","Author Contributions","
K.W., Y.L., J.X., and J.L. contributed equally to this work. N.T., C.C. and A.H. conceived and designed the study. K.W., Y.L., J.X., and J.L. performed most experiments and analyzed the data. K.W. provided suggestions and designed primer sequence. H.L. constructed plasmids. F.X., D.L., D.N., and X.T. assisted with mice experiments. C.C. provided technical assistance. K.W. and N.T. wrote the manuscript with all authors providing feedback. The order of the co‐first authors was determined on the basis of their relative contributions to the study.
Liver tissue samples from healthy controls (n = 14) and cirrhotic patients (n = 20) were obtained from the Second Affiliated Hospital of Chongqing Medical University. Fasting serum samples were collected from healthy controls (n = 18) and cirrhotic patients (n = 18) recruited from the Second Affiliated Hospital of Chongqing Medical University. Fasting blood specimens were collected from all volunteers and centrifuged at 2000 g for 10 min at 4 °C. The resulting sera were collected and stored at −80 °C until analysis. Clinical characteristics of participants were summarized in Table ##SUPPL##0##S1## (Supporting Information). Informed consents were obtained from all involved participants. This study was approved by the Research Ethics Committee of Chongqing Medical University (Approval number: 2022054).
\",\n \"Cell Cultures and Reagents\",\n \"
Human hepatic stellate cell line LX‐2 was obtained from the China Center for Type Culture Collection (CCTC, Wuhan, China). An immortalized human liver cell line MIHA was kindly provided by Prof. Ben C. B. Ko (The Hong Kong Polytechnic University, Hong Kong, China).[\\n##REF##25708728##\\n58\\n##\\n] Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Corning, NY, USA), and 1% penicillin‐streptomycin (MedChemExpress, NJ, USA) at 37 °C containing 5% CO2.
\",\n \"
For the bile acid treatment experiments, LX‐2 cells were seeded in 6‐well plates, and cells were stimulated by fresh 2% FBS medium with or without individual BAs (Sigma‐Aldrich, St Louis, MO, USA) at 25, 50, 75, and 100 µм for 48 h, and DMSO were used as a vehicle control.
\",\n \"
For co‐culture experiments, LX‐2 cells were seeded in 12‐well plates at densities of 50000 cells cm−2. MIHA cells were seeded and adhered to the porous polyester (PET) membrane surface of trans‐well inserts (0.4 µm pores, BIOFIL, Guangzhou, China) at a density of 100000 cells cm−2. Inserts containing MIHA cells were incubated overnight to adapt to the new conditions before being placed in 12‐well plates containing HSCs. The co‐culture system was incubated for 48 h in DMEM medium with 2% FBS. Trans‐well inserts were then carefully removed and the LX‐2 cells in 12‐well plates were immediately collected for analysis.
\",\n \"Animals\",\n \"
Heterozygous C57BL/6N‐Slc27a5em1cyagen mice were created using the CRISPR‐Cas9 technology by Cyagen Biosciences (Suzhou, China) and were crossed to breed wild‐type (WT) and Slc27a5\\n−/− mice. The genotype of WT and Slc27a5\\n−/− mice was confirmed by PCR amplification of tail DNA (PCR primers were listed in Table ##SUPPL##0##S2##, Supporting Information). All mice were maintained under specific pathogen‐free conditions in the laboratory animal center of Chongqing Medical University. All animal experiments were performed under the guidelines of the institutional Animal Care and Use Committee at Chongqing Medical University. All animal procedures were also approved by the Animal Experimentation Ethics Committees of Chongqing Medical University (Approval number: 2022054).
\",\n \"
For the carbon tetrachloride (CCl4) model of liver fibrosis, 6‐ to 8‐week‐old male WT and Slc27a5\\n−/− mice were given intraperitoneal (i.p.) injections of CCl4 (1.0 mL kg−1 body weight, dissolved in corn oil at a ratio of 1:9) (Macklin, Shanghai, China) or vehicle (corn oil) twice a week for 6 weeks (n = 5 per group). For the thioacetamide (TAA) model of liver fibrosis, 6‐ to 8‐week‐old male WT and Slc27a5\\n−/− mice were intraperitoneally injected with phosphate‐buffered saline (PBS) or TAA (100 mg kg−1 body weight) (Macklin, Shanghai, China) three times a week for 8 weeks (n = 5 per group). The mice were starved overnight and sacrificed 2 days after the final injection. The mouse livers and serum were collected for subsequent experiments. Mouse serum ALT, AST, ALP. and TBil were detected using an automatic biochemical analyzer (Catalyst One, IDEXX, USA).
\",\n \"
For overexpression of SLC27A5, AAV8‐TBG‐control or AAV8‐TBG‐Slc27a5 (OBiO Technology, Corp., Ltd. Shanghai, China) was injected via tail vein of WT and Slc27a5\\n−/− male mice at 8 weeks of age (2 × 1011 genome copies per mouse). After 8 weeks of CCI4 injection, mice were starved overnight and sacrificed for analysis.
\",\n \"
For inhibition of the hepatic CA accumulation, 8‐week‐old male WT and Slc27a5\\n\\n−/−\\n mice were subjected to the CCl4 injection for 6 weeks and treated with vehicle or bile acid transporter inhibitor A4250 (10 mg kg−1 body weight) (HY‐109120, MedChemExpress) by daily gavages in the last 4 weeks. After 4 weeks of A4250 treatment, mice were starved overnight and sacrificed for analysis.
\",\n \"
For treatment of BSH‐IN‐1, Adult male WT or Slc27a5\\n\\n−/−\\n mice were gavaged with a single dose of BSH‐IN‐1 (10 mg kg−1, TargetMol, USA) or vehicle control (n = 3 per group). The primary mice hepatocytes were isolated after 24 h of the gavage to perform the co‐culture experiment.
\",\n \"Extraction and Profiling of Bile Acids\",\n \"
Serum bile acids extraction was performed by mixing 50 µL of serum with 500 µL of methanol containing 20 µL of 1 µg mL−1 of Cholic acid‐d4 (CDN Isotopes Inc, Pointe‐Claire, Canada) used as the internal standard. For liver bile acid extraction, ≈60 mg of frozen liver samples was weighed and homogenized in 600 mL of methanol. An amount of 500 µL of liver homogenate was spiked with 20 µL of internal standards. The serum or liver mixture was then vortexed at 12000 g at 4 °C for 30 seconds and incubated for 30 min at 65 °C. The samples were then heated on boiling water bath for 3 min and cooled to room temperature. The mixture was centrifuged at 12,000 g for 10 min at 4 °C, and the supernatant was collected. The pellet was resuspended in 500 µL of methanol, vortexed for 2 min, and centrifuged at 12,000 g for 10 min at 4 °C. The supernatant was combined with that collected earlier, and dried under vacuum. The residue was redissolved with 50% methanol to a final volume of 100 µL. After centrifugation at 12000 g at for 10 min at 4 °C, an aliquot of 70 µL of supernatant was used for liquid chromatography‐tandem mass spectrometry (LC‐MS) analysis as previously described.[\\n##REF##36379100##\\n59\\n##\\n] Briefly, the separation and detection were performed on a Waters ACQUITY UPLC CSH C18 column (2.1 ×100 mm, 1.7 µm) with an Agilent 1290—6495 C ultra performance liquid chromatography‐triple quadrupole tandem mass spectrometry system. Ammonium formate solution of 5 mм with 0.1% formic acid was used as mobile phase A, and methanol was chosen as mobile phase B. The dynamic multiple reaction monitoring (DMRM) was used for MS analysis.
\",\n \"
For bile acid quantitation, calibration curves of standard samples containing various bile acids were used according to recently published methods.[\\n##REF##36379100##\\n59\\n##\\n] Serial standard mixtures were obtained by gradient dilution (4 ×). The standard mixtures were then mixed with internal standard and underwent the same sample preparation as the test samples. Bile acid quantitation was based on the peak area ratio of the targeted bile acids to Cholic acid‐d4. The bile acids concentrations of test samples were quantified using slopes, but not intercepts, of the calibration curves to deduct background signals.
\",\n \"Primary Mouse Liver Cell Isolation and Culture\",\n \"
Primary murine hepatocytes were isolated from the livers of male WT and Slc27a5\\n−/− mice aged 8–12 weeks according to a reported protocol [\\n##UREF##4##\\n60\\n##\\n] with some modification that includes the following steps: in situ pronase (P5147, Sigma‐Aldrich) /collagenase (V900893, Sigma‐Aldrich) perfusion of mouse liver, perfused livers were minced, filtered through 70 µm cell strainer (BS‐70‐XBS, Biosharp, Beijing, China), and centrifuged at 40 g for 5 min at 4 °C to separate hepatocytes. Hepatocytes were plated in Dulbecco's modified Eagle's medium with 1 g L−1 glucose (DMEM‐low glucose, SH30021.01, HyClone) supplemented with 5% FBS, 15 mmol L−1 HEPES (H1095, Solarbio, Beijing, China), and 1% penicillin‐streptomycin. Cells were maintained overnight in serum‐free DMEM containing 1 g L−1 glucose. After 48 h of cell seeding in coated plates, medium was collected and concentrated by a speed‐vacuum to measure bile acids by LC‐MS.
\",\n \"
HSCs were isolated according to a previously published method,[\\n##REF##25612230##\\n61\\n##\\n] and the supernatant was further centrifuged at 580 g for 10 min at 4 °C, resuspended in density gradient‐based Nycodenz (1002424‐1, Alere Technologios AS), and centrifuged at 1400 g for 17 min at 4 °C. HSCs were collected from the interface and cultured in DMEM with 10% FBS and 1% penicillin‐streptomycin.
\",\n \"
The 48 h culture supernatant from primary hepatocytes of WT or Slc27a5\\n\\n−/−\\n mice was collected and centrifuged at 2000 g for 5 min to prepare it for the conditional culture. The supernatant was collected and labelled as conditioned medium. Twenty‐four hours post isolation, the medium of the primary HSCs was replaced by the conditioned medium from WT or Slc27a5\\n\\n−/−\\n mice primary hepatocytes and incubated for another 48 h. For TGFβ1 treatment, the primary HSCs after 24 h isolation were treated with and without TGFβ1 (4 ng mL−1, Novoprotein, Beijing, China) for another 48 h. For CA treatment, the primary HSCs after 24 h isolation were treated with CA (50 µм) for another 48 h.
\",\n \"Western Blotting\",\n \"
Proteins were extracted from cells or liver tissues in the lysis buffer (Beyotime, Shanghai, China) consisting of protease inhibitor cocktail (1:100, TargetMol, MA, USA). The concentration of proteins was determined using BCA Protein Assay kit (Beyotime). The extracted proteins were separated by 10% SDS/PAGE and electro‐transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking in 5% milk for 1 h, the membranes were probed with primary antibodies against SLC27A5 (1:1000, NBP2‐37412, Novus Biologicals, CO, USA), α‐SMA (1:1000, ER1003, Huabio, Hangzhou, China), COL1A1 (1:1000, WL0088, Wanleibio, Shenyang, China), COL3A1 (1:1000, 22734‐1‐AP, Proteintech, IL, USA), RUNX2 (1:1000, 20700‐1‐AP, Proteintech, IL, USA), EGR3 (1:500, sc‐390967, Santa Cruz Biotechnology, USA), β‐actin (1:3000, BL005B, Biosharp), or GAPDH (1:3000, AG019‐1, Beyotime, Shanghai, China) at 4 °C overnight. Membranes were then incubated with horseradish peroxidase‐conjugated secondary antibody (Abcam, Cambridge, UK). The staining was visualized using ClarityTM Western ECL Substrate (Bio‐Rad, CA, USA).
\",\n \"Liver histological and Immunohistological (IHC) Staining\",\n \"
Liver specimens were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 µm sections. Then, the specimens were deparaffinized, hydrated and stained by standard methods. To examine hepatic morphology and assess liver fibrosis, H&E and Sirius Red staining were performed. Sections were immune‐stained for SLC27A5 (1:500, NBP2‐37412, Novus Biologicals, CO, USA), α‐SMA (1:300, 19245T, CST, MA, USA), F4/80 (1:300, 70076T, CST), or RUNX2 (1:300, 20700‐1‐AP, Proteintech, IL, USA) overnight at 4 °C. Sections were then incubated with a secondary anti‐rabbit or anti‐mouse IgG (ZSGB‐BIO, Beijing, China) and stained using 3,3′‐diaminobenzidine (ZSGB‐BIO). Stained slides were scanned with a Pannoramic Scan 250 Flash and images were acquired using Pannoramic Viewer 1.15.2 (3DHistech, Budapest, Hungary). Immunostaining and Sirius Red staining were quantified by threshold analysis using the NIH ImageJ software. The immunohistochemical staining of SLC27A5 and RUNX2 was semi‐quantitatively analyzed using the immunoreactive scoring system.[\\n##REF##34921145##\\n62\\n##\\n]\\n
The HYP levels of fresh liver samples of mice were quantified. Concentration was calculated by a standard curve using the HYP Content Assay Kit (BC0255, Solarbio, China) according to the manufacturer protocol.
\",\n \"Immunofluorescence\",\n \"
HSCs were seeded and treated with CA (50 µм) in slide chambers. After being fixed in 4% paraformaldehyde, cells were permeabilized in 0.5% Triton X‐100 for 10 min. After being blocked in 5% goat serum for 1 h, slides were incubated with primary antibody against α‐SMA (1:300, 19245T, CST) overnight at 4 °C. The slides were then washed 5 times in PBS and incubated with goat anti‐rabbit conjugated with Alexa Fluor 488 or 552 secondary antibody (1:100, ZF‐0311, ZSGB‐bio) for 1 h at 37 °C. Nucleus was stained using 1 µg mL−1 4′,6‐diamidino‐2‐phenylindole (DAPI,1:2000, Roche, Basel, Switzerland). Stained sections were observed by a laser‐scanning confocal microscope (Leica TCS SP8, Solms, Germany).
Total RNA of tissue or cells was isolated with Trizol reagent (Invitrogen, Rockville, MD). Reverse transcription (RT) reactions were performed using PrimeScript RT Reagent Kit (RR047A, TaKaRa, TKY, Japan). Real‐time PCR was carried out using Bio‐Rad CFX96 machine (Bio‐Rad, Hercules, CA, USA). The level of GAPDH or β‐actin RNA expression was used to normalize the data. The sequences of qRT‐PCR primers were listed in Table ##SUPPL##0##S2## (Supporting Information).
\",\n \"Construction of Plasmids\",\n \"
The expression vector of human RUNX2 was inserted into the Sal I and Xba I sites of the shuttle vector pAdTrack‐TO4 (from Dr T‐C He, University of Chicago, USA). The 5′‐flanking region (from −2023 to +366 nt) of SLC27A5 gene was inserted into the Hind III and Xho I sites of the pGL3‐Basic vector (Promega, Madison, WI, USA), named pGL3‐P1. To construct another length of luciferase reporter plasmid of SLC27A5, the region from −1001 to +182 nt of SLC27A5 gene was inserted into the pGL3‐Basic vector, named pGL3‐P2. Oligonucleotide sequences were listed in Table ##SUPPL##0##S2## (Supporting Information).
The small double‐strand hairpin shRNA expressing constructs (shRUNX2, shEGR3, and shEgr3) were designed and annealed into the Hpa I and Xho I sites of pLL3.7 lentivirus vector (kindly provided by Prof. Bing Sun, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China). A negative control construct (shCon) was also generated. The lentiviral supernatants were produced in HEK293T cells as previously described.[\\n##REF##34650217##\\n63\\n##\\n] Oligonucleotide sequences were listed in Table ##SUPPL##0##S2## (Supporting Information).
SLC27A5‐knockout cells were constructed by the CRISPR‐Cas9 system provided by Prof. Ding Xue (Tsinghua University, Beijing, China), as previously described.[\\n##REF##31367013##\\n32\\n##\\n] The Knockout of SLC27A5 in MIHA cells were validated by immunoblotting. The sgRNA target sequences were listed in Table ##SUPPL##0##S2## (Supporting Information).
\",\n \"Luciferase Reporter Assay\",\n \"
MIHA cells were transfected in twelve‐well plates containing 4 µL of Lipofectamine 8000, 0.5 µg of pADTrack‐TO4 control or pADTrack‐RUNX2 overexpression vectors, 0.5 µg of each luciferase reporter plasmids pGL3‐basic (Promega, Madison, WI, USA), pGL3‐P1 (from −2023 to +366 nt of the promoter region of the SLC27A5 gene), or pGL3‐P2 (from −1001 to +182 nt of the promoter region of the SLC27A5 gene), and 10 ng of pRL‐TK‐Renilla (as transfection control) for 48 h. Cells were harvested and assayed for luciferase activity using the Dual Luciferase Assay Kit (Promega, Madison, WI, USA). All experiments were performed at least three times and expressed as mean ± SEM.
MIHA cells of 6 × 106 were cross‐linked using 1% paraformaldehyde for 8 min at 37 °C. Cell lysates were sonicated at 30% power for 10 cycles (15 seconds ON and 15 seconds OFF). Supernatants were separated and incubated with anti‐RUNX2 (20700‐1‐AP, Proteintech, IL, USA), anti‐EGR3 (sc‐390967, Santa Cruz Biotechnology, USA), or control IgG overnight at 4 °C. Chromatin‐antibody complexes were collected by protein A/G agarose beads (Millipore), washed and then eluted. Real‐time PCR was used to analyze the RUNX2‐binding DNA fragments from ChIP assays. The ChIP and ChIP‐qPCR assays were performed as previously described.[\\n##REF##34650217##\\n63\\n##\\n] Primers were listed in Table ##SUPPL##0##S2## (Supporting Information).
\",\n \"RNA‐Seq Analysis\",\n \"
Total RNA was extracted in LX‐2 cells treated with CA (50 µм) or vehicle for 48 h using Trizol reagent and subjected to library preparation. RNA‐seq was performed by Majorbio Bio‐pharm Technology Co.,Ltd (Shanghai, China). Differential expression genes (DEGs) were analyzed using the DESeq2. Heatmap of DEGs was performed using the “ggplot2” packages in R (v4.2.1, The R Project for Statistical Computing, Vienna, Austria).
\",\n \"Online Database Analysis\",\n \"
UCSC (https://genome.ucsc.edu/) was used to predict transcriptional factors (TFs) that could affect SLC27A5.[\\n##REF##36420891##\\n64\\n##\\n] JASPAR (http://jaspar.genereg.net/) was used to predict the potential binding sites of TFs on SLC27A5 promoter.[\\n##REF##34850907##\\n21\\n##\\n]\\n
\",\n \"Gene Expression Omnibus Database Mining\",\n \"
Four data sets from GEO database (https://www.ncbi.nlm.nih.gov/geo/) were analyzed with GEO2R to profile gene expression between different samples, such as mild fibrosis versus advanced fibrosis in NAFLD patients (GSE31803), control versus NASH patients (GSE48452), normal versus cirrhosis patients (GSE25097), and different stage of fibrosis in HBV infected patients (GSE84044).
\",\n \"Statistical Analysis\",\n \"
All data were presented as the means ± SEM. Tests used to examine the differences between groups include Student's t test, one‐way ANOVA and with the Tukey's post hoc test, and two‐way ANOVA with Tukey's multiple comparisons test. Pearson correlation coefficient (r) was used to test the linear correlation. P‐values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were conducted using GraphPad Prism 8.0 software (La Jolla, CA, USA).
\",\n \"Ethics Approval Statement\",\n \"
This study was approved by the Research Ethics Committee of Chongqing Medical University (approval number: 2022054). All animal experiments were performed under the guidelines of the institutional Animal Care and Use Committee at Chongqing Medical University.
\",\n \"Patient Consent Statement\",\n \"
Informed consents were obtained from all involved participants.
\",\n \"Conflict of Interest\",\n \"
The authors declare no conflict of interest.
\",\n \"Author Contributions\",\n \"
K.W., Y.L., J.X., and J.L. contributed equally to this work. N.T., C.C. and A.H. conceived and designed the study. K.W., Y.L., J.X., and J.L. performed most experiments and analyzed the data. K.W. provided suggestions and designed primer sequence. H.L. constructed plasmids. F.X., D.L., D.N., and X.T. assisted with mice experiments. C.C. provided technical assistance. K.W. and N.T. wrote the manuscript with all authors providing feedback. The order of the co‐first authors was determined on the basis of their relative contributions to the study.
The authors thank Dr. T.‐C He (University of Chicago, USA) for providing the pAdEasy system. The authors were grateful that Prof. Ding Xue (Tsinghua University, Beijing, China) supplied the CRISPR‐Cas9 system. This work was supported by the China National Natural Science Foundation (grant no. U20A20392, 82272975, 82072286, 82071671), the 111 Project (No. D20028), the Innovative and Entrepreneurial Team of Chongqing Talents Plan, Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023DBXM007), Senior Medical Talents Program of Chongqing for Young and Middle‐aged, the Kuanren talents program and the DengFeng program of the second affiliated hospital of Chongqing Medical University, and the Future Medical Youth Innovation Team of Chongqing Medical University (W0036, W0101).
","Data Availability Statement","
The data that support the findings of this study are available from the corresponding author upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
The authors thank Dr. T.‐C He (University of Chicago, USA) for providing the pAdEasy system. The authors were grateful that Prof. Ding Xue (Tsinghua University, Beijing, China) supplied the CRISPR‐Cas9 system. This work was supported by the China National Natural Science Foundation (grant no. U20A20392, 82272975, 82072286, 82071671), the 111 Project (No. D20028), the Innovative and Entrepreneurial Team of Chongqing Talents Plan, Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023DBXM007), Senior Medical Talents Program of Chongqing for Young and Middle‐aged, the Kuanren talents program and the DengFeng program of the second affiliated hospital of Chongqing Medical University, and the Future Medical Youth Innovation Team of Chongqing Medical University (W0036, W0101).
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available from the corresponding author upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
SLC27A5 expression is generally down‐regulated in the livers of cirrhosis patients and CCI4‐treated mice A) Box plots of relative SLC27A5 mRNA levels in GSE31803, GSE48452, GSE25097, and GSE84044 datasets. B) Representative liver histology of H&E, Sirius Red staining, SLC27A5, and α‐SMA IHC in consecutive sections of normal and cirrhotic human livers, and its statistical summary (n = 10 per group). Scale bar, 100 µm. C) The Runx2, Hnf4a, Rxra, Rest, and Nr2c2 expression levels were detected using qRT‐PCR in WT mice livers subjected to CCl4 treatment (n = 4 per group). D) Representative H&E and RUNX2 IHC in liver sections from healthy controls and patients with cirrhosis, and its statistical summary (n = 10 per group). Scale bar: 50 µm. E) Putative binding sites of RUNX2 (black spots) in the SLC27A5 gene promoter (−2023/+366). F) MIHA cells were co‐transfected with the SLC27A5 promoter luciferase reporter and expression plasmids for RUNX2, and the luciferase activity was monitored as described in the panel (n = 3 per group). G) ChIP‐qPCR analysis to determine the binding of RUNX2 protein to the SLC27A5 promoter in MIHA cells. Diagram of the SLC27A5 gene promoter (−1001/+182) depicting the location of the amplified region (−396/−280) (n = 3 per group). H) MIHA cells were transfected with vector or RUNX2‐overexpressing plasmid. The protein levels of RUNX2 and SLC27A5 were analyzed using Western blotting. I) MIHA cells were transfected with shControl or shRUNX2. The expression of RUNX2 and SLC27A5 was detected. J) Box plots of relative mRNA levels of RUNX2 in the GSE25097 dataset. K) Correlation of hepatic RUNX2 mRNA with SLC27A5 in patients with cirrhosis (n = 40 for GSE25097). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant. Data in (A) (left three panels), (B–D), (F–G), and (J) were analyzed using two‐tailed unpaired Student's t‐test. One‐way ANOVA analyzed data in (A) (right panel) with Tukey's post hoc test. Data in (K) was analyzed using Pearson correlation coefficient analysis.
","
SLC27A5 deficiency in mice develops liver fibrosis after 24 months of age A) Representative pictures of livers from 24‐month‐old WT and Slc27a5\n−/− mice. Scale bar, 2 mm. B) Serum ALT, AST, and ALP levels from 24‐month‐old WT and Slc27a5\n−/− mice (n = 5 per group). C) Representative images of H&E and Sirius Red staining from liver tissues of WT and Slc27a5\n−/− mice at 12, 18, and 24 months (n = 5 per group). Scale bar, 50 µm. D) Quantification of Sirius red staining is described in (C). E,F) Relative mRNA levels of profibrotic genes (E) and inflammatory genes (F) of liver tissues from 24‐month‐old WT and Slc27a5\n−/− mice (n = 5 per group). G) The hepatic mRNA levels of gene expression of bile acids synthesis in 24‐month‐old WT and Slc27a5\n−/− mice (n = 5 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant, two‐tailed Student's t test.
","
SLC27A5 deficiency promotes CCl4‐ and TAA‐induced liver fibrosis in mice A–D) Eight‐week‐old male WT and Slc27a5\n−/− mice were injected with CCl4 for six weeks (n = 5 per group). E–H) Eight‐week‐old male WT and Slc27a5\n−/− mice were injected with TAA for eight weeks (n = 5 per group). (A,E) The experimental approach of the animal model establishment. (B,F) Representative histology of H&E, Sirius Red, and IHC staining (α‐SMA, F4/80) from each group. Scale bar, 50 µm. (C) Quantification of Sirius red, F4/80, and α‐SMA IHC staining are described in (B). G) Quantification of Sirius red, F4/80, and α‐SMA IHC staining are described in (F). (D,H) Serum levels of ALT, AST, and ALP were measured. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t test.
","
SLC27A5 loss in hepatocytes promotes HSCs activation in vitro. A) Primary mouse HSCs were isolated from WT and Slc27a5\n−/− mice and culture‐activated for the indicated duration. Cell morphology as observed under light‐field microscopy. Scale bar: 50 µm. B–D) Primary HSCs isolated from WT and Slc27a5\n−/− mice were culture‐activated for 72 h (n = 3 per group). mRNA expression of fibrogenic genes (B), protein expression of α‐SMA, COL1A1, and COL3A1 (C), and immunofluorescence of α‐SMA (D) are displayed. Scale bar, 25 µm. DAPI, 4′,6‐diamidino‐2‐phenylindole. E) Schematic of co‐culture experiments. F–H) Primary HSCs from WT mice were incubated with the supernatant from WT or Slc27a5\n−/− PMHs for 48 h. mRNA expression of fibrogenic genes (F), protein expression of α‐SMA, COL1A1, and COL3A1 (G), and immunofluorescence of α‐SMA (H) are displayed. Scale bar, 25 µm. I) Schematic flow chart of co‐culture models. (J‐K) LX‐2 cells were co‐cultured with parental and SLC27A5‐KO MIHA cells for 48 h. Protein expression of α‐SMA, COL1A1, and COL3A1 J), and immunofluorescence of α‐SMA K) are displayed. Scale bar, 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t test.
","
Elevation of unconjugated CA promotes HSCs activation. A) Serum CA and DCA levels in Slc27a5\n−/− and WT mice at 24 months of age (n = 5 per group). B) Liver CA and DCA levels in Slc27a5\n−/− and WT mice at 24 months of age (n = 5 per group). C) LX‐2 cells were treated with CA at 25, 50, and 100 µм for 48 h. The expression of α‐SMA was measured using immunostaining. D–F) WT mice HSCs were treated with CA at 50 µм for 48 h. mRNA expression of fibrogenic genes (D), protein expression of α‐SMA, COL1A1, and COL3A1 (E), and α‐SMA immunostaining (F) are displayed. G) The serum levels of CA and DCA in healthy individuals and patients with cirrhosis (n = 18 per group). H) CA levels were measured in media from parental and SLC27A5‐KO MIHA cells (n = 3 per group). I) CA levels were measured in PMHs supernatant from WT and Slc27a5\n−/− mice (n = 3 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t test.
","
CA‐triggered activation of HSC is dependent on EGR3. A) Expression profile of regulatory genes of fibrosis in LX‐2 cells treated with 50 µм CA or vehicle controls based on RNA‐seq data (n = 3 per group). B) qRT‐PCR was performed to validate the expression of fibrosis‐regulated genes in vehicle‐ and CA‐treated LX‐2 cells (n = 3 per group). C–E) LX‐2 cells were transfected with Control (shCon) or shEGR3 plasmid in the presence of 50 µм CA for 48 h. Expression of the fibrotic genes was analyzed using qRT‐PCR (C), Western blotting (D), or immunofluorescence (E). Scale bar, 25 µm. F–H) Primary mouse HSCs were isolated from WT mice and transfected with Control (shCon) or shEgr3 plasmid in 50 µм CA for 48 h. Expression of the fibrotic genes was analyzed using qRT‐PCR (F), Western blotting (G), or immunofluorescence (H). Scale bar, 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant. Two‐tailed unpaired Student's t test was used to analyze data in (B). Data in (C) and (F) were analyzed using one‐way ANOVA with Tukey's post hoc test.
","
Overexpression of SLC27A5 or inhibition of intestinal bile acid absorption ameliorates CCl4‐induced liver fibrosis A–H) Eight‐week‐old male WT and Slc27a5\n\n−/−\n (KO) mice were subjected to CCl4 treatment and injected with AAV‐Control (AAV‐Con) or AAV‐Slc27a5 (n = 5 per group). I–P) Eight‐week‐old male WT and Slc27a5\n\n−/−\n (KO) mice were subjected to CCl4 model and treated with vehicle or A4250 by daily gavages as outlined in (I) (n = 5 per group). (A,I) The experimental approach of the animal model establishment. (B,J) Representative liver histology of H&E, Sirius Red, and α‐SMA IHC staining. Scale Bar, 50 µm. (C,D,K,L) Quantification of Sirius red (C,K) and α‐SMA IHC (D,L) staining described in (B,J). (E,M) Hepatic hydroxyproline content was measured. (F,N) Serum levels of ALT, AST, and ALP were measured. (G,H,O,P) CA levels in serum (G,O) and liver (H,P). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant, one‐way ANOVA with Tukey's post hoc test.
","
Underlying mechanisms of SLC27A5 deficiency promotes HSCs activation and fibrosis via CA. SLC27A5 was downregulated in liver fibrosis tissues via RUNX2. The loss of SLC27A5 in mice resulted in accumulating unconjugated CA in hepatocytes and subsequently activated HSCs via EGR3. Re‐expression of SLC27A5 or inhibition of CA accumulation may represent a potential strategy for liver fibrosis treatment. SLC27A5, solute carrier family 27 member 5; RUNX2, RUNX family transcription factor 2; CA, cholic acid; TCA, taurocholic acid; BSH, bile salt hydrolases; ASBT, apical sodium‐dependent bile acid transporter; EGR3, early growth response protein 3; HSC, hepatic stellate cell.
"],"string":"[\n \"
SLC27A5 expression is generally down‐regulated in the livers of cirrhosis patients and CCI4‐treated mice A) Box plots of relative SLC27A5 mRNA levels in GSE31803, GSE48452, GSE25097, and GSE84044 datasets. B) Representative liver histology of H&E, Sirius Red staining, SLC27A5, and α‐SMA IHC in consecutive sections of normal and cirrhotic human livers, and its statistical summary (n = 10 per group). Scale bar, 100 µm. C) The Runx2, Hnf4a, Rxra, Rest, and Nr2c2 expression levels were detected using qRT‐PCR in WT mice livers subjected to CCl4 treatment (n = 4 per group). D) Representative H&E and RUNX2 IHC in liver sections from healthy controls and patients with cirrhosis, and its statistical summary (n = 10 per group). Scale bar: 50 µm. E) Putative binding sites of RUNX2 (black spots) in the SLC27A5 gene promoter (−2023/+366). F) MIHA cells were co‐transfected with the SLC27A5 promoter luciferase reporter and expression plasmids for RUNX2, and the luciferase activity was monitored as described in the panel (n = 3 per group). G) ChIP‐qPCR analysis to determine the binding of RUNX2 protein to the SLC27A5 promoter in MIHA cells. Diagram of the SLC27A5 gene promoter (−1001/+182) depicting the location of the amplified region (−396/−280) (n = 3 per group). H) MIHA cells were transfected with vector or RUNX2‐overexpressing plasmid. The protein levels of RUNX2 and SLC27A5 were analyzed using Western blotting. I) MIHA cells were transfected with shControl or shRUNX2. The expression of RUNX2 and SLC27A5 was detected. J) Box plots of relative mRNA levels of RUNX2 in the GSE25097 dataset. K) Correlation of hepatic RUNX2 mRNA with SLC27A5 in patients with cirrhosis (n = 40 for GSE25097). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant. Data in (A) (left three panels), (B–D), (F–G), and (J) were analyzed using two‐tailed unpaired Student's t‐test. One‐way ANOVA analyzed data in (A) (right panel) with Tukey's post hoc test. Data in (K) was analyzed using Pearson correlation coefficient analysis.
\",\n \"
SLC27A5 deficiency in mice develops liver fibrosis after 24 months of age A) Representative pictures of livers from 24‐month‐old WT and Slc27a5\\n−/− mice. Scale bar, 2 mm. B) Serum ALT, AST, and ALP levels from 24‐month‐old WT and Slc27a5\\n−/− mice (n = 5 per group). C) Representative images of H&E and Sirius Red staining from liver tissues of WT and Slc27a5\\n−/− mice at 12, 18, and 24 months (n = 5 per group). Scale bar, 50 µm. D) Quantification of Sirius red staining is described in (C). E,F) Relative mRNA levels of profibrotic genes (E) and inflammatory genes (F) of liver tissues from 24‐month‐old WT and Slc27a5\\n−/− mice (n = 5 per group). G) The hepatic mRNA levels of gene expression of bile acids synthesis in 24‐month‐old WT and Slc27a5\\n−/− mice (n = 5 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant, two‐tailed Student's t test.
\",\n \"
SLC27A5 deficiency promotes CCl4‐ and TAA‐induced liver fibrosis in mice A–D) Eight‐week‐old male WT and Slc27a5\\n−/− mice were injected with CCl4 for six weeks (n = 5 per group). E–H) Eight‐week‐old male WT and Slc27a5\\n−/− mice were injected with TAA for eight weeks (n = 5 per group). (A,E) The experimental approach of the animal model establishment. (B,F) Representative histology of H&E, Sirius Red, and IHC staining (α‐SMA, F4/80) from each group. Scale bar, 50 µm. (C) Quantification of Sirius red, F4/80, and α‐SMA IHC staining are described in (B). G) Quantification of Sirius red, F4/80, and α‐SMA IHC staining are described in (F). (D,H) Serum levels of ALT, AST, and ALP were measured. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t test.
\",\n \"
SLC27A5 loss in hepatocytes promotes HSCs activation in vitro. A) Primary mouse HSCs were isolated from WT and Slc27a5\\n−/− mice and culture‐activated for the indicated duration. Cell morphology as observed under light‐field microscopy. Scale bar: 50 µm. B–D) Primary HSCs isolated from WT and Slc27a5\\n−/− mice were culture‐activated for 72 h (n = 3 per group). mRNA expression of fibrogenic genes (B), protein expression of α‐SMA, COL1A1, and COL3A1 (C), and immunofluorescence of α‐SMA (D) are displayed. Scale bar, 25 µm. DAPI, 4′,6‐diamidino‐2‐phenylindole. E) Schematic of co‐culture experiments. F–H) Primary HSCs from WT mice were incubated with the supernatant from WT or Slc27a5\\n−/− PMHs for 48 h. mRNA expression of fibrogenic genes (F), protein expression of α‐SMA, COL1A1, and COL3A1 (G), and immunofluorescence of α‐SMA (H) are displayed. Scale bar, 25 µm. I) Schematic flow chart of co‐culture models. (J‐K) LX‐2 cells were co‐cultured with parental and SLC27A5‐KO MIHA cells for 48 h. Protein expression of α‐SMA, COL1A1, and COL3A1 J), and immunofluorescence of α‐SMA K) are displayed. Scale bar, 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t test.
\",\n \"
Elevation of unconjugated CA promotes HSCs activation. A) Serum CA and DCA levels in Slc27a5\\n−/− and WT mice at 24 months of age (n = 5 per group). B) Liver CA and DCA levels in Slc27a5\\n−/− and WT mice at 24 months of age (n = 5 per group). C) LX‐2 cells were treated with CA at 25, 50, and 100 µм for 48 h. The expression of α‐SMA was measured using immunostaining. D–F) WT mice HSCs were treated with CA at 50 µм for 48 h. mRNA expression of fibrogenic genes (D), protein expression of α‐SMA, COL1A1, and COL3A1 (E), and α‐SMA immunostaining (F) are displayed. G) The serum levels of CA and DCA in healthy individuals and patients with cirrhosis (n = 18 per group). H) CA levels were measured in media from parental and SLC27A5‐KO MIHA cells (n = 3 per group). I) CA levels were measured in PMHs supernatant from WT and Slc27a5\\n−/− mice (n = 3 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed Student's t test.
\",\n \"
CA‐triggered activation of HSC is dependent on EGR3. A) Expression profile of regulatory genes of fibrosis in LX‐2 cells treated with 50 µм CA or vehicle controls based on RNA‐seq data (n = 3 per group). B) qRT‐PCR was performed to validate the expression of fibrosis‐regulated genes in vehicle‐ and CA‐treated LX‐2 cells (n = 3 per group). C–E) LX‐2 cells were transfected with Control (shCon) or shEGR3 plasmid in the presence of 50 µм CA for 48 h. Expression of the fibrotic genes was analyzed using qRT‐PCR (C), Western blotting (D), or immunofluorescence (E). Scale bar, 25 µm. F–H) Primary mouse HSCs were isolated from WT mice and transfected with Control (shCon) or shEgr3 plasmid in 50 µм CA for 48 h. Expression of the fibrotic genes was analyzed using qRT‐PCR (F), Western blotting (G), or immunofluorescence (H). Scale bar, 25 µm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant. Two‐tailed unpaired Student's t test was used to analyze data in (B). Data in (C) and (F) were analyzed using one‐way ANOVA with Tukey's post hoc test.
\",\n \"
Overexpression of SLC27A5 or inhibition of intestinal bile acid absorption ameliorates CCl4‐induced liver fibrosis A–H) Eight‐week‐old male WT and Slc27a5\\n\\n−/−\\n (KO) mice were subjected to CCl4 treatment and injected with AAV‐Control (AAV‐Con) or AAV‐Slc27a5 (n = 5 per group). I–P) Eight‐week‐old male WT and Slc27a5\\n\\n−/−\\n (KO) mice were subjected to CCl4 model and treated with vehicle or A4250 by daily gavages as outlined in (I) (n = 5 per group). (A,I) The experimental approach of the animal model establishment. (B,J) Representative liver histology of H&E, Sirius Red, and α‐SMA IHC staining. Scale Bar, 50 µm. (C,D,K,L) Quantification of Sirius red (C,K) and α‐SMA IHC (D,L) staining described in (B,J). (E,M) Hepatic hydroxyproline content was measured. (F,N) Serum levels of ALT, AST, and ALP were measured. (G,H,O,P) CA levels in serum (G,O) and liver (H,P). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns., not significant, one‐way ANOVA with Tukey's post hoc test.
\",\n \"
Underlying mechanisms of SLC27A5 deficiency promotes HSCs activation and fibrosis via CA. SLC27A5 was downregulated in liver fibrosis tissues via RUNX2. The loss of SLC27A5 in mice resulted in accumulating unconjugated CA in hepatocytes and subsequently activated HSCs via EGR3. Re‐expression of SLC27A5 or inhibition of CA accumulation may represent a potential strategy for liver fibrosis treatment. SLC27A5, solute carrier family 27 member 5; RUNX2, RUNX family transcription factor 2; CA, cholic acid; TCA, taurocholic acid; BSH, bile salt hydrolases; ASBT, apical sodium‐dependent bile acid transporter; EGR3, early growth response protein 3; HSC, hepatic stellate cell.
Epilepsy, a common neurological disorder,[\n##UREF##0##\n1\n##\n] is caused by the failure of neuronal inhibition, which disperses epileptic responses over broad brain regions.[\n##REF##30317911##\n2\n##\n] The primary features of antiepileptic therapies, such as antiepileptic drugs (AEDs),[\n##REF##16581409##\n3\n##, ##REF##31348240##\n4\n##\n] vagus nerve stimulation (VNS),[\n##REF##29670050##\n5\n##, ##REF##31085961##\n6\n##\n] deep brain stimulation (DBS),[\n##REF##29670050##\n5\n##, ##REF##31697763##\n7\n##\n] transcranial magnetic stimulation (TMS), and transcranial direct/alternating current stimulation (tD/ACS),[\n##REF##29670050##\n5\n##, ##REF##19332316##\n8\n##, ##UREF##1##\n9\n##, ##REF##35989912##\n10\n##\n] involve alleviation of cortical hyper‐excitabilities by either enhancing γ‐Aminobutyric acid (GABA)ergic neurons or blocking excitatory amino acid neurotransmission. Optimal stimulation sequences for these conventional therapeutic approaches have been empirically investigated because their performance in seizure suppression relies on stimulation parameters, such as stimulus strength, length, and intervals. In addition, certain combinations of these parameters in AEDs,[\n##REF##30875668##\n11\n##, ##REF##20067500##\n12\n##\n] VNS,[\n##REF##19427651##\n13\n##\n] DBS,[\n##UREF##2##\n14\n##\n] and TMS[\n##REF##7106219##\n15\n##, ##REF##28359831##\n16\n##, ##REF##26642974##\n17\n##\n] can exacerbate epileptic symptoms. Meanwhile, transcranial‐focused ultrasound (tFUS) is a recent therapeutic approach that has gained significant attention for its antiepileptic effects in drug‐induced small animal epilepsy models,[\n##REF##21144673##\n18\n##, ##REF##21375781##\n19\n##, ##REF##31575487##\n20\n##, ##UREF##3##\n21\n##, ##REF##30166265##\n22\n##, ##REF##31002886##\n23\n##, ##UREF##4##\n24\n##, ##REF##31109842##\n25\n##\n] and there are expectations that tFUS may be more efficient in antiepileptic therapeutic approach owing to its ability to stimulate deep brain structures within millimeter‐scale lesions. Despite the successful induction of suppressive effects and several efforts to optimize the stimulation parameters for tFUS, its efficacy in prior studies remains vague. In addition, some studies have indicated that the reliability of the neuromodulation effects may not be sufficient for clinical use because the sonication time for tFUS is too short to cause persistent modulation effects.[\n##UREF##3##\n21\n##, ##REF##31002886##\n23\n##, ##UREF##4##\n24\n##\n] Furthermore, similar to other stimulation approaches, bidirectional neuromodulation can be induced by tFUS. Care should be taken to avoid side effects because ultrasound stimulation also works on inhibitory pathways by activating GABAergic neurons or blocking ion channels, like other conventional stimulant methods,[\n##UREF##3##\n21\n##, ##REF##30166265##\n22\n##, ##REF##31002886##\n23\n##, ##UREF##4##\n24\n##\n] and may exacerbate epileptic symptoms.
","
In the present study, repetitive tFUS (rtFUS) having the longer repetition ratio of tens of seconds had been explored to empirically demonstrate significant suppressive effects on epileptiform activities in a pentylenetetrazole (PTZ)‐induced acute generalized epilepsy model.[\n##UREF##5##\n26\n##\n] As repetitive TMS (rTMS) exerts longer neuromodulatory effects than single‐pulse TMS in the treatment of major depressive disorders,[\n##REF##35396023##\n27\n##, ##UREF##6##\n28\n##, ##REF##32973560##\n29\n##\n] similar repetitive stimulus sequences in ultrasound brain stimulation have recently been reported to have long‐lasting inhibitory effects on the anti‐saccade motion of non‐human primates.[\n##REF##32973560##\n29\n##, ##REF##30747105##\n30\n##\n] Instead of a single stimulation burst, burst trains can be more effective in suppressing epileptiform activities, eventually resulting in symptoms that are comparable to those in the baseline. Therefore, the prolonged sustainability of the antiepileptic effects of rtFUS can be compared with that of single‐burst tFUS. Furthermore, paradoxical proconvulsive effects and even a transition from anticonvulsive to proconvulsive effects may be discovered by navigating changes in rtFUS parameters, such as the interval between stimulus bursts, burst duration, and strength because these parameters are known to affect GABA secretion and induce bidirectional epileptiform activities in other stimulation methods (such as tDCS and TMS[\n##REF##30317911##\n2\n##, ##UREF##2##\n14\n##, ##UREF##7##\n31\n##, ##REF##23882212##\n32\n##, ##UREF##8##\n33\n##, ##UREF##9##\n34\n##\n]). The exploratory studies for comparison in epileptic activities between different acoustic parameters of rtFUS (e.g., stronger vs weaker bursts, longer vs shorter burst length, or burst interval) will help in identifying the optimal transmission conditions to perform antiepileptic effects.
","
Furthermore, we assessed the sustainability and bidirectional modulations in epileptic activities with rtFUS using electroencephalography (EEG) and optical measurements of cerebral blood volume (CBV) changes in the prefrontal cortex, where epileptiform activities in the hippocampus are reflected through the limbic system.[\n##REF##24445927##\n35\n##\n] Immunohistochemistry (IHC) was performed to visualize the excitatory and inhibitory activation of neurons in the hippocampus. In addition, changes in the amounts of excitatory (glutamate) and inhibitory (GABA) neurotransmitters during bidirectional modulation of epileptiform activities were assessed through multitemporal harvesting of interstitial fluid (ISF) from stimulated locations via microdialysis.
"],"string":"[\n \"Introduction\",\n \"
Epilepsy, a common neurological disorder,[\\n##UREF##0##\\n1\\n##\\n] is caused by the failure of neuronal inhibition, which disperses epileptic responses over broad brain regions.[\\n##REF##30317911##\\n2\\n##\\n] The primary features of antiepileptic therapies, such as antiepileptic drugs (AEDs),[\\n##REF##16581409##\\n3\\n##, ##REF##31348240##\\n4\\n##\\n] vagus nerve stimulation (VNS),[\\n##REF##29670050##\\n5\\n##, ##REF##31085961##\\n6\\n##\\n] deep brain stimulation (DBS),[\\n##REF##29670050##\\n5\\n##, ##REF##31697763##\\n7\\n##\\n] transcranial magnetic stimulation (TMS), and transcranial direct/alternating current stimulation (tD/ACS),[\\n##REF##29670050##\\n5\\n##, ##REF##19332316##\\n8\\n##, ##UREF##1##\\n9\\n##, ##REF##35989912##\\n10\\n##\\n] involve alleviation of cortical hyper‐excitabilities by either enhancing γ‐Aminobutyric acid (GABA)ergic neurons or blocking excitatory amino acid neurotransmission. Optimal stimulation sequences for these conventional therapeutic approaches have been empirically investigated because their performance in seizure suppression relies on stimulation parameters, such as stimulus strength, length, and intervals. In addition, certain combinations of these parameters in AEDs,[\\n##REF##30875668##\\n11\\n##, ##REF##20067500##\\n12\\n##\\n] VNS,[\\n##REF##19427651##\\n13\\n##\\n] DBS,[\\n##UREF##2##\\n14\\n##\\n] and TMS[\\n##REF##7106219##\\n15\\n##, ##REF##28359831##\\n16\\n##, ##REF##26642974##\\n17\\n##\\n] can exacerbate epileptic symptoms. Meanwhile, transcranial‐focused ultrasound (tFUS) is a recent therapeutic approach that has gained significant attention for its antiepileptic effects in drug‐induced small animal epilepsy models,[\\n##REF##21144673##\\n18\\n##, ##REF##21375781##\\n19\\n##, ##REF##31575487##\\n20\\n##, ##UREF##3##\\n21\\n##, ##REF##30166265##\\n22\\n##, ##REF##31002886##\\n23\\n##, ##UREF##4##\\n24\\n##, ##REF##31109842##\\n25\\n##\\n] and there are expectations that tFUS may be more efficient in antiepileptic therapeutic approach owing to its ability to stimulate deep brain structures within millimeter‐scale lesions. Despite the successful induction of suppressive effects and several efforts to optimize the stimulation parameters for tFUS, its efficacy in prior studies remains vague. In addition, some studies have indicated that the reliability of the neuromodulation effects may not be sufficient for clinical use because the sonication time for tFUS is too short to cause persistent modulation effects.[\\n##UREF##3##\\n21\\n##, ##REF##31002886##\\n23\\n##, ##UREF##4##\\n24\\n##\\n] Furthermore, similar to other stimulation approaches, bidirectional neuromodulation can be induced by tFUS. Care should be taken to avoid side effects because ultrasound stimulation also works on inhibitory pathways by activating GABAergic neurons or blocking ion channels, like other conventional stimulant methods,[\\n##UREF##3##\\n21\\n##, ##REF##30166265##\\n22\\n##, ##REF##31002886##\\n23\\n##, ##UREF##4##\\n24\\n##\\n] and may exacerbate epileptic symptoms.
\",\n \"
In the present study, repetitive tFUS (rtFUS) having the longer repetition ratio of tens of seconds had been explored to empirically demonstrate significant suppressive effects on epileptiform activities in a pentylenetetrazole (PTZ)‐induced acute generalized epilepsy model.[\\n##UREF##5##\\n26\\n##\\n] As repetitive TMS (rTMS) exerts longer neuromodulatory effects than single‐pulse TMS in the treatment of major depressive disorders,[\\n##REF##35396023##\\n27\\n##, ##UREF##6##\\n28\\n##, ##REF##32973560##\\n29\\n##\\n] similar repetitive stimulus sequences in ultrasound brain stimulation have recently been reported to have long‐lasting inhibitory effects on the anti‐saccade motion of non‐human primates.[\\n##REF##32973560##\\n29\\n##, ##REF##30747105##\\n30\\n##\\n] Instead of a single stimulation burst, burst trains can be more effective in suppressing epileptiform activities, eventually resulting in symptoms that are comparable to those in the baseline. Therefore, the prolonged sustainability of the antiepileptic effects of rtFUS can be compared with that of single‐burst tFUS. Furthermore, paradoxical proconvulsive effects and even a transition from anticonvulsive to proconvulsive effects may be discovered by navigating changes in rtFUS parameters, such as the interval between stimulus bursts, burst duration, and strength because these parameters are known to affect GABA secretion and induce bidirectional epileptiform activities in other stimulation methods (such as tDCS and TMS[\\n##REF##30317911##\\n2\\n##, ##UREF##2##\\n14\\n##, ##UREF##7##\\n31\\n##, ##REF##23882212##\\n32\\n##, ##UREF##8##\\n33\\n##, ##UREF##9##\\n34\\n##\\n]). The exploratory studies for comparison in epileptic activities between different acoustic parameters of rtFUS (e.g., stronger vs weaker bursts, longer vs shorter burst length, or burst interval) will help in identifying the optimal transmission conditions to perform antiepileptic effects.
\",\n \"
Furthermore, we assessed the sustainability and bidirectional modulations in epileptic activities with rtFUS using electroencephalography (EEG) and optical measurements of cerebral blood volume (CBV) changes in the prefrontal cortex, where epileptiform activities in the hippocampus are reflected through the limbic system.[\\n##REF##24445927##\\n35\\n##\\n] Immunohistochemistry (IHC) was performed to visualize the excitatory and inhibitory activation of neurons in the hippocampus. In addition, changes in the amounts of excitatory (glutamate) and inhibitory (GABA) neurotransmitters during bidirectional modulation of epileptiform activities were assessed through multitemporal harvesting of interstitial fluid (ISF) from stimulated locations via microdialysis.
Ultrasound stimulation was applied using a custom‐made focused ultrasound transducer over the right hemisphere of the midbrain to target the anterior thalamic nucleus (ATN). EEG was performed before, during, and after sonication. IHC was performed on brain samples harvested from the model animals. Optical measurements of CBV changes and microdialysis for proteomic analysis of neurotransmitters were performed in separate trials to measure the responses to the stimulation. The detailed repetitive transcranial ultrasound stimulation transmission sequences used in the experiment are shown in Figure\n##FIG##0##\n1\n##. The detailed experimental configurations are described in the Experimental Section and Supporting Information figures (Figures ##SUPPL##0##S1## and ##SUPPL##0##S2##, Supporting Information).
","Repeated Transcranial Low‐Intensity Elongated Ultrasound Stimulations for Sustainable Full Suppression of Epileptiform Activities","
Changes in epileptiform activities were compared between the two stimulant sequences. The first sequence (TXH_0.25_30) comprised eleven repetitions of a stronger (1 MPa) and shorter (0.25 s) burst with a burst interval of 30 s. The second sequence (TXL_40_40) consisted of six repetitions of weaker (0.25 MPa) but longer (40 s) burst with a burst interval of 40 s (Figure ##FIG##0##1##). First, epileptiform activities were reliably induced in a PTZ‐control group that received PTZ, a drug used for acute seizure induction, without ultrasound stimulation. While sparsely located discharge peaks in the EEG signal were identified during the baseline period, the signal was transformed into ictal shapes that demonstrated repetitive high‐frequency discharge peaks in the PTZ‐control group (Figure\n##FIG##1##\n2A##). The relative number of epileptic spikes increased 2.6‐fold in the first 10 min after PTZ injection in the PTZ‐control group. The number then decreased for 7 min and saturated at 2.3 times the baseline (Figure ##FIG##1##2B##). Although the number decreased slightly from 10 to 17 min after the PTZ injection, this decrease was not statistically significant at 17 min after the PTZ injection compared to 10 min after the PTZ injection (p = 0.588; two‐tailed paired t‐test) where the number of epileptic spikes ratio still remained significantly different at 17 min after the PTZ injection compared to the baseline period (p = 0.014; two‐tailed paired t‐test). Following stimulation with TXH_0.25_30, the amplitude of the discharge pattern slightly reduced but gradually became comparable to that of the PTZ‐control group. During the period between 0 and 10 min post‐stimulation (10AS) and 20 and 30 min post‐stimulation (30AS), the number of epileptic spikes ratio in the TXH_0.25_30 group was not significantly different from that of the PTZ‐control (p = 0.524 and 0.724 at 10AS and 30AS, respectively; two‐tailed Mann–Whitney U test) (Figure ##FIG##1##2C##). Meanwhile, the discharging patterns following TXL_40_40 became similar to those of the baseline, and the suppressive effect lasted until the end of the EEG recording. TXL_40_40 administration resulted in the maintenance of the number of epileptic spikes ratio similar to that of the baseline, and a suppressive effect was still found at 10AS and 30AS (p = 0.030 and 0.030 at 10AS and 30AS compared with that of PTZ‐control, respectively; two‐tailed Mann–Whitney U test). In the delta band between 1 and 3 Hz, the spectral power with TXH_0.25_30 at 30AS was not significantly different from that of the PTZ control (p = 0.833; two‐tailed Mann‐Whitney U test). However, the relative spectral power followed by TXL_40_40 was 1.19 at 30AS, indicating that this level was close to the baseline. TXL_40_40 also demonstrated strong suppressive effects on the theta band between 4 and 7 Hz (p = 0.019 at 30AS compared with that of PTZ‐control; two‐tailed Mann–Whitney U test), whereas TXH_0.25_30 demonstrated mild effects (p = 0.093 at 30AS compared with that of PTZ‐control; two‐tailed Mann–Whitney U test) (Figure ##FIG##1##2D##). However, with a single transmission of an elongated (40 s), low pressure (0.25 MPa) burst, the number of epileptic spikes ratio became similar to that of the PTZ‐control group, although the suppressive effects were briefly observed for 5 min immediately after the stimulation (Figure ##FIG##1##2B,C##). In the CBV measurements, while the total hemoglobin amount (Δ[Hb]) barely changed in the normal control group (that did not receive PTZ or ultrasound stimulations), Δ[Hb] sharply increased in the PTZ‐control group after drug administration. The increase in Δ[Hb] was as low as that in the normal control group when TXL_40_40 was used (p = 0.699 at 20AS compared with that of normal control; two‐tailed Mann–Whitney U‐test), while the low Δ[Hb] level was maintained until the end of the recording. Following TXH_0.25_30, the Δ[Hb] level was similar to that of the PTZ control (p = 0.180 at 20AS; two‐tailed Mann–Whitney U test), although a gradual decay pattern was observed after the sonication was completed (Figure ##FIG##1##2E##).
","Efficacy of rtFUS for Epilepsy Treatment is Dependent on the Burst Length in the rtFUS Sequence","
To investigate the effects of burst length changes in rtFUS, the modulation effects were compared for the sequences with burst lengths of 10 s (TXL_10_50), 20 s (TXL_20_40), and 40 s (TXL_40_40). The burst was repeated six times at intervals of 50, 40, and 40 s for TXL_10_50, TXL_20_40, and TXL_40_40, respectively (Figure ##FIG##0##1##). For TXL_10_50, the frequency of the discharge peaks increased in the EEG waveform (Figure\n##FIG##2##\n3A##). The number of epileptic spikes with TXL_10_50 was statistically comparable with that in the PTZ‐control group at the post‐stimulation periods from 10AS to 30AS (p = 0.435, 0.354, and 0.435 at 10AS, 20AS, and 30AS, respectively; two‐tailed Mann–Whitney U test). As the stimulation length elongated from 10 to 40 s (TXL_10_50, TXL_20_40, TXL_40_40), the number of epileptic spikes decreased from 2.63, 1.94, 1.07 times the baseline, respectively, at 10AS while the difference in the number of epileptic spikes ratio between that of the PTZ‐control was only significant in the TXL_40_40 group (p = 0.435, 0.943 and 0.030 at 10AS for TXL_10_50, TXL_20_40, and TXL_40_40, respectively; two‐tailed Mann–Whitney U test) (Figure ##FIG##2##3B,C##). In the delta band, the spectral power of the EEG for the TXL_10_50 condition was comparable to that for the PTZ‐control group (p = 0.524 at 30AS; two‐tailed Mann–Whitney U test) while that of the EEG for TXL_40_40 condition was lower than that for the PTZ‐control group. (p = 0.045 at 30AS; two‐tailed Mann–Whitney U test) (Figure ##FIG##2##3D##). In the theta band, the spectral power of the EEG for the TXL_10_50 condition was comparable to that for the PTZ‐control group (p = 0.524 at 30AS; two‐tailed Mann–Whitney U test). However, the trend of reduction in the theta power was significant as the burst length was increased to 20 and 40 s (p = 0.171 and 0.019 at 30AS for TXL_20_40 and TXL_40_40, respectively, compared to PTZ‐control; two‐tailed Mann–Whitney U test) (Figure ##FIG##2##3D##). In the CBV curve, with TXL_10_50, Δ[Hb] was sharply increased by + 12.89% at 20AS compared to that in the baseline and was maintained at a higher level than that in the PTZ‐control group (p = 0.699 at 20AS; two‐tailed Mann‐Whitney U‐test), while the CBV with TXL_40_40 was similar to that of the normal control (p = 0.699 at 20AS; two‐tailed Mann–Whitney U‐test) (Figure ##FIG##2##3E##).
","Upregulation and Downregulation of Epileptiform Activities Selected by Adjusting the Interval Between Bursts in the rtFUS Sequence","
The epileptiform activity changes by TXL_40_40 were compared with that by TXL_40_20, which also had a burst length of 40 s; however, the interval between the bursts was reduced from 40 to 20 s (Figure ##FIG##0##1##). TXL_40_20 stimulation demonstrated rebound excitatory effects in the post‐stimulus period (Figure\n##FIG##3##\n4A##). TXL_40_20 generated a similar number of epileptic spikes as that in the PTZ‐control group (p = 0.943, 0.833, and 0.943 at 10AS, 20AS, and 30AS, respectively; two‐tailed Mann–Whitney U test). In contrast, TXL_40_40 stimulation demonstrated prominent suppressive effects both during and after stimulation (Figure ##FIG##3##4B,C##). The spectral density in the delta band was higher with TXL_40_20 at 30AS than that in the PTZ control. However, the difference was not significant owing to large deviations with TXL_40_20 (p = 0.833; two‐tailed Mann–Whitney U test) (Figure ##FIG##3##4D##). While the CBV response of TXL_40_20 was similar to that of TXL_40_40 during the stimulation period, the Δ[Hb] sharply increased after TXL_40_20, unlike the CBV responses after TXL_40_40, ultimately becoming higher than that of the PTZ control (Figure ##FIG##3##4E##).
","Transformation from Suppressive to Excitatory States Through a Hybrid Sequence","
As shown in previous studies, TXL_40_40 demonstrated strong suppressive effects. In contrast, the insignificant suppressive effects observed with TXH_0.25_30 – that showed suppressive effects with low statistical significance [19.91% less epileptic spikes ratio (p = 0.724), 16.61% less delta PSD (p = 0.833) and 38.79% less theta PSD (p = 0.093) than that of the PTZ‐control at 30AS]. Although the single use of TXL_40_40 maintained the suppressive effect for more than 30 min, the epileptiform activities were sharply transformed into the excitatory state with the use of TXH_0.25_30, followed by a 3 min resting time after TXL_40_40 (TX_HYBRID, Figure\n##FIG##4##\n5A##). The number of epileptic spikes ratio in the TX_HYBRID group was always comparable with the PTZ‐control group across all post‐stimulation periods at 10AS, 20AS, and 30AS (p = 0.943, 0.943, and 0.622, respectively; two‐tailed Mann–Whitney U test) (Figure ##FIG##4##5B,C##). The elevations of the power spectral densities in the delta and theta band after TX_HYBRID (30AS) were comparable to that of the PTZ‐control group (Figure ##FIG##4##5D##) (p = 1.000 and 0.622 at 30AS, respectively; two‐tailed Mann–Whitney U test). Regarding CBV changes, the Δ[Hb] was reduced after TXL_40_40 sonication, whereas TXH_0.25_30 increased the blood volume, approaching that of the PTZ control (Figure ##FIG##4##5E##).
","Immunohistochemistry Results","
\nFigure\n##FIG##5##\n6A## shows the immunohistochemical results of the samples harvested from sacrificed animals immediately after each experiment. While c‐Fos‐expressed cells were rarely found in the normal control group, c‐Fos‐labeled cells were abundant in the PTZ‐control group (p = 0.002; two‐tailed Mann–Whitney U test). The increase of the proportion of c‐Fos‐positive cells in the TXL_10_50 group was trending toward significance to that in the PTZ‐control group (p = 0.093; two‐tailed Mann–Whitney U test). As shown in the bar graph, 82.58% (standard error of the mean [SEM] = 1.83%) of neuronal cells expressed c‐Fos with TXL_10_50, and 77.14% (SEM = 2.12%) of cells expressed c‐Fos in the PTZ‐control group. TXL_20_40 (p = 0.240; two‐tailed Mann–Whitney U test), TXL_40_20 (p = 0.394; two‐tailed Mann–Whitney U test) and TXH_0.25_30 (p = 0.394; two‐tailed Mann–Whitney U test) demonstrated c‐Fos‐positive cell ratios comparable to those of the PTZ‐control group. Furthermore, TX_HYBRID presented an even higher value of ≈87.47% (SEM = 1.25%) than that of PTZ‐control (p = 0.004; two‐tailed Mann–Whitney U test). In contrast, the expression ratio was drastically reduced to 11.11% with TXL_40_40 compared with the PTZ‐control group (p = 0.002; two‐tailed Mann‐Whitney U test).
","
Although the normal and PTZ‐control cases barely exhibited glutamic acid decarboxylase 65‐kilodalton isoform (GAD65)‐positive cells, the sonicated conditions exhibited a greater number of GAD65‐expressed cells. The GAD65‐positive cell density with TXL_40_40 (mean = 15.88 cells mm−2; SEM = 1.53 cells mm−2) was ≈6.2‐fold higher than that in the PTZ‐control group (mean = 2.56 cells mm−2; SEM = 0.42 cells mm−2). Although sharing the same burst duration, TXL_40_20 exhibited a lower expression of GAD65‐positive cells (mean = 7.27 cells mm−2; SEM = 0.61 cells mm−2) than that of TXL_40_40 (mean = 15.88 cells mm−2; SEM = 1.53 cells mm−2), while both groups showed statistically significant difference compared to the PTZ‐control (p = 0.002 and 0.002, respectively; two‐tailed Mann–Whitney U test). The GAD65‐expressed cell densities for TXL_10_50 (mean = 5.60 cells mm−2; SEM = 0.71 cells mm−2) and TXL_40_20 (mean 7.27 cells mm−2; SEM = 0.61 cells mm−2) were lower than the cell density with TXL_40_40 (mean = 15.88 cells mm−2; SEM = 1.53 cells mm−2) and TX_HYBRID (mean = 14.89 cells mm−2; SEM = 0.81 cells mm−2).
","
While the ionized calcium‐binding adaptor molecule 1 (Iba1)‐expressed cell density in the normal control group was 39.30 cells mm−2 (SEM = 0.89 cells mm−2), the number increased to 55.62 cells mm−2 (SEM = 1.79 cells mm−2) in the PTZ‐control group. The density of activated cells with TXL_40_40 was 49.36 cells mm−2 (SEM = 0.59 cells mm−2), which was significantly lower than that of the PTZ‐control group (p = 0.009, two‐tailed Mann–Whitney U test). The Iba1‐expressed cell densities in TXH_0.25_30, TXL_10_50, TXL_20_40 and TXL_40_20 were 54.13 cells mm−2 (SEM = 1.68 cells mm−2), 60.50 cells mm−2 (SEM = 2.61 cells mm−2), 54.07 cells mm−2 (SEM = 1.58 cells mm−2) and 57.91 cells mm−2 (SEM = 2.95 cells mm−2), respectively. However, those values were not statistically different (p = 0.394, 0.240, 0.699, 0.589 for TXH_0.25_30, TXL_10_50, TXL_20_40 and TXL_40_20, respectively, two‐tailed Mann–Whitney U test) from the one with PTZ‐control. TX_HYBRID induced the highest Iba1‐expression level at 69.72 cells mm−2 (SEM = 3.51 cells mm−2) among all the experimental groups, and the increase was significant compared with PTZ‐control group (p = 0.009, two‐tailed Mann–Whitney U test).
","
In the glial fibrillary acidic protein (GFAP) responses indicating astrocytic reactivity, GFAP density increased to 51.76 cells mm−2 (SEM = 1.96 cells mm−2) in the PTZ‐control group, unlike the normal control group, which presented a density of 39.71 cells mm−2 (SEM = 1.23 cells mm−2) (p = 0.002; two‐tailed Mann–Whitney U test). Furthermore, the GFAP‐positive cell densities of TXL_10_50, TXL_20_40, and TXL_40_40 gradually decreased to 62.58 (SEM = 2.52), 52.59 (SEM = 0.82), and 47.30 cells mm−2 (SEM = 1.82 cells mm−2), respectively, while the GFAP‐positive cell densities with TXL_20_40 and TXL_40_40 were comparable with that with PTZ‐control (p = 0.004, 0.937 and 0.132, respectively, compared to that of PTZ‐control; two‐tailed Mann–Whitney U test). In contrast, TXL_40_20 demonstrated a higher density of 58.85 cells mm−2 (SEM = 2.06 cells mm−2) than that of PTZ‐control (p = 0.041; two‐tailed Mann–Whitney U test). The degree of GFAP expression with TXH_0.25_30 was similar to that with PTZ‐control (p = 0.485; two‐tailed Mann–Whitney U test), demonstrating a reduced level of 50.18 cells mm−2 (SEM = 2.08 cells mm−2). TX_HYBRID presented a higher GFAP‐density level of 66.43 cells mm−2 (SEM = 2.36 cells mm−2) than the PTZ‐control group (p = 0.002; two‐tailed Mann–Whitney U test).
","Multitemporal Neurotransmitter Analysis Through Microdialysis","
Changes in neurotransmitters through bidirectional neuromodulation were measured using proteomic analysis of ISF samples at baseline and 0, 8, and 16 min after sonication (Figure ##SUPPL##0##S2A–C##, Supporting Information). In hybrid transmission, an additional ISF sample was acquired between the two transmissions. For comparison, the changes in the neurotransmitter levels were divided by the baseline values. In the PTZ‐control group, the levels of glutamate were observed to be increased, while that of GABA was observed to be decreased (Figure ##FIG##5##6B##) compared to the baseline levels after PTZ injection. In contrast, the use of TXL_40_40 significantly reversed the pattern of GABA concentration changes observed in the PTZ‐control (p = 0.030; two‐tailed Mann–Whitny U test) while a decrease in glutamate was not significant compared with that in the PTZ‐control group (p = 0.530; two‐tailed Mann–Whitney U test). After the stimulation, GABA levels became similar with that in the PTZ‐control group (p = 0.343 and p = 1.000 at 0 – 8 min and 20 – 28 min after rtFUS, respectively; two‐tailed Mann–Whitny U test). The glutamate concentration decreased during TXL_40_20 was trending toward significance (p = 0.073 compared to that of PTZ‐control; two‐tailed Mann–Whitny U test) while the GABA decrease during TXL_40_20 application was not significant (p = 0.202 compared to that of PTZ‐control; two‐tailed Mann–Whitny U test). However, the GABA concentration in TXL_40_20 group at 20–29 min after rtFUS dropped significantly compared to that in the PTZ‐control group (p = 0.018, compared to that of the PTZ‐control; two‐tailed Mann–Whitny U test) while glutamate levels were similar between two groups. With hybrid transmission, both GABA and glutamate levels became lower during the second transmission (TXH_0.25_30) than PTZ‐control (p = 0.030 and 0.048 for GABA and glutamate, respectively, compared to that of the PTZ‐control; two‐tailed Mann–Whitny U test), but immediately became similar with that in PTZ‐control group (p = 0.876 and 0.343 for GABA and glutamate, respectively, compared to that of the PTZ‐control; two‐tailed Mann–Whitny U test).
"],"string":"[\n \"Results\",\n \"
Ultrasound stimulation was applied using a custom‐made focused ultrasound transducer over the right hemisphere of the midbrain to target the anterior thalamic nucleus (ATN). EEG was performed before, during, and after sonication. IHC was performed on brain samples harvested from the model animals. Optical measurements of CBV changes and microdialysis for proteomic analysis of neurotransmitters were performed in separate trials to measure the responses to the stimulation. The detailed repetitive transcranial ultrasound stimulation transmission sequences used in the experiment are shown in Figure\\n##FIG##0##\\n1\\n##. The detailed experimental configurations are described in the Experimental Section and Supporting Information figures (Figures ##SUPPL##0##S1## and ##SUPPL##0##S2##, Supporting Information).
\",\n \"Repeated Transcranial Low‐Intensity Elongated Ultrasound Stimulations for Sustainable Full Suppression of Epileptiform Activities\",\n \"
Changes in epileptiform activities were compared between the two stimulant sequences. The first sequence (TXH_0.25_30) comprised eleven repetitions of a stronger (1 MPa) and shorter (0.25 s) burst with a burst interval of 30 s. The second sequence (TXL_40_40) consisted of six repetitions of weaker (0.25 MPa) but longer (40 s) burst with a burst interval of 40 s (Figure ##FIG##0##1##). First, epileptiform activities were reliably induced in a PTZ‐control group that received PTZ, a drug used for acute seizure induction, without ultrasound stimulation. While sparsely located discharge peaks in the EEG signal were identified during the baseline period, the signal was transformed into ictal shapes that demonstrated repetitive high‐frequency discharge peaks in the PTZ‐control group (Figure\\n##FIG##1##\\n2A##). The relative number of epileptic spikes increased 2.6‐fold in the first 10 min after PTZ injection in the PTZ‐control group. The number then decreased for 7 min and saturated at 2.3 times the baseline (Figure ##FIG##1##2B##). Although the number decreased slightly from 10 to 17 min after the PTZ injection, this decrease was not statistically significant at 17 min after the PTZ injection compared to 10 min after the PTZ injection (p = 0.588; two‐tailed paired t‐test) where the number of epileptic spikes ratio still remained significantly different at 17 min after the PTZ injection compared to the baseline period (p = 0.014; two‐tailed paired t‐test). Following stimulation with TXH_0.25_30, the amplitude of the discharge pattern slightly reduced but gradually became comparable to that of the PTZ‐control group. During the period between 0 and 10 min post‐stimulation (10AS) and 20 and 30 min post‐stimulation (30AS), the number of epileptic spikes ratio in the TXH_0.25_30 group was not significantly different from that of the PTZ‐control (p = 0.524 and 0.724 at 10AS and 30AS, respectively; two‐tailed Mann–Whitney U test) (Figure ##FIG##1##2C##). Meanwhile, the discharging patterns following TXL_40_40 became similar to those of the baseline, and the suppressive effect lasted until the end of the EEG recording. TXL_40_40 administration resulted in the maintenance of the number of epileptic spikes ratio similar to that of the baseline, and a suppressive effect was still found at 10AS and 30AS (p = 0.030 and 0.030 at 10AS and 30AS compared with that of PTZ‐control, respectively; two‐tailed Mann–Whitney U test). In the delta band between 1 and 3 Hz, the spectral power with TXH_0.25_30 at 30AS was not significantly different from that of the PTZ control (p = 0.833; two‐tailed Mann‐Whitney U test). However, the relative spectral power followed by TXL_40_40 was 1.19 at 30AS, indicating that this level was close to the baseline. TXL_40_40 also demonstrated strong suppressive effects on the theta band between 4 and 7 Hz (p = 0.019 at 30AS compared with that of PTZ‐control; two‐tailed Mann–Whitney U test), whereas TXH_0.25_30 demonstrated mild effects (p = 0.093 at 30AS compared with that of PTZ‐control; two‐tailed Mann–Whitney U test) (Figure ##FIG##1##2D##). However, with a single transmission of an elongated (40 s), low pressure (0.25 MPa) burst, the number of epileptic spikes ratio became similar to that of the PTZ‐control group, although the suppressive effects were briefly observed for 5 min immediately after the stimulation (Figure ##FIG##1##2B,C##). In the CBV measurements, while the total hemoglobin amount (Δ[Hb]) barely changed in the normal control group (that did not receive PTZ or ultrasound stimulations), Δ[Hb] sharply increased in the PTZ‐control group after drug administration. The increase in Δ[Hb] was as low as that in the normal control group when TXL_40_40 was used (p = 0.699 at 20AS compared with that of normal control; two‐tailed Mann–Whitney U‐test), while the low Δ[Hb] level was maintained until the end of the recording. Following TXH_0.25_30, the Δ[Hb] level was similar to that of the PTZ control (p = 0.180 at 20AS; two‐tailed Mann–Whitney U test), although a gradual decay pattern was observed after the sonication was completed (Figure ##FIG##1##2E##).
\",\n \"Efficacy of rtFUS for Epilepsy Treatment is Dependent on the Burst Length in the rtFUS Sequence\",\n \"
To investigate the effects of burst length changes in rtFUS, the modulation effects were compared for the sequences with burst lengths of 10 s (TXL_10_50), 20 s (TXL_20_40), and 40 s (TXL_40_40). The burst was repeated six times at intervals of 50, 40, and 40 s for TXL_10_50, TXL_20_40, and TXL_40_40, respectively (Figure ##FIG##0##1##). For TXL_10_50, the frequency of the discharge peaks increased in the EEG waveform (Figure\\n##FIG##2##\\n3A##). The number of epileptic spikes with TXL_10_50 was statistically comparable with that in the PTZ‐control group at the post‐stimulation periods from 10AS to 30AS (p = 0.435, 0.354, and 0.435 at 10AS, 20AS, and 30AS, respectively; two‐tailed Mann–Whitney U test). As the stimulation length elongated from 10 to 40 s (TXL_10_50, TXL_20_40, TXL_40_40), the number of epileptic spikes decreased from 2.63, 1.94, 1.07 times the baseline, respectively, at 10AS while the difference in the number of epileptic spikes ratio between that of the PTZ‐control was only significant in the TXL_40_40 group (p = 0.435, 0.943 and 0.030 at 10AS for TXL_10_50, TXL_20_40, and TXL_40_40, respectively; two‐tailed Mann–Whitney U test) (Figure ##FIG##2##3B,C##). In the delta band, the spectral power of the EEG for the TXL_10_50 condition was comparable to that for the PTZ‐control group (p = 0.524 at 30AS; two‐tailed Mann–Whitney U test) while that of the EEG for TXL_40_40 condition was lower than that for the PTZ‐control group. (p = 0.045 at 30AS; two‐tailed Mann–Whitney U test) (Figure ##FIG##2##3D##). In the theta band, the spectral power of the EEG for the TXL_10_50 condition was comparable to that for the PTZ‐control group (p = 0.524 at 30AS; two‐tailed Mann–Whitney U test). However, the trend of reduction in the theta power was significant as the burst length was increased to 20 and 40 s (p = 0.171 and 0.019 at 30AS for TXL_20_40 and TXL_40_40, respectively, compared to PTZ‐control; two‐tailed Mann–Whitney U test) (Figure ##FIG##2##3D##). In the CBV curve, with TXL_10_50, Δ[Hb] was sharply increased by + 12.89% at 20AS compared to that in the baseline and was maintained at a higher level than that in the PTZ‐control group (p = 0.699 at 20AS; two‐tailed Mann‐Whitney U‐test), while the CBV with TXL_40_40 was similar to that of the normal control (p = 0.699 at 20AS; two‐tailed Mann–Whitney U‐test) (Figure ##FIG##2##3E##).
\",\n \"Upregulation and Downregulation of Epileptiform Activities Selected by Adjusting the Interval Between Bursts in the rtFUS Sequence\",\n \"
The epileptiform activity changes by TXL_40_40 were compared with that by TXL_40_20, which also had a burst length of 40 s; however, the interval between the bursts was reduced from 40 to 20 s (Figure ##FIG##0##1##). TXL_40_20 stimulation demonstrated rebound excitatory effects in the post‐stimulus period (Figure\\n##FIG##3##\\n4A##). TXL_40_20 generated a similar number of epileptic spikes as that in the PTZ‐control group (p = 0.943, 0.833, and 0.943 at 10AS, 20AS, and 30AS, respectively; two‐tailed Mann–Whitney U test). In contrast, TXL_40_40 stimulation demonstrated prominent suppressive effects both during and after stimulation (Figure ##FIG##3##4B,C##). The spectral density in the delta band was higher with TXL_40_20 at 30AS than that in the PTZ control. However, the difference was not significant owing to large deviations with TXL_40_20 (p = 0.833; two‐tailed Mann–Whitney U test) (Figure ##FIG##3##4D##). While the CBV response of TXL_40_20 was similar to that of TXL_40_40 during the stimulation period, the Δ[Hb] sharply increased after TXL_40_20, unlike the CBV responses after TXL_40_40, ultimately becoming higher than that of the PTZ control (Figure ##FIG##3##4E##).
\",\n \"Transformation from Suppressive to Excitatory States Through a Hybrid Sequence\",\n \"
As shown in previous studies, TXL_40_40 demonstrated strong suppressive effects. In contrast, the insignificant suppressive effects observed with TXH_0.25_30 – that showed suppressive effects with low statistical significance [19.91% less epileptic spikes ratio (p = 0.724), 16.61% less delta PSD (p = 0.833) and 38.79% less theta PSD (p = 0.093) than that of the PTZ‐control at 30AS]. Although the single use of TXL_40_40 maintained the suppressive effect for more than 30 min, the epileptiform activities were sharply transformed into the excitatory state with the use of TXH_0.25_30, followed by a 3 min resting time after TXL_40_40 (TX_HYBRID, Figure\\n##FIG##4##\\n5A##). The number of epileptic spikes ratio in the TX_HYBRID group was always comparable with the PTZ‐control group across all post‐stimulation periods at 10AS, 20AS, and 30AS (p = 0.943, 0.943, and 0.622, respectively; two‐tailed Mann–Whitney U test) (Figure ##FIG##4##5B,C##). The elevations of the power spectral densities in the delta and theta band after TX_HYBRID (30AS) were comparable to that of the PTZ‐control group (Figure ##FIG##4##5D##) (p = 1.000 and 0.622 at 30AS, respectively; two‐tailed Mann–Whitney U test). Regarding CBV changes, the Δ[Hb] was reduced after TXL_40_40 sonication, whereas TXH_0.25_30 increased the blood volume, approaching that of the PTZ control (Figure ##FIG##4##5E##).
\",\n \"Immunohistochemistry Results\",\n \"
\\nFigure\\n##FIG##5##\\n6A## shows the immunohistochemical results of the samples harvested from sacrificed animals immediately after each experiment. While c‐Fos‐expressed cells were rarely found in the normal control group, c‐Fos‐labeled cells were abundant in the PTZ‐control group (p = 0.002; two‐tailed Mann–Whitney U test). The increase of the proportion of c‐Fos‐positive cells in the TXL_10_50 group was trending toward significance to that in the PTZ‐control group (p = 0.093; two‐tailed Mann–Whitney U test). As shown in the bar graph, 82.58% (standard error of the mean [SEM] = 1.83%) of neuronal cells expressed c‐Fos with TXL_10_50, and 77.14% (SEM = 2.12%) of cells expressed c‐Fos in the PTZ‐control group. TXL_20_40 (p = 0.240; two‐tailed Mann–Whitney U test), TXL_40_20 (p = 0.394; two‐tailed Mann–Whitney U test) and TXH_0.25_30 (p = 0.394; two‐tailed Mann–Whitney U test) demonstrated c‐Fos‐positive cell ratios comparable to those of the PTZ‐control group. Furthermore, TX_HYBRID presented an even higher value of ≈87.47% (SEM = 1.25%) than that of PTZ‐control (p = 0.004; two‐tailed Mann–Whitney U test). In contrast, the expression ratio was drastically reduced to 11.11% with TXL_40_40 compared with the PTZ‐control group (p = 0.002; two‐tailed Mann‐Whitney U test).
\",\n \"
Although the normal and PTZ‐control cases barely exhibited glutamic acid decarboxylase 65‐kilodalton isoform (GAD65)‐positive cells, the sonicated conditions exhibited a greater number of GAD65‐expressed cells. The GAD65‐positive cell density with TXL_40_40 (mean = 15.88 cells mm−2; SEM = 1.53 cells mm−2) was ≈6.2‐fold higher than that in the PTZ‐control group (mean = 2.56 cells mm−2; SEM = 0.42 cells mm−2). Although sharing the same burst duration, TXL_40_20 exhibited a lower expression of GAD65‐positive cells (mean = 7.27 cells mm−2; SEM = 0.61 cells mm−2) than that of TXL_40_40 (mean = 15.88 cells mm−2; SEM = 1.53 cells mm−2), while both groups showed statistically significant difference compared to the PTZ‐control (p = 0.002 and 0.002, respectively; two‐tailed Mann–Whitney U test). The GAD65‐expressed cell densities for TXL_10_50 (mean = 5.60 cells mm−2; SEM = 0.71 cells mm−2) and TXL_40_20 (mean 7.27 cells mm−2; SEM = 0.61 cells mm−2) were lower than the cell density with TXL_40_40 (mean = 15.88 cells mm−2; SEM = 1.53 cells mm−2) and TX_HYBRID (mean = 14.89 cells mm−2; SEM = 0.81 cells mm−2).
\",\n \"
While the ionized calcium‐binding adaptor molecule 1 (Iba1)‐expressed cell density in the normal control group was 39.30 cells mm−2 (SEM = 0.89 cells mm−2), the number increased to 55.62 cells mm−2 (SEM = 1.79 cells mm−2) in the PTZ‐control group. The density of activated cells with TXL_40_40 was 49.36 cells mm−2 (SEM = 0.59 cells mm−2), which was significantly lower than that of the PTZ‐control group (p = 0.009, two‐tailed Mann–Whitney U test). The Iba1‐expressed cell densities in TXH_0.25_30, TXL_10_50, TXL_20_40 and TXL_40_20 were 54.13 cells mm−2 (SEM = 1.68 cells mm−2), 60.50 cells mm−2 (SEM = 2.61 cells mm−2), 54.07 cells mm−2 (SEM = 1.58 cells mm−2) and 57.91 cells mm−2 (SEM = 2.95 cells mm−2), respectively. However, those values were not statistically different (p = 0.394, 0.240, 0.699, 0.589 for TXH_0.25_30, TXL_10_50, TXL_20_40 and TXL_40_20, respectively, two‐tailed Mann–Whitney U test) from the one with PTZ‐control. TX_HYBRID induced the highest Iba1‐expression level at 69.72 cells mm−2 (SEM = 3.51 cells mm−2) among all the experimental groups, and the increase was significant compared with PTZ‐control group (p = 0.009, two‐tailed Mann–Whitney U test).
\",\n \"
In the glial fibrillary acidic protein (GFAP) responses indicating astrocytic reactivity, GFAP density increased to 51.76 cells mm−2 (SEM = 1.96 cells mm−2) in the PTZ‐control group, unlike the normal control group, which presented a density of 39.71 cells mm−2 (SEM = 1.23 cells mm−2) (p = 0.002; two‐tailed Mann–Whitney U test). Furthermore, the GFAP‐positive cell densities of TXL_10_50, TXL_20_40, and TXL_40_40 gradually decreased to 62.58 (SEM = 2.52), 52.59 (SEM = 0.82), and 47.30 cells mm−2 (SEM = 1.82 cells mm−2), respectively, while the GFAP‐positive cell densities with TXL_20_40 and TXL_40_40 were comparable with that with PTZ‐control (p = 0.004, 0.937 and 0.132, respectively, compared to that of PTZ‐control; two‐tailed Mann–Whitney U test). In contrast, TXL_40_20 demonstrated a higher density of 58.85 cells mm−2 (SEM = 2.06 cells mm−2) than that of PTZ‐control (p = 0.041; two‐tailed Mann–Whitney U test). The degree of GFAP expression with TXH_0.25_30 was similar to that with PTZ‐control (p = 0.485; two‐tailed Mann–Whitney U test), demonstrating a reduced level of 50.18 cells mm−2 (SEM = 2.08 cells mm−2). TX_HYBRID presented a higher GFAP‐density level of 66.43 cells mm−2 (SEM = 2.36 cells mm−2) than the PTZ‐control group (p = 0.002; two‐tailed Mann–Whitney U test).
\",\n \"Multitemporal Neurotransmitter Analysis Through Microdialysis\",\n \"
Changes in neurotransmitters through bidirectional neuromodulation were measured using proteomic analysis of ISF samples at baseline and 0, 8, and 16 min after sonication (Figure ##SUPPL##0##S2A–C##, Supporting Information). In hybrid transmission, an additional ISF sample was acquired between the two transmissions. For comparison, the changes in the neurotransmitter levels were divided by the baseline values. In the PTZ‐control group, the levels of glutamate were observed to be increased, while that of GABA was observed to be decreased (Figure ##FIG##5##6B##) compared to the baseline levels after PTZ injection. In contrast, the use of TXL_40_40 significantly reversed the pattern of GABA concentration changes observed in the PTZ‐control (p = 0.030; two‐tailed Mann–Whitny U test) while a decrease in glutamate was not significant compared with that in the PTZ‐control group (p = 0.530; two‐tailed Mann–Whitney U test). After the stimulation, GABA levels became similar with that in the PTZ‐control group (p = 0.343 and p = 1.000 at 0 – 8 min and 20 – 28 min after rtFUS, respectively; two‐tailed Mann–Whitny U test). The glutamate concentration decreased during TXL_40_20 was trending toward significance (p = 0.073 compared to that of PTZ‐control; two‐tailed Mann–Whitny U test) while the GABA decrease during TXL_40_20 application was not significant (p = 0.202 compared to that of PTZ‐control; two‐tailed Mann–Whitny U test). However, the GABA concentration in TXL_40_20 group at 20–29 min after rtFUS dropped significantly compared to that in the PTZ‐control group (p = 0.018, compared to that of the PTZ‐control; two‐tailed Mann–Whitny U test) while glutamate levels were similar between two groups. With hybrid transmission, both GABA and glutamate levels became lower during the second transmission (TXH_0.25_30) than PTZ‐control (p = 0.030 and 0.048 for GABA and glutamate, respectively, compared to that of the PTZ‐control; two‐tailed Mann–Whitny U test), but immediately became similar with that in PTZ‐control group (p = 0.876 and 0.343 for GABA and glutamate, respectively, compared to that of the PTZ‐control; two‐tailed Mann–Whitny U test).
In previous studies, tFUS that sonicated a brain region a single time was more or less effective in lowering epileptiform activities. In the current study, rtFUS, which involves the repetitive transmission of elongated bursts, was first introduced. In contrast to tFUS, rtFUS was able to fully suppress epileptic discharge, resulting in a state comparable to baseline brain responses as measured by EEG and CBV. Surprisingly, the c‐Fos response in the IHC study was barely expressed in the brain samples receiving TXL_40_40 stimulation, whereas the response rate of GAD65 was highest with TXL_40_40 that repeated to transmit a 40 s long low‐intensity burst six times with an interval of 40 s. In microdialysis studies, GABA levels during stimulation with TXL_40_40 were prominently increased, and this observation could support the highest number of GAD65 stains after stimulation with TXL_40_40. Meanwhile, the use of a single transmission of a 40 s long burst suppressed epileptiform activities for a brief period and eventually resulting in symptoms similar to those of the PTZ‐control group. These results suggested that repetitive ultrasonic stimulation with elongated bursts can provide reliable and sustainable suppressive effects by modulating GABA release.
","
In contrast to TXL_40_40, which induced seizure‐suppressive effects, shorter bursting intervals of 20 s (TXL_40_20) could exacerbate epileptiform activities, as shown by EEG, CBV, and IHC. The interval between ultrasound stimulation might not be sufficient for 20 s and needs to be longer than 40 s to maintain a reasonable GABA level for suppression. This is supported by previous findings that reported an excretion time for GABA with electrical stimulation exceeding 40 s in a mouse model.[\n##REF##32728074##\n36\n##\n] This assumption was verified by performing multitemporal measurements of neurotransmitters because the GABA level after TXL_40_20 was continuously decreased and eventually became significantly lower than that with PTZ‐control (Figure ##FIG##5##6B##). Furthermore, in immunohistochemical studies, the number of c‐Fos‐expressing cells was reduced by 85.6% in samples treated with TXL_40_40 compared to the PTZ‐control. In comparison, GAD65 expression increased by 520% compared to that in the PTZ‐control group (Figure ##FIG##5##6A##). However, with TXL_40_20, the expression of GAD65 was significantly reduced by 54.2% compared to that with TXL_40_40 whereas c‐Fos expression was comparable to that in the PTZ‐control group.
","
Our results further demonstrated that burst pressure and length also influenced these suppressive effects. rtFUS using a sequence repeating a shorter (0.25 s) but stronger pressure (1 MPa) burst at every 30 s for eleven times (TXH_0.25_30) demonstrated only mild antiepileptic effects. Considering previous studies that reported neuronal activation with high‐pressure stimulation, TXH_0.25_30 may simultaneously activate excitatory and inhibitory neurons and reduce the net suppressive effect. As shown in the IHC results, the expression ratio of GAD65 with TXH_0.25_30 was 2.74 times higher than that of the PTZ‐Control, while the expression levels of c‐Fos, Iba‐1 and GFAP with TXH_0.25_30 were comparable with those in the PTZ‐control. Interestingly, TXH_0.25_30 induced an excitatory response when the transmit sequence was applied after TXL_40_40. Therefore, controlling the progressive direction of epileptiform activities with short‐ and high‐intensity stimulation was challenging. In contrast, burst lengths longer than 40 s clearly demonstrated seizure suppressive effects. c‐Fos expression decreased drastically, and GAD65 expression gradually increased as the burst length increased from 10 to 40 s with low stimulation pressure levels. These results indicated that the burst pressure and length of rtFUS could be key parameters in determining the progressive direction of epileptiform activities in animal models of drug‐induced epilepsy.
","
In addition to generating different epileptiform activities for different burst lengths or intervals in rtFUS, the suppressive effects could be cleared and translated to excitatory states, worsening epileptic activity by hybrid transmission. The application of TX_HYBRID, comprising a back‐to‐back transmission of TXL_40_40 and TXH_0.25_30 with a 3 min interval, induced the transformation of the suppressant effect into a paradoxically proconvulsant cortical response (Figure ##FIG##4##5B##), although TXL_40_40 and TXH_0.25_30 alone induced strong and mild suppressive effects (Figure ##FIG##1##2B##). In the IHC experiments, the expression rates of GAD65 and c‐Fos with TX_HYBRID were higher than those in PTZ‐control. In the multitemporal neurotransmitter analysis, the elevated GABA level induced by TXL_40_40 also immediately decreased after TXH_0.25_30 treatment, becoming similar to that of the PTZ‐control. Therefore, both inhibitory and excitatory responses were induced by highly conjugated and compensatory regulation, which may also be explained by an increase in GAD65 and c‐Fos expression. Amplified excitation by TX_HYBRID also induced more extensive hippocampal damages than the responses in other animals receiving sole stimulation types. The numbers of both Iba1‐ and GFAP‐expressing cells were the highest in the TX_HYBRID group compared to that in the PTZ‐control group.
","
Although the EEG and CBV measurement of brain activities for temporal lobe epilepsy were not located around the hippocampus, but mostly in the prefrontal cortex, the measured signals should reflect deep brain activities because of the prefrontal limbic network connection to deep brain structures.[\n##UREF##10##\n37\n##\n] Because a four‐channel EEG can measure both the frontal and midbrain regions in both hemispheres, a simple topography revealing the number of discharge peaks could be acquired by linear interpolation among the numbers from the four channels. As shown in the Supporting Information figure (Figure ##SUPPL##0##S3##, Supporting Information) and videos, the excitatory brain response to PTZ injection was initially identified in the prefrontal region, and the signal was stronger than that in the midbrain region. Furthermore, the GABA and glutamate levels measured in the deep brain region using microdialysis demonstrated temporal patterns similar to epidural electrical signal changes. In future work, deep brain electrode recordings may help acquire direct proof of the seizure modulation capability of rtFUS.
","
The number of Iba1‐positive cells, which are reactive near‐damaged neuronal cells sharply increased in the PTZ control group compared to that of the normal control group. The amount of activation increased with TX_HYBRID and decreased with TXL_40_40 compared with PTZ‐control. Therefore, rtFUS sequences that induce more frequent EEG discharges could induce higher cellular damage than that of other sequences. In contrast, other sequences suppressing epileptiform activities could preserve neuronal cells, although they could not fully recover from the damage caused by suppressive treatments. To confirm that the damage to neuronal cells was caused by the proconvulsant effect and not by the delivery of ultrasonic energy, Cresyl Violet staining was performed on the normal control and PTZ‐/rtFUS+ groups. The Nissl‐stained brain slice images demonstrated no damage to the rat brain after TXH_0.25_30 and TXL_40_40 stimulations, which may be the worst cases for sonication amplitude and time, respectively (Figure ##SUPPL##0##S4##, Supporting Information).
","
Bidirectional neuromodulation was achieved by adjusting the acoustic parameters of stimulation strength, length, and the interval of stimulus repetition. For instance, post‐stimulus extracellular GABA level was lowered by shorter intervals of longer stimulation length (TXL_40_20) that might cause GABA depletion because post‐stimulus GABA release took tens of seconds to occur, as demonstrated in a prior study using electrical stimulation[\n##UREF##4##\n24\n##\n] and the stimulation before the GABA recovery might reduce extracellular GABA. Meanwhile, a longer interval (TXL_40_40) could elevate extracellular GABA by allowing sufficient post‐stimulus GABA recovery time. Although we demonstrated the changes in GABA over time using protein analysis with microdialysis, further analysis using a device that presents the changes in GABA with higher time resolution, such as an implantable electrochemical GABA sensor,[\n##REF##32728074##\n36\n##\n] may be needed to prove the depletion and recovery of post‐stimulus GABA over time. Moreover, molecular mechanistic studies are required to extend bidirectional control to other applications. A study has shown that the astrocytic mechanosensitive TRPA1 channel plays a significant role in inducing neuronal activation and eventually releasing glutamate by low‐intensity ultrasound stimulation,[\n##REF##32155416##\n38\n##\n] while there are only limited molecular mechanistic studies investigating pathways of GABA release in response to ultrasound stimulation. The navigation of possible pathways that activate GABA excretion with ultrasound stimulation should be planned to clearly understand bidirectional neuromodulation.
","
We demonstrated the effectiveness of rtFUS in modulating acutely elevated neuronal responses in a chemically induced epilepsy model. As both anticonvulsive and proconvulsive effects were immediately acquired and sustained for at least 30 min using low‐intensity rtFUS, the pulse sequence may be applied to treat epilepsy in the clinic. To achieve this, a seizure detection system, such as a closed‐loop system with EEG, will need to be combined with rtFUS to rapidly suppress signals by sonication as soon as epileptiform activities are detected. Furthermore, this technology may be applicable to other neuronal disorders originating from a dysfunction in the control of neurotransmitters, such as depression and drug addiction.[\n##REF##30946828##\n39\n##, ##REF##37041107##\n40\n##, ##REF##30569209##\n41\n##, ##UREF##11##\n42\n##\n] As introduced in a previous review,[\n##UREF##12##\n43\n##\n] ultrasound neuromodulations with tFUS are highlighted to be effective in brain therapeutics while the efficacies in some results were mild. The use of different ultrasound pulse sequences modulates different outcomes in treating Alzheimer's disease.[\n##UREF##13##\n44\n##\n] Therefore, we expect that the efficacies of ultrasound neuromodulation can be amplified and prolonged by using rtFUS.
"],"string":"[\n \"Discussion\",\n \"
In previous studies, tFUS that sonicated a brain region a single time was more or less effective in lowering epileptiform activities. In the current study, rtFUS, which involves the repetitive transmission of elongated bursts, was first introduced. In contrast to tFUS, rtFUS was able to fully suppress epileptic discharge, resulting in a state comparable to baseline brain responses as measured by EEG and CBV. Surprisingly, the c‐Fos response in the IHC study was barely expressed in the brain samples receiving TXL_40_40 stimulation, whereas the response rate of GAD65 was highest with TXL_40_40 that repeated to transmit a 40 s long low‐intensity burst six times with an interval of 40 s. In microdialysis studies, GABA levels during stimulation with TXL_40_40 were prominently increased, and this observation could support the highest number of GAD65 stains after stimulation with TXL_40_40. Meanwhile, the use of a single transmission of a 40 s long burst suppressed epileptiform activities for a brief period and eventually resulting in symptoms similar to those of the PTZ‐control group. These results suggested that repetitive ultrasonic stimulation with elongated bursts can provide reliable and sustainable suppressive effects by modulating GABA release.
\",\n \"
In contrast to TXL_40_40, which induced seizure‐suppressive effects, shorter bursting intervals of 20 s (TXL_40_20) could exacerbate epileptiform activities, as shown by EEG, CBV, and IHC. The interval between ultrasound stimulation might not be sufficient for 20 s and needs to be longer than 40 s to maintain a reasonable GABA level for suppression. This is supported by previous findings that reported an excretion time for GABA with electrical stimulation exceeding 40 s in a mouse model.[\\n##REF##32728074##\\n36\\n##\\n] This assumption was verified by performing multitemporal measurements of neurotransmitters because the GABA level after TXL_40_20 was continuously decreased and eventually became significantly lower than that with PTZ‐control (Figure ##FIG##5##6B##). Furthermore, in immunohistochemical studies, the number of c‐Fos‐expressing cells was reduced by 85.6% in samples treated with TXL_40_40 compared to the PTZ‐control. In comparison, GAD65 expression increased by 520% compared to that in the PTZ‐control group (Figure ##FIG##5##6A##). However, with TXL_40_20, the expression of GAD65 was significantly reduced by 54.2% compared to that with TXL_40_40 whereas c‐Fos expression was comparable to that in the PTZ‐control group.
\",\n \"
Our results further demonstrated that burst pressure and length also influenced these suppressive effects. rtFUS using a sequence repeating a shorter (0.25 s) but stronger pressure (1 MPa) burst at every 30 s for eleven times (TXH_0.25_30) demonstrated only mild antiepileptic effects. Considering previous studies that reported neuronal activation with high‐pressure stimulation, TXH_0.25_30 may simultaneously activate excitatory and inhibitory neurons and reduce the net suppressive effect. As shown in the IHC results, the expression ratio of GAD65 with TXH_0.25_30 was 2.74 times higher than that of the PTZ‐Control, while the expression levels of c‐Fos, Iba‐1 and GFAP with TXH_0.25_30 were comparable with those in the PTZ‐control. Interestingly, TXH_0.25_30 induced an excitatory response when the transmit sequence was applied after TXL_40_40. Therefore, controlling the progressive direction of epileptiform activities with short‐ and high‐intensity stimulation was challenging. In contrast, burst lengths longer than 40 s clearly demonstrated seizure suppressive effects. c‐Fos expression decreased drastically, and GAD65 expression gradually increased as the burst length increased from 10 to 40 s with low stimulation pressure levels. These results indicated that the burst pressure and length of rtFUS could be key parameters in determining the progressive direction of epileptiform activities in animal models of drug‐induced epilepsy.
\",\n \"
In addition to generating different epileptiform activities for different burst lengths or intervals in rtFUS, the suppressive effects could be cleared and translated to excitatory states, worsening epileptic activity by hybrid transmission. The application of TX_HYBRID, comprising a back‐to‐back transmission of TXL_40_40 and TXH_0.25_30 with a 3 min interval, induced the transformation of the suppressant effect into a paradoxically proconvulsant cortical response (Figure ##FIG##4##5B##), although TXL_40_40 and TXH_0.25_30 alone induced strong and mild suppressive effects (Figure ##FIG##1##2B##). In the IHC experiments, the expression rates of GAD65 and c‐Fos with TX_HYBRID were higher than those in PTZ‐control. In the multitemporal neurotransmitter analysis, the elevated GABA level induced by TXL_40_40 also immediately decreased after TXH_0.25_30 treatment, becoming similar to that of the PTZ‐control. Therefore, both inhibitory and excitatory responses were induced by highly conjugated and compensatory regulation, which may also be explained by an increase in GAD65 and c‐Fos expression. Amplified excitation by TX_HYBRID also induced more extensive hippocampal damages than the responses in other animals receiving sole stimulation types. The numbers of both Iba1‐ and GFAP‐expressing cells were the highest in the TX_HYBRID group compared to that in the PTZ‐control group.
\",\n \"
Although the EEG and CBV measurement of brain activities for temporal lobe epilepsy were not located around the hippocampus, but mostly in the prefrontal cortex, the measured signals should reflect deep brain activities because of the prefrontal limbic network connection to deep brain structures.[\\n##UREF##10##\\n37\\n##\\n] Because a four‐channel EEG can measure both the frontal and midbrain regions in both hemispheres, a simple topography revealing the number of discharge peaks could be acquired by linear interpolation among the numbers from the four channels. As shown in the Supporting Information figure (Figure ##SUPPL##0##S3##, Supporting Information) and videos, the excitatory brain response to PTZ injection was initially identified in the prefrontal region, and the signal was stronger than that in the midbrain region. Furthermore, the GABA and glutamate levels measured in the deep brain region using microdialysis demonstrated temporal patterns similar to epidural electrical signal changes. In future work, deep brain electrode recordings may help acquire direct proof of the seizure modulation capability of rtFUS.
\",\n \"
The number of Iba1‐positive cells, which are reactive near‐damaged neuronal cells sharply increased in the PTZ control group compared to that of the normal control group. The amount of activation increased with TX_HYBRID and decreased with TXL_40_40 compared with PTZ‐control. Therefore, rtFUS sequences that induce more frequent EEG discharges could induce higher cellular damage than that of other sequences. In contrast, other sequences suppressing epileptiform activities could preserve neuronal cells, although they could not fully recover from the damage caused by suppressive treatments. To confirm that the damage to neuronal cells was caused by the proconvulsant effect and not by the delivery of ultrasonic energy, Cresyl Violet staining was performed on the normal control and PTZ‐/rtFUS+ groups. The Nissl‐stained brain slice images demonstrated no damage to the rat brain after TXH_0.25_30 and TXL_40_40 stimulations, which may be the worst cases for sonication amplitude and time, respectively (Figure ##SUPPL##0##S4##, Supporting Information).
\",\n \"
Bidirectional neuromodulation was achieved by adjusting the acoustic parameters of stimulation strength, length, and the interval of stimulus repetition. For instance, post‐stimulus extracellular GABA level was lowered by shorter intervals of longer stimulation length (TXL_40_20) that might cause GABA depletion because post‐stimulus GABA release took tens of seconds to occur, as demonstrated in a prior study using electrical stimulation[\\n##UREF##4##\\n24\\n##\\n] and the stimulation before the GABA recovery might reduce extracellular GABA. Meanwhile, a longer interval (TXL_40_40) could elevate extracellular GABA by allowing sufficient post‐stimulus GABA recovery time. Although we demonstrated the changes in GABA over time using protein analysis with microdialysis, further analysis using a device that presents the changes in GABA with higher time resolution, such as an implantable electrochemical GABA sensor,[\\n##REF##32728074##\\n36\\n##\\n] may be needed to prove the depletion and recovery of post‐stimulus GABA over time. Moreover, molecular mechanistic studies are required to extend bidirectional control to other applications. A study has shown that the astrocytic mechanosensitive TRPA1 channel plays a significant role in inducing neuronal activation and eventually releasing glutamate by low‐intensity ultrasound stimulation,[\\n##REF##32155416##\\n38\\n##\\n] while there are only limited molecular mechanistic studies investigating pathways of GABA release in response to ultrasound stimulation. The navigation of possible pathways that activate GABA excretion with ultrasound stimulation should be planned to clearly understand bidirectional neuromodulation.
\",\n \"
We demonstrated the effectiveness of rtFUS in modulating acutely elevated neuronal responses in a chemically induced epilepsy model. As both anticonvulsive and proconvulsive effects were immediately acquired and sustained for at least 30 min using low‐intensity rtFUS, the pulse sequence may be applied to treat epilepsy in the clinic. To achieve this, a seizure detection system, such as a closed‐loop system with EEG, will need to be combined with rtFUS to rapidly suppress signals by sonication as soon as epileptiform activities are detected. Furthermore, this technology may be applicable to other neuronal disorders originating from a dysfunction in the control of neurotransmitters, such as depression and drug addiction.[\\n##REF##30946828##\\n39\\n##, ##REF##37041107##\\n40\\n##, ##REF##30569209##\\n41\\n##, ##UREF##11##\\n42\\n##\\n] As introduced in a previous review,[\\n##UREF##12##\\n43\\n##\\n] ultrasound neuromodulations with tFUS are highlighted to be effective in brain therapeutics while the efficacies in some results were mild. The use of different ultrasound pulse sequences modulates different outcomes in treating Alzheimer's disease.[\\n##UREF##13##\\n44\\n##\\n] Therefore, we expect that the efficacies of ultrasound neuromodulation can be amplified and prolonged by using rtFUS.
In our study, the bidirectional modulation of epileptiform brain activity was first demonstrated by changing the burst duration, interval, and amplitude of rtFUS. A stimulation train repeating elongated bursts with sufficiently long intervals was shown to effectively suppress epileptiform activities. In contrast, stimulation transmitting the same burst length with a shorter interval worsened the epileptic responses. Furthermore, the suppressive effect achieved by the long burst could be reversed using a short, strong burst train, leading to a shift from a suppressive to an excitatory state. The IHC and proteomic analyses conducted in this study allowed us to conclude that bidirectional modulation of epileptiform activity with rtFUS was achieved by controlling the excretion of GABA in sonicated brain tissue.
"],"string":"[\n \"Conclusion\",\n \"
In our study, the bidirectional modulation of epileptiform brain activity was first demonstrated by changing the burst duration, interval, and amplitude of rtFUS. A stimulation train repeating elongated bursts with sufficiently long intervals was shown to effectively suppress epileptiform activities. In contrast, stimulation transmitting the same burst length with a shorter interval worsened the epileptic responses. Furthermore, the suppressive effect achieved by the long burst could be reversed using a short, strong burst train, leading to a shift from a suppressive to an excitatory state. The IHC and proteomic analyses conducted in this study allowed us to conclude that bidirectional modulation of epileptiform activity with rtFUS was achieved by controlling the excretion of GABA in sonicated brain tissue.
Repetitive stimulation procedures are used in neuromodulation techniques to induce persistent excitatory or inhibitory brain activity. The directivity of modulation is empirically regulated by modifying the stimulation length, interval, and strength. However, bidirectional neuronal modulations using ultrasound stimulations are rarely reported. This study presents bidirectional control of epileptiform activities with repetitive transcranial‐focused ultrasound stimulations in a rat model of drug‐induced acute epilepsy. It is found that repeated transmission of elongated (40 s), ultra‐low pressure (0.25 MPa) ultrasound can fully suppress epileptic activities in electro‐encephalography and cerebral blood volume measurements, while the change in bursting intervals from 40 to 20 s worsens epileptic activities even with the same burst length. Furthermore, the suppression induced by 40 s long bursts is transformed to excitatory states by a subsequent transmission. Bidirectional modulation of epileptic seizures with repeated ultrasound stimulation is achieved by regulating the changes in glutamate and γ‐Aminobutyric acid levels, as confirmed by measurements of expressed c‐Fos and GAD65 and multitemporal analysis of neurotransmitters in the interstitial fluid obtained via microdialysis.
","
Bidirectional control of epileptiform activities can be achieved by changing stimulus parameters for repetitive transcranial focused ultrasound. The repeated sonication of elongated burst with longer intervals can increase extracellular GABA and induce suppressive effects while the same burst with shorter intervals returns opposite responses. Furthermore, the suppressed epileptiform transforms the excitatory state by following stronger short bursts.\n\n
","
\n\n\nT.\nChoi\n, \nM.\nKoo\n, \nJ.\nJoo\n, \nT.\nKim\n, \nY.‐M.\nShon\n, \nJ.\nPark\n, Bidirectional Neuronal Control of Epileptiform Activity by Repetitive Transcranial Focused Ultrasound Stimulations. Adv. Sci.\n2024, 11, 2302404. 10.1002/advs.202302404\n\n
"],"string":"[\n \"Abstract\",\n \"
Repetitive stimulation procedures are used in neuromodulation techniques to induce persistent excitatory or inhibitory brain activity. The directivity of modulation is empirically regulated by modifying the stimulation length, interval, and strength. However, bidirectional neuronal modulations using ultrasound stimulations are rarely reported. This study presents bidirectional control of epileptiform activities with repetitive transcranial‐focused ultrasound stimulations in a rat model of drug‐induced acute epilepsy. It is found that repeated transmission of elongated (40 s), ultra‐low pressure (0.25 MPa) ultrasound can fully suppress epileptic activities in electro‐encephalography and cerebral blood volume measurements, while the change in bursting intervals from 40 to 20 s worsens epileptic activities even with the same burst length. Furthermore, the suppression induced by 40 s long bursts is transformed to excitatory states by a subsequent transmission. Bidirectional modulation of epileptic seizures with repeated ultrasound stimulation is achieved by regulating the changes in glutamate and γ‐Aminobutyric acid levels, as confirmed by measurements of expressed c‐Fos and GAD65 and multitemporal analysis of neurotransmitters in the interstitial fluid obtained via microdialysis.
\",\n \"
Bidirectional control of epileptiform activities can be achieved by changing stimulus parameters for repetitive transcranial focused ultrasound. The repeated sonication of elongated burst with longer intervals can increase extracellular GABA and induce suppressive effects while the same burst with shorter intervals returns opposite responses. Furthermore, the suppressed epileptiform transforms the excitatory state by following stronger short bursts.\\n\\n
\",\n \"
\\n\\n\\nT.\\nChoi\\n, \\nM.\\nKoo\\n, \\nJ.\\nJoo\\n, \\nT.\\nKim\\n, \\nY.‐M.\\nShon\\n, \\nJ.\\nPark\\n, Bidirectional Neuronal Control of Epileptiform Activity by Repetitive Transcranial Focused Ultrasound Stimulations. Adv. Sci.\\n2024, 11, 2302404. 10.1002/advs.202302404\\n\\n
All animal experiments were performed in accordance with the Korean Ministry of Food and Drug Safety Guide for the Care and Use of Laboratory Animals and were conducted according to protocols approved by the Institutional Animal Care and Use Committees (IACUC). This study was reviewed and approved by the IACUC of Samsung Biomedical Research Institute (SBRI) and WOOJUNG BIO (Approval Numbers: SBRIIACUC20200109003 and IACUC2301‐025). SBRI and WOOJUNG BIO are Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accredited facility and abide by the Institute of Laboratory Animal Resources (ILAR) guide.
","Animal Preparation and Epileptic Induction","
Fifty‐nine 8‐week‐old male Sprague–Dawley rats weighing 300–320 g (Orient Bio, Inc.) were used in the experiments. For the EEG, eight rats were chosen for the non‐stimulated PTZ+/tFUS‐control (PTZ‐control) group, and five rats were chosen for each stimulated experimental group. IHC was performed within the EEG group for five rats in each of the PTZ‐control and stimulated experimental groups, and five additional rats were evaluated as the PTZ‐/tFUS‐control (normal control) group. For wide‐field optical imaging, six rats each from the PTZ control, normal control, and stimulated experimental groups were evaluated. For the microdialysis, eight rats were chosen for PTZ‐control, and five rats were chosen for each group of TXL_40_40, TXL_40_20 and TX_Hybrid. (Table\n##TAB##0##\n1\n##) Animals were anesthetized using urethane (1.25 g kg−1 body weight in 1.25 g 4 mL−1 saline; Sigma–Aldrich, U‐2500‐500G) and pentylenetetrazol (100 mg kg−1 body weight in 10 mg 0.1 mL−1 saline; Sigma–Aldrich, P6500‐25G), administered acutely and intraperitoneally to induce epileptic seizures.
","Ultrasound Transducer Fabrication","
A lead zirconate titanate (Del Piezo Specialties LLC., DL‐47) 690 kHz single‐element ring transducer with inner and outer diameters of 10 and 31 mm, respectively, was fabricated with a focal distance of 45 mm and a bandwidth of 25%. An acoustic coupling gel‐filled cone‐shaped beam guide was attached to the front side of the transducer. The beam was focused 5 mm ahead of the tip of the collimator with −3 dB beam sizes of 4.7 and 2.2 mm along the axial and lateral directions, respectively (Figure ##SUPPL##0##S1A##, Supporting Information). By placing the tip of the transducer above the parietal bone, the acoustic beam was delivered to the right anterior thalamic nuclei (ATN) (AP = −1.5 mm, ML = + 1.5 mm, DV = 5.0–5.5 mm; AP denotes the anterior (+) or posterior (−) distance from the bregma; ML denotes the lateral distance from the bregma; and DV denotes the dorsoventral distance from the bregma). The beam pathway was confirmed by overlaying the beam profile on the Paxinos rat brain atlas[\n##UREF##14##\n45\n##\n] along the sagittal and coronal planes (Figure ##SUPPL##0##S1B,C##, Supporting Information).
","Sonication and Monitoring Protocol","
The stimuli were classified into two types to simplify the ultrasonic conditions and mimic electrical stimulations generating rebound excitations: one with a low‐pressure (0.25 MPa) consecutive burst train (TXL sequences), and the other with shorter, high‐pressure (1.0 MPa) bursts (TXH sequence) with a 50% duty cycle of 100 Hz pulse repetition frequency. TX_HYBRID, a back‐to‐back transmission of TXL_40_40 and TXH_0.25_30 was generated assuming that rebound excitation could be artificially induced using brief excitation followed by inhibitory stimulation.
","
Figure ##SUPPL##0##S1D## (Supporting Information) depicts the entire experimental platform for delivering ultrasonic energy to the target region and recording EEG signals. The rats were anchored to a custom‐made stereotaxic frame (Scitech Korea, Inc.), and ultrasound pulsation was generated using two function generators. The first function generator (Tektronix, AFG3252) sent out a pulse train of 100 Hz, and the second function generator (Agilent, 33250A) transmitted a 5 ms, 3450 cycle, 690 kHz burst to secure a 50% duty cycle (Figure ##SUPPL##0##S1D##, Supporting Information) at every rising edge in the pulse train from the first function generator. The generated waveform was then passed through a radiofrequency power amplifier (Electronics and Innovation, 350 L) and applied to the transducer. The experimental protocol involved: 1) anesthesia; 2) pre‐tFUS (baseline) monitoring; 3) PTZ injection and sonication; 4) post‐tFUS monitoring; and 5) euthanization and brain tissue harvesting. Figure ##FIG##0##1## shows the overall timeline of sonication and data acquisition.
","EEG Recording","
The rats were stereotactically implanted with five cortical screw electrodes (Fine Science Tools, 19010‐00 screws) to record the EEG signals (Figure ##SUPPL##0##S1A##, Supporting Information). The implantation coordinates were adopted based on a stereotactic atlas[\n##UREF##14##\n45\n##, ##UREF##15##\n46\n##\n]: C4/C3 (primary motor cortex; A = + 2.2 mm, L = ± 3.2 mm) and T6/T5 (temporal association cortex; A = −8.3 mm, L = ± 5.8 mm). A reference ground electrode was implanted on the posterior side of the lambda. EEG was performed to monitor changes in neuronal activity using a four‐channel commercial EEG acquisition system (Natus, NicoletOne vEEG) at a sampling rate of 500 Hz. The ratio of the number of epileptic spikes at baseline to the number at specific time points was calculated to assess the epileptic severity. Epileptic spikes were defined as local maximum points with amplitudes greater than twice the standard deviation from individual baseline EEG activities lasting 70 ms. The discharge peaks of the EEG signal were counted every 30 s in a 1 min segment across the monitoring period and averaged every 10 min for each animal. The power spectral density values were computed for every 1 min segment using Welch's method and also averaged every 10 min for each animal. Pairwise comparison was made at each time period for the statistical analysis where each value represented a single value from a single animal.
","Immunohistochemistry","
IHC was performed using c‐Fos, GAD65, Iba‐1, and GFAP staining of rat brain sections. The rats were euthanized after the EEG recording and transcardially perfused with saline to obtain brain tissue samples. The ratio of c‐Fos‐positive cells to total cells in the granular layer of the dentate gyrus was quantified using ImageJ software (National Institutes of Health, 1.53c) to compare the degree of excitatory responses generated under ultrasonic stimulus conditions. To compare inhibitory synaptic transmission, GAD65‐positive cell densities were measured in the molecular and granular layers of the dentate gyrus. Staining for Iba1 and GFAP was performed to investigate the reactivity of microglia and astrocytes, respectively, by computing the Iba1‐ and GFAP‐positive cell densities. All values were measured three times independently from a different person in a random order to avoid biased erroneous measurement and averaged in a single value for each animal. A pairwise comparison was made where each value represented a single value from a single antibody‐stained brain slice from a single animal.
","Cerebral Blood Volume Measurement","
Craniotomies were performed on isoflurane‐anesthetized rats secured in a custom‐made stereotaxic frame. The cranial window region was created above the left ATN (A = −1.5 mm, L = −1.5 mm), such that the rtFUS stimulus could be applied on the right ATN while simultaneously imaging the cranial window (Figure ##SUPPL##0##S2C,D##, Supporting Information). After an overnight recovery, urethane (1.25 mg kg−1 body weight) was injected intraperitoneally for wide‐field optical imaging.
","
Optical measurement was conducted for the normal control, PTZ‐control, and tFUS‐stimulated groups to examine the regional hemodynamic response of cerebral blood volume (CBV). The imaging system consisted of a macro‐zoom microscope (Olympus, MVX10), an sCMOS camera (Andor, Zyla 5.5), and a light‐emitting diode source (Figure ##SUPPL##0##S2C##, Supporting Information). For the experiment, raw reflectance data were collected under green (530 nm) light, as 530 nm is primarily sensitive to changes in the local total blood volume, denoted as the HbT.[\n##REF##31834545##\n47\n##, ##UREF##16##\n48\n##\n] Each imaging trial was recorded for 30 min, including a 2 min baseline (pre‐PTZ/tFUS).
","Microdialysis","
Microdialysis analysis was conducted to determine the amount of neurotransmitter secreted during the alterations in epileptiform activities generated by the rtFUS transmission sequences (Figure ##SUPPL##0##S2A##, Supporting Information) to demonstrate the cause of bidirectional modulation in animal models of drug‐induced epilepsy. The guide cannula was implanted in twenty male Sprague‐Dawley rats weighing 300–320 g (Koatech, Inc.) under respiratory anesthesia using a hand drill at the target coordinates of the hippocampal region CA2/CA3 (Coordinates −3.80 mm posterior (AP), −4.40 mm lateral (ML), and −2.20 mm ventral (DV), 26 degrees lateral) (Figure ##SUPPL##0##S2B##, Supporting Information). After the guide cannula reached the target coordinates, dental cement was applied for fixation, avoiding the position of the ultrasound transducer, and the incision site was sutured with a silk suture for the recovery period. For proteomic analysis, five rats were chosen for the PTZ‐control group (PTZ+/tFUS‐) and five rats were chosen for each experimental group: TXL_40_40, TXL_40_20, and TX_HYBRID.
","
Two days after the guide cannula insertion surgery, the recovered rats were intraperitoneally anesthetized with urethane (1.25 g kg−1 body weight in 1.25 g 4 mL−1 saline; Sigma–Aldrich, U‐2500‐500G). After the anesthetized rats were secured to the stereotaxic frame, the dummy was removed from the guide cannula and a CMA12 1‐mm membrane probe (CMA Microdialysis) was inserted. A 5 mL glass syringe filled with aCSF (M dialysis AB) was mounted on a syringe pump (KD Scientific) and connected to the probe inlet. The tubing was connected to the probe outlet to collect the interstitial fluid (ISF) in a sample collector (BASi, U.S.A). After ≈1 h of stabilization (1 point 8 min−1), Interstitial fluid (ISF) was collected by sampling for 1 h. For microdialysis, samples were collected for 8 min at a flow rate of 1.3 µL min−1 in order to obtain a sufficient amount (minimum 10 µL) of ISF for the neurotransmitter analysis. The sample collection time was minimized to match the sonication duration (440 s/340 s), but due to the fixed flow rate and the minimum required collection volume, the sample collection time was inevitably longer than the sonication duration. Therefore, a portion of the baseline (40 s for TXL_40_40 and 140 s for TXL_40_20) was collected together when analyzing the neurotransmitter concentration during the rtFUS. All samples were stored in a freezer at −80 °C.
","
Neurotransmitter analysis of the microdialysis samples was conducted using LC‐MS/MS in NeuroVIS (Chungcheongnam‐do, Republic of Korea). Table\n##TAB##1##\n2\n## presents the analytical conditions of the LC‐MS/MS instrument. To separate GABA and glutamate in the LC‐MS/MS system composed of ExionLC Series UHPLC, AB SCIEX Triple Quadrupole 6500+, and ESI (SCIEX, Framingham), an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm, Waters, Milford, MA, USA) at 50 °C was used. The mobile phase consisted of 1) water containing 0.1% FA and 5 mm ammonium formate and 2) a 1:1 mixture of methanol and acetonitrile containing 5 mm ammonium formate. The flow rate was adjusted at 0.3 mL min−1, and a 7 µL per sample was injected in LC‐MS/MS. Multiple reaction monitoring (MRM) was performed in the positive mode for each NTs. The experiment was conducted at an ion transfer temperature of 500 °C, and a positive ion spray voltage of 5000 V.
","Statistical Analyses","
All experimental data were analyzed using statistics software (IBM, SPSS Statistics 27). In order to determine whether the data from each group follows a normal distribution, the Shapiro‐Wilk test[\n##UREF##17##\n49\n##\n] was performed. Once the normality was confirmed, the statistical significance of the difference between a sonicated group and the PTZ‐control group was assessed by t‐test. For the t‐test, a paired t‐test was employed in case the data were compared by time within the same group. If the distribution of the data departed significantly from the normality, a non‐parametric Mann–Whitney U test was performed to compare the significance of the difference. The type of test used for the statistical analysis was presented along with the p‐values throughout the paper. The purpose of experiments in this paper was to find either upregulating or downregulating effects for each stimulation sequence independently by comparing the effect with the control group. The neuromodulation effect for each stimulation sequence was only observed from a rat group of PTZ‐control that did not receive other stimulation sequences. Therefore, two‐sided Mann–Whitney U test was employed without considering multiple comparison corrections. p <0.05 was considered statistically significant and was marked as ** p <0.10 was considered trending toward statistical significance and was marked as *p > 0.10 was considered non‐significant and was marked as NS throughout the paper. Although p < 0.05 was the typical standard significance threshold in the general scientific community, the differences p <0.10 were also provisioned on the figures as providing the information may still identify potential trends – especially considering the small sample sizes – and suggest a potentially meaningful relationship that warrants further study.[\n##UREF##18##\n50\n##\n] Effects with significance of p <0.10 were termed as “mild” throughout the paper. The sample sizes were determined by using the resource equation method.[\n##UREF##19##\n51\n##, ##REF##24250214##\n52\n##\n]\n
All animal experiments were performed in accordance with the Korean Ministry of Food and Drug Safety Guide for the Care and Use of Laboratory Animals and were conducted according to protocols approved by the Institutional Animal Care and Use Committees (IACUC). This study was reviewed and approved by the IACUC of Samsung Biomedical Research Institute (SBRI) and WOOJUNG BIO (Approval Numbers: SBRIIACUC20200109003 and IACUC2301‐025). SBRI and WOOJUNG BIO are Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) accredited facility and abide by the Institute of Laboratory Animal Resources (ILAR) guide.
\",\n \"Animal Preparation and Epileptic Induction\",\n \"
Fifty‐nine 8‐week‐old male Sprague–Dawley rats weighing 300–320 g (Orient Bio, Inc.) were used in the experiments. For the EEG, eight rats were chosen for the non‐stimulated PTZ+/tFUS‐control (PTZ‐control) group, and five rats were chosen for each stimulated experimental group. IHC was performed within the EEG group for five rats in each of the PTZ‐control and stimulated experimental groups, and five additional rats were evaluated as the PTZ‐/tFUS‐control (normal control) group. For wide‐field optical imaging, six rats each from the PTZ control, normal control, and stimulated experimental groups were evaluated. For the microdialysis, eight rats were chosen for PTZ‐control, and five rats were chosen for each group of TXL_40_40, TXL_40_20 and TX_Hybrid. (Table\\n##TAB##0##\\n1\\n##) Animals were anesthetized using urethane (1.25 g kg−1 body weight in 1.25 g 4 mL−1 saline; Sigma–Aldrich, U‐2500‐500G) and pentylenetetrazol (100 mg kg−1 body weight in 10 mg 0.1 mL−1 saline; Sigma–Aldrich, P6500‐25G), administered acutely and intraperitoneally to induce epileptic seizures.
\",\n \"Ultrasound Transducer Fabrication\",\n \"
A lead zirconate titanate (Del Piezo Specialties LLC., DL‐47) 690 kHz single‐element ring transducer with inner and outer diameters of 10 and 31 mm, respectively, was fabricated with a focal distance of 45 mm and a bandwidth of 25%. An acoustic coupling gel‐filled cone‐shaped beam guide was attached to the front side of the transducer. The beam was focused 5 mm ahead of the tip of the collimator with −3 dB beam sizes of 4.7 and 2.2 mm along the axial and lateral directions, respectively (Figure ##SUPPL##0##S1A##, Supporting Information). By placing the tip of the transducer above the parietal bone, the acoustic beam was delivered to the right anterior thalamic nuclei (ATN) (AP = −1.5 mm, ML = + 1.5 mm, DV = 5.0–5.5 mm; AP denotes the anterior (+) or posterior (−) distance from the bregma; ML denotes the lateral distance from the bregma; and DV denotes the dorsoventral distance from the bregma). The beam pathway was confirmed by overlaying the beam profile on the Paxinos rat brain atlas[\\n##UREF##14##\\n45\\n##\\n] along the sagittal and coronal planes (Figure ##SUPPL##0##S1B,C##, Supporting Information).
\",\n \"Sonication and Monitoring Protocol\",\n \"
The stimuli were classified into two types to simplify the ultrasonic conditions and mimic electrical stimulations generating rebound excitations: one with a low‐pressure (0.25 MPa) consecutive burst train (TXL sequences), and the other with shorter, high‐pressure (1.0 MPa) bursts (TXH sequence) with a 50% duty cycle of 100 Hz pulse repetition frequency. TX_HYBRID, a back‐to‐back transmission of TXL_40_40 and TXH_0.25_30 was generated assuming that rebound excitation could be artificially induced using brief excitation followed by inhibitory stimulation.
\",\n \"
Figure ##SUPPL##0##S1D## (Supporting Information) depicts the entire experimental platform for delivering ultrasonic energy to the target region and recording EEG signals. The rats were anchored to a custom‐made stereotaxic frame (Scitech Korea, Inc.), and ultrasound pulsation was generated using two function generators. The first function generator (Tektronix, AFG3252) sent out a pulse train of 100 Hz, and the second function generator (Agilent, 33250A) transmitted a 5 ms, 3450 cycle, 690 kHz burst to secure a 50% duty cycle (Figure ##SUPPL##0##S1D##, Supporting Information) at every rising edge in the pulse train from the first function generator. The generated waveform was then passed through a radiofrequency power amplifier (Electronics and Innovation, 350 L) and applied to the transducer. The experimental protocol involved: 1) anesthesia; 2) pre‐tFUS (baseline) monitoring; 3) PTZ injection and sonication; 4) post‐tFUS monitoring; and 5) euthanization and brain tissue harvesting. Figure ##FIG##0##1## shows the overall timeline of sonication and data acquisition.
\",\n \"EEG Recording\",\n \"
The rats were stereotactically implanted with five cortical screw electrodes (Fine Science Tools, 19010‐00 screws) to record the EEG signals (Figure ##SUPPL##0##S1A##, Supporting Information). The implantation coordinates were adopted based on a stereotactic atlas[\\n##UREF##14##\\n45\\n##, ##UREF##15##\\n46\\n##\\n]: C4/C3 (primary motor cortex; A = + 2.2 mm, L = ± 3.2 mm) and T6/T5 (temporal association cortex; A = −8.3 mm, L = ± 5.8 mm). A reference ground electrode was implanted on the posterior side of the lambda. EEG was performed to monitor changes in neuronal activity using a four‐channel commercial EEG acquisition system (Natus, NicoletOne vEEG) at a sampling rate of 500 Hz. The ratio of the number of epileptic spikes at baseline to the number at specific time points was calculated to assess the epileptic severity. Epileptic spikes were defined as local maximum points with amplitudes greater than twice the standard deviation from individual baseline EEG activities lasting 70 ms. The discharge peaks of the EEG signal were counted every 30 s in a 1 min segment across the monitoring period and averaged every 10 min for each animal. The power spectral density values were computed for every 1 min segment using Welch's method and also averaged every 10 min for each animal. Pairwise comparison was made at each time period for the statistical analysis where each value represented a single value from a single animal.
\",\n \"Immunohistochemistry\",\n \"
IHC was performed using c‐Fos, GAD65, Iba‐1, and GFAP staining of rat brain sections. The rats were euthanized after the EEG recording and transcardially perfused with saline to obtain brain tissue samples. The ratio of c‐Fos‐positive cells to total cells in the granular layer of the dentate gyrus was quantified using ImageJ software (National Institutes of Health, 1.53c) to compare the degree of excitatory responses generated under ultrasonic stimulus conditions. To compare inhibitory synaptic transmission, GAD65‐positive cell densities were measured in the molecular and granular layers of the dentate gyrus. Staining for Iba1 and GFAP was performed to investigate the reactivity of microglia and astrocytes, respectively, by computing the Iba1‐ and GFAP‐positive cell densities. All values were measured three times independently from a different person in a random order to avoid biased erroneous measurement and averaged in a single value for each animal. A pairwise comparison was made where each value represented a single value from a single antibody‐stained brain slice from a single animal.
\",\n \"Cerebral Blood Volume Measurement\",\n \"
Craniotomies were performed on isoflurane‐anesthetized rats secured in a custom‐made stereotaxic frame. The cranial window region was created above the left ATN (A = −1.5 mm, L = −1.5 mm), such that the rtFUS stimulus could be applied on the right ATN while simultaneously imaging the cranial window (Figure ##SUPPL##0##S2C,D##, Supporting Information). After an overnight recovery, urethane (1.25 mg kg−1 body weight) was injected intraperitoneally for wide‐field optical imaging.
\",\n \"
Optical measurement was conducted for the normal control, PTZ‐control, and tFUS‐stimulated groups to examine the regional hemodynamic response of cerebral blood volume (CBV). The imaging system consisted of a macro‐zoom microscope (Olympus, MVX10), an sCMOS camera (Andor, Zyla 5.5), and a light‐emitting diode source (Figure ##SUPPL##0##S2C##, Supporting Information). For the experiment, raw reflectance data were collected under green (530 nm) light, as 530 nm is primarily sensitive to changes in the local total blood volume, denoted as the HbT.[\\n##REF##31834545##\\n47\\n##, ##UREF##16##\\n48\\n##\\n] Each imaging trial was recorded for 30 min, including a 2 min baseline (pre‐PTZ/tFUS).
\",\n \"Microdialysis\",\n \"
Microdialysis analysis was conducted to determine the amount of neurotransmitter secreted during the alterations in epileptiform activities generated by the rtFUS transmission sequences (Figure ##SUPPL##0##S2A##, Supporting Information) to demonstrate the cause of bidirectional modulation in animal models of drug‐induced epilepsy. The guide cannula was implanted in twenty male Sprague‐Dawley rats weighing 300–320 g (Koatech, Inc.) under respiratory anesthesia using a hand drill at the target coordinates of the hippocampal region CA2/CA3 (Coordinates −3.80 mm posterior (AP), −4.40 mm lateral (ML), and −2.20 mm ventral (DV), 26 degrees lateral) (Figure ##SUPPL##0##S2B##, Supporting Information). After the guide cannula reached the target coordinates, dental cement was applied for fixation, avoiding the position of the ultrasound transducer, and the incision site was sutured with a silk suture for the recovery period. For proteomic analysis, five rats were chosen for the PTZ‐control group (PTZ+/tFUS‐) and five rats were chosen for each experimental group: TXL_40_40, TXL_40_20, and TX_HYBRID.
\",\n \"
Two days after the guide cannula insertion surgery, the recovered rats were intraperitoneally anesthetized with urethane (1.25 g kg−1 body weight in 1.25 g 4 mL−1 saline; Sigma–Aldrich, U‐2500‐500G). After the anesthetized rats were secured to the stereotaxic frame, the dummy was removed from the guide cannula and a CMA12 1‐mm membrane probe (CMA Microdialysis) was inserted. A 5 mL glass syringe filled with aCSF (M dialysis AB) was mounted on a syringe pump (KD Scientific) and connected to the probe inlet. The tubing was connected to the probe outlet to collect the interstitial fluid (ISF) in a sample collector (BASi, U.S.A). After ≈1 h of stabilization (1 point 8 min−1), Interstitial fluid (ISF) was collected by sampling for 1 h. For microdialysis, samples were collected for 8 min at a flow rate of 1.3 µL min−1 in order to obtain a sufficient amount (minimum 10 µL) of ISF for the neurotransmitter analysis. The sample collection time was minimized to match the sonication duration (440 s/340 s), but due to the fixed flow rate and the minimum required collection volume, the sample collection time was inevitably longer than the sonication duration. Therefore, a portion of the baseline (40 s for TXL_40_40 and 140 s for TXL_40_20) was collected together when analyzing the neurotransmitter concentration during the rtFUS. All samples were stored in a freezer at −80 °C.
\",\n \"
Neurotransmitter analysis of the microdialysis samples was conducted using LC‐MS/MS in NeuroVIS (Chungcheongnam‐do, Republic of Korea). Table\\n##TAB##1##\\n2\\n## presents the analytical conditions of the LC‐MS/MS instrument. To separate GABA and glutamate in the LC‐MS/MS system composed of ExionLC Series UHPLC, AB SCIEX Triple Quadrupole 6500+, and ESI (SCIEX, Framingham), an ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 µm, Waters, Milford, MA, USA) at 50 °C was used. The mobile phase consisted of 1) water containing 0.1% FA and 5 mm ammonium formate and 2) a 1:1 mixture of methanol and acetonitrile containing 5 mm ammonium formate. The flow rate was adjusted at 0.3 mL min−1, and a 7 µL per sample was injected in LC‐MS/MS. Multiple reaction monitoring (MRM) was performed in the positive mode for each NTs. The experiment was conducted at an ion transfer temperature of 500 °C, and a positive ion spray voltage of 5000 V.
\",\n \"Statistical Analyses\",\n \"
All experimental data were analyzed using statistics software (IBM, SPSS Statistics 27). In order to determine whether the data from each group follows a normal distribution, the Shapiro‐Wilk test[\\n##UREF##17##\\n49\\n##\\n] was performed. Once the normality was confirmed, the statistical significance of the difference between a sonicated group and the PTZ‐control group was assessed by t‐test. For the t‐test, a paired t‐test was employed in case the data were compared by time within the same group. If the distribution of the data departed significantly from the normality, a non‐parametric Mann–Whitney U test was performed to compare the significance of the difference. The type of test used for the statistical analysis was presented along with the p‐values throughout the paper. The purpose of experiments in this paper was to find either upregulating or downregulating effects for each stimulation sequence independently by comparing the effect with the control group. The neuromodulation effect for each stimulation sequence was only observed from a rat group of PTZ‐control that did not receive other stimulation sequences. Therefore, two‐sided Mann–Whitney U test was employed without considering multiple comparison corrections. p <0.05 was considered statistically significant and was marked as ** p <0.10 was considered trending toward statistical significance and was marked as *p > 0.10 was considered non‐significant and was marked as NS throughout the paper. Although p < 0.05 was the typical standard significance threshold in the general scientific community, the differences p <0.10 were also provisioned on the figures as providing the information may still identify potential trends – especially considering the small sample sizes – and suggest a potentially meaningful relationship that warrants further study.[\\n##UREF##18##\\n50\\n##\\n] Effects with significance of p <0.10 were termed as “mild” throughout the paper. The sample sizes were determined by using the resource equation method.[\\n##UREF##19##\\n51\\n##, ##REF##24250214##\\n52\\n##\\n]\\n
T.C. and M.K. contributed equally to this work. This work was supported in part by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MIST) NRF‐2020R1A2C2011808 (J.P.), NRF‐2021R1A4A1028713 (J.P.), and NRF‐2019M3C1B8090805 (J.P.). Also, this research was partially supported by the K‐Brain Project of National Research Foundation (NRF) funded by the Korean government (MIST) (RS‐2023‐00265524) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HR21C0885)
","Data Availability Statement","
The data that support the findings of this study are available from the corresponding author upon reasonable request.
"],"string":"[\n \"Acknowledgements\",\n \"
T.C. and M.K. contributed equally to this work. This work was supported in part by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MIST) NRF‐2020R1A2C2011808 (J.P.), NRF‐2021R1A4A1028713 (J.P.), and NRF‐2019M3C1B8090805 (J.P.). Also, this research was partially supported by the K‐Brain Project of National Research Foundation (NRF) funded by the Korean government (MIST) (RS‐2023‐00265524) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HR21C0885)
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available from the corresponding author upon reasonable request.
\"\n]"},"figure":{"kind":"list like","value":["
Sonication and EEG recording protocol. A) Summary of the overall experimental protocols for sonication and EEG recording. B) Detailed timeline of rtFUS stimulation and EEG recording for all transmit condition groups. TXH_0.25_30 is a sequence repeating eleven times of 1 MPa, 0.25 s bursts with 30 s intervals. TXL_40_40 is a sequence repeating six times of 0.25 MPa, 40 s bursts with 40 s intervals. TXL_10_50 is a sequence repeating six times of 0.25 MPa, 10 s bursts with 50 s intervals. TXL_20_40 is a sequence repeating six times of 0.25 MPa, 20 s bursts with 40 s intervals. TXL_40_20 is a sequence repeating six times of 0.25 MPa, 40 s bursts with 20 s intervals. TX_HYBRID is a sequence that consecutively applies TXL_40_40 and TXH_0.25_30 with 3 min resting period in between the two sequences.
","
Changes in epileptiform activities during and after TXH_0.25_30, TXL_40_40, and TXL_40_Single. A) Time courses of EEG signals in the PTZ‐control and stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, \n**\n : p <0.05, * : p <0.10, NS : p> 0.10. p = ① 0.036 ② 0.030 ③ 0.030 ④ 0.045 ⑤ 0.019 ⑥ 0.093.
","
Changes in epileptiform activities during and after TXL_10_50, TXL_20_40, and TXL_40_40. A) Time courses of EEG signals in the PTZ‐control and stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann‐Whitney U test, ** : p <0.05, * : p <0.10, NS : p> 0.10. p = ① 0.030 ② 0.030 ③ 0.045 ④ 0.019.
","
Changes in epileptiform activities during and after TXL_40_20 and TXL_40_40. A) Time courses of EEG signals in the PTZ‐control and stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, ** : p <0.05, * : p <0.10, NS : p> 0.10. p = ① 0.030 ② 0.030 ③ 0.045 ④ 0.019.
","
Changes in epileptiform activities during and after TX_HYBRID (consecutive transmits of TXL_40_40 and TXH_0.25_30). A) Time courses of EEG signals in the PTZ‐control and the stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, ** : p <0.05, * : p <0.10, NS : p> 0.10.
","
Immunohistochemistry results and multitemporal neurotransmitter analysis from microdialysis. A) Representative slice images and quantitative measurements from c‐Fos‐, GAD65‐, Iba1‐, and GFAP‐stained brain tissue at the dentate gyrus. Quantitative measurements were made in the stained cell ratio for c‐Fos and the stain‐positive cell density for GAD65, Iba1, and GFAP (PTZ‐control: n = 6, TX groups: n = 6). B) Changes in the normalized glutamate and GABA concentration (%) during and after TXL_40_40, TX_40_20 and TX_HYBRID compared to the baseline (PTZ‐control: n = 8, TX groups: n = 5). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, ** : p <0.05, * : p < 0.10, NS : p> 0.10. p = ① 0.002 ② 0.002 ③ 0.093 ④ 0.004 ⑤ 0.004 ⑥ 0.002 ⑦ 0.015 ⑧ 0.002 ⑨ 0.002 ⑩ 0.002 ⑪ 0.002 ⑫ 0.009 ⑬ 0.009 ⑭ 0.002 ⑮ 0.004 ⑯ 0.041 ⑰ 0.002 ⑱ 0.048 ⑲ 0.073 ⑳ 0.062 ㉑ 0.088 ㉒ 0.073 ㉓ 0.003 ㉔ 0.030 ㉕ 0.018.
"],"string":"[\n \"
Sonication and EEG recording protocol. A) Summary of the overall experimental protocols for sonication and EEG recording. B) Detailed timeline of rtFUS stimulation and EEG recording for all transmit condition groups. TXH_0.25_30 is a sequence repeating eleven times of 1 MPa, 0.25 s bursts with 30 s intervals. TXL_40_40 is a sequence repeating six times of 0.25 MPa, 40 s bursts with 40 s intervals. TXL_10_50 is a sequence repeating six times of 0.25 MPa, 10 s bursts with 50 s intervals. TXL_20_40 is a sequence repeating six times of 0.25 MPa, 20 s bursts with 40 s intervals. TXL_40_20 is a sequence repeating six times of 0.25 MPa, 40 s bursts with 20 s intervals. TX_HYBRID is a sequence that consecutively applies TXL_40_40 and TXH_0.25_30 with 3 min resting period in between the two sequences.
\",\n \"
Changes in epileptiform activities during and after TXH_0.25_30, TXL_40_40, and TXL_40_Single. A) Time courses of EEG signals in the PTZ‐control and stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, \\n**\\n : p <0.05, * : p <0.10, NS : p> 0.10. p = ① 0.036 ② 0.030 ③ 0.030 ④ 0.045 ⑤ 0.019 ⑥ 0.093.
\",\n \"
Changes in epileptiform activities during and after TXL_10_50, TXL_20_40, and TXL_40_40. A) Time courses of EEG signals in the PTZ‐control and stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann‐Whitney U test, ** : p <0.05, * : p <0.10, NS : p> 0.10. p = ① 0.030 ② 0.030 ③ 0.045 ④ 0.019.
\",\n \"
Changes in epileptiform activities during and after TXL_40_20 and TXL_40_40. A) Time courses of EEG signals in the PTZ‐control and stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, ** : p <0.05, * : p <0.10, NS : p> 0.10. p = ① 0.030 ② 0.030 ③ 0.045 ④ 0.019.
\",\n \"
Changes in epileptiform activities during and after TX_HYBRID (consecutive transmits of TXL_40_40 and TXH_0.25_30). A) Time courses of EEG signals in the PTZ‐control and the stimulated experimental groups. B) Time courses of the number of epileptic spikes ratio in each group (PTZ‐control: n = 8, TX groups: n = 5). C) Ratio of the number of epileptic spikes measured at time periods of 0–10 min (10AS) and 10–20 min (20AS) (PTZ‐control: n = 8, TX groups: n = 5). D) Power spectral density ratio of EEG signals at the delta (0.5–3 Hz) and theta (4–7 Hz) frequency bands (PTZ‐control: n = 8, TX groups: n = 5). E) Changes in the cerebral blood volume [Hb] calculated from 530 nm reflectance data (PTZ‐control: n = 6, TX groups: n = 6). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, ** : p <0.05, * : p <0.10, NS : p> 0.10.
\",\n \"
Immunohistochemistry results and multitemporal neurotransmitter analysis from microdialysis. A) Representative slice images and quantitative measurements from c‐Fos‐, GAD65‐, Iba1‐, and GFAP‐stained brain tissue at the dentate gyrus. Quantitative measurements were made in the stained cell ratio for c‐Fos and the stain‐positive cell density for GAD65, Iba1, and GFAP (PTZ‐control: n = 6, TX groups: n = 6). B) Changes in the normalized glutamate and GABA concentration (%) during and after TXL_40_40, TX_40_20 and TX_HYBRID compared to the baseline (PTZ‐control: n = 8, TX groups: n = 5). Data presented as mean ± SEM, p‐values calculated via two‐tailed Mann–Whitney U test, ** : p <0.05, * : p < 0.10, NS : p> 0.10. p = ① 0.002 ② 0.002 ③ 0.093 ④ 0.004 ⑤ 0.004 ⑥ 0.002 ⑦ 0.015 ⑧ 0.002 ⑨ 0.002 ⑩ 0.002 ⑪ 0.002 ⑫ 0.009 ⑬ 0.009 ⑭ 0.002 ⑮ 0.004 ⑯ 0.041 ⑰ 0.002 ⑱ 0.048 ⑲ 0.073 ⑳ 0.062 ㉑ 0.088 ㉒ 0.073 ㉓ 0.003 ㉔ 0.030 ㉕ 0.018.
\"\n]"},"table":{"kind":"list like","value":["
Animal Allocation for Each Experimental Group and Studies.
Normal Control
PTZ‐Control
TXH_0.25_30
TXL_10_50
TXL_20_40
TXL_40_40
TXL_40_20
TX_HYBRID
EEG/IHC\na)\n\n
n = 0/6
n = 8/6
n = 5/6
n = 5/6
n = 5/6
n = 5/6
n = 5/6
n = 5/6
CBV
n = 6
n = 6
n = 6
n = 6
‐
n = 6
n = 6
n = 6
Microdialysis
‐
n = 8
‐
‐
‐
n = 5
n = 5
n = 5
John Wiley & Sons, Ltd.","
MRM transitions, retention times, and other conditions of GABA and glutamate.
Analyte
Q1 Mass (m/z)
Q3 Mass (m/z)
CE (V)
CXP (V)
Retention time (min)
Ionization polarity
GABA
104
87
15
10
0.97
Positive
Glutamate
148
84
25
10
0.98
Positive
John Wiley & Sons, Ltd."],"string":"[\n \"
Animal Allocation for Each Experimental Group and Studies.
Normal Control
PTZ‐Control
TXH_0.25_30
TXL_10_50
TXL_20_40
TXL_40_40
TXL_40_20
TX_HYBRID
EEG/IHC\\na)\\n\\n
n = 0/6
n = 8/6
n = 5/6
n = 5/6
n = 5/6
n = 5/6
n = 5/6
n = 5/6
CBV
n = 6
n = 6
n = 6
n = 6
‐
n = 6
n = 6
n = 6
Microdialysis
‐
n = 8
‐
‐
‐
n = 5
n = 5
n = 5
John Wiley & Sons, Ltd.\",\n \"
MRM transitions, retention times, and other conditions of GABA and glutamate.
Analyte
Q1 Mass (m/z)
Q3 Mass (m/z)
CE (V)
CXP (V)
Retention time (min)
Ionization polarity
GABA
104
87
15
10
0.97
Positive
Glutamate
148
84
25
10
0.98
Positive
John Wiley & Sons, Ltd.\"\n]"},"formula":{"kind":"list like","value":[],"string":"[]"},"box":{"kind":"list like","value":[""],"string":"[\n \"\"\n]"},"code":{"kind":"list like","value":[],"string":"[]"},"quote":{"kind":"list like","value":[],"string":"[]"},"chemical":{"kind":"list like","value":[],"string":"[]"},"supplementary":{"kind":"list like","value":["
Supporting Information
","
Supplemental Video 1
","
Supplemental Video 2
","
Supplemental Video 3
","
Supplemental Video 4
","
Supplemental Video 5
","
Supplemental Video 6
","
Supplemental Video 7
"],"string":"[\n \"
Supporting Information
\",\n \"
Supplemental Video 1
\",\n \"
Supplemental Video 2
\",\n \"
Supplemental Video 3
\",\n \"
Supplemental Video 4
\",\n \"
Supplemental Video 5
\",\n \"
Supplemental Video 6
\",\n \"
Supplemental Video 7
\"\n]"},"footnote":{"kind":"list like","value":["
Immunohistochemical analyses were performed with n = 6 for each staining method. Additionally, the safety of focused ultrasound stimulation was evaluated in the PTZ‐/rtFUS+ group (n = 3), where TXH_0.25_30 was applied to the left hemisphere and TXL_40_40 was applied to the right hemisphere (Figure ##SUPPL##0##S4##, Supporting Information).
"],"string":"[\n \"
Immunohistochemical analyses were performed with n = 6 for each staining method. Additionally, the safety of focused ultrasound stimulation was evaluated in the PTZ‐/rtFUS+ group (n = 3), where TXH_0.25_30 was applied to the left hemisphere and TXL_40_40 was applied to the right hemisphere (Figure ##SUPPL##0##S4##, Supporting Information).
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, with high mortality rate and poor prognosis.[\n##REF##30970190##\n1\n##, ##REF##33479224##\n2\n##\n] The five‐year overall survival rate exceeds 70% for early‐stage HCC, while the median survival of advanced (metastatic) HCC patients is ≈1.5 years.[\n##REF##33479224##\n2\n##\n] Metastatic HCC is less effective for surgical treatment and more resistant to drug therapy.[\n##REF##32017145##\n3\n##, ##REF##24700742##\n4\n##\n] Metastasis is a pivotal process, frequently accompanied by dynamic metabolic alterations.[\n##REF##35821160##\n5\n##, ##REF##33899000##\n6\n##\n] During the progress of metastasis, cancer cells utilize multiple mechanisms including lipid accumulation to favor cancer cell survival and provide energy to distant metastasis.[\n##REF##33462499##\n7\n##\n] Lipid metabolism reprogramming is actually another cancer hallmark, which is a complex cascade controlled by various factors, and AMP‐activated protein kinase (AMPK) and acetyl‐CoA carboxylase (ACC) are two key regulators among them.[\n##REF##35022204##\n8\n##, ##UREF##0##\n9\n##, ##REF##23446547##\n10\n##\n] AMPK is a metabolic sensor in mammals activated by the increased cellular ratio of AMP/ATP.[\n##REF##36316383##\n11\n##\n] ACC is the rate‐limiting enzyme for fatty acid synthesis, and can be inhibited by AMPK.[\n##UREF##0##\n9\n##, ##REF##36316383##\n11\n##, ##REF##30244972##\n12\n##\n]\n
","
Circular RNAs (circRNAs) are a class of endogenous RNA transcripts, generated by back‐splicing from linear precursor RNAs or other RNA circularization mechanisms.[\n##REF##34865956##\n13\n##, ##REF##31395983##\n14\n##, ##REF##31973951##\n15\n##, ##REF##32048164##\n16\n##, ##REF##25664725##\n17\n##\n] Most circRNAs localize to the cytoplasm in an Exportin 4 dependent mechanism,[\n##REF##36182935##\n18\n##\n] and cytoplasmic circRNAs mainly function through sponging miRNAs, modulating RNA binding proteins (RBPs), and serving as translation templates.[\n##REF##34865956##\n13\n##, ##REF##23446348##\n19\n##, ##UREF##1##\n20\n##, ##REF##31619685##\n21\n##, ##REF##33664496##\n22\n##, ##REF##31027518##\n23\n##\n] Compiling studies have been carried out to investigate the molecular and cellular functions of circRNAs, although only a handful of circRNAs have been studied for the physiological roles with genetically manipulated mice, in which the expression of the corresponding circRNAs is missing or at a low level.[\n##REF##34865956##\n13\n##, ##REF##31395983##\n14\n##, ##REF##31973951##\n15\n##\n] The mammalian conserved circRNA CDR1as impacts brain development and function by sequestering miR‐7.[\n##REF##23446348##\n19\n##, ##UREF##1##\n20\n##\n]\nCircBoule, a circRNA conserved in metazoan, plays roles in the antistress reaction of sperm by regulating the stability of heat shock proteins.[\n##UREF##2##\n24\n##\n]\nCia‐cGAS regulates the differentiation of hematopoietic stem cells by binding and inhibiting cGAS in bone marrow.[\n##REF##29625897##\n25\n##\n]\n
","
In cancers, an array of circRNAs have been shown to play regulatory roles.[\n##REF##34912049##\n26\n##, ##UREF##3##\n27\n##\n] For example, circNSUN2 interacts with IGF2BP2 to form a circNSUN2‐IGF2BP2‐HMGA2 ternary complex to promote liver metastasis of colorectal cancer.[\n##REF##31619685##\n21\n##\n] The circRNA FECR1 induces extensive DNA demethylation in the FLI1 genomic locus, thereby enhancing its expression to promote metastasis of breast cancer.[\n##REF##30537986##\n28\n##\n]\nCircTP63 upregulates FOXM1 by competitively binding to miR‐873‐3p to facilitate cell cycle progression in lung squamous cell carcinoma.[\n##REF##31324812##\n29\n##\n] Multiple circRNAs have also been reported to play roles in HCC.[\n##REF##31027518##\n23\n##, ##REF##28520103##\n30\n##, ##UREF##4##\n31\n##\n] For example, circMTO1 suppresses HCC progression by serving as an miR‐9 sponge to promote the expression of p21.[\n##REF##28520103##\n30\n##\n]\nCircPABPC1 represses both intrahepatic and distant metastases in HCC through the degradation of ITGB1 in a ubiquitination‐independent manner.[\n##UREF##4##\n31\n##\n]\nCircβ‐catenin promotes the HCC cell growth and metastasis via encoding a small protein, which stabilizes the full‐length β‐catenin to activate the Wnt pathway.[\n##REF##31027518##\n23\n##\n] CircRNAs can participate in HCC in multiple ways,[\n##REF##31027518##\n23\n##, ##REF##28520103##\n30\n##, ##UREF##4##\n31\n##\n] although there is so far no circRNA identified to regulate HCC metastasis by modulating lipid metabolism.
","
Most of the studies in cancer circRNAs have been carried out with tumor cell lines, with associated clinical specimens and data, and sometimes with nude mice models.[\n##REF##34912049##\n26\n##, ##UREF##3##\n27\n##, ##UREF##5##\n32\n##\n] Lots of circRNAs with identified functions in cancer are not conserved between human and mice, which in some way hampers in‐depth investigations for roles of circRNAs and the underlying mechanisms with animal models. Identification of conserved circRNAs with critical roles in HCC yields meaningful insights into potential biomarkers and therapeutic targets.
","
In an effort to explore the roles of circRNAs in HCC metastasis, we have identified a mammalian conserved circRNA circLARP1B. With a series of bioinformatics, molecular, biochemical, and cellular analyses, and very importantly by using genetically modified mice, we have provided data to support roles of circLARP1B in HCC. We have uncovered that circLARP1B disturbs heterogeneous nuclear ribonucleoprotein D (HNRNPD) from the binding to 3′ UTR of liver kinase B1 (LKB1), which leads to destabilizing LKB1 mRNA and then modulating a pathway pivotal to HCC metastasis and lipid metabolism.
"],"string":"[\n \"Introduction\",\n \"
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, with high mortality rate and poor prognosis.[\\n##REF##30970190##\\n1\\n##, ##REF##33479224##\\n2\\n##\\n] The five‐year overall survival rate exceeds 70% for early‐stage HCC, while the median survival of advanced (metastatic) HCC patients is ≈1.5 years.[\\n##REF##33479224##\\n2\\n##\\n] Metastatic HCC is less effective for surgical treatment and more resistant to drug therapy.[\\n##REF##32017145##\\n3\\n##, ##REF##24700742##\\n4\\n##\\n] Metastasis is a pivotal process, frequently accompanied by dynamic metabolic alterations.[\\n##REF##35821160##\\n5\\n##, ##REF##33899000##\\n6\\n##\\n] During the progress of metastasis, cancer cells utilize multiple mechanisms including lipid accumulation to favor cancer cell survival and provide energy to distant metastasis.[\\n##REF##33462499##\\n7\\n##\\n] Lipid metabolism reprogramming is actually another cancer hallmark, which is a complex cascade controlled by various factors, and AMP‐activated protein kinase (AMPK) and acetyl‐CoA carboxylase (ACC) are two key regulators among them.[\\n##REF##35022204##\\n8\\n##, ##UREF##0##\\n9\\n##, ##REF##23446547##\\n10\\n##\\n] AMPK is a metabolic sensor in mammals activated by the increased cellular ratio of AMP/ATP.[\\n##REF##36316383##\\n11\\n##\\n] ACC is the rate‐limiting enzyme for fatty acid synthesis, and can be inhibited by AMPK.[\\n##UREF##0##\\n9\\n##, ##REF##36316383##\\n11\\n##, ##REF##30244972##\\n12\\n##\\n]\\n
\",\n \"
Circular RNAs (circRNAs) are a class of endogenous RNA transcripts, generated by back‐splicing from linear precursor RNAs or other RNA circularization mechanisms.[\\n##REF##34865956##\\n13\\n##, ##REF##31395983##\\n14\\n##, ##REF##31973951##\\n15\\n##, ##REF##32048164##\\n16\\n##, ##REF##25664725##\\n17\\n##\\n] Most circRNAs localize to the cytoplasm in an Exportin 4 dependent mechanism,[\\n##REF##36182935##\\n18\\n##\\n] and cytoplasmic circRNAs mainly function through sponging miRNAs, modulating RNA binding proteins (RBPs), and serving as translation templates.[\\n##REF##34865956##\\n13\\n##, ##REF##23446348##\\n19\\n##, ##UREF##1##\\n20\\n##, ##REF##31619685##\\n21\\n##, ##REF##33664496##\\n22\\n##, ##REF##31027518##\\n23\\n##\\n] Compiling studies have been carried out to investigate the molecular and cellular functions of circRNAs, although only a handful of circRNAs have been studied for the physiological roles with genetically manipulated mice, in which the expression of the corresponding circRNAs is missing or at a low level.[\\n##REF##34865956##\\n13\\n##, ##REF##31395983##\\n14\\n##, ##REF##31973951##\\n15\\n##\\n] The mammalian conserved circRNA CDR1as impacts brain development and function by sequestering miR‐7.[\\n##REF##23446348##\\n19\\n##, ##UREF##1##\\n20\\n##\\n]\\nCircBoule, a circRNA conserved in metazoan, plays roles in the antistress reaction of sperm by regulating the stability of heat shock proteins.[\\n##UREF##2##\\n24\\n##\\n]\\nCia‐cGAS regulates the differentiation of hematopoietic stem cells by binding and inhibiting cGAS in bone marrow.[\\n##REF##29625897##\\n25\\n##\\n]\\n
\",\n \"
In cancers, an array of circRNAs have been shown to play regulatory roles.[\\n##REF##34912049##\\n26\\n##, ##UREF##3##\\n27\\n##\\n] For example, circNSUN2 interacts with IGF2BP2 to form a circNSUN2‐IGF2BP2‐HMGA2 ternary complex to promote liver metastasis of colorectal cancer.[\\n##REF##31619685##\\n21\\n##\\n] The circRNA FECR1 induces extensive DNA demethylation in the FLI1 genomic locus, thereby enhancing its expression to promote metastasis of breast cancer.[\\n##REF##30537986##\\n28\\n##\\n]\\nCircTP63 upregulates FOXM1 by competitively binding to miR‐873‐3p to facilitate cell cycle progression in lung squamous cell carcinoma.[\\n##REF##31324812##\\n29\\n##\\n] Multiple circRNAs have also been reported to play roles in HCC.[\\n##REF##31027518##\\n23\\n##, ##REF##28520103##\\n30\\n##, ##UREF##4##\\n31\\n##\\n] For example, circMTO1 suppresses HCC progression by serving as an miR‐9 sponge to promote the expression of p21.[\\n##REF##28520103##\\n30\\n##\\n]\\nCircPABPC1 represses both intrahepatic and distant metastases in HCC through the degradation of ITGB1 in a ubiquitination‐independent manner.[\\n##UREF##4##\\n31\\n##\\n]\\nCircβ‐catenin promotes the HCC cell growth and metastasis via encoding a small protein, which stabilizes the full‐length β‐catenin to activate the Wnt pathway.[\\n##REF##31027518##\\n23\\n##\\n] CircRNAs can participate in HCC in multiple ways,[\\n##REF##31027518##\\n23\\n##, ##REF##28520103##\\n30\\n##, ##UREF##4##\\n31\\n##\\n] although there is so far no circRNA identified to regulate HCC metastasis by modulating lipid metabolism.
\",\n \"
Most of the studies in cancer circRNAs have been carried out with tumor cell lines, with associated clinical specimens and data, and sometimes with nude mice models.[\\n##REF##34912049##\\n26\\n##, ##UREF##3##\\n27\\n##, ##UREF##5##\\n32\\n##\\n] Lots of circRNAs with identified functions in cancer are not conserved between human and mice, which in some way hampers in‐depth investigations for roles of circRNAs and the underlying mechanisms with animal models. Identification of conserved circRNAs with critical roles in HCC yields meaningful insights into potential biomarkers and therapeutic targets.
\",\n \"
In an effort to explore the roles of circRNAs in HCC metastasis, we have identified a mammalian conserved circRNA circLARP1B. With a series of bioinformatics, molecular, biochemical, and cellular analyses, and very importantly by using genetically modified mice, we have provided data to support roles of circLARP1B in HCC. We have uncovered that circLARP1B disturbs heterogeneous nuclear ribonucleoprotein D (HNRNPD) from the binding to 3′ UTR of liver kinase B1 (LKB1), which leads to destabilizing LKB1 mRNA and then modulating a pathway pivotal to HCC metastasis and lipid metabolism.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results","\n<italic toggle=\"yes\">CircLARP1B</italic> as a Mammalian Conserved CircRNA Is Identified in HCC Metastasis","
In order to investigate the functional roles of conserved circRNAs in HCC, we performed ribosomal RNA depleted RNA‐sequencing (RNA‐seq) of six HCC specimens, among which three HCC patients were metastatic and the other three were nonmetastatic (Figure ##FIG##0##\n1a##). 4614 circRNAs with back‐splicing junction (BSJ) reads >2 were detected, and 48 of them were with over fourfold changes in expression levels between the two groups (Figure ##FIG##0##1a##; Table ##SUPPL##1##S1##, Supporting Information). 38 of these circRNAs were with lower levels, and 10 out of the 48 were with higher levels in metastatic HCC (Figure ##FIG##0##1a##). Then we aligned the 48 circRNA sequences to annotated murine circRNAs from the circAtlas database to identify conserved circRNAs with a stringent criterion (full‐length and BSJ sequences >75% identical).[\n##REF##32345360##\n33\n##\n]\nCircLARP1B was the most differentially expressed and also the only metastasis upregulated circRNA among the five conserved circRNAs identified (Figure ##FIG##0##1a##). CircLARP1B is highly conserved with ≈86% sequence identity between human and mice, and consists of exons 2–4 from the La ribonucleoprotein 1B (LARP1B) gene, with a deduced size of 294 nt in both human and mice (Figure ##FIG##0##1b##; Figure ##SUPPL##0##S1a##, Supporting Information). CircLARP1B and LARP1B mRNA exhibited distinct expression patterns in human tissues analyzed from the NCBI and circAtlas databases; for example, LARP1B mRNA but not circLARP1B was expressed in thyroid tissue (Figure ##SUPPL##0##S1b##, Supporting Information). LARP1B itself is an RBP that regulates the translation of certain mRNAs.[\n##REF##28650797##\n34\n##\n] The overall survival rate showed no significant difference between the two groups of HCC patients with higher LARP1B expression and lower expression (Figure ##SUPPL##0##S1c##, Supporting Information), indicating that LARP1B mRNA may not be a key regulator in HCC. The circLARP1B BSJ was validated by PCR amplification and confirmed by Sanger sequencing in a human HCC cell line PLC and mouse liver, to verify the circular nature of circLARP1B (Figure ##SUPPL##0##S1d##, Supporting Information). Then, the actinomycin D chase assay revealed that circLARP1B was stable compared to LARP1B mRNA (Figure ##SUPPL##0##S1e##, Supporting Information). By Northern blotting, bands corresponding to the deduced size of circLARP1B were detected, and the estimated copy numbers per cell in two HCC cell lines PLC and HepG2 were ≈136 and ≈158, respectively (Figure ##FIG##0##1b##; Figure ##SUPPL##0##S1f##, Supporting Information). Copy numbers of more than 100 are high for circRNAs or even for mRNAs. Single molecular fluorescence in situ hybridization (smFISH) of circLARP1B in PLC cells revealed that this circRNA localized predominately in the cytoplasm (Figure ##FIG##0##1c##; Figure ##SUPPL##0##S1g,h##, Supporting Information). The specificity of smFISH to circLARP1B was demonstrated with significantly decreased smFISH signals upon shcircLARP1B knockdown of the circRNA (Figure ##SUPPL##0##S1i–k##, Supporting Information). With a thyroid cell line (K1 cells), which exhibited considerable LARP1B mRNA levels but no circLARP1B expression, the specificity of smFISH to circLARP1B or LARP1B mRNA was further proofed (Figure ##SUPPL##0##S1l##, Supporting Information). More than 85% of circLARP1B were found to be cytoplasmic in one murine HCC cell line Hepa1‐6 and the two human HCC cell lines (Figure ##FIG##0##1d##). Furthermore, smFISH of circLARP1B in mouse liver also showed cytoplasmic enrichment (Figure ##FIG##0##1e##).
","
Higher circLARP1B levels in metastatic compared to nonmetastatic HCC specimens were revealed by smFISH analysis (Figure ##FIG##0##1f##). We collected HCC specimens from 90 patients, and found that higher circLARP1B levels were associated with 23 metastatic specimens as against the 67 nonmetastatic specimens (Figure ##FIG##0##1g##). Except 5 specimens without information of prognostic stage, these clinical samples could be divided into 65 specimens with prognostic TNM stage I–II and 20 specimens with TNM stage III–IV, and higher circLARP1B levels were significantly correlated with advanced TNM stages (III–IV) (Figure ##FIG##0##1h##). HCC patients with higher circLARP1B levels in tumors had significantly lower five‐year overall survival (OS) and disease‐free survival (DFS) rates (Figure ##FIG##0##1i,j##).
","
Collectively, these results demonstrated that circLARP1B as a mammalian conserved and highly expressed circRNA might play promoting roles in HCC metastasis as supported by data from clinical specimens.
","\n<italic toggle=\"yes\">CircLARP1B</italic> Regulates Cell Invasion and Lipid Accumulation via Fatty Acid Synthesis","
To explore the cellular functions of circLARP1B, we applied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to generate circLARP1B deficient (circLARP1B‐Def) PLC cells (Figure ##FIG##1##\n2a##). Flanking introns of circularized exons contain Alu elements and B1 repeats in human and mice, respectively, which both belong to reverse complementary sequences, known to facilitate circRNA biogenesis (Figure ##SUPPL##0##S2a##, Supporting Information).[\n##REF##34865956##\n13\n##, ##REF##33514913##\n35\n##\n] To generate circLARP1B‐Def PLC cells, repeat sequences including four proximal Alu elements in the fourth intron of human LARP1B were deleted, but functional intronic elements such as the splice sites and pyrimidine tract remained unaltered (Figure ##FIG##1##2a##). The resulted circLARP1B‐Def PLC cells exhibited significantly decreased circLARP1B levels with no alteration in LARP1B mRNA levels, compared to the wildtype (WT) PLC cells (Figure ##FIG##1##2b##).
","
CircLARP1B‐Def PLC cells compared to WT cells showed significantly reduced invasion, when examined with Transwell invasion assay (Figure ##FIG##1##2c##). No significant difference in growth curve and colony formation was observed between the circLARP1B‐Def and WT cells (Figure ##SUPPL##0##S2b–d##, Supporting Information). Invasion ability is directly related to metastasis, and growth curve and colony formation are associated with cell growth and proliferation.
","
We then assessed the potential metabolic alterations in circLARP1B‐Def PLC cells by liquid chromatography‐mass spectrometry (LC‐MS) untargeted metabolomics (Figure ##FIG##1##2d##). A total of 366 metabolites and lipid classes (including 44 lipid classes) were detected, and 105 of them, 42 increased and 63 decreased, were significantly dysregulated (variable importance in the projection, VIP >1; P‐value <0.05) upon circLARP1B deficiency in PLC cells (Figure ##SUPPL##0##S2e##; Table ##SUPPL##2##S2##, Supporting Information). KEGG pathway analysis of these dysregulated metabolites revealed that lipid metabolism, among the 6 metabolic pathways significantly affected, was perturbed the most (Figure ##FIG##1##2e##). For the 24 lipid classes enriched in lipid metabolism of KEGG analysis, 18 ones were decreased, and 6 were increased in circLARP1B‐Def cells (Figure ##SUPPL##0##S2e##, Supporting Information). We then performed lipidomics to examine lipid class composition, and identified 2709 lipid species that compose the 24 dysregulated lipid classes detected in the metabolomics. 273 lipid species covering all these 24 lipid classes were significantly changed upon circLARP1B deficiency (Figure ##FIG##1##2f##; Table ##SUPPL##2##S2##, Supporting Information). Components of lipid droplets (LDs) including lipid classes such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), diglyceride (DG), triglyceride (TG), and phosphatidylinositol (PI) were among the metabolites with lower levels in circLARP1B‐Def cells (Figure ##SUPPL##0##S2e##, Supporting Information).[\n##REF##24220094##\n36\n##\n] LDs examined by Nile red staining were markedly decreased in circLARP1B‐Def cells compared to WT cells (Figure ##FIG##1##2g##).
","
To further explore the mechanism of lipid accumulation promoted by circLARP1B, we performed metabolic labeling with 13C‐labeled targeted metabolic flux analysis to assess the incorporation of 13C‐labeled glucose and glutamine into lipid in circLARP1B‐Def and WT PLC cells (Figure ##FIG##1##2h,i##).[\n##REF##32101748##\n37\n##\n] Targeted metabolic flux analyses were performed, and we totally detected 17 kinds of fatty acids with 13C labeling, and found that all of them were significantly reduced in circLARP1B‐Def cells (Figure ##FIG##1##2h##; Table ##SUPPL##3##S3##, Supporting Information), indicating a promoting effect of circLARP1B in fatty acid synthesis (FAS). Glucose and glutamine are first converted to two‐carbon acetyl‐CoA, which serves as a substrate for ACC1 enzyme in FAS, and 13C‐labeled two‐carbon units from 13C‐glucose or 13C‐glutamine into palmitate can thus be examined to reveal FAS from glucose or glutamine. As expected, the distributions of 13C‐labeled palmitate isotopologs (M12, M14, and M16) in circLARP1B‐Def cells, compared to WT cells were significantly lower (Figure ##FIG##1##2i##). We further evaluated the involvement of circLARP1B in fatty acid oxidation (FAO) with oxygen consumption rate (OCR) assays after supplementing cells with exogenous free fatty acids (FFAs; palmitate was supplied). CircLARP1B‐Def cells showed similar OCR compared with WT cells (Figure ##SUPPL##0##S2f##, Supporting Information). Unc‐51 like autophagy activating kinase 1 (ULK1) is a key regulator in lipophagy initiation, and phosphorylation of Ser555 of ULK1 induces lipophagy.[\n##REF##32863214##\n38\n##\n] The ULK1 and phosphorylated ULK1 (p‐ULK1) levels exhibited no significant alternation between circLARP1B‐Def and WT cells (Figure ##FIG##1##2j##). Together, these findings suggested that circLARP1B facilitated lipid accumulation by FAS, not through regulating FAO or lipophagy.
","
The activity of ACC1 is inhibited by phosphorylation of Ser79,[\n##UREF##0##\n9\n##, ##REF##36316383##\n11\n##\n] and we observed that the phosphorylated ACC1 (p‐ACC1) levels were substantially upregulated in circLARP1B‐Def cells (Figure ##FIG##1##2j##). AMPK is the major kinase that catalyzes the phosphorylation of ACC1, and AMPK itself is initially activated by phosphorylation at Thr172.[\n##REF##36316383##\n11\n##\n] The phosphorylated AMPK (p‐AMPK) levels were also significantly higher in circLARP1B‐Def cells than WT cells (Figure ##FIG##1##2j##). Therefore, higher levels of p‐AMPK and then p‐ACC1 would repress lipogenesis and lead to decreased amount of LDs in circLARP1B‐Def cells. AMPK activation also causes a range of metabolites changed in HCC.[\n##REF##28355557##\n39\n##, ##REF##33690219##\n40\n##, ##REF##30468782##\n41\n##\n] From the literature we searched, 12 of circLARP1B‐modulated metabolites have been reported to be regulated by AMPK signaling in HCC,[\n##REF##28355557##\n39\n##, ##REF##33690219##\n40\n##, ##REF##30468782##\n41\n##\n] and 8 of these metabolites exhibit consistent changes between circLARP1B deficiency and AMPK activation (Figure ##SUPPL##0##S2g##, Supporting Information).
","
Additionally, we examined the effects of circLARP1B on the other downstream processes known to be regulated by AMPK, such as the mTOR pathway, angiogenesis, TGF‐β, and mitochondrial activity.[\n##REF##36316383##\n11\n##, ##REF##22576211##\n42\n##, ##REF##31974393##\n43\n##, ##REF##30425336##\n44\n##\n] AMPK activation led to phosphorylation of the mTORC1 subunit RAPTOR to inhibit mTOR pathway,[\n##REF##36316383##\n11\n##\n] and significantly increased phosphorylated RAPTOR (p‐RAPTOR) levels were observed in circLARP1B‐Def cells compared to WT cells (Figure ##SUPPL##0##S2h##, Supporting Information). Once activated, AMPK is reported to increase the protein level of VEGFA (an angiogenesis driver) and inhibit TGF‐β signaling,[\n##REF##36316383##\n11\n##, ##REF##22576211##\n42\n##, ##REF##31974393##\n43\n##\n] and the VEGFA and TGF‐β protein levels did not demonstrate significant change in circLARP1B‐Def cells compared to WT cells (Figure ##SUPPL##0##S2h##, Supporting Information). As another substrate of AMPK, mitochondrial fission factor (MFF) is phosphorylated by AMPK to promote mitochondrial fission,[\n##REF##36316383##\n11\n##\n] and the MFF/p‐MFF protein levels remained unaltered upon circLARP1B deficiency in PLC cells (Figure ##SUPPL##0##S2h##, Supporting Information). Moreover, basal respiration, ATP production, and maximum respiration revealing mitochondrial activity examined by OCR assays demonstrated no significant difference between circLARP1B‐Def cells and WT cells (Figure ##SUPPL##0##S2i##, Supporting Information). Our results demonstrated that both ACC1 and the mTOR signaling directly regulated by AMPK were modulated by circLARP1B through a mechanism awaiting characterization in PLC cells. Both ACC1 and the mTOR signaling are known to promote fatty acid synthesis,[\n##UREF##0##\n9\n##, ##REF##30425336##\n44\n##\n] we focused on examining ACC1/p‐ACC1 as a readout of downstream effects of AMPK, owing to the fact that ACC1 is the rate‐limiting enzyme for FAS.[\n##UREF##0##\n9\n##\n]\n
","
We also manipulated levels of circLARP1B in trans by either RNA interference knockdown or plasmid overexpression, and in human PLC cells or murine Hepa1‐6 cells. ShRNA‐mediated circLARP1B knockdown suppressed cell invasion and formation of LDs, and increased p‐AMPK and p‐ACC1 levels in both PLC and Hepa1‐6 cells (Figure ##SUPPL##0##S3a–h##, Supporting Information). CircLARP1B overexpression promoted cell invasion and LDs formation, and decreased the p‐AMPK and p‐ACC1 levels in PLC cells (Figure ##SUPPL##0##S3i–l##, Supporting Information).
","
Roles of lipid metabolism in the promoting effects of circLARP1B on HCC cell invasion were then investigated. Fatty acid synthase (FASN), which is the human lipogenic enzyme for de novo fatty acid synthesis,[\n##UREF##0##\n9\n##\n] was overexpressed in circLARP1B‐Def PLC cells (Figure ##FIG##1##2k,l##). FASN overexpression enhanced formation of LDs in circLARP1B‐Def PLC cells, and rescued the inhibitory effect on cell invasion caused by circLARP1B deficiency (Figure ##FIG##1##2k,l##). Then, we treated circLARP1B‐overexpressing PLC cells with IPI‐9119, a potent FASN inhibitor,[\n##REF##30578319##\n45\n##\n] and found that FASN inhibition blocked the promoting effect of circLARP1B on the formation of LDs and cell invasion (Figure ##SUPPL##0##S3m,n##, Supporting Information).
","
Taken together, these findings demonstrated that circLARP1B stimulated HCC invasion through remodeling lipid metabolism at the cellular level, and promoted FAS via modulating the AMPK and its downstream targets including ACC1.
","\n<italic toggle=\"yes\">CircLARP1B</italic> Interacts with HNRNPD Protein","
We set out to elucidate the functional mechanism of circLARP1B through inspecting its potential interacting molecules. Several putative miRNA binding sites on circLARP1B were predicted using CircInteractome (Figure ##SUPPL##0##S4a##, Supporting Information),[\n##REF##26669964##\n46\n##\n] however, RNA immunoprecipitation (RIP) of AGO2, which mediates the interaction between target RNA and miRNA,[\n##REF##34865956##\n13\n##\n] showed no enrichment of circLARP1B in PLC cells (Figure ##SUPPL##0##S4b##, Supporting Information). Furthermore, we applied ribosome profiling assay and found no obvious binding of circLARP1B to polysomes (Figure ##SUPPL##0##S4c##, Supporting Information), indicating that circLARP1B did not function as a template for protein translation. Both human and murine circLARP1B also displayed low coding possibilities in CPAT predicting tool (Figure ##SUPPL##0##S4d##, Supporting Information).[\n##REF##23335781##\n47\n##\n] Therefore, circLARP1B is noncoding and does not function as miRNA sponge.
","
We then performed RNA pull‐down with the cytoplasmic fraction of PLC cells with a biotinylated oligo against the BSJ of circLARP1B, which showed effective and specific capture of circLARP1B (Figure ##FIG##2##\n3a##). Cytoplasmic fraction was used due to the predominately cytoplasmic localization of circLARP1B (Figure ##FIG##0##1c–e##). The proteins co‐pulled down with circLARP1B were separated with SDS‐PAGE, followed by silver staining and mass spectrometry (MS) to disclose the specific circLARP1B binding band (Figure ##FIG##2##3a##). HNRNPD, also known as AU‐rich element RNA‐binding factor 1 (AUF1), was identified as the circLARP1B‐interacting protein in human cells (Figure ##FIG##2##3a##; Figure ##SUPPL##0##S4e##, Supporting Information). HNRNPD is an RBP with both cytoplasmic and nuclear roles.[\n##REF##1901943##\n48\n##, ##REF##19033365##\n49\n##\n] In the cytoplasm, HNRNPD regulates the stability of some mRNAs, e.g., CCND1 mRNA, through binding to the AU‐rich region in 3′ UTR.[\n##REF##1901943##\n48\n##, ##REF##19033365##\n49\n##, ##REF##33444453##\n50\n##\n] HNRNPD undergoes alternative splicing to produce four protein variants (p37, p40, p42, and p45) that are expressed with cell type‐specificity,[\n##REF##19033365##\n49\n##, ##REF##21956942##\n51\n##\n] and two variants (p37 and p40) were further confirmed as major HNRNPD isoforms in PLC cells (Figure ##SUPPL##0##S4f##, Supporting Information). RNA pull‐down of circLARP1B and HNRNPD RIP assays using the cytoplasmic fraction of PLC cells further verified their interaction (Figure ##FIG##2##3b,c##). Using mouse liver as the experimental material, RNA pull‐down of murine circLARP1B could co‐pull down Hnrnpd, and Hnrnpd RIP acquired circLARP1B (Figure ##FIG##2##3d,e##). Therefore, circLARP1B and HNRNPD interact in both human and murine cells.
","
To map the binding sites of HNRNPD in circLARP1B, we uncovered two motifs (motif 1 and motif 2) conserved in human and murine circLARP1B via RBPmap (Figure ##FIG##2##3f##), a tool for predicting RBP binding sites in an RNA sequence.[\n##REF##24829458##\n52\n##\n] Both sites (Positions 143–151 and 158–166 from BSJ) are also AU‐rich, consistent with the HNRNPD binding preference to AU‐rich region.[\n##REF##19033365##\n49\n##\n] Mutation of either motif 1 or motif 2 in circLARP1B decreased the interaction between circLARP1B and HNRNPD in PLC cells (Figure ##FIG##2##3g##). Mutation of both motifs (doublemut) abolished the interaction (Figure ##FIG##2##3g##). The 5′ portion of LARP1B mRNA possessing the same nucleic acid sequences as circLARP1B also has the HNRNPD binding site sequences present in circLARP1B, although HNRNPD RIP followed by semiquantitative RT‐PCR and real‐time quantification PCR (RT‐qPCR) analyses revealed that HNRNPD did not bind to LARP1B mRNA in PLC cells and mouse liver (Figure ##FIG##2##3c,e##; Figure ##SUPPL##0##S5a##, Supporting Information). The 5′ portion of LARP1B mRNA is heavily bound by ribosomes through analyzing available ribosome profiling sequencing (Ribo‐seq) data from HCC cell lines (GSE125757, GSE128320, and GSE147840) (Figure ##SUPPL##0##S5b##, Supporting Information).[\n##REF##31240521##\n53\n##, ##REF##34728628##\n54\n##, ##REF##35609991##\n55\n##\n] One possibility is that LARP1B mRNA is actively involved in translation, and the HNRNPD sites present in its 5′ are thus not available for HNRNPD binding.
","
HNRNPD binding to mRNA would modulate RNA stability,[\n##REF##1901943##\n48\n##, ##REF##33444453##\n50\n##, ##REF##10702317##\n56\n##\n] and we found out that knocking down of HNRNPD with siRNA did not affect levels of circLARP1B, indicating that HNRNPD did not regulate the stability of circLARP1B (Figure ##SUPPL##0##S5c##, Supporting Information). Knocking down of circLARP1B with shRNA also did not affect levels of HNRNPD mRNA and protein (Figure ##SUPPL##0##S5d,e##, Supporting Information). Consistent with previous reports,[\n##REF##10702317##\n56\n##, ##REF##30799487##\n57\n##, ##REF##22633954##\n58\n##\n] immunofluorescence (IF) analysis demonstrated that most HNRNPD localized in the nucleus, and ≈20% HNRNPD localized in the cytoplasm in PLC cells, which affected better visualization of cytoplasmic HNRNPD signals (Figure ##FIG##2##3h##). By using a condition to reduce the nuclear permeability to decrease the nuclear signals and thus to highlight the cytoplasmic HNRNPD signals,[\n##REF##34549195##\n59\n##, ##REF##31508410##\n60\n##\n] we observed that more than 70% circLARP1B signals overlapped with HNRNPD protein signals in the cytoplasm (Figure ##FIG##2##3i##). A binding assay with FLAG‐tagged HNRNPD protein purified from HEK293 cells and synthesized circLARP1B RNA demonstrated the direct binding between circLARP1B and HNRNPD in vitro (Figure ##FIG##2##3j##). With over ≈100 copies in the cytoplasm, circLARP1B with two functional sites can bind to a substantial portion of cytoplasmic HNRNPD protein. Collectively, we concluded that circLARP1B with two functional sites and reasonable abundance could interact with a substantial portion of cytoplasmic HNRNPD, and circLARP1B and HNRNPD did not reciprocally regulate their expression or stability.
","HNRNPD and HNRNPD Binding Are Essential for <italic toggle=\"yes\">CircLARP1B</italic> Functions","
We then set out to investigate the roles of HNRNPD binding in the functionality of circLARP1B. Overexpression of WT but not doublemut\ncircLARP1B could rescue the phenotypes in cell invasion and formation of LDs of circLARP1B‐Def cells (Figure ##FIG##3##\n4a,b##; Figure ##SUPPL##0##S5f##, Supporting Information). At the molecular level, overexpression of WT but not doublemut\ncircLARP1B restored the p‐AMPK and p‐ACC1 levels in circLARP1B‐Def cells (Figure ##FIG##3##4c##). Overexpression of HNRNPD inhibited cell invasion and formation of LDs (Figure ##FIG##3##4d,e##; Figure ##SUPPL##0##S5g##, Supporting Information), and levels of p‐AMPK and p‐ACC1 were also increased in PLC cells (Figure ##FIG##3##4f##). WT but not doublemut\ncircLARP1B that overexpressed together with HNRNPD blocked the effects of HNRNPD on cell invasion, formation of LDs, and levels of p‐AMPK and p‐ACC1 (Figure ##FIG##3##4d–f##). Taking these results together, we concluded that the interaction between HNRNPD and circLARP1B was essential to regulatory functions of this circRNA, and it is possible that circLARP1B might function through binding to and modulating HNRNPD.
","\n<italic toggle=\"yes\">CircLARP1B</italic> Destabilizes <italic toggle=\"yes\">LKB1</italic> mRNA via Perturbing HNRNPD","
To provide further insights into the molecular mechanisms of circLARP1B and HNRNPD in HCC cells, we extracted cytoplasmic fractions of PLC cells and performed HNRNPD RIP of endogenously expressed HNRNPD, and the RNAs from RIP were subjected to RNA‐seq (RIP‐seq) (Figure ##SUPPL##0##S6a,b##, Supporting Information). Distribution of RIP‐seq reads on mRNA targets revealed predominate 3′ UTR counts (Figure ##SUPPL##0##S6a##, Supporting Information), consistent with the functional roles of cytoplasmic HNRNPD in regulating mRNA stability by binding to 3′ UTR.[\n##REF##1901943##\n48\n##, ##REF##33444453##\n50\n##, ##REF##10702317##\n56\n##\n] Comparison of RIP‐seq data from circLARP1B‐Def cells and circLARP1B overexpression cells to those from the corresponding control cells, identified 216 mRNAs with contrast changes of HNRNPD bindings on their 3′ UTR upon circLARP1B deficiency or overexpression (Figure ##FIG##4##\n5a##; Table ##SUPPL##4##S4##, Supporting Information). We have reanalyzed the HNRNPD PAR‐CLIP data (GSE52977) from HEK293 cells,[\n##REF##25366541##\n61\n##\n] and found that the 216 mRNAs sensitive to circLARP1B had significantly lower HNRNPD PAR‐CLIP binding signals in the 3′ UTRs, compared to the other HNRNPD PAR‐CLIP targets (Figure ##SUPPL##0##S6c##, Supporting Information). These results indicated that the subset of mRNAs impacted by changes in circLARP1B levels might be with disadvantage in competing for the pool of HNRNPD, due to either relatively weaker binding ability to the protein or unfavorable cellular distribution and/or ratio of the corresponding mRNA/HNRNPD protein.
","
Gene ontology (GO) analysis for these target genes illustrated biological processes such as peptidyl‐Thr phosphorylation, protein stability, and mRNA metabolic process (Figure ##FIG##4##5b##). Peptidyl‐Thr phosphorylation was the most significantly (with the lowest P‐value) enriched process, among which LKB1 also known as serine/threonine kinase 11 (STK11) showed the most overall HNRNPD binding signals (the sum of binding signals from all four types of cells) on the 3′ UTR (Figure ##FIG##4##5c##). This indicated that its 3′ UTR occupied the most HNRNPD protein out of the 3′ UTRs of the six genes enriched in this GO term. With mouse liver, we found that Hnrnpd RIP could also pull down Lkb1 mRNA (Figure ##SUPPL##0##S6d##, Supporting Information). LKB1 is well known to directly catalyze the phosphorylation of Thr172 of AMPK, which is required for AMPK activation.[\n##REF##12847291##\n62\n##, ##REF##14985505##\n63\n##, ##REF##14614828##\n64\n##\n] Directly based on the RIP‐seq data, circLARP1B deficiency resulted in more HNRNPD bindings, and circLARP1B overexpression led to less HNRNPD bindings, to the 3′ UTR of LKB1 mRNA (Figure ##FIG##4##5d##). We also found that the endogenous HNRNPD protein bound more circLARP1B upon circLARP1B overexpression and less circLARP1B in circLARP1B‐Def cells (Figure ##FIG##4##5e##). Furthermore, HNRNPD RIP revealed that circLARP1B deficiency but not knockdown of LARP1B mRNA resulted in significantly more HNRNPD binding to the 3′ UTR of LKB1 mRNA (Figure ##SUPPL##0##S6e–g##, Supporting Information). A recent study demonstrated that HNRNPD bound to the c‐MYC 3′ UTR and promoted the c‐MYC expression in colorectal cancer.[\n##REF##35940521##\n65\n##\n] HNRNPD RIP‐qPCR revealed that HNRNPD also bound to the c‐MYC 3′ UTR, and siRNA‐mediated HNRNPD knockdown significantly increased the c‐MYC protein levels in PLC cells (Figure ##SUPPL##0##S6h,i##, Supporting Information). c‐MYC was not in the 216 targets sensitive to circLARP1B (Figure ##FIG##4##5a##; Table ##SUPPL##4##S4##, Supporting Information), and the opposite effect of HNRNPD on c‐MYC expression in colorectal cancer and HCC cells required further investigation.
","
We examined the effect of LKB1 in HCC cells directly by overexpression, and we found that the overexpressed LKB1 blocked the effect of circLARP1B in promoting cell invasion and formation of LDs (Figure ##FIG##4##5f,g##). Overexpressed LKB1 also nearly abolished the effects of circLARP1B on p‐AMPK and p‐ACC1 levels (Figure ##FIG##4##5h##). Lower LKB1 protein levels were observed in metastatic compared to nonmetastatic HCC specimens (Figure ##FIG##4##5i##). These results indicated that LKB1 mRNA was a downstream target of circLARP1B with robust contributions to the effects of circLARP1B.
","\n<italic toggle=\"yes\">CircLARP1B</italic> Competes with <italic toggle=\"yes\">LKB1</italic> mRNA for HNRNPD Binding and Leads to <italic toggle=\"yes\">LKB1</italic> mRNA Instability","
HNRNPD has complex regulatory effects on the stability of mRNAs, with some stabilized and the others destabilized.[\n##REF##1901943##\n48\n##, ##REF##33444453##\n50\n##, ##REF##10702317##\n56\n##\n] We observed that the steady levels but not nascent levels of LKB1 mRNA were significantly increased, upon circLARP1B deficiency or knockdown in human and murine cells (Figure ##SUPPL##0##S6j,k##, Supporting Information). We then examined whether and how HNRNPD and circLARP1B regulated the steady levels of LKB1 mRNA. Knocking down HNRNPD with siRNA resulted in significant decrease in half‐life of LKB1 mRNA and also LKB1 protein levels in PLC and Hepa1‐6 cells (Figure ##FIG##5##\n6a–d##), indicating that HNRNPD stabilized LKB1 mRNA. HNRNPD has been reported to bind to the 3′ UTR and simultaneously interact with translation initiation factor eIF4G1 and cap‐binding protein eIF4E to form a ternary complex, which could bring the 5′ and 3′ ends of the mRNA together to form a loop.[\n##REF##21956942##\n51\n##\n] We wondered whether HNRNPD looped LKB1 mRNA through this mechanism. We performed eIF4G1 and eIF4E RIP followed by RT‐qPCR with specific primers to detect the 3′ UTR of LKB1 mRNA in PLC cells with or without HNRNPD knockdown.[\n##REF##25826658##\n66\n##, ##REF##27437580##\n67\n##\n] HNRNPD knockdown resulted in decreased binding signal of both eIF4G1 and eIF4E to the LKB1 3′ UTR (Figure ##FIG##5##6e,f##). These results demonstrated that HNRNPD enhanced the mRNA looping and stabilized LKB1 mRNA.
","
In circLARP1B‐Def PLC cells, compared to the WT cells, LKB1 mRNA stability was increased, and the steady levels of LKB1 protein were also increased (Figure ##FIG##5##6g,h##). Overexpression of circLARP1B in PLC cells destabilized LKB1 mRNA, and led to lower levels of LKB1 protein (Figure ##SUPPL##0##S6l,m##, Supporting Information). In mouse Hepa1‐6 cells, the stability of Lkb1 mRNA was markedly enhanced, and the steady levels of Lkb1 protein were significantly increased upon circLARP1B knockdown with shRNA (Figure ##FIG##5##6i,j##). Dual‐luciferase reporter using the 3′ UTR of LKB1 mRNA as the 3′ UTR of firefly luciferase mRNA showed that WT circLARP1B but not the doublemut\ncircLARP1B could partially block the promoting effect on HNRNPD‐mediated stabilization of LKB1 3′ UTR (Figure ##SUPPL##0##S7a##, Supporting Information). Fluorescent in situ hybridization (FISH) of LKB1 mRNA and the HNRNPD IF staining revealed that circLARP1B‐Def compared to WT PLC cells, exhibited significantly more colocalization of LKB1 mRNA signals and HNRNPD protein signals in the cytoplasm (Figure ##SUPPL##0##S7b##, Supporting Information). ≈459 and ≈663 copies of LKB1 mRNA per cell were estimated in PLC and HepG2 cells, respectively (Figure ##SUPPL##0##S7c##, Supporting Information). The cytoplasmic copies of circLARP1B and LKB1 mRNA were evaluated in PLC cells, and the molecular ratio was ≈1:3 (Figure ##SUPPL##0##S7d##, Supporting Information). A competing assay, in which purified HNRNPD was incubated with equal moles of in vitro synthesized circLARP1B and LKB1 3′ UTR, was set up (Figure ##FIG##5##6k##). It was found that HNRNPD had much higher (≈18‐fold) binding affinity to circLARP1B than to the 3′ UTR of LKB1 mRNA (Figure ##FIG##5##6k##).
","
A 24‐nt antisense oligodeoxynucleotide (ODN) complementary to the two HNRNPD binding sites in circLARP1B was synthesized to examine as an inhibitor of circLARP1B. The antisense ODN was modified with phosphorothioate (PS) and 2′‐O‐methyl (2′‐OMe),[\n##REF##15064360##\n68\n##\n] and termed ODN‐AS. ODN‐AS transfection in PLC cells increased the HNRNPD binding to LKB1 3′ UTR and upregulated LKB1 protein levels (Figure ##FIG##5##6l,m##). 5‐methylcytosine (m5C) modified sense single‐stranded RNA oligos (m5C‐ssRNA) containing the HNRNPD binding sites of circLARP1B were also tested.[\n##UREF##6##\n69\n##\n] m5C‐ssRNA did not affect either the HNRNPD binding to LKB1 3′ UTR or LKB1 protein levels (Figure ##SUPPL##0##S7e,f##, Supporting Information). These results indicated that the small m5C‐ssRNA oligos somehow could not function as a circLARP1B mimics in cells. On the other hand, the interaction between HNRNPD and circLARP1B could be blocked by ODN‐AS, and the potential effects of ODN‐AS in lipid metabolism, HCC metastasis, and even therapeutics could be further investigated.
","\n<italic toggle=\"yes\">CircLARP1B</italic> Deficiency in Mice Causes Liver Changes Attributable to Lkb1 Surplus","
To directly investigate the physiological roles of circLARP1B in animals, we used CRISPR/Cas9 to generate circLARP1B deficient mice, in which repeat sequences in intron 4 of murine Larp1b gene were deleted (circLARP1B−/−\n), with functional intronic elements such as the splice sites and pyrimidine tract remained unaltered (Figure ##FIG##6##\n7a##). The mutant mice did not show statistically significant difference in body weight or litter size, when compared age‐matched WT mice (Figure ##SUPPL##0##S8a,b##, Supporting Information). The circLARP1B expression was significantly decreased in livers of circLARP1B−/−\n mice compared to WT mice, while the total and nascent levels of Larp1b mRNA were unaltered (Figure ##FIG##6##7b##; Figure ##SUPPL##0##S8c##, Supporting Information). The levels of HNRNPD mRNA and protein also showed no significant difference in livers of WT and circLARP1B−/−\n mice (Figure ##SUPPL##0##S8d##, Supporting Information). Liver was the focus due to that it is the organ giving rise to HCC, and a central organ of metabolism in the body.
","
\nCircLARP1B−/−\n mice compared to WT mice had significantly reduced LDs in livers, examined by Oil red O staining (Figure ##FIG##6##7c##). Protein levels of Lkb1, p‐Ampk, and p‐Acc1 were significantly increased in circLARP1B−/−\n livers (Figure ##FIG##6##7d,e##; Figure ##SUPPL##0##S8e##, Supporting Information). Further, we performed tail vein injection of 8‐week‐old circLARP1B−/−\n mice with hepatocyte‐directed adeno‐associated virus 8 (AAV8) to express shRNA against Lkb1 (AAV8‐shLkb1) or the corresponding control (AAV8‐shCtrl). When examined 3 weeks after the infection, AAV8‐shLkb1 resulted in effective Lkb1 knockdown in circLARP1B−/−\n livers, and Lkb1 depletion recovered the LDs levels in circLARP1B−/−\n livers to the levels in WT livers (Figure ##FIG##6##7f,g##). The increased p‐Ampk and p‐Acc1 levels in circLARP1B−/−\n liver were also restored nearly to the levels in WT liver (Figure ##FIG##6##7h,i##; Figure ##SUPPL##0##S8f##, Supporting Information).
","\n<italic toggle=\"yes\">CircLARP1B</italic> Deficiency in Mice Impedes HCC Metastasis and Lipid Accumulation","
To examine physiological roles of circLARP1B in HCC at the whole organismal level, we induced HCC in mice with diethylnitrosamine (DEN), which is well established to cause severe liver damage and eventually hepatocarcinogenesis (Figure ##FIG##7##\n8a##).[\n##REF##22265403##\n70\n##\n] After DEN induction for 10 weeks, liver inflammation examined by H&E staining was significantly lower in livers of circLARP1B−/−\n mice than that of WT mice (Figure ##SUPPL##0##S8g##, Supporting Information). mRNA levels of Il6 and Tnf (as inflammation indicators) were also decreased in livers of circLARP1B−/−\n mice (Figure ##SUPPL##0##S8h##, Supporting Information). The presence of bridging fibrosis was also examined, and it was found that circLARP1B−/−\n mice had fewer Masson and Sirius Red signals in the portal areas (Figure ##SUPPL##0##S8i,j##, Supporting Information). Consistently, mRNA levels of fibrosis‐related markers, Col1a1, Tgfb1, and Mmp2 were decreased in livers of circLARP1B−/−\n mice (Figure ##SUPPL##0##S8k##, Supporting Information). After DEN induction for 18 weeks (HCC mice), circLARP1B−/−\n mice exhibited fewer and smaller tumor nodes in livers, and significantly smaller lung metastatic nodes, compared with WT mice (Figure ##FIG##7##8b,c##). HNRNPD levels demonstrated no significant difference in liver tumors between WT and circLARP1B−/−\n HCC mice, while in HCC liver tumors of both genotypes, Hnrnpd levels were significantly higher than livers from age‐matched WT mice (Figure ##SUPPL##0##S8l,m##, Supporting Information). Immunohistochemistry (IHC) of liver tumor nodes revealed that the prometastasis marker Vimentin (Vim) was lower and the antimetastasis marker E‐cadherin (Ecad) was higher in circLARP1B−/−\n HCC mice (Figure ##FIG##7##8d##). In consistence with HCC severity, the serum concentrations of alpha‐fetoprotein (AFP), a biomarker for HCC, was significantly lower in circLARP1B−/−\n HCC mice (Figure ##FIG##7##8e##). Alanine transaminase (ALT) and aspartate transaminase (AST), two markers of liver injury, were also significantly lower in the serum of circLARP1B−/−\n HCC mice (Figure ##FIG##7##8f,g##). CircLARP1B−/−\n HCC mice exhibited lower triglyceride (TG) and total cholesterol (TC) levels in serum, and significantly fewer LDs in liver tumor nodes (Figure ##FIG##7##8h–j##). Meanwhile, levels of Lkb1, p‐Ampk, and p‐Acc1 were significantly higher in liver tumor nodes from circLARP1B−/−\n HCC mice (Figure ##FIG##7##8k,l##; Figure ##SUPPL##0##S9a##, Supporting Information).
","
To provide further insights for HCC metastatic roles of circLARP1B in liver cells, we infused hepatocyte‐directed AAV8 expressing shRNA against circLARP1B (AAV8‐shcircLARP1B) or the corresponding negative control (AAV8‐shCtrl) into WT mice after DEN induction for 10 weeks (Figure ##FIG##8##\n9a##; Figure ##SUPPL##0##S3e##, Supporting Information).[\n##REF##12192090##\n71\n##\n] In livers of WT mice infused with AAV8‐shcircLARP1B, effective knockdown of circLARP1B was observed, while the Larp1b mRNA was unaltered (Figure ##FIG##8##9b##). Consistent with results from circLARP1B−/−\n mice, AAV8‐shcircLARP1B injection resulted in fewer and smaller tumor nodes in livers, and smaller lung metastatic nodes, compared to AAV8‐shCtrl injection in WT mice (Figure ##FIG##8##9c,d##). The Vim IHC signals were lower, and the Ecad IHC signals were higher in liver tumors of WT mice with AAV8‐shcircLARP1B (Figure ##FIG##8##9e##). Serum AFP, ALT, AST, TG, and TC levels were also significantly lower in HCC mice with AAV8‐shcircLARP1B (Figure ##FIG##8##9f–j##). WT HCC mice infused with AAV8‐shcircLARP1B exhibited significantly fewer LDs and markedly higher levels of Lkb1, p‐Ampk, and p‐Acc1 in liver tumor nodes (Figure ##FIG##8##9k–m##; Figure ##SUPPL##0##S9b##, Supporting Information). Even these results were based on AAV8‐mediated hepatocyte specific circLARP1B knockdown, in the real case of HCC, effects of the niche, immune cells, and other unaccounted parameters could still contribute to the overall effects of circLARP1B on metastasis.
","
Taken together, these results demonstrated that lower circLARP1B levels were unfavorable to HCC progression, lipid accumulation, and metastasis in mouse models, by affecting the same circLARP1B–HNRNPD–LKB1–AMPK regulatory pathway identified in HCC cell lines.
","Lkb1 Is the Key Factor for the Effect of <italic toggle=\"yes\">CircLARP1B</italic> in HCC Mouse Model","
We then performed tail vein injection with AAV8‐shLkb1 or the AAV8‐shCtrl once in circLARP1B−/−\n HCC mice also at the 10th week after DEN induction. Hepatic Lkb1 protein was successfully knocked down (Figure ##SUPPL##0##S9c##, Supporting Information). We found that AAV8‐shLkb1 injection led to more and larger liver tumors, and larger lung metastatic nodes compared to AAV8‐shCtrl injection in circLARP1B−/−\n HCC mice (Figure ##FIG##9##\n10a,b##). In circLARP1B−/−\n HCC mice upon AAV8‐shLkb1 application, liver tumor number and size, and lung metastases were as worse as what happened in the WT HCC mice (Figure ##FIG##9##10a,b##), indicating strongly that Lkb1 was the key downstream factor in hepatocytes for the effects of circLARP1B.
","
Lkb1 knockdown led to the increased Vim levels and the decreased Ecad levels in liver tumor nodes in circLARP1B−/−\n HCC mice (Figure ##FIG##9##10c##). Meanwhile, Lkb1 knockdown reversed these changes in Vim and Ecad levels caused by circLARP1B deficiency (Figure ##FIG##9##10c##). Lkb1 silencing nearly restored the circLARP1B‐deficient mediated suppressive effects on the levels of serum markers AFP, ALT, and AST in circLARP1B−/−\n HCC mice (Figure ##SUPPL##0##S9d–f##, Supporting Information). Furthermore, Lkb1 depletion almost completely restored the inhibitory effects of circLARP1B deficiency on lipid accumulation in liver tumor nodes and serum TG and TC levels (Figure ##FIG##9##10d##; Figure ##SUPPL##0##S9g,h##, Supporting Information). Increased levels of p‐Ampk and p‐Acc1 observed in the liver of circLARP1B−/−\n HCC mice were also eradicated upon Lkb1 knockdown (Figure ##FIG##9##10e##; Figure ##SUPPL##0##S9i,j##, Supporting Information). Therefore, Lkb1 was the key factor in hepatocytes for the molecular regulatory pathway of circLARP1B in HCC lipid metabolism and metastasis.
","Clinical Significance of HNRNPD, LKB1, and AMPK in HCC","
Levels of HNRNPD mRNA were significantly higher in the 23 metastatic specimens compared to 67 nonmetastatic specimens we collected (Figure ##SUPPL##0##S10a##, Supporting Information), and higher HNRNPD mRNA levels were positively associated with shorter OS and DFS analyzed based on these 90 HCC specimens (Figure ##FIG##9##10f##). HCC patients with higher LKB1 mRNA levels in tumors had significantly longer OS and DFS (Figure ##FIG##9##10g##). On the other hand, levels of LKB1 mRNA showed no statistically significant difference in metastatic versus nonmetastatic specimens (Figure ##SUPPL##0##S10b##, Supporting Information). Presumably upstream mechanisms might upregulate both HNRNPD and circLARP1B in metastatic HCC (Figure ##FIG##0##1g##), and the two regulators had opposite effects on the levels of LKB1 mRNA, with HNRNPD enhancing and circLARP1B destabilizing LKB1 mRNA. Therefore, these regulations might lead to the balance of LKB1 mRNA levels in metastatic versus nonmetastatic HCC. The AMPK mRNA levels demonstrated statistically significant increase in metastatic HCC, but its levels had no significant correlation to the OS and DFS of HCC patients (Figure ##SUPPL##0##S10c,d##, Supporting Information). AMPK was reported to either promote or suppress HCC,[\n##REF##36316383##\n11\n##, ##REF##32631382##\n72\n##, ##REF##23942093##\n73\n##, ##REF##30853530##\n74\n##\n] and in the circLARP1B–HNRNPD–LKB1–AMPK axis, the levels of p‐AMPK rather than the overall AMPK levels would be more relevant to HCC.
"],"string":"[\n \"Results\",\n \"\\n<italic toggle=\\\"yes\\\">CircLARP1B</italic> as a Mammalian Conserved CircRNA Is Identified in HCC Metastasis\",\n \"
In order to investigate the functional roles of conserved circRNAs in HCC, we performed ribosomal RNA depleted RNA‐sequencing (RNA‐seq) of six HCC specimens, among which three HCC patients were metastatic and the other three were nonmetastatic (Figure ##FIG##0##\\n1a##). 4614 circRNAs with back‐splicing junction (BSJ) reads >2 were detected, and 48 of them were with over fourfold changes in expression levels between the two groups (Figure ##FIG##0##1a##; Table ##SUPPL##1##S1##, Supporting Information). 38 of these circRNAs were with lower levels, and 10 out of the 48 were with higher levels in metastatic HCC (Figure ##FIG##0##1a##). Then we aligned the 48 circRNA sequences to annotated murine circRNAs from the circAtlas database to identify conserved circRNAs with a stringent criterion (full‐length and BSJ sequences >75% identical).[\\n##REF##32345360##\\n33\\n##\\n]\\nCircLARP1B was the most differentially expressed and also the only metastasis upregulated circRNA among the five conserved circRNAs identified (Figure ##FIG##0##1a##). CircLARP1B is highly conserved with ≈86% sequence identity between human and mice, and consists of exons 2–4 from the La ribonucleoprotein 1B (LARP1B) gene, with a deduced size of 294 nt in both human and mice (Figure ##FIG##0##1b##; Figure ##SUPPL##0##S1a##, Supporting Information). CircLARP1B and LARP1B mRNA exhibited distinct expression patterns in human tissues analyzed from the NCBI and circAtlas databases; for example, LARP1B mRNA but not circLARP1B was expressed in thyroid tissue (Figure ##SUPPL##0##S1b##, Supporting Information). LARP1B itself is an RBP that regulates the translation of certain mRNAs.[\\n##REF##28650797##\\n34\\n##\\n] The overall survival rate showed no significant difference between the two groups of HCC patients with higher LARP1B expression and lower expression (Figure ##SUPPL##0##S1c##, Supporting Information), indicating that LARP1B mRNA may not be a key regulator in HCC. The circLARP1B BSJ was validated by PCR amplification and confirmed by Sanger sequencing in a human HCC cell line PLC and mouse liver, to verify the circular nature of circLARP1B (Figure ##SUPPL##0##S1d##, Supporting Information). Then, the actinomycin D chase assay revealed that circLARP1B was stable compared to LARP1B mRNA (Figure ##SUPPL##0##S1e##, Supporting Information). By Northern blotting, bands corresponding to the deduced size of circLARP1B were detected, and the estimated copy numbers per cell in two HCC cell lines PLC and HepG2 were ≈136 and ≈158, respectively (Figure ##FIG##0##1b##; Figure ##SUPPL##0##S1f##, Supporting Information). Copy numbers of more than 100 are high for circRNAs or even for mRNAs. Single molecular fluorescence in situ hybridization (smFISH) of circLARP1B in PLC cells revealed that this circRNA localized predominately in the cytoplasm (Figure ##FIG##0##1c##; Figure ##SUPPL##0##S1g,h##, Supporting Information). The specificity of smFISH to circLARP1B was demonstrated with significantly decreased smFISH signals upon shcircLARP1B knockdown of the circRNA (Figure ##SUPPL##0##S1i–k##, Supporting Information). With a thyroid cell line (K1 cells), which exhibited considerable LARP1B mRNA levels but no circLARP1B expression, the specificity of smFISH to circLARP1B or LARP1B mRNA was further proofed (Figure ##SUPPL##0##S1l##, Supporting Information). More than 85% of circLARP1B were found to be cytoplasmic in one murine HCC cell line Hepa1‐6 and the two human HCC cell lines (Figure ##FIG##0##1d##). Furthermore, smFISH of circLARP1B in mouse liver also showed cytoplasmic enrichment (Figure ##FIG##0##1e##).
\",\n \"
Higher circLARP1B levels in metastatic compared to nonmetastatic HCC specimens were revealed by smFISH analysis (Figure ##FIG##0##1f##). We collected HCC specimens from 90 patients, and found that higher circLARP1B levels were associated with 23 metastatic specimens as against the 67 nonmetastatic specimens (Figure ##FIG##0##1g##). Except 5 specimens without information of prognostic stage, these clinical samples could be divided into 65 specimens with prognostic TNM stage I–II and 20 specimens with TNM stage III–IV, and higher circLARP1B levels were significantly correlated with advanced TNM stages (III–IV) (Figure ##FIG##0##1h##). HCC patients with higher circLARP1B levels in tumors had significantly lower five‐year overall survival (OS) and disease‐free survival (DFS) rates (Figure ##FIG##0##1i,j##).
\",\n \"
Collectively, these results demonstrated that circLARP1B as a mammalian conserved and highly expressed circRNA might play promoting roles in HCC metastasis as supported by data from clinical specimens.
\",\n \"\\n<italic toggle=\\\"yes\\\">CircLARP1B</italic> Regulates Cell Invasion and Lipid Accumulation via Fatty Acid Synthesis\",\n \"
To explore the cellular functions of circLARP1B, we applied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to generate circLARP1B deficient (circLARP1B‐Def) PLC cells (Figure ##FIG##1##\\n2a##). Flanking introns of circularized exons contain Alu elements and B1 repeats in human and mice, respectively, which both belong to reverse complementary sequences, known to facilitate circRNA biogenesis (Figure ##SUPPL##0##S2a##, Supporting Information).[\\n##REF##34865956##\\n13\\n##, ##REF##33514913##\\n35\\n##\\n] To generate circLARP1B‐Def PLC cells, repeat sequences including four proximal Alu elements in the fourth intron of human LARP1B were deleted, but functional intronic elements such as the splice sites and pyrimidine tract remained unaltered (Figure ##FIG##1##2a##). The resulted circLARP1B‐Def PLC cells exhibited significantly decreased circLARP1B levels with no alteration in LARP1B mRNA levels, compared to the wildtype (WT) PLC cells (Figure ##FIG##1##2b##).
\",\n \"
CircLARP1B‐Def PLC cells compared to WT cells showed significantly reduced invasion, when examined with Transwell invasion assay (Figure ##FIG##1##2c##). No significant difference in growth curve and colony formation was observed between the circLARP1B‐Def and WT cells (Figure ##SUPPL##0##S2b–d##, Supporting Information). Invasion ability is directly related to metastasis, and growth curve and colony formation are associated with cell growth and proliferation.
\",\n \"
We then assessed the potential metabolic alterations in circLARP1B‐Def PLC cells by liquid chromatography‐mass spectrometry (LC‐MS) untargeted metabolomics (Figure ##FIG##1##2d##). A total of 366 metabolites and lipid classes (including 44 lipid classes) were detected, and 105 of them, 42 increased and 63 decreased, were significantly dysregulated (variable importance in the projection, VIP >1; P‐value <0.05) upon circLARP1B deficiency in PLC cells (Figure ##SUPPL##0##S2e##; Table ##SUPPL##2##S2##, Supporting Information). KEGG pathway analysis of these dysregulated metabolites revealed that lipid metabolism, among the 6 metabolic pathways significantly affected, was perturbed the most (Figure ##FIG##1##2e##). For the 24 lipid classes enriched in lipid metabolism of KEGG analysis, 18 ones were decreased, and 6 were increased in circLARP1B‐Def cells (Figure ##SUPPL##0##S2e##, Supporting Information). We then performed lipidomics to examine lipid class composition, and identified 2709 lipid species that compose the 24 dysregulated lipid classes detected in the metabolomics. 273 lipid species covering all these 24 lipid classes were significantly changed upon circLARP1B deficiency (Figure ##FIG##1##2f##; Table ##SUPPL##2##S2##, Supporting Information). Components of lipid droplets (LDs) including lipid classes such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), diglyceride (DG), triglyceride (TG), and phosphatidylinositol (PI) were among the metabolites with lower levels in circLARP1B‐Def cells (Figure ##SUPPL##0##S2e##, Supporting Information).[\\n##REF##24220094##\\n36\\n##\\n] LDs examined by Nile red staining were markedly decreased in circLARP1B‐Def cells compared to WT cells (Figure ##FIG##1##2g##).
\",\n \"
To further explore the mechanism of lipid accumulation promoted by circLARP1B, we performed metabolic labeling with 13C‐labeled targeted metabolic flux analysis to assess the incorporation of 13C‐labeled glucose and glutamine into lipid in circLARP1B‐Def and WT PLC cells (Figure ##FIG##1##2h,i##).[\\n##REF##32101748##\\n37\\n##\\n] Targeted metabolic flux analyses were performed, and we totally detected 17 kinds of fatty acids with 13C labeling, and found that all of them were significantly reduced in circLARP1B‐Def cells (Figure ##FIG##1##2h##; Table ##SUPPL##3##S3##, Supporting Information), indicating a promoting effect of circLARP1B in fatty acid synthesis (FAS). Glucose and glutamine are first converted to two‐carbon acetyl‐CoA, which serves as a substrate for ACC1 enzyme in FAS, and 13C‐labeled two‐carbon units from 13C‐glucose or 13C‐glutamine into palmitate can thus be examined to reveal FAS from glucose or glutamine. As expected, the distributions of 13C‐labeled palmitate isotopologs (M12, M14, and M16) in circLARP1B‐Def cells, compared to WT cells were significantly lower (Figure ##FIG##1##2i##). We further evaluated the involvement of circLARP1B in fatty acid oxidation (FAO) with oxygen consumption rate (OCR) assays after supplementing cells with exogenous free fatty acids (FFAs; palmitate was supplied). CircLARP1B‐Def cells showed similar OCR compared with WT cells (Figure ##SUPPL##0##S2f##, Supporting Information). Unc‐51 like autophagy activating kinase 1 (ULK1) is a key regulator in lipophagy initiation, and phosphorylation of Ser555 of ULK1 induces lipophagy.[\\n##REF##32863214##\\n38\\n##\\n] The ULK1 and phosphorylated ULK1 (p‐ULK1) levels exhibited no significant alternation between circLARP1B‐Def and WT cells (Figure ##FIG##1##2j##). Together, these findings suggested that circLARP1B facilitated lipid accumulation by FAS, not through regulating FAO or lipophagy.
\",\n \"
The activity of ACC1 is inhibited by phosphorylation of Ser79,[\\n##UREF##0##\\n9\\n##, ##REF##36316383##\\n11\\n##\\n] and we observed that the phosphorylated ACC1 (p‐ACC1) levels were substantially upregulated in circLARP1B‐Def cells (Figure ##FIG##1##2j##). AMPK is the major kinase that catalyzes the phosphorylation of ACC1, and AMPK itself is initially activated by phosphorylation at Thr172.[\\n##REF##36316383##\\n11\\n##\\n] The phosphorylated AMPK (p‐AMPK) levels were also significantly higher in circLARP1B‐Def cells than WT cells (Figure ##FIG##1##2j##). Therefore, higher levels of p‐AMPK and then p‐ACC1 would repress lipogenesis and lead to decreased amount of LDs in circLARP1B‐Def cells. AMPK activation also causes a range of metabolites changed in HCC.[\\n##REF##28355557##\\n39\\n##, ##REF##33690219##\\n40\\n##, ##REF##30468782##\\n41\\n##\\n] From the literature we searched, 12 of circLARP1B‐modulated metabolites have been reported to be regulated by AMPK signaling in HCC,[\\n##REF##28355557##\\n39\\n##, ##REF##33690219##\\n40\\n##, ##REF##30468782##\\n41\\n##\\n] and 8 of these metabolites exhibit consistent changes between circLARP1B deficiency and AMPK activation (Figure ##SUPPL##0##S2g##, Supporting Information).
\",\n \"
Additionally, we examined the effects of circLARP1B on the other downstream processes known to be regulated by AMPK, such as the mTOR pathway, angiogenesis, TGF‐β, and mitochondrial activity.[\\n##REF##36316383##\\n11\\n##, ##REF##22576211##\\n42\\n##, ##REF##31974393##\\n43\\n##, ##REF##30425336##\\n44\\n##\\n] AMPK activation led to phosphorylation of the mTORC1 subunit RAPTOR to inhibit mTOR pathway,[\\n##REF##36316383##\\n11\\n##\\n] and significantly increased phosphorylated RAPTOR (p‐RAPTOR) levels were observed in circLARP1B‐Def cells compared to WT cells (Figure ##SUPPL##0##S2h##, Supporting Information). Once activated, AMPK is reported to increase the protein level of VEGFA (an angiogenesis driver) and inhibit TGF‐β signaling,[\\n##REF##36316383##\\n11\\n##, ##REF##22576211##\\n42\\n##, ##REF##31974393##\\n43\\n##\\n] and the VEGFA and TGF‐β protein levels did not demonstrate significant change in circLARP1B‐Def cells compared to WT cells (Figure ##SUPPL##0##S2h##, Supporting Information). As another substrate of AMPK, mitochondrial fission factor (MFF) is phosphorylated by AMPK to promote mitochondrial fission,[\\n##REF##36316383##\\n11\\n##\\n] and the MFF/p‐MFF protein levels remained unaltered upon circLARP1B deficiency in PLC cells (Figure ##SUPPL##0##S2h##, Supporting Information). Moreover, basal respiration, ATP production, and maximum respiration revealing mitochondrial activity examined by OCR assays demonstrated no significant difference between circLARP1B‐Def cells and WT cells (Figure ##SUPPL##0##S2i##, Supporting Information). Our results demonstrated that both ACC1 and the mTOR signaling directly regulated by AMPK were modulated by circLARP1B through a mechanism awaiting characterization in PLC cells. Both ACC1 and the mTOR signaling are known to promote fatty acid synthesis,[\\n##UREF##0##\\n9\\n##, ##REF##30425336##\\n44\\n##\\n] we focused on examining ACC1/p‐ACC1 as a readout of downstream effects of AMPK, owing to the fact that ACC1 is the rate‐limiting enzyme for FAS.[\\n##UREF##0##\\n9\\n##\\n]\\n
\",\n \"
We also manipulated levels of circLARP1B in trans by either RNA interference knockdown or plasmid overexpression, and in human PLC cells or murine Hepa1‐6 cells. ShRNA‐mediated circLARP1B knockdown suppressed cell invasion and formation of LDs, and increased p‐AMPK and p‐ACC1 levels in both PLC and Hepa1‐6 cells (Figure ##SUPPL##0##S3a–h##, Supporting Information). CircLARP1B overexpression promoted cell invasion and LDs formation, and decreased the p‐AMPK and p‐ACC1 levels in PLC cells (Figure ##SUPPL##0##S3i–l##, Supporting Information).
\",\n \"
Roles of lipid metabolism in the promoting effects of circLARP1B on HCC cell invasion were then investigated. Fatty acid synthase (FASN), which is the human lipogenic enzyme for de novo fatty acid synthesis,[\\n##UREF##0##\\n9\\n##\\n] was overexpressed in circLARP1B‐Def PLC cells (Figure ##FIG##1##2k,l##). FASN overexpression enhanced formation of LDs in circLARP1B‐Def PLC cells, and rescued the inhibitory effect on cell invasion caused by circLARP1B deficiency (Figure ##FIG##1##2k,l##). Then, we treated circLARP1B‐overexpressing PLC cells with IPI‐9119, a potent FASN inhibitor,[\\n##REF##30578319##\\n45\\n##\\n] and found that FASN inhibition blocked the promoting effect of circLARP1B on the formation of LDs and cell invasion (Figure ##SUPPL##0##S3m,n##, Supporting Information).
\",\n \"
Taken together, these findings demonstrated that circLARP1B stimulated HCC invasion through remodeling lipid metabolism at the cellular level, and promoted FAS via modulating the AMPK and its downstream targets including ACC1.
\",\n \"\\n<italic toggle=\\\"yes\\\">CircLARP1B</italic> Interacts with HNRNPD Protein\",\n \"
We set out to elucidate the functional mechanism of circLARP1B through inspecting its potential interacting molecules. Several putative miRNA binding sites on circLARP1B were predicted using CircInteractome (Figure ##SUPPL##0##S4a##, Supporting Information),[\\n##REF##26669964##\\n46\\n##\\n] however, RNA immunoprecipitation (RIP) of AGO2, which mediates the interaction between target RNA and miRNA,[\\n##REF##34865956##\\n13\\n##\\n] showed no enrichment of circLARP1B in PLC cells (Figure ##SUPPL##0##S4b##, Supporting Information). Furthermore, we applied ribosome profiling assay and found no obvious binding of circLARP1B to polysomes (Figure ##SUPPL##0##S4c##, Supporting Information), indicating that circLARP1B did not function as a template for protein translation. Both human and murine circLARP1B also displayed low coding possibilities in CPAT predicting tool (Figure ##SUPPL##0##S4d##, Supporting Information).[\\n##REF##23335781##\\n47\\n##\\n] Therefore, circLARP1B is noncoding and does not function as miRNA sponge.
\",\n \"
We then performed RNA pull‐down with the cytoplasmic fraction of PLC cells with a biotinylated oligo against the BSJ of circLARP1B, which showed effective and specific capture of circLARP1B (Figure ##FIG##2##\\n3a##). Cytoplasmic fraction was used due to the predominately cytoplasmic localization of circLARP1B (Figure ##FIG##0##1c–e##). The proteins co‐pulled down with circLARP1B were separated with SDS‐PAGE, followed by silver staining and mass spectrometry (MS) to disclose the specific circLARP1B binding band (Figure ##FIG##2##3a##). HNRNPD, also known as AU‐rich element RNA‐binding factor 1 (AUF1), was identified as the circLARP1B‐interacting protein in human cells (Figure ##FIG##2##3a##; Figure ##SUPPL##0##S4e##, Supporting Information). HNRNPD is an RBP with both cytoplasmic and nuclear roles.[\\n##REF##1901943##\\n48\\n##, ##REF##19033365##\\n49\\n##\\n] In the cytoplasm, HNRNPD regulates the stability of some mRNAs, e.g., CCND1 mRNA, through binding to the AU‐rich region in 3′ UTR.[\\n##REF##1901943##\\n48\\n##, ##REF##19033365##\\n49\\n##, ##REF##33444453##\\n50\\n##\\n] HNRNPD undergoes alternative splicing to produce four protein variants (p37, p40, p42, and p45) that are expressed with cell type‐specificity,[\\n##REF##19033365##\\n49\\n##, ##REF##21956942##\\n51\\n##\\n] and two variants (p37 and p40) were further confirmed as major HNRNPD isoforms in PLC cells (Figure ##SUPPL##0##S4f##, Supporting Information). RNA pull‐down of circLARP1B and HNRNPD RIP assays using the cytoplasmic fraction of PLC cells further verified their interaction (Figure ##FIG##2##3b,c##). Using mouse liver as the experimental material, RNA pull‐down of murine circLARP1B could co‐pull down Hnrnpd, and Hnrnpd RIP acquired circLARP1B (Figure ##FIG##2##3d,e##). Therefore, circLARP1B and HNRNPD interact in both human and murine cells.
\",\n \"
To map the binding sites of HNRNPD in circLARP1B, we uncovered two motifs (motif 1 and motif 2) conserved in human and murine circLARP1B via RBPmap (Figure ##FIG##2##3f##), a tool for predicting RBP binding sites in an RNA sequence.[\\n##REF##24829458##\\n52\\n##\\n] Both sites (Positions 143–151 and 158–166 from BSJ) are also AU‐rich, consistent with the HNRNPD binding preference to AU‐rich region.[\\n##REF##19033365##\\n49\\n##\\n] Mutation of either motif 1 or motif 2 in circLARP1B decreased the interaction between circLARP1B and HNRNPD in PLC cells (Figure ##FIG##2##3g##). Mutation of both motifs (doublemut) abolished the interaction (Figure ##FIG##2##3g##). The 5′ portion of LARP1B mRNA possessing the same nucleic acid sequences as circLARP1B also has the HNRNPD binding site sequences present in circLARP1B, although HNRNPD RIP followed by semiquantitative RT‐PCR and real‐time quantification PCR (RT‐qPCR) analyses revealed that HNRNPD did not bind to LARP1B mRNA in PLC cells and mouse liver (Figure ##FIG##2##3c,e##; Figure ##SUPPL##0##S5a##, Supporting Information). The 5′ portion of LARP1B mRNA is heavily bound by ribosomes through analyzing available ribosome profiling sequencing (Ribo‐seq) data from HCC cell lines (GSE125757, GSE128320, and GSE147840) (Figure ##SUPPL##0##S5b##, Supporting Information).[\\n##REF##31240521##\\n53\\n##, ##REF##34728628##\\n54\\n##, ##REF##35609991##\\n55\\n##\\n] One possibility is that LARP1B mRNA is actively involved in translation, and the HNRNPD sites present in its 5′ are thus not available for HNRNPD binding.
\",\n \"
HNRNPD binding to mRNA would modulate RNA stability,[\\n##REF##1901943##\\n48\\n##, ##REF##33444453##\\n50\\n##, ##REF##10702317##\\n56\\n##\\n] and we found out that knocking down of HNRNPD with siRNA did not affect levels of circLARP1B, indicating that HNRNPD did not regulate the stability of circLARP1B (Figure ##SUPPL##0##S5c##, Supporting Information). Knocking down of circLARP1B with shRNA also did not affect levels of HNRNPD mRNA and protein (Figure ##SUPPL##0##S5d,e##, Supporting Information). Consistent with previous reports,[\\n##REF##10702317##\\n56\\n##, ##REF##30799487##\\n57\\n##, ##REF##22633954##\\n58\\n##\\n] immunofluorescence (IF) analysis demonstrated that most HNRNPD localized in the nucleus, and ≈20% HNRNPD localized in the cytoplasm in PLC cells, which affected better visualization of cytoplasmic HNRNPD signals (Figure ##FIG##2##3h##). By using a condition to reduce the nuclear permeability to decrease the nuclear signals and thus to highlight the cytoplasmic HNRNPD signals,[\\n##REF##34549195##\\n59\\n##, ##REF##31508410##\\n60\\n##\\n] we observed that more than 70% circLARP1B signals overlapped with HNRNPD protein signals in the cytoplasm (Figure ##FIG##2##3i##). A binding assay with FLAG‐tagged HNRNPD protein purified from HEK293 cells and synthesized circLARP1B RNA demonstrated the direct binding between circLARP1B and HNRNPD in vitro (Figure ##FIG##2##3j##). With over ≈100 copies in the cytoplasm, circLARP1B with two functional sites can bind to a substantial portion of cytoplasmic HNRNPD protein. Collectively, we concluded that circLARP1B with two functional sites and reasonable abundance could interact with a substantial portion of cytoplasmic HNRNPD, and circLARP1B and HNRNPD did not reciprocally regulate their expression or stability.
\",\n \"HNRNPD and HNRNPD Binding Are Essential for <italic toggle=\\\"yes\\\">CircLARP1B</italic> Functions\",\n \"
We then set out to investigate the roles of HNRNPD binding in the functionality of circLARP1B. Overexpression of WT but not doublemut\\ncircLARP1B could rescue the phenotypes in cell invasion and formation of LDs of circLARP1B‐Def cells (Figure ##FIG##3##\\n4a,b##; Figure ##SUPPL##0##S5f##, Supporting Information). At the molecular level, overexpression of WT but not doublemut\\ncircLARP1B restored the p‐AMPK and p‐ACC1 levels in circLARP1B‐Def cells (Figure ##FIG##3##4c##). Overexpression of HNRNPD inhibited cell invasion and formation of LDs (Figure ##FIG##3##4d,e##; Figure ##SUPPL##0##S5g##, Supporting Information), and levels of p‐AMPK and p‐ACC1 were also increased in PLC cells (Figure ##FIG##3##4f##). WT but not doublemut\\ncircLARP1B that overexpressed together with HNRNPD blocked the effects of HNRNPD on cell invasion, formation of LDs, and levels of p‐AMPK and p‐ACC1 (Figure ##FIG##3##4d–f##). Taking these results together, we concluded that the interaction between HNRNPD and circLARP1B was essential to regulatory functions of this circRNA, and it is possible that circLARP1B might function through binding to and modulating HNRNPD.
To provide further insights into the molecular mechanisms of circLARP1B and HNRNPD in HCC cells, we extracted cytoplasmic fractions of PLC cells and performed HNRNPD RIP of endogenously expressed HNRNPD, and the RNAs from RIP were subjected to RNA‐seq (RIP‐seq) (Figure ##SUPPL##0##S6a,b##, Supporting Information). Distribution of RIP‐seq reads on mRNA targets revealed predominate 3′ UTR counts (Figure ##SUPPL##0##S6a##, Supporting Information), consistent with the functional roles of cytoplasmic HNRNPD in regulating mRNA stability by binding to 3′ UTR.[\\n##REF##1901943##\\n48\\n##, ##REF##33444453##\\n50\\n##, ##REF##10702317##\\n56\\n##\\n] Comparison of RIP‐seq data from circLARP1B‐Def cells and circLARP1B overexpression cells to those from the corresponding control cells, identified 216 mRNAs with contrast changes of HNRNPD bindings on their 3′ UTR upon circLARP1B deficiency or overexpression (Figure ##FIG##4##\\n5a##; Table ##SUPPL##4##S4##, Supporting Information). We have reanalyzed the HNRNPD PAR‐CLIP data (GSE52977) from HEK293 cells,[\\n##REF##25366541##\\n61\\n##\\n] and found that the 216 mRNAs sensitive to circLARP1B had significantly lower HNRNPD PAR‐CLIP binding signals in the 3′ UTRs, compared to the other HNRNPD PAR‐CLIP targets (Figure ##SUPPL##0##S6c##, Supporting Information). These results indicated that the subset of mRNAs impacted by changes in circLARP1B levels might be with disadvantage in competing for the pool of HNRNPD, due to either relatively weaker binding ability to the protein or unfavorable cellular distribution and/or ratio of the corresponding mRNA/HNRNPD protein.
\",\n \"
Gene ontology (GO) analysis for these target genes illustrated biological processes such as peptidyl‐Thr phosphorylation, protein stability, and mRNA metabolic process (Figure ##FIG##4##5b##). Peptidyl‐Thr phosphorylation was the most significantly (with the lowest P‐value) enriched process, among which LKB1 also known as serine/threonine kinase 11 (STK11) showed the most overall HNRNPD binding signals (the sum of binding signals from all four types of cells) on the 3′ UTR (Figure ##FIG##4##5c##). This indicated that its 3′ UTR occupied the most HNRNPD protein out of the 3′ UTRs of the six genes enriched in this GO term. With mouse liver, we found that Hnrnpd RIP could also pull down Lkb1 mRNA (Figure ##SUPPL##0##S6d##, Supporting Information). LKB1 is well known to directly catalyze the phosphorylation of Thr172 of AMPK, which is required for AMPK activation.[\\n##REF##12847291##\\n62\\n##, ##REF##14985505##\\n63\\n##, ##REF##14614828##\\n64\\n##\\n] Directly based on the RIP‐seq data, circLARP1B deficiency resulted in more HNRNPD bindings, and circLARP1B overexpression led to less HNRNPD bindings, to the 3′ UTR of LKB1 mRNA (Figure ##FIG##4##5d##). We also found that the endogenous HNRNPD protein bound more circLARP1B upon circLARP1B overexpression and less circLARP1B in circLARP1B‐Def cells (Figure ##FIG##4##5e##). Furthermore, HNRNPD RIP revealed that circLARP1B deficiency but not knockdown of LARP1B mRNA resulted in significantly more HNRNPD binding to the 3′ UTR of LKB1 mRNA (Figure ##SUPPL##0##S6e–g##, Supporting Information). A recent study demonstrated that HNRNPD bound to the c‐MYC 3′ UTR and promoted the c‐MYC expression in colorectal cancer.[\\n##REF##35940521##\\n65\\n##\\n] HNRNPD RIP‐qPCR revealed that HNRNPD also bound to the c‐MYC 3′ UTR, and siRNA‐mediated HNRNPD knockdown significantly increased the c‐MYC protein levels in PLC cells (Figure ##SUPPL##0##S6h,i##, Supporting Information). c‐MYC was not in the 216 targets sensitive to circLARP1B (Figure ##FIG##4##5a##; Table ##SUPPL##4##S4##, Supporting Information), and the opposite effect of HNRNPD on c‐MYC expression in colorectal cancer and HCC cells required further investigation.
\",\n \"
We examined the effect of LKB1 in HCC cells directly by overexpression, and we found that the overexpressed LKB1 blocked the effect of circLARP1B in promoting cell invasion and formation of LDs (Figure ##FIG##4##5f,g##). Overexpressed LKB1 also nearly abolished the effects of circLARP1B on p‐AMPK and p‐ACC1 levels (Figure ##FIG##4##5h##). Lower LKB1 protein levels were observed in metastatic compared to nonmetastatic HCC specimens (Figure ##FIG##4##5i##). These results indicated that LKB1 mRNA was a downstream target of circLARP1B with robust contributions to the effects of circLARP1B.
\",\n \"\\n<italic toggle=\\\"yes\\\">CircLARP1B</italic> Competes with <italic toggle=\\\"yes\\\">LKB1</italic> mRNA for HNRNPD Binding and Leads to <italic toggle=\\\"yes\\\">LKB1</italic> mRNA Instability\",\n \"
HNRNPD has complex regulatory effects on the stability of mRNAs, with some stabilized and the others destabilized.[\\n##REF##1901943##\\n48\\n##, ##REF##33444453##\\n50\\n##, ##REF##10702317##\\n56\\n##\\n] We observed that the steady levels but not nascent levels of LKB1 mRNA were significantly increased, upon circLARP1B deficiency or knockdown in human and murine cells (Figure ##SUPPL##0##S6j,k##, Supporting Information). We then examined whether and how HNRNPD and circLARP1B regulated the steady levels of LKB1 mRNA. Knocking down HNRNPD with siRNA resulted in significant decrease in half‐life of LKB1 mRNA and also LKB1 protein levels in PLC and Hepa1‐6 cells (Figure ##FIG##5##\\n6a–d##), indicating that HNRNPD stabilized LKB1 mRNA. HNRNPD has been reported to bind to the 3′ UTR and simultaneously interact with translation initiation factor eIF4G1 and cap‐binding protein eIF4E to form a ternary complex, which could bring the 5′ and 3′ ends of the mRNA together to form a loop.[\\n##REF##21956942##\\n51\\n##\\n] We wondered whether HNRNPD looped LKB1 mRNA through this mechanism. We performed eIF4G1 and eIF4E RIP followed by RT‐qPCR with specific primers to detect the 3′ UTR of LKB1 mRNA in PLC cells with or without HNRNPD knockdown.[\\n##REF##25826658##\\n66\\n##, ##REF##27437580##\\n67\\n##\\n] HNRNPD knockdown resulted in decreased binding signal of both eIF4G1 and eIF4E to the LKB1 3′ UTR (Figure ##FIG##5##6e,f##). These results demonstrated that HNRNPD enhanced the mRNA looping and stabilized LKB1 mRNA.
\",\n \"
In circLARP1B‐Def PLC cells, compared to the WT cells, LKB1 mRNA stability was increased, and the steady levels of LKB1 protein were also increased (Figure ##FIG##5##6g,h##). Overexpression of circLARP1B in PLC cells destabilized LKB1 mRNA, and led to lower levels of LKB1 protein (Figure ##SUPPL##0##S6l,m##, Supporting Information). In mouse Hepa1‐6 cells, the stability of Lkb1 mRNA was markedly enhanced, and the steady levels of Lkb1 protein were significantly increased upon circLARP1B knockdown with shRNA (Figure ##FIG##5##6i,j##). Dual‐luciferase reporter using the 3′ UTR of LKB1 mRNA as the 3′ UTR of firefly luciferase mRNA showed that WT circLARP1B but not the doublemut\\ncircLARP1B could partially block the promoting effect on HNRNPD‐mediated stabilization of LKB1 3′ UTR (Figure ##SUPPL##0##S7a##, Supporting Information). Fluorescent in situ hybridization (FISH) of LKB1 mRNA and the HNRNPD IF staining revealed that circLARP1B‐Def compared to WT PLC cells, exhibited significantly more colocalization of LKB1 mRNA signals and HNRNPD protein signals in the cytoplasm (Figure ##SUPPL##0##S7b##, Supporting Information). ≈459 and ≈663 copies of LKB1 mRNA per cell were estimated in PLC and HepG2 cells, respectively (Figure ##SUPPL##0##S7c##, Supporting Information). The cytoplasmic copies of circLARP1B and LKB1 mRNA were evaluated in PLC cells, and the molecular ratio was ≈1:3 (Figure ##SUPPL##0##S7d##, Supporting Information). A competing assay, in which purified HNRNPD was incubated with equal moles of in vitro synthesized circLARP1B and LKB1 3′ UTR, was set up (Figure ##FIG##5##6k##). It was found that HNRNPD had much higher (≈18‐fold) binding affinity to circLARP1B than to the 3′ UTR of LKB1 mRNA (Figure ##FIG##5##6k##).
\",\n \"
A 24‐nt antisense oligodeoxynucleotide (ODN) complementary to the two HNRNPD binding sites in circLARP1B was synthesized to examine as an inhibitor of circLARP1B. The antisense ODN was modified with phosphorothioate (PS) and 2′‐O‐methyl (2′‐OMe),[\\n##REF##15064360##\\n68\\n##\\n] and termed ODN‐AS. ODN‐AS transfection in PLC cells increased the HNRNPD binding to LKB1 3′ UTR and upregulated LKB1 protein levels (Figure ##FIG##5##6l,m##). 5‐methylcytosine (m5C) modified sense single‐stranded RNA oligos (m5C‐ssRNA) containing the HNRNPD binding sites of circLARP1B were also tested.[\\n##UREF##6##\\n69\\n##\\n] m5C‐ssRNA did not affect either the HNRNPD binding to LKB1 3′ UTR or LKB1 protein levels (Figure ##SUPPL##0##S7e,f##, Supporting Information). These results indicated that the small m5C‐ssRNA oligos somehow could not function as a circLARP1B mimics in cells. On the other hand, the interaction between HNRNPD and circLARP1B could be blocked by ODN‐AS, and the potential effects of ODN‐AS in lipid metabolism, HCC metastasis, and even therapeutics could be further investigated.
\",\n \"\\n<italic toggle=\\\"yes\\\">CircLARP1B</italic> Deficiency in Mice Causes Liver Changes Attributable to Lkb1 Surplus\",\n \"
To directly investigate the physiological roles of circLARP1B in animals, we used CRISPR/Cas9 to generate circLARP1B deficient mice, in which repeat sequences in intron 4 of murine Larp1b gene were deleted (circLARP1B−/−\\n), with functional intronic elements such as the splice sites and pyrimidine tract remained unaltered (Figure ##FIG##6##\\n7a##). The mutant mice did not show statistically significant difference in body weight or litter size, when compared age‐matched WT mice (Figure ##SUPPL##0##S8a,b##, Supporting Information). The circLARP1B expression was significantly decreased in livers of circLARP1B−/−\\n mice compared to WT mice, while the total and nascent levels of Larp1b mRNA were unaltered (Figure ##FIG##6##7b##; Figure ##SUPPL##0##S8c##, Supporting Information). The levels of HNRNPD mRNA and protein also showed no significant difference in livers of WT and circLARP1B−/−\\n mice (Figure ##SUPPL##0##S8d##, Supporting Information). Liver was the focus due to that it is the organ giving rise to HCC, and a central organ of metabolism in the body.
\",\n \"
\\nCircLARP1B−/−\\n mice compared to WT mice had significantly reduced LDs in livers, examined by Oil red O staining (Figure ##FIG##6##7c##). Protein levels of Lkb1, p‐Ampk, and p‐Acc1 were significantly increased in circLARP1B−/−\\n livers (Figure ##FIG##6##7d,e##; Figure ##SUPPL##0##S8e##, Supporting Information). Further, we performed tail vein injection of 8‐week‐old circLARP1B−/−\\n mice with hepatocyte‐directed adeno‐associated virus 8 (AAV8) to express shRNA against Lkb1 (AAV8‐shLkb1) or the corresponding control (AAV8‐shCtrl). When examined 3 weeks after the infection, AAV8‐shLkb1 resulted in effective Lkb1 knockdown in circLARP1B−/−\\n livers, and Lkb1 depletion recovered the LDs levels in circLARP1B−/−\\n livers to the levels in WT livers (Figure ##FIG##6##7f,g##). The increased p‐Ampk and p‐Acc1 levels in circLARP1B−/−\\n liver were also restored nearly to the levels in WT liver (Figure ##FIG##6##7h,i##; Figure ##SUPPL##0##S8f##, Supporting Information).
\",\n \"\\n<italic toggle=\\\"yes\\\">CircLARP1B</italic> Deficiency in Mice Impedes HCC Metastasis and Lipid Accumulation\",\n \"
To examine physiological roles of circLARP1B in HCC at the whole organismal level, we induced HCC in mice with diethylnitrosamine (DEN), which is well established to cause severe liver damage and eventually hepatocarcinogenesis (Figure ##FIG##7##\\n8a##).[\\n##REF##22265403##\\n70\\n##\\n] After DEN induction for 10 weeks, liver inflammation examined by H&E staining was significantly lower in livers of circLARP1B−/−\\n mice than that of WT mice (Figure ##SUPPL##0##S8g##, Supporting Information). mRNA levels of Il6 and Tnf (as inflammation indicators) were also decreased in livers of circLARP1B−/−\\n mice (Figure ##SUPPL##0##S8h##, Supporting Information). The presence of bridging fibrosis was also examined, and it was found that circLARP1B−/−\\n mice had fewer Masson and Sirius Red signals in the portal areas (Figure ##SUPPL##0##S8i,j##, Supporting Information). Consistently, mRNA levels of fibrosis‐related markers, Col1a1, Tgfb1, and Mmp2 were decreased in livers of circLARP1B−/−\\n mice (Figure ##SUPPL##0##S8k##, Supporting Information). After DEN induction for 18 weeks (HCC mice), circLARP1B−/−\\n mice exhibited fewer and smaller tumor nodes in livers, and significantly smaller lung metastatic nodes, compared with WT mice (Figure ##FIG##7##8b,c##). HNRNPD levels demonstrated no significant difference in liver tumors between WT and circLARP1B−/−\\n HCC mice, while in HCC liver tumors of both genotypes, Hnrnpd levels were significantly higher than livers from age‐matched WT mice (Figure ##SUPPL##0##S8l,m##, Supporting Information). Immunohistochemistry (IHC) of liver tumor nodes revealed that the prometastasis marker Vimentin (Vim) was lower and the antimetastasis marker E‐cadherin (Ecad) was higher in circLARP1B−/−\\n HCC mice (Figure ##FIG##7##8d##). In consistence with HCC severity, the serum concentrations of alpha‐fetoprotein (AFP), a biomarker for HCC, was significantly lower in circLARP1B−/−\\n HCC mice (Figure ##FIG##7##8e##). Alanine transaminase (ALT) and aspartate transaminase (AST), two markers of liver injury, were also significantly lower in the serum of circLARP1B−/−\\n HCC mice (Figure ##FIG##7##8f,g##). CircLARP1B−/−\\n HCC mice exhibited lower triglyceride (TG) and total cholesterol (TC) levels in serum, and significantly fewer LDs in liver tumor nodes (Figure ##FIG##7##8h–j##). Meanwhile, levels of Lkb1, p‐Ampk, and p‐Acc1 were significantly higher in liver tumor nodes from circLARP1B−/−\\n HCC mice (Figure ##FIG##7##8k,l##; Figure ##SUPPL##0##S9a##, Supporting Information).
\",\n \"
To provide further insights for HCC metastatic roles of circLARP1B in liver cells, we infused hepatocyte‐directed AAV8 expressing shRNA against circLARP1B (AAV8‐shcircLARP1B) or the corresponding negative control (AAV8‐shCtrl) into WT mice after DEN induction for 10 weeks (Figure ##FIG##8##\\n9a##; Figure ##SUPPL##0##S3e##, Supporting Information).[\\n##REF##12192090##\\n71\\n##\\n] In livers of WT mice infused with AAV8‐shcircLARP1B, effective knockdown of circLARP1B was observed, while the Larp1b mRNA was unaltered (Figure ##FIG##8##9b##). Consistent with results from circLARP1B−/−\\n mice, AAV8‐shcircLARP1B injection resulted in fewer and smaller tumor nodes in livers, and smaller lung metastatic nodes, compared to AAV8‐shCtrl injection in WT mice (Figure ##FIG##8##9c,d##). The Vim IHC signals were lower, and the Ecad IHC signals were higher in liver tumors of WT mice with AAV8‐shcircLARP1B (Figure ##FIG##8##9e##). Serum AFP, ALT, AST, TG, and TC levels were also significantly lower in HCC mice with AAV8‐shcircLARP1B (Figure ##FIG##8##9f–j##). WT HCC mice infused with AAV8‐shcircLARP1B exhibited significantly fewer LDs and markedly higher levels of Lkb1, p‐Ampk, and p‐Acc1 in liver tumor nodes (Figure ##FIG##8##9k–m##; Figure ##SUPPL##0##S9b##, Supporting Information). Even these results were based on AAV8‐mediated hepatocyte specific circLARP1B knockdown, in the real case of HCC, effects of the niche, immune cells, and other unaccounted parameters could still contribute to the overall effects of circLARP1B on metastasis.
\",\n \"
Taken together, these results demonstrated that lower circLARP1B levels were unfavorable to HCC progression, lipid accumulation, and metastasis in mouse models, by affecting the same circLARP1B–HNRNPD–LKB1–AMPK regulatory pathway identified in HCC cell lines.
\",\n \"Lkb1 Is the Key Factor for the Effect of <italic toggle=\\\"yes\\\">CircLARP1B</italic> in HCC Mouse Model\",\n \"
We then performed tail vein injection with AAV8‐shLkb1 or the AAV8‐shCtrl once in circLARP1B−/−\\n HCC mice also at the 10th week after DEN induction. Hepatic Lkb1 protein was successfully knocked down (Figure ##SUPPL##0##S9c##, Supporting Information). We found that AAV8‐shLkb1 injection led to more and larger liver tumors, and larger lung metastatic nodes compared to AAV8‐shCtrl injection in circLARP1B−/−\\n HCC mice (Figure ##FIG##9##\\n10a,b##). In circLARP1B−/−\\n HCC mice upon AAV8‐shLkb1 application, liver tumor number and size, and lung metastases were as worse as what happened in the WT HCC mice (Figure ##FIG##9##10a,b##), indicating strongly that Lkb1 was the key downstream factor in hepatocytes for the effects of circLARP1B.
\",\n \"
Lkb1 knockdown led to the increased Vim levels and the decreased Ecad levels in liver tumor nodes in circLARP1B−/−\\n HCC mice (Figure ##FIG##9##10c##). Meanwhile, Lkb1 knockdown reversed these changes in Vim and Ecad levels caused by circLARP1B deficiency (Figure ##FIG##9##10c##). Lkb1 silencing nearly restored the circLARP1B‐deficient mediated suppressive effects on the levels of serum markers AFP, ALT, and AST in circLARP1B−/−\\n HCC mice (Figure ##SUPPL##0##S9d–f##, Supporting Information). Furthermore, Lkb1 depletion almost completely restored the inhibitory effects of circLARP1B deficiency on lipid accumulation in liver tumor nodes and serum TG and TC levels (Figure ##FIG##9##10d##; Figure ##SUPPL##0##S9g,h##, Supporting Information). Increased levels of p‐Ampk and p‐Acc1 observed in the liver of circLARP1B−/−\\n HCC mice were also eradicated upon Lkb1 knockdown (Figure ##FIG##9##10e##; Figure ##SUPPL##0##S9i,j##, Supporting Information). Therefore, Lkb1 was the key factor in hepatocytes for the molecular regulatory pathway of circLARP1B in HCC lipid metabolism and metastasis.
\",\n \"Clinical Significance of HNRNPD, LKB1, and AMPK in HCC\",\n \"
Levels of HNRNPD mRNA were significantly higher in the 23 metastatic specimens compared to 67 nonmetastatic specimens we collected (Figure ##SUPPL##0##S10a##, Supporting Information), and higher HNRNPD mRNA levels were positively associated with shorter OS and DFS analyzed based on these 90 HCC specimens (Figure ##FIG##9##10f##). HCC patients with higher LKB1 mRNA levels in tumors had significantly longer OS and DFS (Figure ##FIG##9##10g##). On the other hand, levels of LKB1 mRNA showed no statistically significant difference in metastatic versus nonmetastatic specimens (Figure ##SUPPL##0##S10b##, Supporting Information). Presumably upstream mechanisms might upregulate both HNRNPD and circLARP1B in metastatic HCC (Figure ##FIG##0##1g##), and the two regulators had opposite effects on the levels of LKB1 mRNA, with HNRNPD enhancing and circLARP1B destabilizing LKB1 mRNA. Therefore, these regulations might lead to the balance of LKB1 mRNA levels in metastatic versus nonmetastatic HCC. The AMPK mRNA levels demonstrated statistically significant increase in metastatic HCC, but its levels had no significant correlation to the OS and DFS of HCC patients (Figure ##SUPPL##0##S10c,d##, Supporting Information). AMPK was reported to either promote or suppress HCC,[\\n##REF##36316383##\\n11\\n##, ##REF##32631382##\\n72\\n##, ##REF##23942093##\\n73\\n##, ##REF##30853530##\\n74\\n##\\n] and in the circLARP1B–HNRNPD–LKB1–AMPK axis, the levels of p‐AMPK rather than the overall AMPK levels would be more relevant to HCC.
Roles of circRNAs in human diseases are a field with extensive studies,[\n##REF##35821160##\n5\n##, ##REF##34865956##\n13\n##, ##REF##31395983##\n14\n##, ##REF##31973951##\n15\n##\n] although further understandings of circRNAs with strong functions and in‐depth circRNA functional mechanisms are still required. In this study, a mammalian conserved circRNA circLARP1B is identified through the inspection of clinical specimens with different metastasis and prognostic features. Each circLARP1B molecule possesses two HNRNPD binding sites, and with more than 100 copies in the cytoplasm, circLARP1B can absolve a considerable portion of HNRNPD, and perturb the HNRNPD from binding to LKB1 mRNA. LKB1 mRNA is remarkably sensitive to low availability of HNRNPD, which leads to instability of this mRNA and then low LKB1 protein level. LKB1 as a kinase activates another mighty kinase AMPK that regulates downstream targets including the phosphorylation of ACC1 to constrain FAS and metastasis in HCC. Through this regulatory pathway, high levels of circLARP1B result in high level of FAS and metastasis in HCC (Figure ##SUPPL##0##S10e##, Supporting Information).
","
CircRNAs are pervasively expressed and can be conserved across species,[\n##REF##34865956##\n13\n##, ##REF##31395983##\n14\n##, ##REF##31973951##\n15\n##, ##REF##32345360##\n33\n##, ##REF##32694641##\n75\n##\n] and several conserved circRNAs such as CDR1as in mammals and circBoule in metazoans have been investigated with animal models.[\n##REF##23446348##\n19\n##, ##UREF##1##\n20\n##, ##UREF##2##\n24\n##\n] As for HCC or even cancers, almost all reported circRNAs are either not conserved or not subjected to examinations with genetically manipulated animal models.[\n##REF##35821160##\n5\n##, ##REF##34865956##\n13\n##\n] A previous report has revealed that the p21 mRNA stability is enhanced by circPCNX, which inhibits HNRNPD's binding to p21 mRNA in human HeLa cells,[\n##REF##33444453##\n50\n##\n] although circPCNX is not conserved in mice. Another circRNA, circURI1 also functions as a protein sponge of HNRNPM to modulate alternative splicing of genes such as VEGF1 to inhibit gastric cancer metastasis,[\n##UREF##5##\n32\n##\n] and circURI1 is again not conserved in mice. The nature that circLARP1B is a mammalian conserved circRNA facilitates our study, especially enables detailed investigations of circLARP1B functions and functional mechanisms at the whole organismal level. It is evident that circLARP1B utilizes a conserved mechanism to play roles in regulating a cascade that dictates lipid synthesis in HCC, and maybe also in other physiological events or disorders.
","
Cancer metastasis is always coupled with lipid metabolism reprogramming.[\n##REF##35022204##\n8\n##, ##UREF##0##\n9\n##, ##REF##23446547##\n10\n##\n] Take LDs as an example, besides other metastasis promoting functions, increased storage of lipids as LDs at least provide energy reservoir for metastatic cloning.[\n##REF##33462499##\n7\n##, ##REF##32506038##\n76\n##\n] A recent study has demonstrated that ND‐654, as a liver‐specific ACC inhibitor, constrains hepatic de novo lipogenesis and HCC development by mimicking the effects of ACC phosphorylation.[\n##REF##30244972##\n12\n##\n] Besides the regulation of ACC activity in lipid biosynthesis, both LKB1 and AMPK are potent kinases, and LKB1/AMPK pathway has an array of known functionalities in HCC and cancers.[\n##REF##36316383##\n11\n##, ##REF##19629071##\n77\n##\n] In addition to ACC1, the mTOR signaling has been known to promote FAS and is relevant to liver tumorigenesis,[\n##UREF##0##\n9\n##, ##REF##30425336##\n44\n##\n] which seems also regulated by circLARP1B through the AMPK pathway (Figure ##SUPPL##0##S2h##, Supporting Information). mTOR has been subjected to extensive investigations, although the involvement of mTOR signaling in the effects of circLARP1B in HCC metastasis and lipid metabolism needs further exploration. Data from PLC cells demonstrate that FAS, but not FAO or lipophagy, is the process that regulated by circLARP1B (Figure ##FIG##1##2d–j##; Figure ##SUPPL##0##S2e–i##, Supporting Information).
","
\nCircLARP1B deficiency leads to the dysregulation of a series of lipid classes (Figure ##FIG##1##2f##). PC, PE, PI, phosphatidylglycerol, and cardiolipin decreased in circLARP1B‐Def PLC cells belong to glycerophospholipids, which are associated with drug‐resistance in multiple cancers.[\n##REF##31846838##\n78\n##\n] For instance, colorectal cancer patients with abnormal biosynthesis of PC are resistant to oxaliplatin and 5‐fluorouracil.[\n##REF##31846838##\n78\n##\n] DG, TG, and monoglyceride that are also decreased in circLARP1B‐Def PLC cells belong to glycerolipids, which function as secondary messengers to activate downstream oncogenic signaling.[\n##REF##35764645##\n79\n##\n] Sphingomyelin (SM), N‐acetylhexosyl ceramide (CerG2GNAc1), ceramides phosphate, and phytosphingosine with increased levels in circLARP1B‐Def cells belong to sphingolipids, which are enriched in the lipid rafts localized in cellular membranes, and play critical roles in tumor metastasis, such as EMT and angiogenesis.[\n##REF##32999001##\n80\n##\n] It is highly possible that these molecules are not just substrates or products of lipid metabolism but also modulators that contribute to the effects of circLARP1B in HCC.
","
We have provided lines of evidence to reveal roles of circLARP1B in hepatocytes with cell lines, knockout mice, and hepatocyte‐directed circLARP1B knockdown mice, although the effects of the niche, immune cells, and other unaccounted parameters could also contribute to the final effects observed in HCC. For the effects of hepatocytic Lkb1 depletion in circLARP1B−/−\n HCC mice (Figure ##FIG##9##10a–c##), the restoration of liver tumor numbers and the Vim and Ecad IHC in liver tumors indicate that Lkb1 depletion in hepatocytes promotes malignant transformation; the restoration of liver tumor size indicates that Lkb1 depletion also promotes tumor growth. Hepatocyte‐directed AAV8‐mediated circLARP1B knockdown significantly inhibits HCC metastasis and relevant traits in mice, providing valuable insights for developing this circRNA as a therapeutic target of HCC.
","
We have provided experimental data from multiple levels to support that LKB1 mRNA is the key downstream target of circLARP1B via perturbing HNRNPD in both human and mice. Even hepatocytic knockdown of LKB1 seems sufficient to abolish effects of circLARP1B absence in HCC (Figure ##FIG##9##10a–e##; Figure ##SUPPL##0##S9c–j##, Supporting Information), it is possible that the other mRNAs sensitive to circLARP1B and HNRNPD may also take part in the regulatory effects of circLARP1B to some degree. HNRNPD, a well‐recognized and conserved RBP with multiple post‐translational modifications, is upregulated in various human cancers including HCC, and is associated with poor prognosis and drug resistance.[\n##REF##1901943##\n48\n##, ##REF##19033365##\n49\n##, ##REF##21956942##\n51\n##, ##REF##35178834##\n81\n##, ##REF##37060820##\n82\n##, ##UREF##7##\n83\n##, ##REF##33016643##\n84\n##\n] In the cytoplasm, HNRNPD can promote the decay of some mRNAs, and on the other hand stabilize some mRNA targets (e.g., PTH and VHL).[\n##REF##1901943##\n48\n##, ##REF##33444453##\n50\n##, ##REF##10702317##\n56\n##\n] Intriguingly, for c‐MYC mRNA, HNRNPD promotes its degradation in human lymphoblast cell line K562 and PLC liver cancer cells, and increases its stability in murine fibroblast 3T3 and human colorectal cancer cell line HCT116 indicating that the effect of HNRNPD may depend on the overall interactions among proteins bound to the mRNA.[\n##REF##35940521##\n65\n##, ##REF##16391004##\n85\n##\n] We have demonstrated clinical significance of circLARP1B, HNRNPD, and LKB1 in HCC. As for AMPK, it may play complex regulatory roles through various downstream signaling networks in cancers. In this study, p‐AMPK has been shown to play critical roles in regulating HCC metastasis through remodeling lipid metabolism, and further investigation concentrating on clinical relevance of the kinase role of AMPK in HCC is required.
","
Starting from screening with clinical specimens collected, we have identified the functional roles and mechanisms of the mammalian conserved circLARP1B in HCC metastasis and lipid metabolism. Data from both human and mice have revealed a pathway of circLARP1B–HNRNPD–LKB1–AMPK with potent regulatory effects in HCC lipid metabolism and metastasis. As it is closely associated with progression and prognosis, circLARP1B may be explored as a diagnostic marker or even a therapeutic target for HCC.
"],"string":"[\n \"Discussion\",\n \"
Roles of circRNAs in human diseases are a field with extensive studies,[\\n##REF##35821160##\\n5\\n##, ##REF##34865956##\\n13\\n##, ##REF##31395983##\\n14\\n##, ##REF##31973951##\\n15\\n##\\n] although further understandings of circRNAs with strong functions and in‐depth circRNA functional mechanisms are still required. In this study, a mammalian conserved circRNA circLARP1B is identified through the inspection of clinical specimens with different metastasis and prognostic features. Each circLARP1B molecule possesses two HNRNPD binding sites, and with more than 100 copies in the cytoplasm, circLARP1B can absolve a considerable portion of HNRNPD, and perturb the HNRNPD from binding to LKB1 mRNA. LKB1 mRNA is remarkably sensitive to low availability of HNRNPD, which leads to instability of this mRNA and then low LKB1 protein level. LKB1 as a kinase activates another mighty kinase AMPK that regulates downstream targets including the phosphorylation of ACC1 to constrain FAS and metastasis in HCC. Through this regulatory pathway, high levels of circLARP1B result in high level of FAS and metastasis in HCC (Figure ##SUPPL##0##S10e##, Supporting Information).
\",\n \"
CircRNAs are pervasively expressed and can be conserved across species,[\\n##REF##34865956##\\n13\\n##, ##REF##31395983##\\n14\\n##, ##REF##31973951##\\n15\\n##, ##REF##32345360##\\n33\\n##, ##REF##32694641##\\n75\\n##\\n] and several conserved circRNAs such as CDR1as in mammals and circBoule in metazoans have been investigated with animal models.[\\n##REF##23446348##\\n19\\n##, ##UREF##1##\\n20\\n##, ##UREF##2##\\n24\\n##\\n] As for HCC or even cancers, almost all reported circRNAs are either not conserved or not subjected to examinations with genetically manipulated animal models.[\\n##REF##35821160##\\n5\\n##, ##REF##34865956##\\n13\\n##\\n] A previous report has revealed that the p21 mRNA stability is enhanced by circPCNX, which inhibits HNRNPD's binding to p21 mRNA in human HeLa cells,[\\n##REF##33444453##\\n50\\n##\\n] although circPCNX is not conserved in mice. Another circRNA, circURI1 also functions as a protein sponge of HNRNPM to modulate alternative splicing of genes such as VEGF1 to inhibit gastric cancer metastasis,[\\n##UREF##5##\\n32\\n##\\n] and circURI1 is again not conserved in mice. The nature that circLARP1B is a mammalian conserved circRNA facilitates our study, especially enables detailed investigations of circLARP1B functions and functional mechanisms at the whole organismal level. It is evident that circLARP1B utilizes a conserved mechanism to play roles in regulating a cascade that dictates lipid synthesis in HCC, and maybe also in other physiological events or disorders.
\",\n \"
Cancer metastasis is always coupled with lipid metabolism reprogramming.[\\n##REF##35022204##\\n8\\n##, ##UREF##0##\\n9\\n##, ##REF##23446547##\\n10\\n##\\n] Take LDs as an example, besides other metastasis promoting functions, increased storage of lipids as LDs at least provide energy reservoir for metastatic cloning.[\\n##REF##33462499##\\n7\\n##, ##REF##32506038##\\n76\\n##\\n] A recent study has demonstrated that ND‐654, as a liver‐specific ACC inhibitor, constrains hepatic de novo lipogenesis and HCC development by mimicking the effects of ACC phosphorylation.[\\n##REF##30244972##\\n12\\n##\\n] Besides the regulation of ACC activity in lipid biosynthesis, both LKB1 and AMPK are potent kinases, and LKB1/AMPK pathway has an array of known functionalities in HCC and cancers.[\\n##REF##36316383##\\n11\\n##, ##REF##19629071##\\n77\\n##\\n] In addition to ACC1, the mTOR signaling has been known to promote FAS and is relevant to liver tumorigenesis,[\\n##UREF##0##\\n9\\n##, ##REF##30425336##\\n44\\n##\\n] which seems also regulated by circLARP1B through the AMPK pathway (Figure ##SUPPL##0##S2h##, Supporting Information). mTOR has been subjected to extensive investigations, although the involvement of mTOR signaling in the effects of circLARP1B in HCC metastasis and lipid metabolism needs further exploration. Data from PLC cells demonstrate that FAS, but not FAO or lipophagy, is the process that regulated by circLARP1B (Figure ##FIG##1##2d–j##; Figure ##SUPPL##0##S2e–i##, Supporting Information).
\",\n \"
\\nCircLARP1B deficiency leads to the dysregulation of a series of lipid classes (Figure ##FIG##1##2f##). PC, PE, PI, phosphatidylglycerol, and cardiolipin decreased in circLARP1B‐Def PLC cells belong to glycerophospholipids, which are associated with drug‐resistance in multiple cancers.[\\n##REF##31846838##\\n78\\n##\\n] For instance, colorectal cancer patients with abnormal biosynthesis of PC are resistant to oxaliplatin and 5‐fluorouracil.[\\n##REF##31846838##\\n78\\n##\\n] DG, TG, and monoglyceride that are also decreased in circLARP1B‐Def PLC cells belong to glycerolipids, which function as secondary messengers to activate downstream oncogenic signaling.[\\n##REF##35764645##\\n79\\n##\\n] Sphingomyelin (SM), N‐acetylhexosyl ceramide (CerG2GNAc1), ceramides phosphate, and phytosphingosine with increased levels in circLARP1B‐Def cells belong to sphingolipids, which are enriched in the lipid rafts localized in cellular membranes, and play critical roles in tumor metastasis, such as EMT and angiogenesis.[\\n##REF##32999001##\\n80\\n##\\n] It is highly possible that these molecules are not just substrates or products of lipid metabolism but also modulators that contribute to the effects of circLARP1B in HCC.
\",\n \"
We have provided lines of evidence to reveal roles of circLARP1B in hepatocytes with cell lines, knockout mice, and hepatocyte‐directed circLARP1B knockdown mice, although the effects of the niche, immune cells, and other unaccounted parameters could also contribute to the final effects observed in HCC. For the effects of hepatocytic Lkb1 depletion in circLARP1B−/−\\n HCC mice (Figure ##FIG##9##10a–c##), the restoration of liver tumor numbers and the Vim and Ecad IHC in liver tumors indicate that Lkb1 depletion in hepatocytes promotes malignant transformation; the restoration of liver tumor size indicates that Lkb1 depletion also promotes tumor growth. Hepatocyte‐directed AAV8‐mediated circLARP1B knockdown significantly inhibits HCC metastasis and relevant traits in mice, providing valuable insights for developing this circRNA as a therapeutic target of HCC.
\",\n \"
We have provided experimental data from multiple levels to support that LKB1 mRNA is the key downstream target of circLARP1B via perturbing HNRNPD in both human and mice. Even hepatocytic knockdown of LKB1 seems sufficient to abolish effects of circLARP1B absence in HCC (Figure ##FIG##9##10a–e##; Figure ##SUPPL##0##S9c–j##, Supporting Information), it is possible that the other mRNAs sensitive to circLARP1B and HNRNPD may also take part in the regulatory effects of circLARP1B to some degree. HNRNPD, a well‐recognized and conserved RBP with multiple post‐translational modifications, is upregulated in various human cancers including HCC, and is associated with poor prognosis and drug resistance.[\\n##REF##1901943##\\n48\\n##, ##REF##19033365##\\n49\\n##, ##REF##21956942##\\n51\\n##, ##REF##35178834##\\n81\\n##, ##REF##37060820##\\n82\\n##, ##UREF##7##\\n83\\n##, ##REF##33016643##\\n84\\n##\\n] In the cytoplasm, HNRNPD can promote the decay of some mRNAs, and on the other hand stabilize some mRNA targets (e.g., PTH and VHL).[\\n##REF##1901943##\\n48\\n##, ##REF##33444453##\\n50\\n##, ##REF##10702317##\\n56\\n##\\n] Intriguingly, for c‐MYC mRNA, HNRNPD promotes its degradation in human lymphoblast cell line K562 and PLC liver cancer cells, and increases its stability in murine fibroblast 3T3 and human colorectal cancer cell line HCT116 indicating that the effect of HNRNPD may depend on the overall interactions among proteins bound to the mRNA.[\\n##REF##35940521##\\n65\\n##, ##REF##16391004##\\n85\\n##\\n] We have demonstrated clinical significance of circLARP1B, HNRNPD, and LKB1 in HCC. As for AMPK, it may play complex regulatory roles through various downstream signaling networks in cancers. In this study, p‐AMPK has been shown to play critical roles in regulating HCC metastasis through remodeling lipid metabolism, and further investigation concentrating on clinical relevance of the kinase role of AMPK in HCC is required.
\",\n \"
Starting from screening with clinical specimens collected, we have identified the functional roles and mechanisms of the mammalian conserved circLARP1B in HCC metastasis and lipid metabolism. Data from both human and mice have revealed a pathway of circLARP1B–HNRNPD–LKB1–AMPK with potent regulatory effects in HCC lipid metabolism and metastasis. As it is closely associated with progression and prognosis, circLARP1B may be explored as a diagnostic marker or even a therapeutic target for HCC.
Circular RNAs (circRNAs) have emerged as crucial regulators in physiology and human diseases. However, evolutionarily conserved circRNAs with potent functions in cancers are rarely reported. In this study, a mammalian conserved circRNA circLARP1B is identified to play critical roles in hepatocellular carcinoma (HCC). Patients with high circLARP1B levels have advanced prognostic stage and poor overall survival. CircLARP1B facilitates cellular metastatic properties and lipid accumulation through promoting fatty acid synthesis in HCC. CircLARP1B deficient mice exhibit reduced metastasis and less lipid accumulation in an induced HCC model. Multiple lines of evidence demonstrate that circLARP1B binds to heterogeneous nuclear ribonucleoprotein D (HNRNPD) in the cytoplasm, and thus affects the binding of HNRNPD to sensitive transcripts including liver kinase B1 (LKB1) mRNA. This regulation causes decreased LKB1 mRNA stability and lower LKB1 protein levels. Antisense oligodeoxynucleotide complementary to theHNRNPD binding sites in circLARP1B increases the HNRNPD binding to LKB1 mRNA. Through the HNRNPD–LKB1–AMPK pathway, circLARP1B promotes HCC metastasis and lipid accumulation. Results from AAV8‐mediated hepatocyte‐directed knockdown of circLARP1B or Lkb1 in mouse models also demonstrate critical roles of hepatocytic circLARP1B regulatory pathway in HCC metastasis and lipid accumulation, and indicate that circLARP1B may be potential target of HCC treatment.
","
The mammalian conserved circLARP1B promotes hepatocellular carcinoma metastasis and lipid accumulation. CircLARP1B functions by sequestering cytoplasmic heterogeneous nuclear ribonucleoprotein D (HNRNPD) to decrease HNRNPD binding to sensitive transcripts including liver kinase B1 (LKB1) mRNA, leading to decreased LKB1 mRNA stability and lower protein levels. Hepatocyte‐directed knockdown of circLARP1B or Lkb1 in mice impedes hepatocellular carcinoma metastasis and lipid accumulation.\n\n
Circular RNAs (circRNAs) have emerged as crucial regulators in physiology and human diseases. However, evolutionarily conserved circRNAs with potent functions in cancers are rarely reported. In this study, a mammalian conserved circRNA circLARP1B is identified to play critical roles in hepatocellular carcinoma (HCC). Patients with high circLARP1B levels have advanced prognostic stage and poor overall survival. CircLARP1B facilitates cellular metastatic properties and lipid accumulation through promoting fatty acid synthesis in HCC. CircLARP1B deficient mice exhibit reduced metastasis and less lipid accumulation in an induced HCC model. Multiple lines of evidence demonstrate that circLARP1B binds to heterogeneous nuclear ribonucleoprotein D (HNRNPD) in the cytoplasm, and thus affects the binding of HNRNPD to sensitive transcripts including liver kinase B1 (LKB1) mRNA. This regulation causes decreased LKB1 mRNA stability and lower LKB1 protein levels. Antisense oligodeoxynucleotide complementary to theHNRNPD binding sites in circLARP1B increases the HNRNPD binding to LKB1 mRNA. Through the HNRNPD–LKB1–AMPK pathway, circLARP1B promotes HCC metastasis and lipid accumulation. Results from AAV8‐mediated hepatocyte‐directed knockdown of circLARP1B or Lkb1 in mouse models also demonstrate critical roles of hepatocytic circLARP1B regulatory pathway in HCC metastasis and lipid accumulation, and indicate that circLARP1B may be potential target of HCC treatment.
\",\n \"
The mammalian conserved circLARP1B promotes hepatocellular carcinoma metastasis and lipid accumulation. CircLARP1B functions by sequestering cytoplasmic heterogeneous nuclear ribonucleoprotein D (HNRNPD) to decrease HNRNPD binding to sensitive transcripts including liver kinase B1 (LKB1) mRNA, leading to decreased LKB1 mRNA stability and lower protein levels. Hepatocyte‐directed knockdown of circLARP1B or Lkb1 in mice impedes hepatocellular carcinoma metastasis and lipid accumulation.\\n\\n
All fresh HCC tumor tissues were collected from Sir Run Run Shaw Hospital, which was approved by the Human Research Ethics Committee of Sir Run Run Shaw Hospital (SRRSHLS2022Y0312). Written informed consent was obtained from each patient for this study. All samples were frozen and stored in liquid nitrogen after removal from the operation followed by washing with PBS twice.
","Mice","
All mice were housed in an enriched environment under the standard conditions (23–25 °C temperature, 40–60% humidity) with a 12‐h light‐dark cycle (lights on from 08:00 to 20:00) at the Specific‐Pathogen‐Free facility with unrestricted access to food and water for the duration of the experiment, unless specified in the corresponding situation. All animal protocols were approved by the Animal Care and Use Committee of the University of Science and Technology of China (USTCACUC212201037). For genetically engineered mice, whole‐body circLARP1B\n−/− mice were generated with the CRISPR/Cas9 system as previously described.[\n##REF##36182935##\n18\n##\n] sgRNAs targeting Larp1b intron 4 were designed by CRISPick web‐server (https://portals.broadinstitute.org/gppx/crispick/public). The synthesized Cas9 mRNA and sgRNAs were coinjected into fertilized embryos of C57BL/6J mice and 100 zygotes were implanted into five ICR strain surrogate mice. All F0 founders were genotyped at 2 weeks after birth with PCR amplification followed by Sanger sequencing. Specific primers for PCR were included in Table ##SUPPL##5##S5## of the Supporting Information. Positive F0 founders were backcrossed to C57BL/6 mice to obtain deficient allele heritable heterozygous F1 mice, which were further intercrossed to generate circLARP1B\n−/− homozygous mice. All circLARP1B\n−/− mice used for analyses were in parallel with age‐ and gender‐matched wildtype littermates (as the control group). Male mice were used for experiments in this study.[\n##REF##37005415##\n86\n##, ##REF##32182125##\n87\n##\n]\n
","Library Preparation for RNA‐seq","
Total RNA from frozen tissues or cultured cells was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's protocols. Libraries were prepared with the TruSeq Ribo Profile Library Prep Kit (Illumina) according to the manufacturer's instructions and then subjected to sequencing in an Illumina Nova‐PE150 system (Novogene). Each library generated ≈100 million 150‐nt paired‐end read pairs.
","CircRNA Identification","
The circRNA candidates were identified with CIRI2 and the BSJ reads per million were used to calculate circRNA levels. Briefly, the adapters were removed with Cutadapt (‐e 0.1 ‐O 5) to obtain clean reads. The reads continuously aligned to the human reference genome (hg19) were filtered with Bowtie (‐v 1) allowing one mismatch. The remaining reads were used to predict circRNA candidates with CIRI2 with default parameters. The differentially expressed circRNAs were determined by DEseq2 with a criterion (fold change >4 or <0.25, P‐value <0.05). For conservation analysis of circRNAs, the circRNA sequences were aligned to annotated murine circRNAs from the circAtlas database with Blast. Conserved circRNAs between human and mice were determined with a stringent cutoff (full‐length and BSJ sequences >75% identical).
","Cell Culture and Cell Transfection","
The PLC/PRF/5 (PLC) cells were kindly provided by Dr. Yide Mei (USTC). HepG2, HEK293T, K1, and Hepa1‐6 cells all originated from the American Type Culture Collection. All above cells were maintained under standard conditions with the DMEM medium containing 10% FBS (CLARK, FB25015) and 1% penicillin/streptomycin (Beyotime, C0222) at 37 °C under 5% CO2. A PCR‐based method and DAPI staining were used to ensure cells without contamination of mycoplasma. All cell lines were authenticated by short‐tandem‐repeat profiling. Transfection of plasmids and siRNAs was conducted with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
Two sgRNAs targeting the intron 4 of human LARP1B were designed based on http://crispr.mit.edu to knockout circLARP1B. After transfection of 2 µg pX330 plasmids expressing Cas9 mRNA and targeted sgRNAs for two days, hundreds of mCherry‐positive single‐cell clones were sorted through the BD FACSAria III cell sorting system. Two weeks later, the colonies were split into two identical plates and one plate was harvested for PCR‐mediated genotype and confirmation by Sanger sequencing. The primer sequences are listed in Table ##SUPPL##5##S5## of the Supporting Information.
","OCR Measurements","
The OCR analyses were performed using the Agilent Seahorse XF Cell Mito Stress Test Kit (Agilent, 103015‐100) as previously described.[\n##REF##33758188##\n88\n##\n] Briefly, 20 000 cells per well were seeded in a 96‐well XF cell culture microplate in the growth medium overnight at 37 °C in 5% CO2. OCR was measured with an XF96 analyzer in XF DMEM medium (Agilent, 103575‐100) containing 1 mm pyruvate, 2 mm glutamine, and 10 mm glucose, followed by sequential addition of 1.5 µm oligomycin (ATP synthase inhibitor), 1.0 µm FCCP (membrane potential uncoupler), 0.5 µm rotenone (Complex I inhibitor), and 0.5 µm antimycin A (Complex III inhibitor). Data were analyzed by the Seahorse XF Cell Mito Stress Test Report Generator package.
","FAO Rate Measurements","
The FAO rate analyses were performed using the Agilent Seahorse XF Cell Mito Stress Test Kit (Agilent, 103015‐100) as previously described.[\n##REF##31155494##\n89\n##\n] Briefly, 15 000 cells per well were seeded in a 96‐well XF cell culture microplate in the growth medium overnight at 37 °C in 5% CO2. Then, cells were starved by incubating with substrate‐limited DMEM supplemented with 0.5 mm glucose, 1 mm glutamate, 0.5 mm carnitine, and 1% FBS. After 24‐h starvation, the medium was replaced by FAO assay medium (111 mm NaCl, 4.7 mm KCl, 1.25 mm CaCl2, 2 mm MgSO4, 1.2 mm NaH2PO4 supplemented with 2.5 mm glucose, 0.5 mm carnitine, and 5 mm HEPES, pH 7.4). Finally, BSA‐conjugated palmitate (Agilent, 102720‐100) was added to a final concentration of 50 mm, followed by sequential addition of 1.5 µm oligomycin, 1.0 µm FCCP, 0.5 µm rotenone, and 0.5 µm antimycin A. Data were analyzed by the Seahorse XF Cell Mito Stress Test Report Generator package.
\n13C‐labeled targeted metabolic flux analysis was carried out as previously described with minor modifications.[\n##REF##32101748##\n37\n##\n] In brief, 107 PLC cells were washed with 0.9% saline three times and incubated in substrate‐limited DMEM supplemented with 15 mm\n13C‐glucose, 4 mm\n13C‐glutamine, and 10% FBS for 24 h before metabolite extraction. After washing with cold 0.9% saline twice, cells were incubated with 500 µL cold extraction buffer (methanol:acetonitrile:water, 2:2:1, v/v/v). Harvested cells were sonicated for 2 min, centrifuged at 14 000 × g for 5 min at 4 °C and cell supernatants were transferred to new tubes. 400 µL chloroform was added and the tubes were vortexed, centrifuged at 14 000 × g for 5 min at 4 °C. The resulting phase separation resulted in an aqueous upper phase containing polar metabolites and an organic lower phase containing nonpolar metabolites. The aqueous upper phase was transferred to tubes and the lower chloroform phase was transferred to a glass tube. For reverse phase liquid chromatography separation, ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 µm, Waters) was used. Mass spectrometric data were acquired with a Q‐Exactive plus hybrid quadrupole–orbitrap mass spectrometer (Thermo) using negative ion electrospray ionization. The scan range was from 50 to 1000 m/z. The scan time for each function was set to 0.2 s. Ion monitoring conditions were defined as a capillary voltage of 2.0 kV, source temperature of 120 °C, and desolvation temperature of 500 °C. Data processing and ion annotation based on accurate mass were performed in TraceFinder 5.0 (Thermo) and Xcalibur 4.0 (Thermo). Metabolite mass isotopomer distribution was determined based on the ratio of the integrated peak areas of the chosen isotopomer to the sum of all the integrated peak areas of the possible isotopomers for the given metabolites.
","Plasmids Construction","
All plasmids were constructed with restriction‐enzyme digestion and ligation or with recombinant methods (Vazyme, c113‐02). For circLARP1B overexpression in human, the circularized exons and the endogenous flanking sequences including one Alu pair were inserted into the pcDNA3 vector. The backbone vector of p3×FLAG‐Myc was used for constructing FLAG‐tagged HNRNPD and LKB1. The shRNA against the BSJ of human or murine circLARP1B was cloned into the vector pLKO.1 (Sigma) and the negative‐control shRNA (shCtrl, MFCD07785395) was obtained from the MISSION shRNA Library (Sigma). The sgRNAs for human or murine circLARP1B were inserted into the Cas9‐ and sgRNA‐expressing backbone (pX330), which was engineered with expressing mCherry and a puromycin selectable marker. All plasmids have been sequenced for confirmation and further information about these plasmids is available upon request. Oligonucleotide sequences for primers used in plasmids construction, along with other oligo sequences used in probe preparation, siRNA, and biotin‐labeled nucleic acids were listed in Table ##SUPPL##5##S5## of the Supporting Information.
","Northern Blotting","
Northern blotting was performed as previously described.[\n##REF##29322446##\n90\n##\n] The digoxigenin‐labeled RNA probe with antisense sequences to the circLARP1B BSJ was synthesized with a DIG Northern Starter Kit (Roche, 12039672910) according to the manufacturer's protocol. The primers for probe preparation were listed in Table ##SUPPL##5##S5## of the Supporting Information.
","Single‐Molecule Fluorescence In Situ Hybridization","
The smFISH was carried out as previously described with minor modifications.[\n##UREF##8##\n91\n##\n] Cells or tissues were fixed with 4% PFA for 10 min at room temperature and permeabilized with ice‐cold 70% ethanol overnight at −20 °C. For hybridization, cells or tissues were prehybridized at 37 °C for 30 min in hybridization buffer (30% formamide, 5× SSC, 9 mm citric acid, pH 6.0, 0.1% Tween‐20, 50 µg mL−1 heparin, 1× Denhardt's solution, and 10% dextran sulfate) after washing with 2× SSC twice. Then, samples were hybridized with 2 pmol probes overnight at 37 °C in the hybridization buffer. For the amplification stage, 18 pmol hairpins were heated at 95 °C for 90 s and cooled to room temperature in a dark drawer for 30 min. Samples were incubated with the denatured probes overnight at room temperature followed by three 5 min washes in 2× SSC buffer. After a 10 min incubation in DAPI (5 mg mL−1), the sections were washed three times for 5 min in 2× SSC buffer. The images were captured using the laser confocal microscopy LSM880 (Zeiss). The ImageJ was used to further process the data, and the ImageJ Plot Profile tool was applied to calculate signal intensity. All probe sequences were included in Table ##SUPPL##5##S5## of the Supporting Information.
","PCR Reactions","
For RT‐PCR, 500 ng total RNA was reverse‐transcribed into complementary DNA (cDNA) with the GoScript Reverse Transcription System (Promega, A5001) according to the manufacturer's protocol. For PCR with genomic DNA (gDNA) as the template, gDNA was isolated by phenol/chloroform extraction. For semiquantitative RT‐PCR gels, 25–30 cycles were carried out. The RT‐qPCR was performed using GoTaq SYBR Green qPCR Master Mix (Promega, A6001) on a PikoReal 96 real‐time PCR system (Thermo). All PCR products were confirmed by Sanger sequencing. All used primer sequences are included in Table ##SUPPL##5##S5## of the Supporting Information.
","Nuclear/Cytosolic Fractionation","
Cellular fractionation was carried out as previously described with minor modifications.[\n##REF##36182935##\n18\n##\n] Briefly, cells were incubated with hypotonic buffer (10 mm Tris‐HCl, pH 8.0, 140 mm NaCl, 1.5 mm MgCl2, 0.5% NP‐40, 1 mm DTT, and 0.1 U µL−1 RNase inhibitor (Promega, N2615)) on ice for 20 min after washing with PBS twice. After centrifugation at 1000 × g for 5 min at 4 °C, the supernatant was collected as the cytoplasmic fraction. The pellets were then resuspended in nuclear resuspension buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm DTT, and 0.1 U µL−1 RNase inhibitor followed by the incubation at 4 °C for 30 min. Nuclear fraction was collected after removing insoluble membrane debris by centrifugation at 12 000 × g for 15 min.
","Quantification of RNA Copy Number per Cell","
The protocol was carried out as previously described.[\n##UREF##5##\n32\n##\n] Total RNA was extracted from 106 PLC and HepG2 cells, and cDNA was synthesized. The standard curve was used to calculate the copy numbers per cell in each cell line based on cell numbers and Ct values.
","Transwell Assay","
Transwell invasion assay was performed using the chamber with Matrigel (BD Biosciences). Cells were seeded into the upper chamber cultured with the serum‐free DMEM medium, and DMEM containing 10% FBS was added to the lower chamber. After 24 h, the migrated cells on the outer membrane were stained with 0.1% crystal violet for 10 min. Images were captured with an inverted microscope (Olympus) and the number of invaded cells was quantified by ImageJ.
","Cell Viability and Number Measurement and Colony Formation","
The cell viability was detected using MTT cell proliferation and Cytotoxicity Detection Kit (KeyGEN, KGA311). Cell numbers were measured by trypan blue assay using Trypan Blue Staining Cell Viability Assay Kit (Beyotime, C0011). For colony formation, cells were seeded into 6‐well plates (500 cells per well) and then were fixed and stained with 0.1% crystal violet for 10 min after two weeks.
","Nile Red Staining","
To visualize lipid droplets, cells were washed with PBS twice, fixed with 4% PFA, and stained with 0.05 mg mL−1 Nile red solution for 10 min. The nuclei were further stained with Hoechst 33342, and images were captured using laser confocal microscopy LSM880 (Zeiss). For FASN inhibition, 50 nm IPI‐9119 (Selleck, E2667) was incubated with PLC cells for 72 h. The Nile red signal is defined as the mean gray value per cell (integrated density/area) quantified by ImageJ.
","Untargeted Metabolomics and Lipidomics","
The untargeted metabolomics and lipidomics were carried out as previously described with minor modifications.[\n##REF##29663480##\n92\n##\n] All cells were maintained under standard conditions with DMEM medium containing 10% FBS for 48 h and then subjected to metabolomics analysis. 107 cells were incubated with 800 µL cold methanol for protein removal and metabolite extraction after two PBS washes. The mixture was collected and centrifuged at 12 000 × g for 20 min, and the supernatant was further dried in a vacuum centrifuge. For LC‐MS analysis, samples were redissolved in 100 µL acetonitrile/water (1:1, v/v) solvent and centrifuged at 14 000 × g at 4 °C for 15 min. Then the supernatant was subjected to AB Triple TOF 6600 (AB SCIEX) and Q Exactive HF‐X (Thermo) for metabolomics and lipidomics, respectively (Shanghai Applied Protein Technology). For statistical analysis, the VIP value of each variable in the orthogonal partial least‐squares discriminant analysis model was calculated to indicate its contribution to the classification. The significantly differential metabolites were identified with the cutoff (VIP >1, P‐value <0.05). P‐values were generated by the two‐tailed Student's t‐test.
","Western Blotting","
For western blots, samples were separated on SDS‐PAGE gels and then transferred to PVDF membranes (Millipore). Membranes were processed according to the ECL western blotting protocol (GE Healthcare). The grayscale statistics of western blotting were performed by ImageJ. The following antibodies were used in western blots: anti‐AMPK (CST, 5832), anti‐p‐AMPK (CST, 2535), anti‐ACC1 (CST, 3676), anti‐p‐ACC1 (CST, 3661), anti‐RAPTOR (CST, 2280), anti‐p‐RAPTOR (CST, 89146), anti‐VEGFA (Proteintech, 9003‐1‐AP), anti‐TGF‐β (Immunoway, YT4632), anti‐MFF (Proteintech, 17090‐1‐AP), anti‐p‐MFF (Immunoway, YP1403), anti‐p‐ULK1 (CST, 5869), anti‐ULK1 (CST, 8054), anti‐LKB1 (Immunoway, YT2573), anti‐HNRNPD (Thermo, PA5‐99469), anti‐AGO2 (Sigma, SAB4200085), anti‐eIF4G1 (CST, 8701), anti‐eIF4E (Abcam, ab33768), anti‐c‐MYC (CST, 9402), and anti‐ACTB (TransGen, HC201). Antibody validation is provided on the manufacturers’ websites.
","RNA Pull‐Down and RNA Immunoprecipitation","
RNA pull‐down with 5′‐biotinylated antisense oligos and RIP were carried out as previously described with minor modifications.[\n##REF##27694840##\n93\n##\n] PLC cells and murine liver cells were cross‐linked (a total of 0.4 J cm−2) in a UV cross‐linker. Cells were harvested in ice‐cold lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor (Promega, N2615)) for 30 min on ice. The supernatant was collected after centrifugation at 1000 × g for 5 min at 4 °C, and subjected to sonication on ice for 5 min with a Sonics Vibra‐Cell (3 s on, 6 s off, 30%). 100 pmol biotinylated AS oligos (for pull‐down) or 2 µg antibody (for RIP) was added to the supernatant. After rotation 4 h at 4 °C, 50 µL M‐280 Streptavidin Dynabeads (Invitrogen, 11206D, for RNA pulldown) or Protein G Dynabeads (Invitrogen, 10004D, for RIP), which were blocked with 500 ng µL−1 yeast total RNA and 1 mg mL−1 BSA for 1 h at room temperature were added. After rotation 4 h at 4 °C, washing once with lysis buffer, twice with high salt lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 500 mm NaCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor). The purified RNAs were analyzed by RT‐qPCR. The purified proteins were analyzed by MS after silver staining using Protein Stains K kit (Sangon Biotech, C500021‐0010) or western blot. The following antibodies were used: anti‐HNRNPD (Thermo, PA5‐99469); anti‐AGO2 (Sigma, SAB4200085). For HNRNPD RIP, CCND1 and Mef2c mRNAs were used as positive controls in human and mice, respectively.[\n##REF##33444453##\n50\n##, ##REF##24891619##\n94\n##\n] For AGO2 RIP, TNFRSF12A mRNA was used as a positive control.[\n##REF##29458013##\n95\n##\n]\n
","Ribosome Profiling and Ribo‐seq Analysis","
The ribosome profiling assay was conducted as previously described with minor modifications.[\n##UREF##5##\n32\n##\n] The curve was generated with optical scanning at 254 nm using a Gradient Profiler (BioComp). GAPDH mRNA is a positive control, and circHIPK3, a negative control, is known to be noncoding.[\n##REF##27050392##\n96\n##\n] For Ribo‐seq analysis from previous studies (GSE147840, GSE125757, and GSE128320), the adapters were trimmed to obtain clean reads. The left reads with lengths ranging from 28 to 32 nt were then aligned to the human genome (hg19) with Bowtie (‐v 1) allowing one mismatch. Ribo‐seq signals shown in Figure ##SUPPL##0##S5b## of the Supporting Information were presented by IGV visualization.
","Protein Mass Spectrometry","
The specific silver‐stained band was cut, cleaned, and digested in gel with the digestion buffer (100 mm NH4HCO3, pH 8.5) containing trypsin (Promega, V5111). The samples were analyzed using an LC‐ESI‐MS/MS system after extraction and purification. Protech's ProtQuest software suite was used to search the mass spectrometric data against the UniProt protein database.
","ImmunofluorescenceStaining","
PLC cells or HCC tissues were fixed with methanol/glacial acetic acid (3:1, v/v) at room temperature for 10 min after washing with PBS twice, and permeabilized with fresh PBS containing 1% Triton X‐100 on ice for 10 min. After blocking with 1% BSA for 30 min at room temperature, samples were incubated with the anti‐HNRNPD antibody (Thermo, PA5‐99469) or anti‐LKB1 antibody (Proteintech, 10746‐1‐AP) (1:200 dilution in 1% BSA) for 4 h at room temperature. With three 5 min washes in PBST buffer (PBS with 0.4% Tween‐20), samples were incubated with Goat Anti‐Rabbit Secondary Antibody Alexa Fluor 488 (Abcam, ab150077) (1:200 diluted in 1% BSA) for 2 h at room temperature, protected from light. After three 5 min washes in PBST buffer, nuclei were stained with DAPI, and the images were captured using laser confocal microscopy LSM880 (Zeiss).
","RIP‐seq and Data Analysis","
RIP‐seq was carried out as previously described with minor modifications.[\n##REF##36182935##\n18\n##, ##UREF##2##\n24\n##\n] 5 × 107 PLC cells were ultraviolet cross‐linked (a total of 0.4 J cm−2) in a UVP cross‐linker and harvested in ice‐cold lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor (Promega, N2615)) for 30 min on ice. The supernatant was collected after centrifugation at 1000 × g for 5 min at 4 °C, and subjected to sonication on ice for 5 min with a Sonics Vibra‐Cell (3 s on, 6 s off, 30%). The HNRNPD‐binding complexes were isolated by anti‐HNRNPD (Thermo, PA5‐99469) coupled with Protein G Dynabeads (Life Technology, 10004D). After one wash with lysis buffer, two washes with high salt lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 500 mm NaCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor) and treatment of proteinase K, the mixture was subjected to RNA extraction with TRIzol reagent according to standard protocol. For RIP‐seq cDNA library preparation, the purified RNAs were ligated to adapters, reverse transcribed, PCR‐amplified for ≈25 cycles, and then subjected to high‐throughput sequencing using an Illumina Novoseq platform with a 150‐nt run length. For data analysis, the adapters were first trimmed to obtain clean reads, and the left reads were then mapped to the human genome (hg19) with Bowtie2. After the alignment, duplicates were removed and MACS2 was used for peak calling. The genome distribution of HNRNPD RIP‐seq signals was annotated according to the gene transfer format file from UCSC. For circLARP1B, all circLARP1B reads including the BSJ reads from the HNRNPD RIP‐seq were used for IGV visualization. For HNRNPD PAR‐CLIP reanalysis from previous studies (GSE52977), HNRNPD signals in the 3′ UTRs of targeted mRNAs were used for comparison.
","KEGG and GO Analysis","
For KEGG pathway analysis, the metabolites were blasted against the online KEGG database (https://www.genome.jp/kegg/) to annotate the corresponding pathways. For GO analysis of HNRNPD binding targets regulated by circLARP1B, GOrilla web‐server (https://cbl‐gorilla.cs.technion.ac.il/) with default parameters was visualized by the ggplot2 package in R software.
","mRNA Stability Assay","
For mRNA stability assay, cells were cultured with fresh DMEM at 37 °C for 12 h before being treated with 5 mg mL−1 Actinomycin D (Sigma, A4262). The samples were harvested at indicated times and then subjected to RT‐qPCR analysis.
","EIF4E and eIF4G1 RIP Assays","
EIF4E and eIF4G1 RIP assays were performed as previously described with minor modifications.[\n##REF##25826658##\n66\n##\n] 5 × 107 PLC cells were ultraviolet cross‐linked (a total of 0.4 J cm−2) in a UV cross‐linker, and harvested in ice‐cold RIPA buffer (50 mm Tris‐HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% NP‐40, 0.1% SDS, 1 mm DTT, 1× Protease Inhibitor Cocktail) for 30 min on ice. The lysate was then subjected to sonication on ice for 5 min with a Sonics Vibra‐Cell (3 s on, 6 s off, 30%) and the supernatant was collected after centrifugation at 12 000 × g for 5 min at 4 °C. After the measurement of the absorbance at 260 nm, the supernatant was incubated with 3 U of RNase I (Invitrogen, AM2294) per A260 unit (Absorbance at 260 nm × volume in mL). An aliquot of the digestion reaction was saved as the input sample. The left supernatant was incubated with Protein G beads preconjugated antibody for the immunoprecipitation procedures. The bead–lysate mixture was rotated at room temperature (25 °C) for 30 min, and the input sample was placed alongside it. After collection on a magnetic rack at 4 °C, beads were rinsed briefly in 1 mL ice‐cold RIPA buffer, then collected and washed twice for 5 min each with rotating. Meanwhile, the bead supernatant and the input sample were aliquoted into two samples each (for RNA and protein isolation) and frozen. The RNA samples were subjected to TRIzol extraction. For further RT‐qPCR detection, primers used were included in Table ##SUPPL##5##S5## of the Supporting Information. The following antibodies were used: anti‐eIF4E (Abcam, ab33768); anti‐eIF4G1 (CST, 8701).
","Dual‐Luciferase Reporter Assay","
The 3′ UTR sequence of LKB1 mRNA was used as the 3′ UTR of firefly luciferase in the vector pGL3 (Promega, HG‐VQP0122). Vector expressing renilla luciferase was used as a loading control. After transfection in HEK293T cells for 24 h, relative luciferase activities were determined using a Dual‐Luciferase Reporter Assay System (Promega, E1910) according to the manufacturer's instructions.
","Immunofluorescence Combined with Fluorescence In Situ Hybridization","
The IF combined with FISH assay was performed as previously described with several modifications.[\n##REF##32048164##\n16\n##, ##REF##31508410##\n60\n##\n] PLC cells were fixed with methanol/glacial acetic acid (3:1, v/v) at room temperature for 10 min after washing with PBS twice and then permeabilized with freshly made PBS containing 0.01% Triton X‐100 (v/v) and 0.1 U µL−1 RNase inhibitor (Promega, N2615) on ice for 20 min. After blocking with 1% BSA for 30 min at room temperature, samples were incubated with the anti‐HNRNPD antibody (Thermo, PA5‐99469) (1:200 dilution in 1% BSA) for 3 h at room temperature. With three 5 min washes in PBST buffer (PBS with 0.4% Tween‐20), samples were incubated with Donkey anti‐Rabbit IgG Secondary Antibody Alexa Fluor 546 (Invitrogen, A10040) (1:200 diluted in 1% BSA) for 1 h at room temperature in dark. For LKB1 mRNA FISH, probes were generated with Transcript Aid T7 High Yield Transcription Kit (Thermo, K0441), and then labeled with Alexa Fluor 488 by using the ULYSIS Nucleic Acid Labeling Kit (Invitrogen, 2161899) according to manufacturer's instructions. RNA probes were denatured at 80 °C for 5 min and placed on ice immediately. After washing with PBST buffer twice and 2× SSC once in dark, samples were incubated with RNA probes mixed with 20 ng µL−1 human Cot‐1 DNA (Invitrogen, 15279011), 500 ng µL−1 yeast total RNA (Invitrogen, AM7118) and 0.1 U µL−1 RNase inhibitor in 2× hybridization buffer (4× SSC, 40% dextran sulfate) at 37 °C for 15–17 h. Slides were washed with 2× SSC containing 0.1% Triton X‐100 at 45 °C. Nuclei were stained with DAPI and the images were captured using laser confocal microscopy LSM880 (Zeiss). The regain of interest (ROI) was drawn as a small rectangle and colocalization percentage of HNRNPD protein and LKB1 mRNA in 15 ROIs was measured by using ImageJ plugin Coloc2.
","In Vitro RNA Circularization","
Linear RNA fragment was generated with Transcript Aid T7 High Yield Transcription Kit (Thermo, K0441). In brief, 1 µg PCR‐amplified template, 2 µL T7 RNA polymerase enzyme, 0.5 mm NTPs, and 2 mm GMP were mixed and incubated for 4 h at 37 °C. After DNase I treatment, transcribed linear RNA was purified using phenol/chloroform (pH 4.5). For in vitro circularization, 50 µg linear RNA was incubated with T4 RNA ligase 1 (NEB, M0204) in 500 µL reaction buffer for overnight at 16 °C. After separation on 5% Urea PAGE gel, circularized RNA was cut in the corresponding band and eluted overnight in elution buffer (20 mm Tris‐HCl, pH 7.5, 250 mm NaOAc, 1 mm EDTA, and 0.25% SDS). The eluted RNA was then purified using phenol/chloroform (pH 4.5).
","In Vitro Binding/Competing Assay","
In vitro synthesized circLARP1B, ACTB fragment, and LKB1 3′ UTR fragment RNAs were heated in RNA‐folding buffer (10 mm HEPES, pH 7.4 and 10 mm MgCl2) for 5 min at 65 °C and slowly cooled down over the course of 40 min to room temperature. For in vitro binding assay, equal amounts (10 pmol) of folded circLARP1B or ACTB RNAs were incubated with 1 µg purified Flag‐tagged HNRNPD protein (OriGene, TP320809) in 0.2 mL Binding buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor) for 2 h at 4 °C. For in vitro competing assay, 5 pmol folded circLARP1B and 5 pmol LKB1 3′ UTR fragment RNA were incubated with 1 µg purified Flag‐tagged HNRNPD protein in 0.2 mL Binding buffer for 2 h at 4 °C. For both assays, one tenth of incubated solution was saved as input. The HNRNPD–RNA complex was purified with Protein G beads (Invitrogen, 10004D) preconjugated anti‐FLAG antibody (Sigma, F1804). After one Binding buffer wash and one high‐salt Binding buffer (50 mm HEPES, pH 7.4, 500 mm NaCl, 10 mm MgCl2, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor) wash at 4 °C for 5 min, the interacted RNA was extracted with TRIzol according to the manufacturer's protocol for further analysis.
","DEN‐Induced HCC Mouse Model","
Diethylnitrosamine (DEN)‐induced HCC mouse model was established as described previously.[\n##REF##22265403##\n70\n##\n] In brief, 2‐week‐old mice were injected with DEN (20 mg k−1g body weight, Sigma, N0258) to start the growth of tumors. The phenobarbital‐like inducer TCPOBOP (3 mg k−1g body weight; ApexBio, B8576) was injected every 2 weeks (a total of 8 times) to accelerate HCC progression after DEN injection for 2 weeks. All mice were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water. For Figure ##SUPPL##0##S8g–k## of the Supporting Information to examine liver inflammation and fibrosis, all data were collected from mice at 9:00 AM. For the other HCC mouse model, mice were fasted for 12 h before bleeding and sacrifice at 9:00 AM.
","Liver‐Directed AAV8‐Mediated CircLARP1B and Lkb1 Silencing","
For preparing AAV8 to silence circLARP1B or Lkb1, shRNA was inserted into the p‐AAV‐MCS plasmid. AAV8‐shcircLARP1B, AAV8‐shLkb1 or AAV8‐shCtrl was generated with the AAV Helper‐Free System and infected mice with tail vein injection (1012 vg AAV8/mouse). For evaluating Lkb1's roles in normal livers as shown in Figure ##FIG##6##7f–i## and Figure ##SUPPL##0##S8f## (Supporting Information), 8‐week‐old circLARP1B−/−\n or WT male mice were injected with AAV8‐shLkb1 or AAV8‐shCtrl, and sacrificed after 3 weeks. For AAV treatment in HCC mice models, AAV8 expressing shcircLARP1B, shLKB1 or shCtrl was injected once at 10 weeks after DEN injection and TCPOBOP was also injected into mice at the time point.
","Nuclear Run‐On Assay","
For nuclear isolation, cells were harvested in ice‐cold solution I (10 mm Tris‐HCl, pH 7.5, 150 mm KCl, 4 mm Mg(OAc)2) and placed on ice for 10 min. After centrifugation at 1000 × g for 10 min, pellets were resuspended in solution II (10 mm Tris‐HCl, pH 7.5, 150 mm KCl, 4 mm Mg(OAc)2, 0.5% NP‐40, 10% glycerol, and 1 U µL−1 RNase inhibitor (Promega, N2615)) followed by intermittent shaking on ice for 20 min. The nuclear run‐on mixture (10 mm ATP, CTP, GTP, BrUTP, and the crude nuclei) was incubated at 30 °C for 5 min in the run‐on buffer (10 mm Tris‐HCl, pH 7.5, 5 mm MgCl2, 150 mm KCl, 1% Sarkosyl, and 2 mm DTT) in the presence of RNase inhibitor. The RNA was isolated with TRIzol reagent (Invitrogen) based on the manufacturer's instructions. Nascent transcripts were immunoprecipitated with anti‐BrdU antibody (Novus, NB500‐235) and converted to cDNA for use in RT‐qPCR assays.
","Oil Red O Staining","
Frozen sections of liver tissues or tumor samples were washed with PBS twice followed by fixing with 4% PFA. After one ddH2O wash, samples were stained with freshly prepared 3 µg mL−1 Oil Red O for 1 h at room temperature. Nuclei were counterstained with hematoxylin. Images were captured using an inverted microscope (Olympus). The percentage of Oil Red O positive area is calculated by image‐Pro plus.
","Immunohistochemistry","
For IHC staining, paraffin sections of murine tissues were deparaffinized with xylene and rehydrated with ethanol at decreasing concentrations. Then, samples were incubated with the indicated antibodies overnight at 4 °C followed by incubating with the correspondent secondary antibodies for 1 h at room temperature. Nuclei were stained with hematoxylin and images were captured using an inverted microscope (Olympus). The following antibodies were used in IHC: anti‐ACC1 (CST, 3676), anti‐p‐ACC1 (CST, 3661), anti‐AMPK (CST, 5832), anti‐p‐AMPK (CST, 2535), anti‐LKB1 (Immunoway, YT2573), anti‐Vim (Bioss, bs‐8533R), anti‐Ecad (Bioss, bs‐10009R), anti‐HNRNPD (Thermo, PA5‐99469). The average optical density (AOD) is calculated by ImageJ to indicate the IHC signal.
","H&E, Masson, and Sirius Red Staining","
The lung or liver tissues were soaked in 10% formalin, dehydrated with graded alcohols, and then embedded in paraffin wax for sections. The paraffin sections were deparaffinized and rehydrated after being immersed in xylene. H&E Staining Kit (Abcam, ab245880) was used for H&E staining. The Fontana‐Masson Stain Kit (Abcam, ab150669) was used for Masson staining. The Picro Sirius Red Stain Kit (Abcam, ab150681) was used for Sirius Red staining. Images were captured using an inverted microscope (Olympus). The percentage of Masson and Sirius Red positive area is calculated by ImageJ.
","Measurement of Serum AFP, ALT, AST, TG, and TC","
The serum AFP level was measured with Alpha‐fetoprotein ELISA Kit (NJJC, H226‐1‐1). The ALT and AST levels in serum were detected with Micro Glutamic‐pyruvic Transaminase Assay Kit (Solarbio, BC1555) and Micro Glutamic‐oxalacetic Transaminase Assay Kit (Solarbio, BC1565), respectively. The serum TG and TC levels were examined by Triglycerides Assay Kit (NJJC, F001‐1‐1) and Total Cholesterol Assay Kit (NJJC, F002‐1‐1), respectively.
","Image Processing","
All images of the Nile Red staining, smFISH, and IF were taken by confocal microscope LSM880 (Zeiss). All images of the Transwell assay, Oil red O staining, and IHC were taken with an inverted fluorescence microscope IX73 (Olympus). Image processing and quantification were performed by ImageJ or Image‐Pro plus (as stated specifically in the corresponding method).
","Statistical Analysis","
Statistical analysis was carried out using GraphPad Prism version 8.0. Either Student's t‐tests, two‐way ANOVA tests were used to calculate P‐values, as indicated in the figure legends. For Student's t‐tests, the values reported in the graphs represent averages with error bars showing S.D. After analysis of variance with F tests, the statistical significance and P‐values were evaluated with Student's t‐tests. Statistical significance was defined as P‐value < 0.05. The detailed statistical analysis applied to each experiment is included in the figure legends. The sample size (N) was represented in the corresponding figure legends.
","Conflict of Interest","
J.L., X.W., and G.S. have an ownership interest in a patent related to this research.
","Author Contributions","
J.L. and X.W. contributed equally to this work. G.S. conceived of and designed this project. J.L., X.W., and S.C. performed experiments. J.L. and X.W. performed bioinformatics analyses of RNA‐seq data, circRNA profiling, and RIP‐seq. X.C., L.S., B.L., and Z.S. provided clinical specimens. X.W., X.C., and G.S. analyzed the results. J.L., X.W., and G.S., wrote the manuscript. All authors have discussed the results and made comments on the manuscript. All authors approved the final manuscript.
All fresh HCC tumor tissues were collected from Sir Run Run Shaw Hospital, which was approved by the Human Research Ethics Committee of Sir Run Run Shaw Hospital (SRRSHLS2022Y0312). Written informed consent was obtained from each patient for this study. All samples were frozen and stored in liquid nitrogen after removal from the operation followed by washing with PBS twice.
\",\n \"Mice\",\n \"
All mice were housed in an enriched environment under the standard conditions (23–25 °C temperature, 40–60% humidity) with a 12‐h light‐dark cycle (lights on from 08:00 to 20:00) at the Specific‐Pathogen‐Free facility with unrestricted access to food and water for the duration of the experiment, unless specified in the corresponding situation. All animal protocols were approved by the Animal Care and Use Committee of the University of Science and Technology of China (USTCACUC212201037). For genetically engineered mice, whole‐body circLARP1B\\n−/− mice were generated with the CRISPR/Cas9 system as previously described.[\\n##REF##36182935##\\n18\\n##\\n] sgRNAs targeting Larp1b intron 4 were designed by CRISPick web‐server (https://portals.broadinstitute.org/gppx/crispick/public). The synthesized Cas9 mRNA and sgRNAs were coinjected into fertilized embryos of C57BL/6J mice and 100 zygotes were implanted into five ICR strain surrogate mice. All F0 founders were genotyped at 2 weeks after birth with PCR amplification followed by Sanger sequencing. Specific primers for PCR were included in Table ##SUPPL##5##S5## of the Supporting Information. Positive F0 founders were backcrossed to C57BL/6 mice to obtain deficient allele heritable heterozygous F1 mice, which were further intercrossed to generate circLARP1B\\n−/− homozygous mice. All circLARP1B\\n−/− mice used for analyses were in parallel with age‐ and gender‐matched wildtype littermates (as the control group). Male mice were used for experiments in this study.[\\n##REF##37005415##\\n86\\n##, ##REF##32182125##\\n87\\n##\\n]\\n
\",\n \"Library Preparation for RNA‐seq\",\n \"
Total RNA from frozen tissues or cultured cells was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's protocols. Libraries were prepared with the TruSeq Ribo Profile Library Prep Kit (Illumina) according to the manufacturer's instructions and then subjected to sequencing in an Illumina Nova‐PE150 system (Novogene). Each library generated ≈100 million 150‐nt paired‐end read pairs.
\",\n \"CircRNA Identification\",\n \"
The circRNA candidates were identified with CIRI2 and the BSJ reads per million were used to calculate circRNA levels. Briefly, the adapters were removed with Cutadapt (‐e 0.1 ‐O 5) to obtain clean reads. The reads continuously aligned to the human reference genome (hg19) were filtered with Bowtie (‐v 1) allowing one mismatch. The remaining reads were used to predict circRNA candidates with CIRI2 with default parameters. The differentially expressed circRNAs were determined by DEseq2 with a criterion (fold change >4 or <0.25, P‐value <0.05). For conservation analysis of circRNAs, the circRNA sequences were aligned to annotated murine circRNAs from the circAtlas database with Blast. Conserved circRNAs between human and mice were determined with a stringent cutoff (full‐length and BSJ sequences >75% identical).
\",\n \"Cell Culture and Cell Transfection\",\n \"
The PLC/PRF/5 (PLC) cells were kindly provided by Dr. Yide Mei (USTC). HepG2, HEK293T, K1, and Hepa1‐6 cells all originated from the American Type Culture Collection. All above cells were maintained under standard conditions with the DMEM medium containing 10% FBS (CLARK, FB25015) and 1% penicillin/streptomycin (Beyotime, C0222) at 37 °C under 5% CO2. A PCR‐based method and DAPI staining were used to ensure cells without contamination of mycoplasma. All cell lines were authenticated by short‐tandem‐repeat profiling. Transfection of plasmids and siRNAs was conducted with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
Two sgRNAs targeting the intron 4 of human LARP1B were designed based on http://crispr.mit.edu to knockout circLARP1B. After transfection of 2 µg pX330 plasmids expressing Cas9 mRNA and targeted sgRNAs for two days, hundreds of mCherry‐positive single‐cell clones were sorted through the BD FACSAria III cell sorting system. Two weeks later, the colonies were split into two identical plates and one plate was harvested for PCR‐mediated genotype and confirmation by Sanger sequencing. The primer sequences are listed in Table ##SUPPL##5##S5## of the Supporting Information.
\",\n \"OCR Measurements\",\n \"
The OCR analyses were performed using the Agilent Seahorse XF Cell Mito Stress Test Kit (Agilent, 103015‐100) as previously described.[\\n##REF##33758188##\\n88\\n##\\n] Briefly, 20 000 cells per well were seeded in a 96‐well XF cell culture microplate in the growth medium overnight at 37 °C in 5% CO2. OCR was measured with an XF96 analyzer in XF DMEM medium (Agilent, 103575‐100) containing 1 mm pyruvate, 2 mm glutamine, and 10 mm glucose, followed by sequential addition of 1.5 µm oligomycin (ATP synthase inhibitor), 1.0 µm FCCP (membrane potential uncoupler), 0.5 µm rotenone (Complex I inhibitor), and 0.5 µm antimycin A (Complex III inhibitor). Data were analyzed by the Seahorse XF Cell Mito Stress Test Report Generator package.
\",\n \"FAO Rate Measurements\",\n \"
The FAO rate analyses were performed using the Agilent Seahorse XF Cell Mito Stress Test Kit (Agilent, 103015‐100) as previously described.[\\n##REF##31155494##\\n89\\n##\\n] Briefly, 15 000 cells per well were seeded in a 96‐well XF cell culture microplate in the growth medium overnight at 37 °C in 5% CO2. Then, cells were starved by incubating with substrate‐limited DMEM supplemented with 0.5 mm glucose, 1 mm glutamate, 0.5 mm carnitine, and 1% FBS. After 24‐h starvation, the medium was replaced by FAO assay medium (111 mm NaCl, 4.7 mm KCl, 1.25 mm CaCl2, 2 mm MgSO4, 1.2 mm NaH2PO4 supplemented with 2.5 mm glucose, 0.5 mm carnitine, and 5 mm HEPES, pH 7.4). Finally, BSA‐conjugated palmitate (Agilent, 102720‐100) was added to a final concentration of 50 mm, followed by sequential addition of 1.5 µm oligomycin, 1.0 µm FCCP, 0.5 µm rotenone, and 0.5 µm antimycin A. Data were analyzed by the Seahorse XF Cell Mito Stress Test Report Generator package.
\\n13C‐labeled targeted metabolic flux analysis was carried out as previously described with minor modifications.[\\n##REF##32101748##\\n37\\n##\\n] In brief, 107 PLC cells were washed with 0.9% saline three times and incubated in substrate‐limited DMEM supplemented with 15 mm\\n13C‐glucose, 4 mm\\n13C‐glutamine, and 10% FBS for 24 h before metabolite extraction. After washing with cold 0.9% saline twice, cells were incubated with 500 µL cold extraction buffer (methanol:acetonitrile:water, 2:2:1, v/v/v). Harvested cells were sonicated for 2 min, centrifuged at 14 000 × g for 5 min at 4 °C and cell supernatants were transferred to new tubes. 400 µL chloroform was added and the tubes were vortexed, centrifuged at 14 000 × g for 5 min at 4 °C. The resulting phase separation resulted in an aqueous upper phase containing polar metabolites and an organic lower phase containing nonpolar metabolites. The aqueous upper phase was transferred to tubes and the lower chloroform phase was transferred to a glass tube. For reverse phase liquid chromatography separation, ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 µm, Waters) was used. Mass spectrometric data were acquired with a Q‐Exactive plus hybrid quadrupole–orbitrap mass spectrometer (Thermo) using negative ion electrospray ionization. The scan range was from 50 to 1000 m/z. The scan time for each function was set to 0.2 s. Ion monitoring conditions were defined as a capillary voltage of 2.0 kV, source temperature of 120 °C, and desolvation temperature of 500 °C. Data processing and ion annotation based on accurate mass were performed in TraceFinder 5.0 (Thermo) and Xcalibur 4.0 (Thermo). Metabolite mass isotopomer distribution was determined based on the ratio of the integrated peak areas of the chosen isotopomer to the sum of all the integrated peak areas of the possible isotopomers for the given metabolites.
\",\n \"Plasmids Construction\",\n \"
All plasmids were constructed with restriction‐enzyme digestion and ligation or with recombinant methods (Vazyme, c113‐02). For circLARP1B overexpression in human, the circularized exons and the endogenous flanking sequences including one Alu pair were inserted into the pcDNA3 vector. The backbone vector of p3×FLAG‐Myc was used for constructing FLAG‐tagged HNRNPD and LKB1. The shRNA against the BSJ of human or murine circLARP1B was cloned into the vector pLKO.1 (Sigma) and the negative‐control shRNA (shCtrl, MFCD07785395) was obtained from the MISSION shRNA Library (Sigma). The sgRNAs for human or murine circLARP1B were inserted into the Cas9‐ and sgRNA‐expressing backbone (pX330), which was engineered with expressing mCherry and a puromycin selectable marker. All plasmids have been sequenced for confirmation and further information about these plasmids is available upon request. Oligonucleotide sequences for primers used in plasmids construction, along with other oligo sequences used in probe preparation, siRNA, and biotin‐labeled nucleic acids were listed in Table ##SUPPL##5##S5## of the Supporting Information.
\",\n \"Northern Blotting\",\n \"
Northern blotting was performed as previously described.[\\n##REF##29322446##\\n90\\n##\\n] The digoxigenin‐labeled RNA probe with antisense sequences to the circLARP1B BSJ was synthesized with a DIG Northern Starter Kit (Roche, 12039672910) according to the manufacturer's protocol. The primers for probe preparation were listed in Table ##SUPPL##5##S5## of the Supporting Information.
\",\n \"Single‐Molecule Fluorescence In Situ Hybridization\",\n \"
The smFISH was carried out as previously described with minor modifications.[\\n##UREF##8##\\n91\\n##\\n] Cells or tissues were fixed with 4% PFA for 10 min at room temperature and permeabilized with ice‐cold 70% ethanol overnight at −20 °C. For hybridization, cells or tissues were prehybridized at 37 °C for 30 min in hybridization buffer (30% formamide, 5× SSC, 9 mm citric acid, pH 6.0, 0.1% Tween‐20, 50 µg mL−1 heparin, 1× Denhardt's solution, and 10% dextran sulfate) after washing with 2× SSC twice. Then, samples were hybridized with 2 pmol probes overnight at 37 °C in the hybridization buffer. For the amplification stage, 18 pmol hairpins were heated at 95 °C for 90 s and cooled to room temperature in a dark drawer for 30 min. Samples were incubated with the denatured probes overnight at room temperature followed by three 5 min washes in 2× SSC buffer. After a 10 min incubation in DAPI (5 mg mL−1), the sections were washed three times for 5 min in 2× SSC buffer. The images were captured using the laser confocal microscopy LSM880 (Zeiss). The ImageJ was used to further process the data, and the ImageJ Plot Profile tool was applied to calculate signal intensity. All probe sequences were included in Table ##SUPPL##5##S5## of the Supporting Information.
\",\n \"PCR Reactions\",\n \"
For RT‐PCR, 500 ng total RNA was reverse‐transcribed into complementary DNA (cDNA) with the GoScript Reverse Transcription System (Promega, A5001) according to the manufacturer's protocol. For PCR with genomic DNA (gDNA) as the template, gDNA was isolated by phenol/chloroform extraction. For semiquantitative RT‐PCR gels, 25–30 cycles were carried out. The RT‐qPCR was performed using GoTaq SYBR Green qPCR Master Mix (Promega, A6001) on a PikoReal 96 real‐time PCR system (Thermo). All PCR products were confirmed by Sanger sequencing. All used primer sequences are included in Table ##SUPPL##5##S5## of the Supporting Information.
\",\n \"Nuclear/Cytosolic Fractionation\",\n \"
Cellular fractionation was carried out as previously described with minor modifications.[\\n##REF##36182935##\\n18\\n##\\n] Briefly, cells were incubated with hypotonic buffer (10 mm Tris‐HCl, pH 8.0, 140 mm NaCl, 1.5 mm MgCl2, 0.5% NP‐40, 1 mm DTT, and 0.1 U µL−1 RNase inhibitor (Promega, N2615)) on ice for 20 min after washing with PBS twice. After centrifugation at 1000 × g for 5 min at 4 °C, the supernatant was collected as the cytoplasmic fraction. The pellets were then resuspended in nuclear resuspension buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm DTT, and 0.1 U µL−1 RNase inhibitor followed by the incubation at 4 °C for 30 min. Nuclear fraction was collected after removing insoluble membrane debris by centrifugation at 12 000 × g for 15 min.
\",\n \"Quantification of RNA Copy Number per Cell\",\n \"
The protocol was carried out as previously described.[\\n##UREF##5##\\n32\\n##\\n] Total RNA was extracted from 106 PLC and HepG2 cells, and cDNA was synthesized. The standard curve was used to calculate the copy numbers per cell in each cell line based on cell numbers and Ct values.
\",\n \"Transwell Assay\",\n \"
Transwell invasion assay was performed using the chamber with Matrigel (BD Biosciences). Cells were seeded into the upper chamber cultured with the serum‐free DMEM medium, and DMEM containing 10% FBS was added to the lower chamber. After 24 h, the migrated cells on the outer membrane were stained with 0.1% crystal violet for 10 min. Images were captured with an inverted microscope (Olympus) and the number of invaded cells was quantified by ImageJ.
\",\n \"Cell Viability and Number Measurement and Colony Formation\",\n \"
The cell viability was detected using MTT cell proliferation and Cytotoxicity Detection Kit (KeyGEN, KGA311). Cell numbers were measured by trypan blue assay using Trypan Blue Staining Cell Viability Assay Kit (Beyotime, C0011). For colony formation, cells were seeded into 6‐well plates (500 cells per well) and then were fixed and stained with 0.1% crystal violet for 10 min after two weeks.
\",\n \"Nile Red Staining\",\n \"
To visualize lipid droplets, cells were washed with PBS twice, fixed with 4% PFA, and stained with 0.05 mg mL−1 Nile red solution for 10 min. The nuclei were further stained with Hoechst 33342, and images were captured using laser confocal microscopy LSM880 (Zeiss). For FASN inhibition, 50 nm IPI‐9119 (Selleck, E2667) was incubated with PLC cells for 72 h. The Nile red signal is defined as the mean gray value per cell (integrated density/area) quantified by ImageJ.
\",\n \"Untargeted Metabolomics and Lipidomics\",\n \"
The untargeted metabolomics and lipidomics were carried out as previously described with minor modifications.[\\n##REF##29663480##\\n92\\n##\\n] All cells were maintained under standard conditions with DMEM medium containing 10% FBS for 48 h and then subjected to metabolomics analysis. 107 cells were incubated with 800 µL cold methanol for protein removal and metabolite extraction after two PBS washes. The mixture was collected and centrifuged at 12 000 × g for 20 min, and the supernatant was further dried in a vacuum centrifuge. For LC‐MS analysis, samples were redissolved in 100 µL acetonitrile/water (1:1, v/v) solvent and centrifuged at 14 000 × g at 4 °C for 15 min. Then the supernatant was subjected to AB Triple TOF 6600 (AB SCIEX) and Q Exactive HF‐X (Thermo) for metabolomics and lipidomics, respectively (Shanghai Applied Protein Technology). For statistical analysis, the VIP value of each variable in the orthogonal partial least‐squares discriminant analysis model was calculated to indicate its contribution to the classification. The significantly differential metabolites were identified with the cutoff (VIP >1, P‐value <0.05). P‐values were generated by the two‐tailed Student's t‐test.
\",\n \"Western Blotting\",\n \"
For western blots, samples were separated on SDS‐PAGE gels and then transferred to PVDF membranes (Millipore). Membranes were processed according to the ECL western blotting protocol (GE Healthcare). The grayscale statistics of western blotting were performed by ImageJ. The following antibodies were used in western blots: anti‐AMPK (CST, 5832), anti‐p‐AMPK (CST, 2535), anti‐ACC1 (CST, 3676), anti‐p‐ACC1 (CST, 3661), anti‐RAPTOR (CST, 2280), anti‐p‐RAPTOR (CST, 89146), anti‐VEGFA (Proteintech, 9003‐1‐AP), anti‐TGF‐β (Immunoway, YT4632), anti‐MFF (Proteintech, 17090‐1‐AP), anti‐p‐MFF (Immunoway, YP1403), anti‐p‐ULK1 (CST, 5869), anti‐ULK1 (CST, 8054), anti‐LKB1 (Immunoway, YT2573), anti‐HNRNPD (Thermo, PA5‐99469), anti‐AGO2 (Sigma, SAB4200085), anti‐eIF4G1 (CST, 8701), anti‐eIF4E (Abcam, ab33768), anti‐c‐MYC (CST, 9402), and anti‐ACTB (TransGen, HC201). Antibody validation is provided on the manufacturers’ websites.
\",\n \"RNA Pull‐Down and RNA Immunoprecipitation\",\n \"
RNA pull‐down with 5′‐biotinylated antisense oligos and RIP were carried out as previously described with minor modifications.[\\n##REF##27694840##\\n93\\n##\\n] PLC cells and murine liver cells were cross‐linked (a total of 0.4 J cm−2) in a UV cross‐linker. Cells were harvested in ice‐cold lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor (Promega, N2615)) for 30 min on ice. The supernatant was collected after centrifugation at 1000 × g for 5 min at 4 °C, and subjected to sonication on ice for 5 min with a Sonics Vibra‐Cell (3 s on, 6 s off, 30%). 100 pmol biotinylated AS oligos (for pull‐down) or 2 µg antibody (for RIP) was added to the supernatant. After rotation 4 h at 4 °C, 50 µL M‐280 Streptavidin Dynabeads (Invitrogen, 11206D, for RNA pulldown) or Protein G Dynabeads (Invitrogen, 10004D, for RIP), which were blocked with 500 ng µL−1 yeast total RNA and 1 mg mL−1 BSA for 1 h at room temperature were added. After rotation 4 h at 4 °C, washing once with lysis buffer, twice with high salt lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 500 mm NaCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor). The purified RNAs were analyzed by RT‐qPCR. The purified proteins were analyzed by MS after silver staining using Protein Stains K kit (Sangon Biotech, C500021‐0010) or western blot. The following antibodies were used: anti‐HNRNPD (Thermo, PA5‐99469); anti‐AGO2 (Sigma, SAB4200085). For HNRNPD RIP, CCND1 and Mef2c mRNAs were used as positive controls in human and mice, respectively.[\\n##REF##33444453##\\n50\\n##, ##REF##24891619##\\n94\\n##\\n] For AGO2 RIP, TNFRSF12A mRNA was used as a positive control.[\\n##REF##29458013##\\n95\\n##\\n]\\n
\",\n \"Ribosome Profiling and Ribo‐seq Analysis\",\n \"
The ribosome profiling assay was conducted as previously described with minor modifications.[\\n##UREF##5##\\n32\\n##\\n] The curve was generated with optical scanning at 254 nm using a Gradient Profiler (BioComp). GAPDH mRNA is a positive control, and circHIPK3, a negative control, is known to be noncoding.[\\n##REF##27050392##\\n96\\n##\\n] For Ribo‐seq analysis from previous studies (GSE147840, GSE125757, and GSE128320), the adapters were trimmed to obtain clean reads. The left reads with lengths ranging from 28 to 32 nt were then aligned to the human genome (hg19) with Bowtie (‐v 1) allowing one mismatch. Ribo‐seq signals shown in Figure ##SUPPL##0##S5b## of the Supporting Information were presented by IGV visualization.
\",\n \"Protein Mass Spectrometry\",\n \"
The specific silver‐stained band was cut, cleaned, and digested in gel with the digestion buffer (100 mm NH4HCO3, pH 8.5) containing trypsin (Promega, V5111). The samples were analyzed using an LC‐ESI‐MS/MS system after extraction and purification. Protech's ProtQuest software suite was used to search the mass spectrometric data against the UniProt protein database.
\",\n \"ImmunofluorescenceStaining\",\n \"
PLC cells or HCC tissues were fixed with methanol/glacial acetic acid (3:1, v/v) at room temperature for 10 min after washing with PBS twice, and permeabilized with fresh PBS containing 1% Triton X‐100 on ice for 10 min. After blocking with 1% BSA for 30 min at room temperature, samples were incubated with the anti‐HNRNPD antibody (Thermo, PA5‐99469) or anti‐LKB1 antibody (Proteintech, 10746‐1‐AP) (1:200 dilution in 1% BSA) for 4 h at room temperature. With three 5 min washes in PBST buffer (PBS with 0.4% Tween‐20), samples were incubated with Goat Anti‐Rabbit Secondary Antibody Alexa Fluor 488 (Abcam, ab150077) (1:200 diluted in 1% BSA) for 2 h at room temperature, protected from light. After three 5 min washes in PBST buffer, nuclei were stained with DAPI, and the images were captured using laser confocal microscopy LSM880 (Zeiss).
\",\n \"RIP‐seq and Data Analysis\",\n \"
RIP‐seq was carried out as previously described with minor modifications.[\\n##REF##36182935##\\n18\\n##, ##UREF##2##\\n24\\n##\\n] 5 × 107 PLC cells were ultraviolet cross‐linked (a total of 0.4 J cm−2) in a UVP cross‐linker and harvested in ice‐cold lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor (Promega, N2615)) for 30 min on ice. The supernatant was collected after centrifugation at 1000 × g for 5 min at 4 °C, and subjected to sonication on ice for 5 min with a Sonics Vibra‐Cell (3 s on, 6 s off, 30%). The HNRNPD‐binding complexes were isolated by anti‐HNRNPD (Thermo, PA5‐99469) coupled with Protein G Dynabeads (Life Technology, 10004D). After one wash with lysis buffer, two washes with high salt lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 500 mm NaCl, 2 mm MgCl2, 0.5% NP‐40, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor) and treatment of proteinase K, the mixture was subjected to RNA extraction with TRIzol reagent according to standard protocol. For RIP‐seq cDNA library preparation, the purified RNAs were ligated to adapters, reverse transcribed, PCR‐amplified for ≈25 cycles, and then subjected to high‐throughput sequencing using an Illumina Novoseq platform with a 150‐nt run length. For data analysis, the adapters were first trimmed to obtain clean reads, and the left reads were then mapped to the human genome (hg19) with Bowtie2. After the alignment, duplicates were removed and MACS2 was used for peak calling. The genome distribution of HNRNPD RIP‐seq signals was annotated according to the gene transfer format file from UCSC. For circLARP1B, all circLARP1B reads including the BSJ reads from the HNRNPD RIP‐seq were used for IGV visualization. For HNRNPD PAR‐CLIP reanalysis from previous studies (GSE52977), HNRNPD signals in the 3′ UTRs of targeted mRNAs were used for comparison.
\",\n \"KEGG and GO Analysis\",\n \"
For KEGG pathway analysis, the metabolites were blasted against the online KEGG database (https://www.genome.jp/kegg/) to annotate the corresponding pathways. For GO analysis of HNRNPD binding targets regulated by circLARP1B, GOrilla web‐server (https://cbl‐gorilla.cs.technion.ac.il/) with default parameters was visualized by the ggplot2 package in R software.
\",\n \"mRNA Stability Assay\",\n \"
For mRNA stability assay, cells were cultured with fresh DMEM at 37 °C for 12 h before being treated with 5 mg mL−1 Actinomycin D (Sigma, A4262). The samples were harvested at indicated times and then subjected to RT‐qPCR analysis.
\",\n \"EIF4E and eIF4G1 RIP Assays\",\n \"
EIF4E and eIF4G1 RIP assays were performed as previously described with minor modifications.[\\n##REF##25826658##\\n66\\n##\\n] 5 × 107 PLC cells were ultraviolet cross‐linked (a total of 0.4 J cm−2) in a UV cross‐linker, and harvested in ice‐cold RIPA buffer (50 mm Tris‐HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% NP‐40, 0.1% SDS, 1 mm DTT, 1× Protease Inhibitor Cocktail) for 30 min on ice. The lysate was then subjected to sonication on ice for 5 min with a Sonics Vibra‐Cell (3 s on, 6 s off, 30%) and the supernatant was collected after centrifugation at 12 000 × g for 5 min at 4 °C. After the measurement of the absorbance at 260 nm, the supernatant was incubated with 3 U of RNase I (Invitrogen, AM2294) per A260 unit (Absorbance at 260 nm × volume in mL). An aliquot of the digestion reaction was saved as the input sample. The left supernatant was incubated with Protein G beads preconjugated antibody for the immunoprecipitation procedures. The bead–lysate mixture was rotated at room temperature (25 °C) for 30 min, and the input sample was placed alongside it. After collection on a magnetic rack at 4 °C, beads were rinsed briefly in 1 mL ice‐cold RIPA buffer, then collected and washed twice for 5 min each with rotating. Meanwhile, the bead supernatant and the input sample were aliquoted into two samples each (for RNA and protein isolation) and frozen. The RNA samples were subjected to TRIzol extraction. For further RT‐qPCR detection, primers used were included in Table ##SUPPL##5##S5## of the Supporting Information. The following antibodies were used: anti‐eIF4E (Abcam, ab33768); anti‐eIF4G1 (CST, 8701).
\",\n \"Dual‐Luciferase Reporter Assay\",\n \"
The 3′ UTR sequence of LKB1 mRNA was used as the 3′ UTR of firefly luciferase in the vector pGL3 (Promega, HG‐VQP0122). Vector expressing renilla luciferase was used as a loading control. After transfection in HEK293T cells for 24 h, relative luciferase activities were determined using a Dual‐Luciferase Reporter Assay System (Promega, E1910) according to the manufacturer's instructions.
\",\n \"Immunofluorescence Combined with Fluorescence In Situ Hybridization\",\n \"
The IF combined with FISH assay was performed as previously described with several modifications.[\\n##REF##32048164##\\n16\\n##, ##REF##31508410##\\n60\\n##\\n] PLC cells were fixed with methanol/glacial acetic acid (3:1, v/v) at room temperature for 10 min after washing with PBS twice and then permeabilized with freshly made PBS containing 0.01% Triton X‐100 (v/v) and 0.1 U µL−1 RNase inhibitor (Promega, N2615) on ice for 20 min. After blocking with 1% BSA for 30 min at room temperature, samples were incubated with the anti‐HNRNPD antibody (Thermo, PA5‐99469) (1:200 dilution in 1% BSA) for 3 h at room temperature. With three 5 min washes in PBST buffer (PBS with 0.4% Tween‐20), samples were incubated with Donkey anti‐Rabbit IgG Secondary Antibody Alexa Fluor 546 (Invitrogen, A10040) (1:200 diluted in 1% BSA) for 1 h at room temperature in dark. For LKB1 mRNA FISH, probes were generated with Transcript Aid T7 High Yield Transcription Kit (Thermo, K0441), and then labeled with Alexa Fluor 488 by using the ULYSIS Nucleic Acid Labeling Kit (Invitrogen, 2161899) according to manufacturer's instructions. RNA probes were denatured at 80 °C for 5 min and placed on ice immediately. After washing with PBST buffer twice and 2× SSC once in dark, samples were incubated with RNA probes mixed with 20 ng µL−1 human Cot‐1 DNA (Invitrogen, 15279011), 500 ng µL−1 yeast total RNA (Invitrogen, AM7118) and 0.1 U µL−1 RNase inhibitor in 2× hybridization buffer (4× SSC, 40% dextran sulfate) at 37 °C for 15–17 h. Slides were washed with 2× SSC containing 0.1% Triton X‐100 at 45 °C. Nuclei were stained with DAPI and the images were captured using laser confocal microscopy LSM880 (Zeiss). The regain of interest (ROI) was drawn as a small rectangle and colocalization percentage of HNRNPD protein and LKB1 mRNA in 15 ROIs was measured by using ImageJ plugin Coloc2.
\",\n \"In Vitro RNA Circularization\",\n \"
Linear RNA fragment was generated with Transcript Aid T7 High Yield Transcription Kit (Thermo, K0441). In brief, 1 µg PCR‐amplified template, 2 µL T7 RNA polymerase enzyme, 0.5 mm NTPs, and 2 mm GMP were mixed and incubated for 4 h at 37 °C. After DNase I treatment, transcribed linear RNA was purified using phenol/chloroform (pH 4.5). For in vitro circularization, 50 µg linear RNA was incubated with T4 RNA ligase 1 (NEB, M0204) in 500 µL reaction buffer for overnight at 16 °C. After separation on 5% Urea PAGE gel, circularized RNA was cut in the corresponding band and eluted overnight in elution buffer (20 mm Tris‐HCl, pH 7.5, 250 mm NaOAc, 1 mm EDTA, and 0.25% SDS). The eluted RNA was then purified using phenol/chloroform (pH 4.5).
\",\n \"In Vitro Binding/Competing Assay\",\n \"
In vitro synthesized circLARP1B, ACTB fragment, and LKB1 3′ UTR fragment RNAs were heated in RNA‐folding buffer (10 mm HEPES, pH 7.4 and 10 mm MgCl2) for 5 min at 65 °C and slowly cooled down over the course of 40 min to room temperature. For in vitro binding assay, equal amounts (10 pmol) of folded circLARP1B or ACTB RNAs were incubated with 1 µg purified Flag‐tagged HNRNPD protein (OriGene, TP320809) in 0.2 mL Binding buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 10 mm MgCl2, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor) for 2 h at 4 °C. For in vitro competing assay, 5 pmol folded circLARP1B and 5 pmol LKB1 3′ UTR fragment RNA were incubated with 1 µg purified Flag‐tagged HNRNPD protein in 0.2 mL Binding buffer for 2 h at 4 °C. For both assays, one tenth of incubated solution was saved as input. The HNRNPD–RNA complex was purified with Protein G beads (Invitrogen, 10004D) preconjugated anti‐FLAG antibody (Sigma, F1804). After one Binding buffer wash and one high‐salt Binding buffer (50 mm HEPES, pH 7.4, 500 mm NaCl, 10 mm MgCl2, 1 mm DTT, 1× Protease Inhibitor Cocktail, and 0.1 U µL−1 RNase inhibitor) wash at 4 °C for 5 min, the interacted RNA was extracted with TRIzol according to the manufacturer's protocol for further analysis.
\",\n \"DEN‐Induced HCC Mouse Model\",\n \"
Diethylnitrosamine (DEN)‐induced HCC mouse model was established as described previously.[\\n##REF##22265403##\\n70\\n##\\n] In brief, 2‐week‐old mice were injected with DEN (20 mg k−1g body weight, Sigma, N0258) to start the growth of tumors. The phenobarbital‐like inducer TCPOBOP (3 mg k−1g body weight; ApexBio, B8576) was injected every 2 weeks (a total of 8 times) to accelerate HCC progression after DEN injection for 2 weeks. All mice were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water. For Figure ##SUPPL##0##S8g–k## of the Supporting Information to examine liver inflammation and fibrosis, all data were collected from mice at 9:00 AM. For the other HCC mouse model, mice were fasted for 12 h before bleeding and sacrifice at 9:00 AM.
\",\n \"Liver‐Directed AAV8‐Mediated CircLARP1B and Lkb1 Silencing\",\n \"
For preparing AAV8 to silence circLARP1B or Lkb1, shRNA was inserted into the p‐AAV‐MCS plasmid. AAV8‐shcircLARP1B, AAV8‐shLkb1 or AAV8‐shCtrl was generated with the AAV Helper‐Free System and infected mice with tail vein injection (1012 vg AAV8/mouse). For evaluating Lkb1's roles in normal livers as shown in Figure ##FIG##6##7f–i## and Figure ##SUPPL##0##S8f## (Supporting Information), 8‐week‐old circLARP1B−/−\\n or WT male mice were injected with AAV8‐shLkb1 or AAV8‐shCtrl, and sacrificed after 3 weeks. For AAV treatment in HCC mice models, AAV8 expressing shcircLARP1B, shLKB1 or shCtrl was injected once at 10 weeks after DEN injection and TCPOBOP was also injected into mice at the time point.
\",\n \"Nuclear Run‐On Assay\",\n \"
For nuclear isolation, cells were harvested in ice‐cold solution I (10 mm Tris‐HCl, pH 7.5, 150 mm KCl, 4 mm Mg(OAc)2) and placed on ice for 10 min. After centrifugation at 1000 × g for 10 min, pellets were resuspended in solution II (10 mm Tris‐HCl, pH 7.5, 150 mm KCl, 4 mm Mg(OAc)2, 0.5% NP‐40, 10% glycerol, and 1 U µL−1 RNase inhibitor (Promega, N2615)) followed by intermittent shaking on ice for 20 min. The nuclear run‐on mixture (10 mm ATP, CTP, GTP, BrUTP, and the crude nuclei) was incubated at 30 °C for 5 min in the run‐on buffer (10 mm Tris‐HCl, pH 7.5, 5 mm MgCl2, 150 mm KCl, 1% Sarkosyl, and 2 mm DTT) in the presence of RNase inhibitor. The RNA was isolated with TRIzol reagent (Invitrogen) based on the manufacturer's instructions. Nascent transcripts were immunoprecipitated with anti‐BrdU antibody (Novus, NB500‐235) and converted to cDNA for use in RT‐qPCR assays.
\",\n \"Oil Red O Staining\",\n \"
Frozen sections of liver tissues or tumor samples were washed with PBS twice followed by fixing with 4% PFA. After one ddH2O wash, samples were stained with freshly prepared 3 µg mL−1 Oil Red O for 1 h at room temperature. Nuclei were counterstained with hematoxylin. Images were captured using an inverted microscope (Olympus). The percentage of Oil Red O positive area is calculated by image‐Pro plus.
\",\n \"Immunohistochemistry\",\n \"
For IHC staining, paraffin sections of murine tissues were deparaffinized with xylene and rehydrated with ethanol at decreasing concentrations. Then, samples were incubated with the indicated antibodies overnight at 4 °C followed by incubating with the correspondent secondary antibodies for 1 h at room temperature. Nuclei were stained with hematoxylin and images were captured using an inverted microscope (Olympus). The following antibodies were used in IHC: anti‐ACC1 (CST, 3676), anti‐p‐ACC1 (CST, 3661), anti‐AMPK (CST, 5832), anti‐p‐AMPK (CST, 2535), anti‐LKB1 (Immunoway, YT2573), anti‐Vim (Bioss, bs‐8533R), anti‐Ecad (Bioss, bs‐10009R), anti‐HNRNPD (Thermo, PA5‐99469). The average optical density (AOD) is calculated by ImageJ to indicate the IHC signal.
\",\n \"H&E, Masson, and Sirius Red Staining\",\n \"
The lung or liver tissues were soaked in 10% formalin, dehydrated with graded alcohols, and then embedded in paraffin wax for sections. The paraffin sections were deparaffinized and rehydrated after being immersed in xylene. H&E Staining Kit (Abcam, ab245880) was used for H&E staining. The Fontana‐Masson Stain Kit (Abcam, ab150669) was used for Masson staining. The Picro Sirius Red Stain Kit (Abcam, ab150681) was used for Sirius Red staining. Images were captured using an inverted microscope (Olympus). The percentage of Masson and Sirius Red positive area is calculated by ImageJ.
\",\n \"Measurement of Serum AFP, ALT, AST, TG, and TC\",\n \"
The serum AFP level was measured with Alpha‐fetoprotein ELISA Kit (NJJC, H226‐1‐1). The ALT and AST levels in serum were detected with Micro Glutamic‐pyruvic Transaminase Assay Kit (Solarbio, BC1555) and Micro Glutamic‐oxalacetic Transaminase Assay Kit (Solarbio, BC1565), respectively. The serum TG and TC levels were examined by Triglycerides Assay Kit (NJJC, F001‐1‐1) and Total Cholesterol Assay Kit (NJJC, F002‐1‐1), respectively.
\",\n \"Image Processing\",\n \"
All images of the Nile Red staining, smFISH, and IF were taken by confocal microscope LSM880 (Zeiss). All images of the Transwell assay, Oil red O staining, and IHC were taken with an inverted fluorescence microscope IX73 (Olympus). Image processing and quantification were performed by ImageJ or Image‐Pro plus (as stated specifically in the corresponding method).
\",\n \"Statistical Analysis\",\n \"
Statistical analysis was carried out using GraphPad Prism version 8.0. Either Student's t‐tests, two‐way ANOVA tests were used to calculate P‐values, as indicated in the figure legends. For Student's t‐tests, the values reported in the graphs represent averages with error bars showing S.D. After analysis of variance with F tests, the statistical significance and P‐values were evaluated with Student's t‐tests. Statistical significance was defined as P‐value < 0.05. The detailed statistical analysis applied to each experiment is included in the figure legends. The sample size (N) was represented in the corresponding figure legends.
\",\n \"Conflict of Interest\",\n \"
J.L., X.W., and G.S. have an ownership interest in a patent related to this research.
\",\n \"Author Contributions\",\n \"
J.L. and X.W. contributed equally to this work. G.S. conceived of and designed this project. J.L., X.W., and S.C. performed experiments. J.L. and X.W. performed bioinformatics analyses of RNA‐seq data, circRNA profiling, and RIP‐seq. X.C., L.S., B.L., and Z.S. provided clinical specimens. X.W., X.C., and G.S. analyzed the results. J.L., X.W., and G.S., wrote the manuscript. All authors have discussed the results and made comments on the manuscript. All authors approved the final manuscript.
The authors thank Dr. Yide Mei (USTC) for providing PLC cells. The authors thank the Bioinformatics Center of the USTC, School of Life Sciences, for providing supercomputing resources, and also thank the Laboratory Animal Research Center of USTC for technique supports. This study was supported by the National Key R&D Program of China (2019YFA0802600) and the National Natural Science Foundation of China (31930019 and 32200431).
","Data Availability Statement","
All high‐throughput sequencing data in this study have been deposited to the Gene Expression Omnibus (GEO) database and are available under Accession No. GSE217403. All metabolomics, lipidomics, and metabolic flux data in this study are available in Metabolomics Workbench with the sproject (DOI: 10.21228/M8CM5D). All experimental materials generated in this study are available upon request.
"],"string":"[\n \"Acknowledgements\",\n \"
The authors thank Dr. Yide Mei (USTC) for providing PLC cells. The authors thank the Bioinformatics Center of the USTC, School of Life Sciences, for providing supercomputing resources, and also thank the Laboratory Animal Research Center of USTC for technique supports. This study was supported by the National Key R&D Program of China (2019YFA0802600) and the National Natural Science Foundation of China (31930019 and 32200431).
\",\n \"Data Availability Statement\",\n \"
All high‐throughput sequencing data in this study have been deposited to the Gene Expression Omnibus (GEO) database and are available under Accession No. GSE217403. All metabolomics, lipidomics, and metabolic flux data in this study are available in Metabolomics Workbench with the sproject (DOI: 10.21228/M8CM5D). All experimental materials generated in this study are available upon request.
\"\n]"},"figure":{"kind":"list like","value":["
Characterization of mammalian‐conserved circLARP1B in HCC metastasis. a) Heatmap and volcano plot illustrating the differentially expressed circRNAs in three metastasis (metas) versus three nonmetastasis (nonmetas) specimens. In the volcano plot, red dots indicate significantly upregulated circRNAs, and blue ones represent downregulated circRNAs. Filled and empty dots indicate conserved and unconserved circRNAs, respectively. Specimens are in situ HCC from patients with or without metastasis. b) Northern blotting analysis of circLARP1B in human cells (PLC and HepG2) and murine Hepa1‐6 cells with or without RNase R treatment. The hybridization probe against the back‐splicing junction (BSJ) of circLARP1B is shown. ACTB mRNA is a control for the effect of RNase R, which digests linear RNAs. c) Representative smFISH images of circLARP1B (green) and LARP1B mRNA (red) in PLC cells. Blue, DAPI staining of nuclei. Scale bar: 10 µm. d) RT‐qPCR analysis of circLARP1B in the nuclear/cytoplasmic fraction of human cells (PLC and HepG2) and murine Hepa1‐6 cells. e) Representative smFISH images of circLARP1B (green) in the liver from wildtype mice. DAPI (nuclei, blue). Scale bar: 10 µm. f) RNA smFISH of circLARP1B (green) in nonmetas and metas specimens. Quantification of positive dots of circLARP1B smFISH is shown (right). N = 15 views. Scale bar: 10 µm. g,h) RT‐qPCR analysis of circLARP1B expressions in HCC specimens. ACTB mRNA is the endogenous control for normalization. i,j) Kaplan–Meier analysis of overall survival (i) and disease‐free survival (j) for 90 HCC patients collected. The red curve indicates survival in patients with higher HCC circLARP1B levels, and the blue line indicates survival in patients with lower HCC circLARP1B levels. (d) Data are shown as mean ± SD from three independent experiments. (f–h) P‐values are from two‐tailed unpaired Student's t‐test. (i,j) P‐values are calculated by the log‐rank test.
","
\nCircLARP1B modulates cell invasion and fatty acid synthesis in mammalian cells. a) Strategy of deleting intronic reverse‐complementary repeats in human LARP1B using CRISPR/Cas9 in PLC cells. Genomic PCR validation of WT and circLARP1B‐Def PLC cells is shown, and the corresponding amplifications are confirmed by Sanger sequencing. WT, wildtype; circLARP1B‐Def, circLARP1B deficient. b) RT‐qPCR analysis of circLARP1B and LARP1B mRNA expressions in WT and circLARP1B‐Def PLC cells. c) Transwell assays of WT and circLARP1B‐Def PLC cells. Quantification of invasive cells is shown (right). Scale bar: 100 µm. d) Schematic procedure of the untargeted metabolomics. All cells were maintained with standard DMEM medium containing 10% FBS for 48 h and then subjected to metabolomics analysis. e) KEGG pathway analysis of dysregulated metabolites in circLARP1B‐Def versus WT PLC cells. The number of significantly changed metabolites in the corresponding pathway is included in the brackets. f) Heatmap of the significantly changed lipid species (VIP >1, P‐value < 0.05) corresponding to 24 lipid classes enriched in lipid metabolism (e) from untargeted lipidomics of WT and circLARP1B‐Def PLC cells. VIP, variable importance in the projection. N = 5 samples for each cell type. g) Representative Nile red (red) and Hoechst 33342 (for nuclei, blue) staining of WT and circLARP1B‐Def PLC cells. N = 15. Scale bar: 20 µm. h) The distribution of 13C‐label fatty acids from 13C‐labeled targeted metabolic flux analysis of WT and circLARP1B‐Def PLC cells measured by liquid chromatography‐mass spectrometry (LC‐MS) following a 24 h 13C‐glucose and 13C‐glutamine incubation. A total of 17 13C‐label fatty acids are detected and three replicates are performed for each group. i) Schematic depicting of the incorporation of uniformly labeled 13C‐glucose or 13C‐glutamine (13C is indicated by gray circle) into the fatty acid palmitate. Two representative palmitate isotopologs are indicated (top). The distribution of 13C‐label in even palmitate isotopologs in WT and circLARP1B‐Def PLC cells measured by LC‐MS is shown (bottom). j) Western blots and the quantification of p‐ULK1, ULK1, p‐ACC1, ACC1, p‐AMPK, and AMPK proteins in WT and circLARP1B‐Def PLC cells. ACTB, Actin b protein used as a loading control. The grayscale statistics of western blotting were performed by ImageJ. k) Representative Nile red (red) and Hoechst 33342 (nuclei, blue) staining of WT or circLARP1B‐Def PLC cells with overexpressing FASN (FASN OE) or not. EV, empty vector. N = 15. Scale bar: 20 µm. l) Transwell assays of WT or circLARP1B‐Def PLC cells with overexpressing FASN or not. Scale bar: 100 µm. (g,k) Nile red signal is defined as the mean gray value per cell quantified by ImageJ. (b,c,i,j,l) Data are shown as mean ± SD from three independent experiments. (b,c,g,i–l) P‐values by two‐tailed unpaired Student's t‐test. (h) P‐values by two‐tailed paired Student's t‐test.
","
\nCircLARP1B interacts with HNRNPD via two binding sites of the circRNA. a) Pull‐down efficiency of circLARP1B in the cytoplasmic fraction of PLC cells (left). Biotinylated oligonucleotide antisense to the circLARP1B back‐splicing junction (BSJ) is indicated. Silver staining shows the proteins co‐pulled down. The red arrow denotes the band identified as HNRNPD by mass spectrometry. Scr, negative control of biotin‐labeled oligo with scrambled sequences; Oligo, biotin‐labeled oligo with antisense sequences to the circLARP1B BSJ. The pull‐down efficiency of circLARP1B is shown with bar figure. b) Western blot of HNRNPD to demonstrate that RNA pull‐down of circLARP1B co‐pulls down HNRNPD in the cytoplasmic fraction of PLC cells. ACTB is a negative control. c) RIP with an antibody against HNRNPD in the cytoplasmic fraction of PLC cells pulls down circLARP1B. Western blots showing efficient pull‐down of HNRNPD with ACTB as a negative control. Gel images demonstrate the semiquantitative RT‐PCR products of RIP RNAs. RT‐qPCR analyses show the enrichment of RIP RNAs. CCND1 mRNA is a known target of HNRNPD and a positive control.[\n##REF##33444453##\n50\n##\n]\nACTB mRNA is a negative control. d,e) circLARP1B RNA pull‐down (d) and Hnrnpd RIP with the cytoplasmic fraction (e) using mouse liver. Mef2c mRNA is a known target of Hnrnpd in mice and a positive control examined in Hnrnpd RIP (e).[\n##REF##24891619##\n94\n##\n] f) Conserved binding motifs of HNRNPD in circLARP1B. The conserved motif 1 (purple) and motif 2 (orange) are mutated separately to motif 1mut and motif 2mut, or mutated together as doublemut. Both sites (Positions 143–151 and 158–166 from the BSJ) are located in the exon 4 of the LARP1B gene. g) Association of circLARP1B examined by RT‐qPCR of HNRNPD RIP in PLC cells with overexpression of the corresponding forms of circLARP1B. Enrichment, normalized to IgG. EV, empty vector. Western blot images indicate successful IP of HNRNPD protein. h) Immunofluorescence (IF) of HNRNPD (green) in PLC cells. Nuclei were stained with DAPI (blue). Quantification of nuclear (Nuc)/cytoplasmic (Cyto) HNRNPD signals is shown as bar figure. N = 15. Scale bar: 10 µm. i) Representative images of circLARP1B FISH together with HNRNPD IF in PLC cells. A permeabilization condition was used to reduce the nuclear HNRNPD signals,[\n##REF##34549195##\n59\n##, ##REF##31508410##\n60\n##\n] thereby highlighting cytoplasmic signals in PLC cells. Boxed areas are enlarged. The colocalization between circLARP1B (G, green) and HNRNPD (R, red) is shown (N = 15 randomly selected areas). R/G, proportion of red signal to green signal colocalization; G/R, proportion of green signal to red signal colocalization. IF, immunofluorescence. Scale bar: 10 and 1 µm (enlarged areas). j) In vitro binding assay of synthesized circLARP1B and FLAG‐tagged HNRNPD protein purified from HEK293 cells. An illustration of the assay was shown (left) and semiquantitative RT‐PCR for circLARP1B or ACTB mRNA fragment was indicated (right). (a–e,g) Data are shown as mean ± SD from three independent experiments; P‐values by two‐tailed unpaired Student's t‐test.
","
Effects of circLARP1B depend on the interaction with HNRNPD. a) Transwell assays of WT or circLARP1B‐Def PLC cells with overexpressing circLARP1B or doublemut\ncircLARP1B. The quantification of invaded cells is analyzed. doublemut, circLARP1B two HNRNPD‐binding sites mutated. Scale bar: 100 µm. b) Representative Nile red (red) and Hoechst 33342 (for nuclei, blue) staining of WT or circLARP1B‐Def PLC cells with overexpressing circLARP1B or doublemut\ncircLARP1B. N = 15. Scale bar: 20 µm. c) Western blot images and the quantification of the indicated proteins in WT or circLARP1B‐Def PLC cells with the overexpression of circLARP1B or doublemut. d) Transwell assays in PLC cells with HNRNPD overexpression. Cells with HNRNPD overexpression are examined upon co‐overexpression of circLARP1B or doublemut\ncircLARP1B. The quantification of invaded cells is shown. Scale bar: 100 µm. e) Representative Nile red (red) and Hoechst 33342 (for nuclei, blue) staining in PLC cells with HNRNPD overexpression. Cells with HNRNPD overexpression are examined upon co‐overexpression of circLARP1B or doublemut\ncircLARP1B. N = 15. Scale bar: 20 µm. f) Western blot images and quantification of the proteins in PLC cells overexpressed HNRNPD, with co‐overexpression of circLARP1B or doublemut. (b,e) The Nile red signal is defined as the mean gray value per cell quantified by ImageJ. (c,f) The grayscale statistics of western blotting was performed by ImageJ. (a,c,d,f) Data are shown as mean ± SD from three independent experiments. (a–f) P‐values by two‐tailed unpaired Student's t‐test. EV, empty vector.
","
\nLKB1 mRNA is a functional downstream target of circLARP1B. a) Heatmap showing HNRNPD RIP‐seq signals on the 3′ UTRs of 216 mRNAs that demonstrate significant sensitivity to circLARP1B levels. EV, PLC cells transfected with empty vector; circLARP1B OE, cells with circLARP1B overexpression; WT, wildtype PLC cells; circLARP1B‐Def, circLARP1B deficient PLC cells. b) Gene ontology (GO) analysis of 216 HNRNPD targets significantly sensitive to changes in circLARP1B levels. c) Total HNRNPD binding signals of four PLC cells (EV, circLARP1B OE, WT, circLARP1B‐Def) for six genes enriched in the biological process of peptidyl‐Thr phosphorylation. d) HNRNPD binding signals on LKB1’s 3′ UTR in four PLC cells. e) HNRNPD binding signals on circLARP1B (All reads including the BSJ reads are counted) in four PLC cells. Enlarged IGV visualization demonstrated HNRNPD binding signals around two functional motifs. f) Transwell assays of PLC cells with circLARP1B overexpression alone or with circLARP1B and LKB1 overexpression together. The quantification of invaded cells by ImageJ is shown. Scale bar: 100 µm. g) Representative Nile red (red) and Hoechst 33342 (nuclei, blue) staining of PLC cells with circLARP1B overexpression or co‐overexpressing of circLARP1B and LKB1. Nile red signal is defined as the mean gray value per cell quantified by ImageJ. N = 15. Scale bar: 20 µm. h) Western blot images and quantification of proteins in PLC cells with circLARP1B overexpression or co‐overexpressing of circLARP1B and LKB1. The grayscale statistics of western blotting was performed by ImageJ. i) Representative immunohistochemistry (IHC) staining and the quantification of LKB1 proteins in nonmetas and metas HCC specimens (N = 3). The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. Scale bar: 50 µm. (f–h) EV, empty vector control. (f,h) Data are shown as mean ± SD from three independent experiments. (f–i) P‐values by two‐tailed unpaired Student's t‐test.
","
\nCircLARP1B destabilizes LKB1 mRNA via perturbing HNRNPD. a–d) Stability assay of LKB1 mRNA and the steady levels of LKB1 protein (examined by western blotting) in human PLC cells (a,b) and murine Hepa1‐6 cells (c,d) treated siRNA against HNRNPD. siNC, negative control siRNA with scrambled sequences. e) Overall experimental strategy of eIF4E and eIF4G1 RIP assays. RNase I is introduced to digest unprotected RNAs across the IP procedure. Western blots showing HNRNPD knockdown efficiency and efficient IPs of eIF4E and eIF4G1 in PLC cells treated with siRNA against HNRNPD (right). ACTB protein acted as the loading control. f) Primers against various regions of LKB1 mRNA (top) and enrichment of LKB1 mRNA regions with eIF4E or eIF4G1 by RT‐qPCR (bottom). g,h) Stability assay of LKB1 mRNA (g) and the steady levels of LKB1 protein (h) in WT or circLARP1B‐Def PLC cells. i,j) Stability assay of Lkb1 mRNA (i) and the steady levels of Lkb1 protein (j) in Hepa1‐6 cells treated with shcircLARP1B or shCtrl. shCtrl, shRNA control that generates siRNA of scrambled sequences; shcircLARP1B, shRNA against the murine circLARP1B BSJ. k) In vitro competing assay of purified HNRNPD protein for equal moles of in vitro synthesized circLARP1B and LKB1 3′ UTR. An illustration of the assay was shown (top). Semiquantitative RT‐PCR gels and RT‐qPCR for circLARP1B and LKB1 3′ UTR were indicated (bottom). LKB1 3′ UTR, in vitro transcribed LKB1 3′ UTR fragments with the HNRNPD binding sequence. l) Association of LKB1 mRNA examined by RT‐qPCR of HNRNPD RIP in PLC cells treated with oligodeoxynucleotide antisense to two HNRNPD binding motifs in circLARP1B (ODN‐AS) or the control (ODN‐Ctrl, ODN with scrambled sequences). Two motifs with reverse complementary sequences were indicated with underlines. PS, phosphorothioate; 2′‐OMe, 2′‐O‐methyl. Western blot images indicate successful IP of HNRNPD protein. Enrichment, normalized to IgG. m) Western blotting and the corresponding quantification showing the steady level of LKB1 protein in PLC cells transfected with ODN‐AS or ODN‐Ctrl. (b,d,h,j,m) The grayscale statistics of western blotting were performed by ImageJ. (a–d,f–m) Data are shown as mean ± SD from three independent experiments. (a,c,g,i) P‐values by two‐way ANOVA test. (b,d,f,h,j,l,m) P‐values by two‐tailed unpaired Student's t‐test.
","
\nCircLARP1B deficiency in mice causes liver changes attributable to Lkb1 surplus. a) Strategy of knockout (KO) reverse‐complementary sequence in mouse Larp1b intron 4 using CRISPR‐Cas9. PCR products of mouse genotyping are shown. WT, wildtype; circLARP1B\n−/−, KO of the intronic reverse‐complementary sequences; B1, mouse B1 repeat. b) RT‐qPCR analysis of the steady levels and nascent levels (with nuclear run‐on assay) of circLARP1B and Larp1b mRNA in WT and circLARP1B\n−/− mouse liver. Data are from three independent experiments. c) Representative Oil Red O staining and the quantification in WT and circLARP1B\n−/− mouse liver (N = 5 per group). Nuclei stained with hematoxylin (blue). Scale bar: 50 µm. d) Representative IHC staining and quantification of the proteins in liver from WT and circLARP1B\n−/− mice (N = 5 per group). Scale bar: 50 µm. AOD, average optical density. e) Western blot of the indicated proteins in livers from WT or circLARP1B\n−/− mice. f) Representative Lkb1 IHC staining in livers from WT or circLARP1B\n−/− mice with the intravenous tail injection of AAV8‐shCtrl or AAV8‐shLkb1 for three weeks (N = 5 per group). Scale bar: 50 µm. g) Representative Oil Red O staining in liver from WT or circLARP1B\n−/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection (N = 5 per group). shCtrl, negative control shRNA construct that gives rise to siRNA with scrambled sequences. Nuclei stained with hematoxylin (blue). Scale bar: 50 µm. h) Representative IHC staining of the indicated proteins in liver from WT or circLARP1B\n−/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection (N = 5 per group). Scale bar: 50 µm. i) Western blot of the indicated proteins in livers from WT or circLARP1B\n−/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection. Data are shown as mean ± SD. (c,g) Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. (d,f,h) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (e,i) The grayscale statistics of western blotting was performed by ImageJ. (c–i) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water; all data were from mice at 9:00 AM. (b–d,f–h) P‐values by two‐tailed unpaired Student's t‐test.
","
\nCircLARP1B deficiency impedes HCC metastasis and lipid accumulation in mice. a) Schematic illustration for generating the DEN‐induced mice HCC model. b) Representative images and H&E staining of liver tumor nodes from WT and circLARP1B\n−/− mice (N = 5 per group) after DEN‐induction for 18 weeks. Statistical analysis of liver tumor node numbers and diameters of the largest tumor node are shown. Scale bar: 2 mm. c) Representative H&E staining and the quantification of lung metastases in WT and circLARP1B\n−/− HCC mice (N = 5 per group). Scale bar: 400 µm. d) Representative Vim (Vimentin, a prometastasis marker) and Ecad (E‐cadherin, an antimetastasis marker) IHC staining of liver tumors from WT and circLARP1B\n−/− HCC mice (N = 5 per group). Scale bar: 50 µm. e–i) The serum AFP (e), ALT (f), AST (g), TG (h), and TC (i) levels in WT and circLARP1B\n−/− HCC mice (N = 5 per group). AFP, alpha‐fetoprotein (a biomarker for HCC); ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; TC, total cholesterol. j) Representative Oil Red O staining images and the quantification in liver tumors from WT and circLARP1B\n−/− HCC mice (N = 5 per group). Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. Scale bar: 100 µm. k) Representative Lkb1, p‐Ampk, and p‐Acc1 IHC staining of liver tumors from WT and circLARP1B\n−/− HCC mice (N = 5 per group). Scale bar: 50 µm. l) Western blot of the indicated proteins of liver tumors from WT and circLARP1B\n−/− HCC mice. The grayscale statistics of western blotting was performed by ImageJ. (b–l) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water, and fasted for 12 h before bleeding and sacrifice at 9:00 AM. All data are shown as mean ± SD. (d,k) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (b–k) P‐values by two‐tailed unpaired Student's t‐test.
","
Hepatocyte‐directed circLARP1B knockdown impedes HCC metastasis and lipid accumulation in mice. a) Schematic illustration for generating the DEN‐induced mice HCC model. Hepatocyte‐directed AAV8 expressing shcircLARP1B and AAV8‐shCtrl were injected after induction for 10 weeks. b) RT‐qPCR analysis of circLARP1B and Larp1b mRNA levels of livers from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. c) Representative images and H&E staining of liver nodes from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection after DEN‐induction for 18 weeks. Statistical analysis of liver tumor node numbers and diameters of the largest tumor node are shown. Scale bar: 2 mm. d) Representative H&E staining and the quantification of lung metastases in WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Scale bar: 400 µm. e) Representative Vim (Vimentin, a prometastasis marker) and Ecad (E‐cadherin, an antimetastasis marker) IHC staining of liver tumors from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Scale bar: 50 µm. f–j) The serum AFP (f), ALT (g), AST (h), TG (i), and TC (j) levels in WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. k) Representative Oil Red O staining images and the quantification in liver tumors from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. Scale bar: 100 µm. l) Representative Lkb1, p‐Ampk, and p‐Acc1 IHC staining of liver tumors from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Scale bar: 50 µm. m) Western blot of the indicated proteins of liver tumors from WT HCC mice with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. The grayscale statistics of western blotting was performed by ImageJ. (b–m) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water, and fasted for 12 h before bleeding and sacrifice at 9:00 AM. Data are shown as mean ± SD. (e,l) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (b–l) P‐values by two‐tailed unpaired Student's t‐test.
","
LKB1 is a key target for the roles of circLARP1B in HCC. a) Representative images and H&E staining of liver tumors from WT and circLARP1B\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection after DEN induction for 10 weeks. Statistical analysis of liver tumor numbers and diameter of the largest tumor is shown. Scale bar: 2 mm. b) Representative H&E staining and the quantification of lung metastases in WT and circLARP1B\n−/− HCC mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection. Scale bar: 400 µm. c) Representative Vim (Vimentin, prometastasis marker) and Ecad (E‐cadherin, antimetastasis marker) IHC staining of liver tumors from WT and circLARP1B\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection. The quantification of IHC is shown. Scale bar: 50 µm. d) Representative Oil Red O staining of liver tumors from WT and circLARP1B\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection. Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. Scale bar: 100 µm. e) Representative p‐Ampk and p‐Acc1 IHC staining of liver tumors from WT and circLARP1B\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection (top). The corresponding quantification is analyzed (bottom). Scale bar: 50 µm. f) Kaplan–Meier analysis of OS (left) and DFS (right) for 90 HCC patients collected. The red curve indicates survival in patients with higher HCC HNRNPD mRNA levels, and the blue line indicates survival in patients with lower HCC HNRNPD mRNA levels. g) Kaplan–Meier analysis of OS (left) and DFS (right) for 90 HCC patients with LKB1 mRNA levels. (a–e) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water, and fasted for 12 h before bleeding and sacrifice at 9:00 AM. Data are shown as mean ± SD. (c,e) The quantification of IHC as average optical density (AOD) calculated by ImageJ is shown. (a–e) P‐values by two‐tailed unpaired Student's t‐test. (f,g) P‐values are calculated by the log‐rank test.
"],"string":"[\n \"
Characterization of mammalian‐conserved circLARP1B in HCC metastasis. a) Heatmap and volcano plot illustrating the differentially expressed circRNAs in three metastasis (metas) versus three nonmetastasis (nonmetas) specimens. In the volcano plot, red dots indicate significantly upregulated circRNAs, and blue ones represent downregulated circRNAs. Filled and empty dots indicate conserved and unconserved circRNAs, respectively. Specimens are in situ HCC from patients with or without metastasis. b) Northern blotting analysis of circLARP1B in human cells (PLC and HepG2) and murine Hepa1‐6 cells with or without RNase R treatment. The hybridization probe against the back‐splicing junction (BSJ) of circLARP1B is shown. ACTB mRNA is a control for the effect of RNase R, which digests linear RNAs. c) Representative smFISH images of circLARP1B (green) and LARP1B mRNA (red) in PLC cells. Blue, DAPI staining of nuclei. Scale bar: 10 µm. d) RT‐qPCR analysis of circLARP1B in the nuclear/cytoplasmic fraction of human cells (PLC and HepG2) and murine Hepa1‐6 cells. e) Representative smFISH images of circLARP1B (green) in the liver from wildtype mice. DAPI (nuclei, blue). Scale bar: 10 µm. f) RNA smFISH of circLARP1B (green) in nonmetas and metas specimens. Quantification of positive dots of circLARP1B smFISH is shown (right). N = 15 views. Scale bar: 10 µm. g,h) RT‐qPCR analysis of circLARP1B expressions in HCC specimens. ACTB mRNA is the endogenous control for normalization. i,j) Kaplan–Meier analysis of overall survival (i) and disease‐free survival (j) for 90 HCC patients collected. The red curve indicates survival in patients with higher HCC circLARP1B levels, and the blue line indicates survival in patients with lower HCC circLARP1B levels. (d) Data are shown as mean ± SD from three independent experiments. (f–h) P‐values are from two‐tailed unpaired Student's t‐test. (i,j) P‐values are calculated by the log‐rank test.
\",\n \"
\\nCircLARP1B modulates cell invasion and fatty acid synthesis in mammalian cells. a) Strategy of deleting intronic reverse‐complementary repeats in human LARP1B using CRISPR/Cas9 in PLC cells. Genomic PCR validation of WT and circLARP1B‐Def PLC cells is shown, and the corresponding amplifications are confirmed by Sanger sequencing. WT, wildtype; circLARP1B‐Def, circLARP1B deficient. b) RT‐qPCR analysis of circLARP1B and LARP1B mRNA expressions in WT and circLARP1B‐Def PLC cells. c) Transwell assays of WT and circLARP1B‐Def PLC cells. Quantification of invasive cells is shown (right). Scale bar: 100 µm. d) Schematic procedure of the untargeted metabolomics. All cells were maintained with standard DMEM medium containing 10% FBS for 48 h and then subjected to metabolomics analysis. e) KEGG pathway analysis of dysregulated metabolites in circLARP1B‐Def versus WT PLC cells. The number of significantly changed metabolites in the corresponding pathway is included in the brackets. f) Heatmap of the significantly changed lipid species (VIP >1, P‐value < 0.05) corresponding to 24 lipid classes enriched in lipid metabolism (e) from untargeted lipidomics of WT and circLARP1B‐Def PLC cells. VIP, variable importance in the projection. N = 5 samples for each cell type. g) Representative Nile red (red) and Hoechst 33342 (for nuclei, blue) staining of WT and circLARP1B‐Def PLC cells. N = 15. Scale bar: 20 µm. h) The distribution of 13C‐label fatty acids from 13C‐labeled targeted metabolic flux analysis of WT and circLARP1B‐Def PLC cells measured by liquid chromatography‐mass spectrometry (LC‐MS) following a 24 h 13C‐glucose and 13C‐glutamine incubation. A total of 17 13C‐label fatty acids are detected and three replicates are performed for each group. i) Schematic depicting of the incorporation of uniformly labeled 13C‐glucose or 13C‐glutamine (13C is indicated by gray circle) into the fatty acid palmitate. Two representative palmitate isotopologs are indicated (top). The distribution of 13C‐label in even palmitate isotopologs in WT and circLARP1B‐Def PLC cells measured by LC‐MS is shown (bottom). j) Western blots and the quantification of p‐ULK1, ULK1, p‐ACC1, ACC1, p‐AMPK, and AMPK proteins in WT and circLARP1B‐Def PLC cells. ACTB, Actin b protein used as a loading control. The grayscale statistics of western blotting were performed by ImageJ. k) Representative Nile red (red) and Hoechst 33342 (nuclei, blue) staining of WT or circLARP1B‐Def PLC cells with overexpressing FASN (FASN OE) or not. EV, empty vector. N = 15. Scale bar: 20 µm. l) Transwell assays of WT or circLARP1B‐Def PLC cells with overexpressing FASN or not. Scale bar: 100 µm. (g,k) Nile red signal is defined as the mean gray value per cell quantified by ImageJ. (b,c,i,j,l) Data are shown as mean ± SD from three independent experiments. (b,c,g,i–l) P‐values by two‐tailed unpaired Student's t‐test. (h) P‐values by two‐tailed paired Student's t‐test.
\",\n \"
\\nCircLARP1B interacts with HNRNPD via two binding sites of the circRNA. a) Pull‐down efficiency of circLARP1B in the cytoplasmic fraction of PLC cells (left). Biotinylated oligonucleotide antisense to the circLARP1B back‐splicing junction (BSJ) is indicated. Silver staining shows the proteins co‐pulled down. The red arrow denotes the band identified as HNRNPD by mass spectrometry. Scr, negative control of biotin‐labeled oligo with scrambled sequences; Oligo, biotin‐labeled oligo with antisense sequences to the circLARP1B BSJ. The pull‐down efficiency of circLARP1B is shown with bar figure. b) Western blot of HNRNPD to demonstrate that RNA pull‐down of circLARP1B co‐pulls down HNRNPD in the cytoplasmic fraction of PLC cells. ACTB is a negative control. c) RIP with an antibody against HNRNPD in the cytoplasmic fraction of PLC cells pulls down circLARP1B. Western blots showing efficient pull‐down of HNRNPD with ACTB as a negative control. Gel images demonstrate the semiquantitative RT‐PCR products of RIP RNAs. RT‐qPCR analyses show the enrichment of RIP RNAs. CCND1 mRNA is a known target of HNRNPD and a positive control.[\\n##REF##33444453##\\n50\\n##\\n]\\nACTB mRNA is a negative control. d,e) circLARP1B RNA pull‐down (d) and Hnrnpd RIP with the cytoplasmic fraction (e) using mouse liver. Mef2c mRNA is a known target of Hnrnpd in mice and a positive control examined in Hnrnpd RIP (e).[\\n##REF##24891619##\\n94\\n##\\n] f) Conserved binding motifs of HNRNPD in circLARP1B. The conserved motif 1 (purple) and motif 2 (orange) are mutated separately to motif 1mut and motif 2mut, or mutated together as doublemut. Both sites (Positions 143–151 and 158–166 from the BSJ) are located in the exon 4 of the LARP1B gene. g) Association of circLARP1B examined by RT‐qPCR of HNRNPD RIP in PLC cells with overexpression of the corresponding forms of circLARP1B. Enrichment, normalized to IgG. EV, empty vector. Western blot images indicate successful IP of HNRNPD protein. h) Immunofluorescence (IF) of HNRNPD (green) in PLC cells. Nuclei were stained with DAPI (blue). Quantification of nuclear (Nuc)/cytoplasmic (Cyto) HNRNPD signals is shown as bar figure. N = 15. Scale bar: 10 µm. i) Representative images of circLARP1B FISH together with HNRNPD IF in PLC cells. A permeabilization condition was used to reduce the nuclear HNRNPD signals,[\\n##REF##34549195##\\n59\\n##, ##REF##31508410##\\n60\\n##\\n] thereby highlighting cytoplasmic signals in PLC cells. Boxed areas are enlarged. The colocalization between circLARP1B (G, green) and HNRNPD (R, red) is shown (N = 15 randomly selected areas). R/G, proportion of red signal to green signal colocalization; G/R, proportion of green signal to red signal colocalization. IF, immunofluorescence. Scale bar: 10 and 1 µm (enlarged areas). j) In vitro binding assay of synthesized circLARP1B and FLAG‐tagged HNRNPD protein purified from HEK293 cells. An illustration of the assay was shown (left) and semiquantitative RT‐PCR for circLARP1B or ACTB mRNA fragment was indicated (right). (a–e,g) Data are shown as mean ± SD from three independent experiments; P‐values by two‐tailed unpaired Student's t‐test.
\",\n \"
Effects of circLARP1B depend on the interaction with HNRNPD. a) Transwell assays of WT or circLARP1B‐Def PLC cells with overexpressing circLARP1B or doublemut\\ncircLARP1B. The quantification of invaded cells is analyzed. doublemut, circLARP1B two HNRNPD‐binding sites mutated. Scale bar: 100 µm. b) Representative Nile red (red) and Hoechst 33342 (for nuclei, blue) staining of WT or circLARP1B‐Def PLC cells with overexpressing circLARP1B or doublemut\\ncircLARP1B. N = 15. Scale bar: 20 µm. c) Western blot images and the quantification of the indicated proteins in WT or circLARP1B‐Def PLC cells with the overexpression of circLARP1B or doublemut. d) Transwell assays in PLC cells with HNRNPD overexpression. Cells with HNRNPD overexpression are examined upon co‐overexpression of circLARP1B or doublemut\\ncircLARP1B. The quantification of invaded cells is shown. Scale bar: 100 µm. e) Representative Nile red (red) and Hoechst 33342 (for nuclei, blue) staining in PLC cells with HNRNPD overexpression. Cells with HNRNPD overexpression are examined upon co‐overexpression of circLARP1B or doublemut\\ncircLARP1B. N = 15. Scale bar: 20 µm. f) Western blot images and quantification of the proteins in PLC cells overexpressed HNRNPD, with co‐overexpression of circLARP1B or doublemut. (b,e) The Nile red signal is defined as the mean gray value per cell quantified by ImageJ. (c,f) The grayscale statistics of western blotting was performed by ImageJ. (a,c,d,f) Data are shown as mean ± SD from three independent experiments. (a–f) P‐values by two‐tailed unpaired Student's t‐test. EV, empty vector.
\",\n \"
\\nLKB1 mRNA is a functional downstream target of circLARP1B. a) Heatmap showing HNRNPD RIP‐seq signals on the 3′ UTRs of 216 mRNAs that demonstrate significant sensitivity to circLARP1B levels. EV, PLC cells transfected with empty vector; circLARP1B OE, cells with circLARP1B overexpression; WT, wildtype PLC cells; circLARP1B‐Def, circLARP1B deficient PLC cells. b) Gene ontology (GO) analysis of 216 HNRNPD targets significantly sensitive to changes in circLARP1B levels. c) Total HNRNPD binding signals of four PLC cells (EV, circLARP1B OE, WT, circLARP1B‐Def) for six genes enriched in the biological process of peptidyl‐Thr phosphorylation. d) HNRNPD binding signals on LKB1’s 3′ UTR in four PLC cells. e) HNRNPD binding signals on circLARP1B (All reads including the BSJ reads are counted) in four PLC cells. Enlarged IGV visualization demonstrated HNRNPD binding signals around two functional motifs. f) Transwell assays of PLC cells with circLARP1B overexpression alone or with circLARP1B and LKB1 overexpression together. The quantification of invaded cells by ImageJ is shown. Scale bar: 100 µm. g) Representative Nile red (red) and Hoechst 33342 (nuclei, blue) staining of PLC cells with circLARP1B overexpression or co‐overexpressing of circLARP1B and LKB1. Nile red signal is defined as the mean gray value per cell quantified by ImageJ. N = 15. Scale bar: 20 µm. h) Western blot images and quantification of proteins in PLC cells with circLARP1B overexpression or co‐overexpressing of circLARP1B and LKB1. The grayscale statistics of western blotting was performed by ImageJ. i) Representative immunohistochemistry (IHC) staining and the quantification of LKB1 proteins in nonmetas and metas HCC specimens (N = 3). The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. Scale bar: 50 µm. (f–h) EV, empty vector control. (f,h) Data are shown as mean ± SD from three independent experiments. (f–i) P‐values by two‐tailed unpaired Student's t‐test.
\",\n \"
\\nCircLARP1B destabilizes LKB1 mRNA via perturbing HNRNPD. a–d) Stability assay of LKB1 mRNA and the steady levels of LKB1 protein (examined by western blotting) in human PLC cells (a,b) and murine Hepa1‐6 cells (c,d) treated siRNA against HNRNPD. siNC, negative control siRNA with scrambled sequences. e) Overall experimental strategy of eIF4E and eIF4G1 RIP assays. RNase I is introduced to digest unprotected RNAs across the IP procedure. Western blots showing HNRNPD knockdown efficiency and efficient IPs of eIF4E and eIF4G1 in PLC cells treated with siRNA against HNRNPD (right). ACTB protein acted as the loading control. f) Primers against various regions of LKB1 mRNA (top) and enrichment of LKB1 mRNA regions with eIF4E or eIF4G1 by RT‐qPCR (bottom). g,h) Stability assay of LKB1 mRNA (g) and the steady levels of LKB1 protein (h) in WT or circLARP1B‐Def PLC cells. i,j) Stability assay of Lkb1 mRNA (i) and the steady levels of Lkb1 protein (j) in Hepa1‐6 cells treated with shcircLARP1B or shCtrl. shCtrl, shRNA control that generates siRNA of scrambled sequences; shcircLARP1B, shRNA against the murine circLARP1B BSJ. k) In vitro competing assay of purified HNRNPD protein for equal moles of in vitro synthesized circLARP1B and LKB1 3′ UTR. An illustration of the assay was shown (top). Semiquantitative RT‐PCR gels and RT‐qPCR for circLARP1B and LKB1 3′ UTR were indicated (bottom). LKB1 3′ UTR, in vitro transcribed LKB1 3′ UTR fragments with the HNRNPD binding sequence. l) Association of LKB1 mRNA examined by RT‐qPCR of HNRNPD RIP in PLC cells treated with oligodeoxynucleotide antisense to two HNRNPD binding motifs in circLARP1B (ODN‐AS) or the control (ODN‐Ctrl, ODN with scrambled sequences). Two motifs with reverse complementary sequences were indicated with underlines. PS, phosphorothioate; 2′‐OMe, 2′‐O‐methyl. Western blot images indicate successful IP of HNRNPD protein. Enrichment, normalized to IgG. m) Western blotting and the corresponding quantification showing the steady level of LKB1 protein in PLC cells transfected with ODN‐AS or ODN‐Ctrl. (b,d,h,j,m) The grayscale statistics of western blotting were performed by ImageJ. (a–d,f–m) Data are shown as mean ± SD from three independent experiments. (a,c,g,i) P‐values by two‐way ANOVA test. (b,d,f,h,j,l,m) P‐values by two‐tailed unpaired Student's t‐test.
\",\n \"
\\nCircLARP1B deficiency in mice causes liver changes attributable to Lkb1 surplus. a) Strategy of knockout (KO) reverse‐complementary sequence in mouse Larp1b intron 4 using CRISPR‐Cas9. PCR products of mouse genotyping are shown. WT, wildtype; circLARP1B\\n−/−, KO of the intronic reverse‐complementary sequences; B1, mouse B1 repeat. b) RT‐qPCR analysis of the steady levels and nascent levels (with nuclear run‐on assay) of circLARP1B and Larp1b mRNA in WT and circLARP1B\\n−/− mouse liver. Data are from three independent experiments. c) Representative Oil Red O staining and the quantification in WT and circLARP1B\\n−/− mouse liver (N = 5 per group). Nuclei stained with hematoxylin (blue). Scale bar: 50 µm. d) Representative IHC staining and quantification of the proteins in liver from WT and circLARP1B\\n−/− mice (N = 5 per group). Scale bar: 50 µm. AOD, average optical density. e) Western blot of the indicated proteins in livers from WT or circLARP1B\\n−/− mice. f) Representative Lkb1 IHC staining in livers from WT or circLARP1B\\n−/− mice with the intravenous tail injection of AAV8‐shCtrl or AAV8‐shLkb1 for three weeks (N = 5 per group). Scale bar: 50 µm. g) Representative Oil Red O staining in liver from WT or circLARP1B\\n−/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection (N = 5 per group). shCtrl, negative control shRNA construct that gives rise to siRNA with scrambled sequences. Nuclei stained with hematoxylin (blue). Scale bar: 50 µm. h) Representative IHC staining of the indicated proteins in liver from WT or circLARP1B\\n−/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection (N = 5 per group). Scale bar: 50 µm. i) Western blot of the indicated proteins in livers from WT or circLARP1B\\n−/− mice with AAV8‐shCtrl or AAV8‐shLkb1 injection. Data are shown as mean ± SD. (c,g) Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. (d,f,h) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (e,i) The grayscale statistics of western blotting was performed by ImageJ. (c–i) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water; all data were from mice at 9:00 AM. (b–d,f–h) P‐values by two‐tailed unpaired Student's t‐test.
\",\n \"
\\nCircLARP1B deficiency impedes HCC metastasis and lipid accumulation in mice. a) Schematic illustration for generating the DEN‐induced mice HCC model. b) Representative images and H&E staining of liver tumor nodes from WT and circLARP1B\\n−/− mice (N = 5 per group) after DEN‐induction for 18 weeks. Statistical analysis of liver tumor node numbers and diameters of the largest tumor node are shown. Scale bar: 2 mm. c) Representative H&E staining and the quantification of lung metastases in WT and circLARP1B\\n−/− HCC mice (N = 5 per group). Scale bar: 400 µm. d) Representative Vim (Vimentin, a prometastasis marker) and Ecad (E‐cadherin, an antimetastasis marker) IHC staining of liver tumors from WT and circLARP1B\\n−/− HCC mice (N = 5 per group). Scale bar: 50 µm. e–i) The serum AFP (e), ALT (f), AST (g), TG (h), and TC (i) levels in WT and circLARP1B\\n−/− HCC mice (N = 5 per group). AFP, alpha‐fetoprotein (a biomarker for HCC); ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; TC, total cholesterol. j) Representative Oil Red O staining images and the quantification in liver tumors from WT and circLARP1B\\n−/− HCC mice (N = 5 per group). Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. Scale bar: 100 µm. k) Representative Lkb1, p‐Ampk, and p‐Acc1 IHC staining of liver tumors from WT and circLARP1B\\n−/− HCC mice (N = 5 per group). Scale bar: 50 µm. l) Western blot of the indicated proteins of liver tumors from WT and circLARP1B\\n−/− HCC mice. The grayscale statistics of western blotting was performed by ImageJ. (b–l) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water, and fasted for 12 h before bleeding and sacrifice at 9:00 AM. All data are shown as mean ± SD. (d,k) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (b–k) P‐values by two‐tailed unpaired Student's t‐test.
\",\n \"
Hepatocyte‐directed circLARP1B knockdown impedes HCC metastasis and lipid accumulation in mice. a) Schematic illustration for generating the DEN‐induced mice HCC model. Hepatocyte‐directed AAV8 expressing shcircLARP1B and AAV8‐shCtrl were injected after induction for 10 weeks. b) RT‐qPCR analysis of circLARP1B and Larp1b mRNA levels of livers from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. c) Representative images and H&E staining of liver nodes from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection after DEN‐induction for 18 weeks. Statistical analysis of liver tumor node numbers and diameters of the largest tumor node are shown. Scale bar: 2 mm. d) Representative H&E staining and the quantification of lung metastases in WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Scale bar: 400 µm. e) Representative Vim (Vimentin, a prometastasis marker) and Ecad (E‐cadherin, an antimetastasis marker) IHC staining of liver tumors from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Scale bar: 50 µm. f–j) The serum AFP (f), ALT (g), AST (h), TG (i), and TC (j) levels in WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. k) Representative Oil Red O staining images and the quantification in liver tumors from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. Scale bar: 100 µm. l) Representative Lkb1, p‐Ampk, and p‐Acc1 IHC staining of liver tumors from WT mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. Scale bar: 50 µm. m) Western blot of the indicated proteins of liver tumors from WT HCC mice with AAV8‐shCtrl or AAV8‐shcircLARP1B injection. The grayscale statistics of western blotting was performed by ImageJ. (b–m) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water, and fasted for 12 h before bleeding and sacrifice at 9:00 AM. Data are shown as mean ± SD. (e,l) The IHC signal is defined as the average optical density (AOD) calculated by ImageJ. (b–l) P‐values by two‐tailed unpaired Student's t‐test.
\",\n \"
LKB1 is a key target for the roles of circLARP1B in HCC. a) Representative images and H&E staining of liver tumors from WT and circLARP1B\\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection after DEN induction for 10 weeks. Statistical analysis of liver tumor numbers and diameter of the largest tumor is shown. Scale bar: 2 mm. b) Representative H&E staining and the quantification of lung metastases in WT and circLARP1B\\n−/− HCC mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection. Scale bar: 400 µm. c) Representative Vim (Vimentin, prometastasis marker) and Ecad (E‐cadherin, antimetastasis marker) IHC staining of liver tumors from WT and circLARP1B\\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection. The quantification of IHC is shown. Scale bar: 50 µm. d) Representative Oil Red O staining of liver tumors from WT and circLARP1B\\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection. Lipid level is defined as the percentage of Oil Red positive area calculated by Image‐Pro plus. Scale bar: 100 µm. e) Representative p‐Ampk and p‐Acc1 IHC staining of liver tumors from WT and circLARP1B\\n−/− mice (N = 5 per group) with AAV8‐shCtrl or AAV8‐shLkb1 injection (top). The corresponding quantification is analyzed (bottom). Scale bar: 50 µm. f) Kaplan–Meier analysis of OS (left) and DFS (right) for 90 HCC patients collected. The red curve indicates survival in patients with higher HCC HNRNPD mRNA levels, and the blue line indicates survival in patients with lower HCC HNRNPD mRNA levels. g) Kaplan–Meier analysis of OS (left) and DFS (right) for 90 HCC patients with LKB1 mRNA levels. (a–e) All animals were kept in a controlled environment (23–25 °C with a 12‐h light‐dark cycle and lights on at 8:00 AM), on normal diet feeding with free access to water, and fasted for 12 h before bleeding and sacrifice at 9:00 AM. Data are shown as mean ± SD. (c,e) The quantification of IHC as average optical density (AOD) calculated by ImageJ is shown. (a–e) P‐values by two‐tailed unpaired Student's t‐test. (f,g) P‐values are calculated by the log‐rank test.
Multidirectional interaction among organ systems is an integral aspect of the development and physiology of mammalian organisms. Factors, including hormones, cytokines, and chemokines, are produced by diverse cells in the body and participate in interorgan communication (Castillo‐Armengol et al., ##REF##31423716##2019##). The mammalian gut is not an exception in the production of factors that mediate interorgan crosstalk for maintaining a healthy homeostatic state and for the normal functioning of several organ systems, including the musculoskeletal and cardiovascular systems (Hu et al., ##REF##35932976##2022##; Lucas et al., ##UREF##2##2018##).
","
The gut microbiome encompasses a diverse array of gene batteries whose products (i.e., postbiotics) are continuously being characterized to participate in important aspects of hosts physiology, including gut (Martin‐Gallausiaux et al., ##REF##32238208##2021##), cardiovascular (Razavi et al., ##REF##31182162##2019##; Yang et al., ##REF##31803054##2019##) and bone health (Lucas et al., ##UREF##2##2018##). More so, gut colonization with microbiota is critical for shaping the mucosal immune system and results in either a homeostatic or abnormal immune reactivity (Al Bander et al., ##REF##33086688##2020##; Brandsma et al., ##REF##30582442##2019##). Importantly, the mammalian gut has evolved to guard against the translocation of unwanted substances, including toxins and pathogens, into the systemic circulation. In addition, ~70% of our immune cells reside in the gut, functioning in antigen sampling, and producing pathogen‐specific immune responses (Wiertsema et al., ##REF##33803407##2021##). Aging‐induced alterations in the mammalian gut microbiome and immune cell function increase chronic inflammation (i.e., inflammaging) in the gut and increase the risk of chronic diseases, including cardiovascular disorders (CVDs) and osteoporosis (Bosco & Noti, ##REF##33875817##2021##; Fasano, ##REF##32742638##2020##; Gibon et al., ##REF##29094003##2017##; Li et al., ##UREF##1##2019##; Liberale et al., ##REF##35210039##2022##).
","
Interleukin (IL)‐10 is a well‐known pleiotropic anti‐inflammatory cytokine (Sabat et al., ##REF##21115385##2010##). The partial or total loss of this cytokine, as in the IL‐10 knockout (KO) mouse, models inflammaging and is associated with spontaneous enterocolitis (Kühn et al., ##REF##8402911##1993##), osteopenia and increased bone fragility (Dresner‐Pollak et al., ##REF##15362035##2004##), and cardiac events including thrombosis, (Caligiuri et al., ##REF##12765335##2003##), atherosclerosis (Mallat et al., ##REF##10521249##1999##), arterial stiffness and cardiac dysfunction (Sikka et al., ##REF##23159957##2013##). Additionally, a role for inflammatory response mediators, including interleukin (IL)‐6 and tumor necrosis factor (TNF) in CVDs and bone resorption have been reported (Epsley et al., ##REF##33584321##2021##; Willerson & Ridker, ##UREF##6##2004##). Research also supports an anti‐inflammatory role for certain postbiotics such as butyrate (Säemann et al., ##REF##11024006##2000##). However, other roles of gut microbiota in reducing inflammation and the crosstalk between the gut, bone, and cardiovascular system are still poorly understood.
","
Fibroblast growth factor (FGF)‐23 and estrogen are hormones derived mainly from the bone and ovaries (testes in males), respectively, and are important regulators of bone metabolism and cardiovascular outcomes (Lu & Hu, ##REF##28785560##2017##; Quarles, ##REF##22421513##2012##). Whereas inflammation, directly and indirectly, increases phosphaturic FGF23 (Francis & David, ##REF##27191351##2016##), an anti‐inflammatory role has been mainly attributed to estrogen (Chakrabarti et al., ##REF##18409173##2008##; Josefsson et al., ##REF##1586960##1992##), a hormone that prevents bone loss (Kameda et al., ##REF##9254647##1997##; Nakamura et al., ##REF##17803905##2007##) and reduces vascular injuries and atherosclerosis (Arnal et al., ##REF##20631350##2010##; Meng et al., ##REF##33364052##2021##; Xing et al., ##REF##19221203##2009##). Egli‐Spichtig et al. (##REF##31301888##2019##) reported increased serum intact FGF23 (iFGF23) in IL‐10 KO mice at 12–14 weeks of age. However, the clinical significance of this finding in relation to skeletal and cardiovascular outcomes has not been investigated.
","
An important aspect of estrogen metabolism is the recycling of its conjugated form by gut bacterial, β‐glucuronidase (Baker et al., ##REF##28778332##2017##). While aging causes an alteration in the metabolic signature of the gut microbiome, the role of inflammation in estrogen recycling and signaling has not been investigated. We aimed to examine the crosstalk between the gut, bone, and cardiovascular system in an IL‐10 KO mouse model. We hypothesized that the anti‐inflammatory cytokine IL‐10 is critical for fine‐tuning gut‐microbial derived factors, including deconjugated estrogen and inflammatory stimuli, which in turn play critical roles in normal bone remodeling and cardiovascular function.
"],"string":"[\n \"INTRODUCTION\",\n \"
Multidirectional interaction among organ systems is an integral aspect of the development and physiology of mammalian organisms. Factors, including hormones, cytokines, and chemokines, are produced by diverse cells in the body and participate in interorgan communication (Castillo‐Armengol et al., ##REF##31423716##2019##). The mammalian gut is not an exception in the production of factors that mediate interorgan crosstalk for maintaining a healthy homeostatic state and for the normal functioning of several organ systems, including the musculoskeletal and cardiovascular systems (Hu et al., ##REF##35932976##2022##; Lucas et al., ##UREF##2##2018##).
\",\n \"
The gut microbiome encompasses a diverse array of gene batteries whose products (i.e., postbiotics) are continuously being characterized to participate in important aspects of hosts physiology, including gut (Martin‐Gallausiaux et al., ##REF##32238208##2021##), cardiovascular (Razavi et al., ##REF##31182162##2019##; Yang et al., ##REF##31803054##2019##) and bone health (Lucas et al., ##UREF##2##2018##). More so, gut colonization with microbiota is critical for shaping the mucosal immune system and results in either a homeostatic or abnormal immune reactivity (Al Bander et al., ##REF##33086688##2020##; Brandsma et al., ##REF##30582442##2019##). Importantly, the mammalian gut has evolved to guard against the translocation of unwanted substances, including toxins and pathogens, into the systemic circulation. In addition, ~70% of our immune cells reside in the gut, functioning in antigen sampling, and producing pathogen‐specific immune responses (Wiertsema et al., ##REF##33803407##2021##). Aging‐induced alterations in the mammalian gut microbiome and immune cell function increase chronic inflammation (i.e., inflammaging) in the gut and increase the risk of chronic diseases, including cardiovascular disorders (CVDs) and osteoporosis (Bosco & Noti, ##REF##33875817##2021##; Fasano, ##REF##32742638##2020##; Gibon et al., ##REF##29094003##2017##; Li et al., ##UREF##1##2019##; Liberale et al., ##REF##35210039##2022##).
\",\n \"
Interleukin (IL)‐10 is a well‐known pleiotropic anti‐inflammatory cytokine (Sabat et al., ##REF##21115385##2010##). The partial or total loss of this cytokine, as in the IL‐10 knockout (KO) mouse, models inflammaging and is associated with spontaneous enterocolitis (Kühn et al., ##REF##8402911##1993##), osteopenia and increased bone fragility (Dresner‐Pollak et al., ##REF##15362035##2004##), and cardiac events including thrombosis, (Caligiuri et al., ##REF##12765335##2003##), atherosclerosis (Mallat et al., ##REF##10521249##1999##), arterial stiffness and cardiac dysfunction (Sikka et al., ##REF##23159957##2013##). Additionally, a role for inflammatory response mediators, including interleukin (IL)‐6 and tumor necrosis factor (TNF) in CVDs and bone resorption have been reported (Epsley et al., ##REF##33584321##2021##; Willerson & Ridker, ##UREF##6##2004##). Research also supports an anti‐inflammatory role for certain postbiotics such as butyrate (Säemann et al., ##REF##11024006##2000##). However, other roles of gut microbiota in reducing inflammation and the crosstalk between the gut, bone, and cardiovascular system are still poorly understood.
\",\n \"
Fibroblast growth factor (FGF)‐23 and estrogen are hormones derived mainly from the bone and ovaries (testes in males), respectively, and are important regulators of bone metabolism and cardiovascular outcomes (Lu & Hu, ##REF##28785560##2017##; Quarles, ##REF##22421513##2012##). Whereas inflammation, directly and indirectly, increases phosphaturic FGF23 (Francis & David, ##REF##27191351##2016##), an anti‐inflammatory role has been mainly attributed to estrogen (Chakrabarti et al., ##REF##18409173##2008##; Josefsson et al., ##REF##1586960##1992##), a hormone that prevents bone loss (Kameda et al., ##REF##9254647##1997##; Nakamura et al., ##REF##17803905##2007##) and reduces vascular injuries and atherosclerosis (Arnal et al., ##REF##20631350##2010##; Meng et al., ##REF##33364052##2021##; Xing et al., ##REF##19221203##2009##). Egli‐Spichtig et al. (##REF##31301888##2019##) reported increased serum intact FGF23 (iFGF23) in IL‐10 KO mice at 12–14 weeks of age. However, the clinical significance of this finding in relation to skeletal and cardiovascular outcomes has not been investigated.
\",\n \"
An important aspect of estrogen metabolism is the recycling of its conjugated form by gut bacterial, β‐glucuronidase (Baker et al., ##REF##28778332##2017##). While aging causes an alteration in the metabolic signature of the gut microbiome, the role of inflammation in estrogen recycling and signaling has not been investigated. We aimed to examine the crosstalk between the gut, bone, and cardiovascular system in an IL‐10 KO mouse model. We hypothesized that the anti‐inflammatory cytokine IL‐10 is critical for fine‐tuning gut‐microbial derived factors, including deconjugated estrogen and inflammatory stimuli, which in turn play critical roles in normal bone remodeling and cardiovascular function.
Animals were maintained at the Oklahoma State University Laboratory Animal Research facility under humidity‐ and temperature‐controlled conditions and a 12‐h light–12‐h dark cycle.
","
Six‐week‐old male and female IL‐10 KO mice (KO) and their wild type (WT) counterpart of C57BL/6 background were (Jackson Laboratories, Bar Harbor, ME) maintained on a semi‐purified rodent growth diet (AIN‐93G) for the first 3 months following weaning and then changed to maintenance diet (AIN‐93M) for the final 3 months of the study. Mice had access to food and water ad libitum. Weekly food intake and body weight were monitored during the study period.
","Sample collection and tissue processing","
Fecal samples were collected per cage and stored at −80°C after 3 and 6 months on the AIN‐93 diet. At the end of each time point, mice were fasted for ~3 h and anesthetized with a ketamine and xylazine cocktail (100 mg and 10 mg, kg−1 body weight, respectively). Whole‐body dual‐energy x‐ray absorptiometry (DXA) scans were performed to determine bone mineral content (BMC) and bone mineral density (BMD) using a whole‐body densitometer (Piximus; GE Medical System Lunar, Madison, WI). Serum was processed from blood collected from the carotid artery and stored at −80°C. The heart, aorta, and white adipose tissue (WAT) were excised and snap‐frozen in liquid nitrogen. Lamina propria (LP) was collected from saline‐flushed ileum and stored in RNALater. A femur from each mouse was flushed with ice cold PBS and the marrow‐free bone was snap‐frozen and cryo‐stored until analyses.
","Serum <styled-content style=\"fixed-case\" toggle=\"no\">17β‐estradiol</styled-content> and cecal content β‐glucuronidase activity","
Serum unconjugated 17β‐estradiol (E2) was assessed using an ELISA kit (Biovision; Milpitas, CA) following the manufacturer's instructions.
","
Bacterial β‐glucuronidase activity in cecal content was assessed with slight modifications to previous protocols (Flores et al., ##REF##23259758##2012##; Walsh et al., ##REF##32146834##2020##). Briefly, approximately 300 mg of frozen cecal content was homogenized in 1 mL ice‐cold PBS. Lysates were centrifuged at 2000 rpm for 5 min at 4°C and the supernatant was further centrifuged at 10,000 rpm for 20 min. Enzyme activity was assessed in the supernatant by mixing 25 μL of supernatant, 50 μL PBS, and 50 μL 4‐nitrophenyl‐β‐d‐glucuronide (1 mM, dissolved in PBS; Sigma‐Aldrich, St. Louis, MO # 10344‐94‐2). The resulting mixture was incubated at 37°C for 15 min, followed by the addition of 125 μL sodium hydroxide (NaOH, 0.5 N, ThermoFisher #BP359) to stop the reaction. The absorbance was read at 405 nm on a Biotek HTX microplate reader. Enzymatic concentrations were extrapolated from a standard curve of a serially diluted pure enzyme purchased from Sigma‐Aldrich (# G7646). Enzyme activities were normalized by the total protein content in each sample.
μCT analyses (μCT40; SCANCO Medical, Brüttisellen, Switzerland) were performed on the lumbar vertebral body (L6), distal femur metaphysis, and the femur mid‐diaphysis to determine alterations in trabecular and cortical bone microarchitecture. Femur specimens were scanned at a high resolution of 2048 × 2048 pixels. Analysis of the distal femur was performed on a volume of interest (VOI) that included a region of secondary spongiosa that was 60 μm from the growth plate and extended in the proximal direction 600 μm (100 images). A 180 μm (30 images) VOI at the mid‐point of the femur was used for cortical bone analysis. Scanning of the vertebra was performed at medium resolution or 1024 × 1024 pixels. The VOI included a region of secondary spongiosa that was approximately 2.56 mm (160 images × 16 μm ea). Analysis of all specimens was performed at the threshold of 360, a sigma of 1.2, and a support setting of 2. The trabecular analysis for the distal femur metaphysis and vertebral body included the following: relative bone volume (BV/TV), trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp), connectivity density (ConnDens), structural model index (SMI), apparent density, material density, and degree of anisotropy. Cortical analyses of the tibial mid‐diaphysis included cortical thickness, cortical area, medullary area, and porosity.
","Gene expression analysis","
Total RNA was isolated from ileum LP, WAT, heart, aorta, ovary, bone marrow, and femur by rupturing tissues in Trizol RNA isolation reagent (ThermoFisher, Waltham, MA, #10‐296‐028) and precipitating chloroform‐separated clear phase in isopropanol as earlier described (Peirson & Butler, ##UREF##3##2007##). cDNA was synthesized following a standardized protocol (Peterson & Freeman, ##UREF##4##2009##), and the relative abundance of genes was determined by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) using SYBR green chemistry (Applied Biosystems, #A25742) on a CFX Opus RT‐PCR System (Bio‐Rad, Hercules, CA). Gene targets included interleukin (Il)‐17a, Il1b, Il6, Tnf, and Tgfb, estrogen receptor (Esr)‐1 and 2, tight junction protein 1 (Tjp1) and occludin (Ocln) in the ileum. Il17a, Tnf, Il6, Il1b, intercellular and vascular cell adhesion molecule 1 (Icam1 and Vcam1), Esr1, adhesion G protein‐coupled receptor E1 (Adgre1), and arginase 1 (Arg1) were assessed in cardiac tissues. Dentin matrix protein 1 (Dmp1), phosphate regulating endopeptidase homolog X‐linked (Phex), Fgf23, Il6, wingless‐type MMTV integration site family member 10B (Wnt10b), osteoprotegerin (Opg), Rankl, collagen type 1 alpha 1 (Col1a1), bone gamma‐carboxyglutamate protein 2 (Bglap2), secreted phosphoprotein 1 (Spp1), and sclerostin (Sost) were assessed in the bone tissue. Esr 1 and 2, Cyp19a1 (aromatase), and Cyp11a1 were assessed in the WAT and ovary. The data were analyzed using the 2−ΔΔCt method (Schmittgen & Livak, ##REF##18546601##2008##), with glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh) serving as the invariant control. Primers were either derived from previous reports in the literature or designed in our laboratory and then validated in our hands before gene expression analyses were performed. Table ##SUPPL##0##S1## contains the list of primer sequences.
","Immunoblotting","
Total protein was extracted and quantified from heart tissue using the radioimmunoprecipitation assay (RIPA) buffer containing 0.5% protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, #P8340 #P0044). Proteins were denatured and separated on polyacrylamide gels (BioRad, #4561093EDU) using SDS‐PAGE before transfer on PVDF membranes (ThermoScientific, Waltham, MA, #PI88585) as previously described (Ojo et al., ##REF##33144228##2021##). Membranes were incubated overnight at 4°C in primary antibodies: (p‐eNOS Ser1119 [ThermoFisher, #PIPA564613]; NOS3 [ThermoFisher, #4904]; p‐IKB [Santa Cruz Biotechnology, Dallas, TX, #SC‐8404]; PI3K [Santa Cruz Biotechnology, #SC‐365290]; IKB [Santa Cruz Biotechnology, #SC‐1643]; p‐NFkBp65 Ser536 [#sc‐136548, Santa Cruz Biotechnology]; NFkBp65 [Santa Cruz Biotechnology, #SC‐8008]; and GAPDH [#60004–1, Proteintech, Rosemont IL]) diluted in 5% bovine serum albumin (BSA). Membranes were washed in PBS and incubated for 1 h in anti‐rabbit (Cell Signaling Technology, Danvers, MA #7074) or anti‐mouse (Cell Signaling Technology, #7076) IgG HRP‐linked antibody diluted in 5% milk solution. Proteins were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific, #34095). Band signals were captured with the ChemiDoc imaging system (BioRad) and quantified with the Image J software, v 1.8.0.
","Cell culture experiments","
Intestinal lymphocytes were isolated from the ileum of 14 month‐old female KO and WT mice (n = 4/strain) maintained on standard chow as previously described with minor modifications (Sheridan & Lefrançois, ##UREF##5##2012##). Briefly, tissues were flushed with RPMI medium (ThermoFisher, #61‐870‐036) supplemented with 2% FBS and 1 mM dithiothreitol (DTT, Sigma‐Aldrich, #D9779). Ileal samples were opened longitudinally and incubated at room temperature in Hanks' balanced salt solution (HBSS) with 2 mM EDTA. Epithelia‐free samples were digested with collagenase type VIII (Sigma‐Aldrich #C2139) and filtered through a 70 μm cell strainer. Lymphocytes were subsequently separated, by differential centrifugation, as an interphase between Percoll gradients (40% and 80%, Sigma‐Aldrich, #GE17‐0891‐01). Cells were collected, washed twice, and incubated for 40 min in a 10% FBS‐supplemented RPMI with or without E2 (100 nM, Sigma‐Aldrich, #E2758).
","
For 3‐dimensional cultures, intestinal crypts were isolated from the ileum of 14 month‐old female KO and WT mice and developed into organoids as previously described (Sato et al., ##REF##19329995##2009##) with minor modifications. Briefly, the small intestine was excised, opened longitudinally and gently flushed with cold PBS. Tissues were cut into small pieces (~2 mm) and gently washed several times in PBS until the supernatant became clear (~20 times). Tissues were digested at room temperature in gentle cell dissociation reagent (Stemcell Technologies, Cambridge, MA, #07174), resuspended in cold 0.1% BSA‐containing PBS, pipetted up and down three times, and allowed to settle under gravity. The wash, digestion, and dissociation step were repeated three times. Crypt‐enriched fractions derived by passing supernatant from the fourth digestion through a 70 μm strainer was centrifuged and resuspended in complete mouse intesticult organoid growth medium (Stemcell Technologies, #06005) and geltrex basement membrane matrix (Thermofisher, #A1413302) before plating in triplicate in a 24‐well plate. Once solidified, organoids were formed in complete mouse intesticult organoid growth medium treated with or without E2 (100 nM).
","Statistical analyses","
Data were evaluated for conformity to normal distribution. Two‐group comparisons of IL‐10 KO and their sex‐matched WT control were performed for all outcome variables using the independent Student t‐test in SAS version 9.4 (SAS Institute, Cary, NC). Data are presented as mean ± standard deviation (SD), and p value <0.05 was considered statistically significant.
"],"string":"[\n \"METHODS\",\n \"Animals\",\n \"
Animals were maintained at the Oklahoma State University Laboratory Animal Research facility under humidity‐ and temperature‐controlled conditions and a 12‐h light–12‐h dark cycle.
\",\n \"
Six‐week‐old male and female IL‐10 KO mice (KO) and their wild type (WT) counterpart of C57BL/6 background were (Jackson Laboratories, Bar Harbor, ME) maintained on a semi‐purified rodent growth diet (AIN‐93G) for the first 3 months following weaning and then changed to maintenance diet (AIN‐93M) for the final 3 months of the study. Mice had access to food and water ad libitum. Weekly food intake and body weight were monitored during the study period.
\",\n \"Sample collection and tissue processing\",\n \"
Fecal samples were collected per cage and stored at −80°C after 3 and 6 months on the AIN‐93 diet. At the end of each time point, mice were fasted for ~3 h and anesthetized with a ketamine and xylazine cocktail (100 mg and 10 mg, kg−1 body weight, respectively). Whole‐body dual‐energy x‐ray absorptiometry (DXA) scans were performed to determine bone mineral content (BMC) and bone mineral density (BMD) using a whole‐body densitometer (Piximus; GE Medical System Lunar, Madison, WI). Serum was processed from blood collected from the carotid artery and stored at −80°C. The heart, aorta, and white adipose tissue (WAT) were excised and snap‐frozen in liquid nitrogen. Lamina propria (LP) was collected from saline‐flushed ileum and stored in RNALater. A femur from each mouse was flushed with ice cold PBS and the marrow‐free bone was snap‐frozen and cryo‐stored until analyses.
Serum unconjugated 17β‐estradiol (E2) was assessed using an ELISA kit (Biovision; Milpitas, CA) following the manufacturer's instructions.
\",\n \"
Bacterial β‐glucuronidase activity in cecal content was assessed with slight modifications to previous protocols (Flores et al., ##REF##23259758##2012##; Walsh et al., ##REF##32146834##2020##). Briefly, approximately 300 mg of frozen cecal content was homogenized in 1 mL ice‐cold PBS. Lysates were centrifuged at 2000 rpm for 5 min at 4°C and the supernatant was further centrifuged at 10,000 rpm for 20 min. Enzyme activity was assessed in the supernatant by mixing 25 μL of supernatant, 50 μL PBS, and 50 μL 4‐nitrophenyl‐β‐d‐glucuronide (1 mM, dissolved in PBS; Sigma‐Aldrich, St. Louis, MO # 10344‐94‐2). The resulting mixture was incubated at 37°C for 15 min, followed by the addition of 125 μL sodium hydroxide (NaOH, 0.5 N, ThermoFisher #BP359) to stop the reaction. The absorbance was read at 405 nm on a Biotek HTX microplate reader. Enzymatic concentrations were extrapolated from a standard curve of a serially diluted pure enzyme purchased from Sigma‐Aldrich (# G7646). Enzyme activities were normalized by the total protein content in each sample.
μCT analyses (μCT40; SCANCO Medical, Brüttisellen, Switzerland) were performed on the lumbar vertebral body (L6), distal femur metaphysis, and the femur mid‐diaphysis to determine alterations in trabecular and cortical bone microarchitecture. Femur specimens were scanned at a high resolution of 2048 × 2048 pixels. Analysis of the distal femur was performed on a volume of interest (VOI) that included a region of secondary spongiosa that was 60 μm from the growth plate and extended in the proximal direction 600 μm (100 images). A 180 μm (30 images) VOI at the mid‐point of the femur was used for cortical bone analysis. Scanning of the vertebra was performed at medium resolution or 1024 × 1024 pixels. The VOI included a region of secondary spongiosa that was approximately 2.56 mm (160 images × 16 μm ea). Analysis of all specimens was performed at the threshold of 360, a sigma of 1.2, and a support setting of 2. The trabecular analysis for the distal femur metaphysis and vertebral body included the following: relative bone volume (BV/TV), trabecular number (TbN), trabecular thickness (TbTh), trabecular separation (TbSp), connectivity density (ConnDens), structural model index (SMI), apparent density, material density, and degree of anisotropy. Cortical analyses of the tibial mid‐diaphysis included cortical thickness, cortical area, medullary area, and porosity.
\",\n \"Gene expression analysis\",\n \"
Total RNA was isolated from ileum LP, WAT, heart, aorta, ovary, bone marrow, and femur by rupturing tissues in Trizol RNA isolation reagent (ThermoFisher, Waltham, MA, #10‐296‐028) and precipitating chloroform‐separated clear phase in isopropanol as earlier described (Peirson & Butler, ##UREF##3##2007##). cDNA was synthesized following a standardized protocol (Peterson & Freeman, ##UREF##4##2009##), and the relative abundance of genes was determined by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR) using SYBR green chemistry (Applied Biosystems, #A25742) on a CFX Opus RT‐PCR System (Bio‐Rad, Hercules, CA). Gene targets included interleukin (Il)‐17a, Il1b, Il6, Tnf, and Tgfb, estrogen receptor (Esr)‐1 and 2, tight junction protein 1 (Tjp1) and occludin (Ocln) in the ileum. Il17a, Tnf, Il6, Il1b, intercellular and vascular cell adhesion molecule 1 (Icam1 and Vcam1), Esr1, adhesion G protein‐coupled receptor E1 (Adgre1), and arginase 1 (Arg1) were assessed in cardiac tissues. Dentin matrix protein 1 (Dmp1), phosphate regulating endopeptidase homolog X‐linked (Phex), Fgf23, Il6, wingless‐type MMTV integration site family member 10B (Wnt10b), osteoprotegerin (Opg), Rankl, collagen type 1 alpha 1 (Col1a1), bone gamma‐carboxyglutamate protein 2 (Bglap2), secreted phosphoprotein 1 (Spp1), and sclerostin (Sost) were assessed in the bone tissue. Esr 1 and 2, Cyp19a1 (aromatase), and Cyp11a1 were assessed in the WAT and ovary. The data were analyzed using the 2−ΔΔCt method (Schmittgen & Livak, ##REF##18546601##2008##), with glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh) serving as the invariant control. Primers were either derived from previous reports in the literature or designed in our laboratory and then validated in our hands before gene expression analyses were performed. Table ##SUPPL##0##S1## contains the list of primer sequences.
\",\n \"Immunoblotting\",\n \"
Total protein was extracted and quantified from heart tissue using the radioimmunoprecipitation assay (RIPA) buffer containing 0.5% protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, #P8340 #P0044). Proteins were denatured and separated on polyacrylamide gels (BioRad, #4561093EDU) using SDS‐PAGE before transfer on PVDF membranes (ThermoScientific, Waltham, MA, #PI88585) as previously described (Ojo et al., ##REF##33144228##2021##). Membranes were incubated overnight at 4°C in primary antibodies: (p‐eNOS Ser1119 [ThermoFisher, #PIPA564613]; NOS3 [ThermoFisher, #4904]; p‐IKB [Santa Cruz Biotechnology, Dallas, TX, #SC‐8404]; PI3K [Santa Cruz Biotechnology, #SC‐365290]; IKB [Santa Cruz Biotechnology, #SC‐1643]; p‐NFkBp65 Ser536 [#sc‐136548, Santa Cruz Biotechnology]; NFkBp65 [Santa Cruz Biotechnology, #SC‐8008]; and GAPDH [#60004–1, Proteintech, Rosemont IL]) diluted in 5% bovine serum albumin (BSA). Membranes were washed in PBS and incubated for 1 h in anti‐rabbit (Cell Signaling Technology, Danvers, MA #7074) or anti‐mouse (Cell Signaling Technology, #7076) IgG HRP‐linked antibody diluted in 5% milk solution. Proteins were detected using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoScientific, #34095). Band signals were captured with the ChemiDoc imaging system (BioRad) and quantified with the Image J software, v 1.8.0.
\",\n \"Cell culture experiments\",\n \"
Intestinal lymphocytes were isolated from the ileum of 14 month‐old female KO and WT mice (n = 4/strain) maintained on standard chow as previously described with minor modifications (Sheridan & Lefrançois, ##UREF##5##2012##). Briefly, tissues were flushed with RPMI medium (ThermoFisher, #61‐870‐036) supplemented with 2% FBS and 1 mM dithiothreitol (DTT, Sigma‐Aldrich, #D9779). Ileal samples were opened longitudinally and incubated at room temperature in Hanks' balanced salt solution (HBSS) with 2 mM EDTA. Epithelia‐free samples were digested with collagenase type VIII (Sigma‐Aldrich #C2139) and filtered through a 70 μm cell strainer. Lymphocytes were subsequently separated, by differential centrifugation, as an interphase between Percoll gradients (40% and 80%, Sigma‐Aldrich, #GE17‐0891‐01). Cells were collected, washed twice, and incubated for 40 min in a 10% FBS‐supplemented RPMI with or without E2 (100 nM, Sigma‐Aldrich, #E2758).
\",\n \"
For 3‐dimensional cultures, intestinal crypts were isolated from the ileum of 14 month‐old female KO and WT mice and developed into organoids as previously described (Sato et al., ##REF##19329995##2009##) with minor modifications. Briefly, the small intestine was excised, opened longitudinally and gently flushed with cold PBS. Tissues were cut into small pieces (~2 mm) and gently washed several times in PBS until the supernatant became clear (~20 times). Tissues were digested at room temperature in gentle cell dissociation reagent (Stemcell Technologies, Cambridge, MA, #07174), resuspended in cold 0.1% BSA‐containing PBS, pipetted up and down three times, and allowed to settle under gravity. The wash, digestion, and dissociation step were repeated three times. Crypt‐enriched fractions derived by passing supernatant from the fourth digestion through a 70 μm strainer was centrifuged and resuspended in complete mouse intesticult organoid growth medium (Stemcell Technologies, #06005) and geltrex basement membrane matrix (Thermofisher, #A1413302) before plating in triplicate in a 24‐well plate. Once solidified, organoids were formed in complete mouse intesticult organoid growth medium treated with or without E2 (100 nM).
\",\n \"Statistical analyses\",\n \"
Data were evaluated for conformity to normal distribution. Two‐group comparisons of IL‐10 KO and their sex‐matched WT control were performed for all outcome variables using the independent Student t‐test in SAS version 9.4 (SAS Institute, Cary, NC). Data are presented as mean ± standard deviation (SD), and p value <0.05 was considered statistically significant.
\"\n]"},"results":{"kind":"list like","value":["RESULTS","Gut inflammation increased in IL‐10 KO mice","
Compared to their WT gender‐matched counterparts, body weight was significantly lower (p < 0.01) in KO male mice from Week 6 to 24 (Figure ##FIG##0##1a##). In contrast, the female KO mice maintained similar body weight as the WT counterparts until Week 19, and then exhibited reduced body weight (p < 0.05) until the end of the experiment (Figure ##FIG##0##1b##). The reduced body weight in the male and female KO mice corresponds with lower food intake (Figure ##SUPPL##0##S1a##) and most likely be due to the increase in gut inflammation observed in these mice.
","
We next examined the relative mRNA abundance of inflammatory cytokines in the ileal LP as an index of inflammation. The anti‐inflammatory cytokine, Tgfb, increased in KO male at 3 months (p = 0.00014), but decreased in KO female at both time points (p < 0.01, Figure ##FIG##0##1c##). There were no significant differences in Il6 gene expressions for either sex compared with WT controls (Figure ##FIG##0##1d##). However, Il17 and Tnf mRNA increased significantly in both male (p < 0.05) and female KO (p < 0.01) mice at both timepoints compared to their WT sex‐matched counterparts (Figure ##FIG##0##1e,f##). As inflammation is crucial to compromising intestinal integrity, we examined tight junction gene expression. At the 3 month timepoint, ileal expression of Tjp1 was lowered in male (p < 0.0001) and Ocln in female (p = 0.027) KO compared to their WT counterpart (Figure ##FIG##0##1g##). However, by the 6 month timepoint, there were no differences in the expression of Tjp1 and Ocln (Figure ##FIG##0##1g,h##).
","Increased inflammation and FGF23 are associated with osteopenia in IL‐10 KO mice","
To confirm the effects of Il10 KO on bone parameters, we performed a whole‐body DXA scan. At 3 and 6 months, both male and female KO mice had lower (p < 0.05) BMD than their WT counterparts (Figure ##FIG##1##2a##). The significant loss of BMC in the KO male mice (Figure ##SUPPL##0##S1b##) is primarily responsible for the reduced BMD since bone mineral area (BMA) was either somewhat reduced or not affected in these mice (Figure ##SUPPL##0##S1c##). However, both BMC and BMA were significantly reduced in female KO mice at both time points (Figure ##SUPPL##0##S1b,c##).
","
Next, we evaluated the alterations in trabecular bone in the distal femur metaphysis and vertebral body using microCT. At the 3 month timepoint, vertebral BV/TV was lower in male and female KO mice by ~20% and 40% compared to their respective WT controls (Figure ##FIG##1##2b##). By 6 months, the magnitude of the difference in vertebral bone of the male and female KO compared to WT controls was even greater, with an ~40% and 60% decline, respectively. The lower vertebral BV/TV was associated with significant reductions (p < 0.05) in TbN and TbTh with a corresponding increase in TbSp in KO mice versus WT controls (Figure ##SUPPL##0##S2a–c##). Similarly, the reduction in BV/TV within the distal femur metaphysis of male KO mice was ~30%–33% at 3 and 6 months, whereas female KO mice exhibited a trend at 3 months (p = 0.065; ~27% reduction) and a statistically significant reduction (p < 0.0001; 44%) at 6 months compared to WT counterpart (Figure ##FIG##1##2c##). The reduced femoral trabecular bone volume was attributed to a corresponding lower TbTh in KO mice in both sexes at both timepoints (Figure ##SUPPL##0##S2d##). Conversely, there were no statistically significant differences in the femur TbN and TbSp for the strains of mice that were compared (Figure ##SUPPL##0##S2e,f##).
","
Cortical bone was also compromised in the IL‐10 KO mice, with ~10% reduction (p < 0.05) in femoral cortical area and thickness at the 3 month timepoint, and ~20% reduction (p < 0.01) in these parameters at the 6 month timepoint (Figure ##FIG##1##2d,e##). At the transcriptional level, the relative abundance of Sost, the Wnt signaling inhibitor, was lower in the femur (p < 0.05) of male KO mice at 6 months and at both timepoints in the female KO mice compared to their WT controls (Figure ##FIG##1##2f##). Interestingly, Wnt10b mRNA was also repressed (p < 0.05) in male KO at both timepoints and upregulated in female KO mice at 6 months versus WT mice (Figure ##SUPPL##0##S2g##). Assessment of the mRNA expression of extracellular matrix proteins revealed similar Col1a1 mRNA level in both WT and KO mice (Figure ##SUPPL##0##S2h##), whereas Opn and Bglap2 had at least ~1.5‐fold increased expression in KO mice compared to gender‐matched WT mice at both the 3 and 6 month timepoints (Figure ##FIG##1##2g,h##). Next, we assessed the gene expression of receptor activator of nuclear factor kappa B ligand (RANKL), which regulates the catabolic activity of bone and OPG, a decoy receptor that inhibits RANKL‐RANK interaction. The relative abundance of RankL was reduced (Figure ##SUPPL##0##S2i##) with a corresponding lower Opg gene expression (p < 0.05) in both male and female KO compared to WT control mice at both timepoints (Figure ##SUPPL##0##S2j##). These data support an attempt to down‐regulate osteolytic activity in the bone of the KO mice.
","
We next examined gene expression of the endopeptidase, phosphate‐regulating endopeptidase X‐linked (PHEX) and its sibling protein, dentine matrix acidic phosphoprotein 1 (DMP1), both produced by osteocytes and participating in bone mineralization (Schaffler & Kennedy, ##REF##22552701##2012##). Phex was significantly reduced at 6 months and Dmp1 was reduced at both timepoints in KO mice compared with sex‐matched WT control (Figure ##FIG##1##2i,j##). DMP1 and Phex are involved in the regulation of Fgf23 (Martin et al., ##REF##21507898##2011##). As expected, Fgf23 mRNA increased (p < 0.05) with reducing Phex and Dmp1 in KO mice at both timepoints compared to their WT control (Figure ##FIG##1##2k##). Importantly, Il6 mRNA tended to increase at 6 months in male KO, and was significantly increased (p < 0.05) at both timepoints in female KO mice compared to WT control (Figure ##FIG##1##2l##).
","Loss of <styled-content style=\"fixed-case\" toggle=\"no\">IL</styled-content>‐10 is associated with increased cardiac inflammation and fibrosis in mice","
Motivated by the reported role of excessive FGF23 in inducing left ventricular hypertrophy, cardiac fibrosis, and vascular calcification (Böckmann et al., ##REF##31540546##2019##; Faul et al., ##REF##21985788##2011##; Hao et al., ##REF##27579618##2016##; Jimbo et al., ##REF##24088960##2014##), we next examined transverse cross‐section of the heart for histological changes. Masson's trichrome staining revealed increased fibrosis in the cardiac tissue of KO mice at 6 months compared with WT control mice (Figure ##FIG##2##3a##). Cardiac gene expression of the inflammatory marker, Tnf, only reached statistical significance at 6 months in female KO mice (Figure ##FIG##2##3b##). Similarly, the Il6 transcript was significantly elevated (p < 0.05) in the KO mice at the 6 months timepoint in both sexes compared to their respective WT control (Figure ##FIG##2##3c##). In contrast, the relative abundance of Il1b expression was higher (p < 0.05) in the cardiac tissue of KO mice at both timepoints (Figure ##FIG##2##3d##). Gene expression of adhesion molecules, including, vascular cell adhesion protein 1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) was increased (p < 0.05) in the cardiac tissue of female KO mice at the 6 month timepoint. (Figure ##SUPPL##0##S3a,b##). Gene expression of the receptor Adgre1, a macrophage marker, increased significantly in the cardiac tissues of KO mice at 6 months (Figure ##FIG##2##3e##). The M2 macrophage marker, Arg1, tended to be reduced (0.05 < p < 0.1) in female KO mice at 3 months, but was reduced (p < 0.05) in the cardiac tissue of male KO mice at both timepoints compared with WT control (Figure ##SUPPL##0##S3c##).
","
Next, we assessed the protein expression/activation of the redox‐sensitive transcription factor, NF‐κB and key components of its signaling pathway. Inhibitor of nuclear factor kappa B (IκB), which prevents the nuclear translocation of NF‐κB, was reduced (p < 0.05) in both sexes of KO mice at the 3 and 6 month timepoints (Figure ##FIG##2##3f,g##), with female KO exhibiting reduced phosphorylation (p < 0.01) of IκB at 6 months (Figure ##FIG##2##3f,h##). The ratio of phosphorylated to total IκB increased (p < 0.05) at both time points in male KO (Figure ##FIG##2##3f,i##). The reduced phosphorylation of IκB at 6 months in female KO mice correlates with a reduced (p < 0.05) phospho‐NFκB (Ser536) in these mice at this timepoint (Figure ##FIG##2##3f,j,k##).
","
We further assessed the protein expression of endothelial nitric oxide synthase (NOS)‐3, whose Ser1119 phosphorylation is critical in nitric oxide‐mediated vasodilation. KO mice had a reduced expression of NOS3 (p < 0.05) at the 6 month timepoint (Figure ##FIG##2##3l,m##). Reduced activation of this enzyme as indicated by its reduced Ser1119 phosphorylation coincided with a reduction in phosphoinositide 3‐kinase (PI3K), which lies downstream of the NOS3 signaling, at both timepoints compared with respective WT control (Figure ##FIG##2##3m–o##).
","Loss of <styled-content style=\"fixed-case\" toggle=\"no\">IL</styled-content>‐10 is associated with reduced estrogen signaling in mice","
Considering the role of estrogen as an activator of NOS3 (Chen et al., ##REF##9927501##1999##) and the role of this hormone in reducing inflammation (Liberale et al., ##REF##35210039##2022##), cardiac dysfunction, and bone loss (Arnal et al., ##REF##20631350##2010##; Meng et al., ##REF##33364052##2021##; Xing et al., ##REF##19221203##2009##), we compared serum 17β‐estradiol (E2) in KO mice with WT control mice. Serum E2 was lower (p < 0.05) in female KO mice and tended to be reduced (0.05 < p < 0.1) in male KO mice at both timepoints compared with WT controls (Figure ##FIG##3##4a##). Following this serendipitous finding of reduced E2 in the KO mice, we assessed estrogen synthesis in the ovaries of 14 month‐old mice. Concurrently, mRNA levels of Cyp11a1 and Cyp19a1, which are involved in estrogen synthesis, were significantly reduced in the ovaries of 14 month‐old KO mice compared with WT counterparts of the same age (Figure ##FIG##3##4b##). Similar reductions in mRNA levels were also observed in the WAT, another source of endogenous E2, of KO mice at the 3 and 6 month timepoints (Figure ##FIG##3##4c,d##). The activity of microbial β‐glucuronidase within the gut, which deconjugates estrogen in the intestinal lumen, was reduced at both time points in KO mice (Figure ##FIG##3##4e##). Estrogen could mediate its effects in several ways, one of which is binding to receptors, including ESR1 and ESR2, to mediate a membrane response or genomic response (Heldring et al., ##REF##17615392##2007##). Esr2 was not expressed in the heart (data not shown), whereas Esr1 mRNA was not different between groups compared at both timepoints (Figure ##SUPPL##0##S4a##). Contrarily, ileal and WAT Esr1 mRNA were significantly reduced (p < 0.05) in KO mice at both timepoints (Figure ##FIG##3##4f##; Figure ##SUPPL##0##S4b##), while the reduction in ileal Esr2 mRNA only reached statistical significance in the ileum tissue at the 6 month timepoint (Figure ##FIG##3##4g##). Esr1 was highly expressed at the 6 month timepoint in the bone marrow of female KO, while Esr2 was repressed within the bone marrow at both timepoints in KO mice compared to WT control mice (Figure ##FIG##3##4h,i##).
","
To further confirm if IL‐10 is required for estrogen to modulate gut inflammation, lymphocytes from the ileum of 14 month‐old‐female WT and KO mice were isolated and treated with conditioned media with or without E2 (100 nM). E2 treatment lowered lymphocytic Tnf and Il17 mRNA in WT, but not in the KO mice (Figure ##FIG##4##5a,b##). We further assessed the effect of E2 treatment (100 nM) on intestinal crypt‐derived organoids from female KO and WT mice. E2‐treated organoids were faster in developing complex structures (crypt domain) in both WT and KO groups (Figure ##FIG##4##5c##). The mRNA expression of Vil1 and Alpi, which are highly expressed in the intestine, were not affected by E2 treatment in both organoid from WT and KO mice (Figure ##SUPPL##0##S5a,b##). In contrast, E2 treatment increased gene expression of Tjp1 in the KO‐derived organoid (Figure ##FIG##4##5d##), and Ocln in organoid from both WT and KO mice (Figure ##FIG##4##5e##). These data support an estrogenic effect on the enterocytes and not the lymphocytes of old IL‐10 KO mice.
"],"string":"[\n \"RESULTS\",\n \"Gut inflammation increased in IL‐10 KO mice\",\n \"
Compared to their WT gender‐matched counterparts, body weight was significantly lower (p < 0.01) in KO male mice from Week 6 to 24 (Figure ##FIG##0##1a##). In contrast, the female KO mice maintained similar body weight as the WT counterparts until Week 19, and then exhibited reduced body weight (p < 0.05) until the end of the experiment (Figure ##FIG##0##1b##). The reduced body weight in the male and female KO mice corresponds with lower food intake (Figure ##SUPPL##0##S1a##) and most likely be due to the increase in gut inflammation observed in these mice.
\",\n \"
We next examined the relative mRNA abundance of inflammatory cytokines in the ileal LP as an index of inflammation. The anti‐inflammatory cytokine, Tgfb, increased in KO male at 3 months (p = 0.00014), but decreased in KO female at both time points (p < 0.01, Figure ##FIG##0##1c##). There were no significant differences in Il6 gene expressions for either sex compared with WT controls (Figure ##FIG##0##1d##). However, Il17 and Tnf mRNA increased significantly in both male (p < 0.05) and female KO (p < 0.01) mice at both timepoints compared to their WT sex‐matched counterparts (Figure ##FIG##0##1e,f##). As inflammation is crucial to compromising intestinal integrity, we examined tight junction gene expression. At the 3 month timepoint, ileal expression of Tjp1 was lowered in male (p < 0.0001) and Ocln in female (p = 0.027) KO compared to their WT counterpart (Figure ##FIG##0##1g##). However, by the 6 month timepoint, there were no differences in the expression of Tjp1 and Ocln (Figure ##FIG##0##1g,h##).
\",\n \"Increased inflammation and FGF23 are associated with osteopenia in IL‐10 KO mice\",\n \"
To confirm the effects of Il10 KO on bone parameters, we performed a whole‐body DXA scan. At 3 and 6 months, both male and female KO mice had lower (p < 0.05) BMD than their WT counterparts (Figure ##FIG##1##2a##). The significant loss of BMC in the KO male mice (Figure ##SUPPL##0##S1b##) is primarily responsible for the reduced BMD since bone mineral area (BMA) was either somewhat reduced or not affected in these mice (Figure ##SUPPL##0##S1c##). However, both BMC and BMA were significantly reduced in female KO mice at both time points (Figure ##SUPPL##0##S1b,c##).
\",\n \"
Next, we evaluated the alterations in trabecular bone in the distal femur metaphysis and vertebral body using microCT. At the 3 month timepoint, vertebral BV/TV was lower in male and female KO mice by ~20% and 40% compared to their respective WT controls (Figure ##FIG##1##2b##). By 6 months, the magnitude of the difference in vertebral bone of the male and female KO compared to WT controls was even greater, with an ~40% and 60% decline, respectively. The lower vertebral BV/TV was associated with significant reductions (p < 0.05) in TbN and TbTh with a corresponding increase in TbSp in KO mice versus WT controls (Figure ##SUPPL##0##S2a–c##). Similarly, the reduction in BV/TV within the distal femur metaphysis of male KO mice was ~30%–33% at 3 and 6 months, whereas female KO mice exhibited a trend at 3 months (p = 0.065; ~27% reduction) and a statistically significant reduction (p < 0.0001; 44%) at 6 months compared to WT counterpart (Figure ##FIG##1##2c##). The reduced femoral trabecular bone volume was attributed to a corresponding lower TbTh in KO mice in both sexes at both timepoints (Figure ##SUPPL##0##S2d##). Conversely, there were no statistically significant differences in the femur TbN and TbSp for the strains of mice that were compared (Figure ##SUPPL##0##S2e,f##).
\",\n \"
Cortical bone was also compromised in the IL‐10 KO mice, with ~10% reduction (p < 0.05) in femoral cortical area and thickness at the 3 month timepoint, and ~20% reduction (p < 0.01) in these parameters at the 6 month timepoint (Figure ##FIG##1##2d,e##). At the transcriptional level, the relative abundance of Sost, the Wnt signaling inhibitor, was lower in the femur (p < 0.05) of male KO mice at 6 months and at both timepoints in the female KO mice compared to their WT controls (Figure ##FIG##1##2f##). Interestingly, Wnt10b mRNA was also repressed (p < 0.05) in male KO at both timepoints and upregulated in female KO mice at 6 months versus WT mice (Figure ##SUPPL##0##S2g##). Assessment of the mRNA expression of extracellular matrix proteins revealed similar Col1a1 mRNA level in both WT and KO mice (Figure ##SUPPL##0##S2h##), whereas Opn and Bglap2 had at least ~1.5‐fold increased expression in KO mice compared to gender‐matched WT mice at both the 3 and 6 month timepoints (Figure ##FIG##1##2g,h##). Next, we assessed the gene expression of receptor activator of nuclear factor kappa B ligand (RANKL), which regulates the catabolic activity of bone and OPG, a decoy receptor that inhibits RANKL‐RANK interaction. The relative abundance of RankL was reduced (Figure ##SUPPL##0##S2i##) with a corresponding lower Opg gene expression (p < 0.05) in both male and female KO compared to WT control mice at both timepoints (Figure ##SUPPL##0##S2j##). These data support an attempt to down‐regulate osteolytic activity in the bone of the KO mice.
\",\n \"
We next examined gene expression of the endopeptidase, phosphate‐regulating endopeptidase X‐linked (PHEX) and its sibling protein, dentine matrix acidic phosphoprotein 1 (DMP1), both produced by osteocytes and participating in bone mineralization (Schaffler & Kennedy, ##REF##22552701##2012##). Phex was significantly reduced at 6 months and Dmp1 was reduced at both timepoints in KO mice compared with sex‐matched WT control (Figure ##FIG##1##2i,j##). DMP1 and Phex are involved in the regulation of Fgf23 (Martin et al., ##REF##21507898##2011##). As expected, Fgf23 mRNA increased (p < 0.05) with reducing Phex and Dmp1 in KO mice at both timepoints compared to their WT control (Figure ##FIG##1##2k##). Importantly, Il6 mRNA tended to increase at 6 months in male KO, and was significantly increased (p < 0.05) at both timepoints in female KO mice compared to WT control (Figure ##FIG##1##2l##).
\",\n \"Loss of <styled-content style=\\\"fixed-case\\\" toggle=\\\"no\\\">IL</styled-content>‐10 is associated with increased cardiac inflammation and fibrosis in mice\",\n \"
Motivated by the reported role of excessive FGF23 in inducing left ventricular hypertrophy, cardiac fibrosis, and vascular calcification (Böckmann et al., ##REF##31540546##2019##; Faul et al., ##REF##21985788##2011##; Hao et al., ##REF##27579618##2016##; Jimbo et al., ##REF##24088960##2014##), we next examined transverse cross‐section of the heart for histological changes. Masson's trichrome staining revealed increased fibrosis in the cardiac tissue of KO mice at 6 months compared with WT control mice (Figure ##FIG##2##3a##). Cardiac gene expression of the inflammatory marker, Tnf, only reached statistical significance at 6 months in female KO mice (Figure ##FIG##2##3b##). Similarly, the Il6 transcript was significantly elevated (p < 0.05) in the KO mice at the 6 months timepoint in both sexes compared to their respective WT control (Figure ##FIG##2##3c##). In contrast, the relative abundance of Il1b expression was higher (p < 0.05) in the cardiac tissue of KO mice at both timepoints (Figure ##FIG##2##3d##). Gene expression of adhesion molecules, including, vascular cell adhesion protein 1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) was increased (p < 0.05) in the cardiac tissue of female KO mice at the 6 month timepoint. (Figure ##SUPPL##0##S3a,b##). Gene expression of the receptor Adgre1, a macrophage marker, increased significantly in the cardiac tissues of KO mice at 6 months (Figure ##FIG##2##3e##). The M2 macrophage marker, Arg1, tended to be reduced (0.05 < p < 0.1) in female KO mice at 3 months, but was reduced (p < 0.05) in the cardiac tissue of male KO mice at both timepoints compared with WT control (Figure ##SUPPL##0##S3c##).
\",\n \"
Next, we assessed the protein expression/activation of the redox‐sensitive transcription factor, NF‐κB and key components of its signaling pathway. Inhibitor of nuclear factor kappa B (IκB), which prevents the nuclear translocation of NF‐κB, was reduced (p < 0.05) in both sexes of KO mice at the 3 and 6 month timepoints (Figure ##FIG##2##3f,g##), with female KO exhibiting reduced phosphorylation (p < 0.01) of IκB at 6 months (Figure ##FIG##2##3f,h##). The ratio of phosphorylated to total IκB increased (p < 0.05) at both time points in male KO (Figure ##FIG##2##3f,i##). The reduced phosphorylation of IκB at 6 months in female KO mice correlates with a reduced (p < 0.05) phospho‐NFκB (Ser536) in these mice at this timepoint (Figure ##FIG##2##3f,j,k##).
\",\n \"
We further assessed the protein expression of endothelial nitric oxide synthase (NOS)‐3, whose Ser1119 phosphorylation is critical in nitric oxide‐mediated vasodilation. KO mice had a reduced expression of NOS3 (p < 0.05) at the 6 month timepoint (Figure ##FIG##2##3l,m##). Reduced activation of this enzyme as indicated by its reduced Ser1119 phosphorylation coincided with a reduction in phosphoinositide 3‐kinase (PI3K), which lies downstream of the NOS3 signaling, at both timepoints compared with respective WT control (Figure ##FIG##2##3m–o##).
\",\n \"Loss of <styled-content style=\\\"fixed-case\\\" toggle=\\\"no\\\">IL</styled-content>‐10 is associated with reduced estrogen signaling in mice\",\n \"
Considering the role of estrogen as an activator of NOS3 (Chen et al., ##REF##9927501##1999##) and the role of this hormone in reducing inflammation (Liberale et al., ##REF##35210039##2022##), cardiac dysfunction, and bone loss (Arnal et al., ##REF##20631350##2010##; Meng et al., ##REF##33364052##2021##; Xing et al., ##REF##19221203##2009##), we compared serum 17β‐estradiol (E2) in KO mice with WT control mice. Serum E2 was lower (p < 0.05) in female KO mice and tended to be reduced (0.05 < p < 0.1) in male KO mice at both timepoints compared with WT controls (Figure ##FIG##3##4a##). Following this serendipitous finding of reduced E2 in the KO mice, we assessed estrogen synthesis in the ovaries of 14 month‐old mice. Concurrently, mRNA levels of Cyp11a1 and Cyp19a1, which are involved in estrogen synthesis, were significantly reduced in the ovaries of 14 month‐old KO mice compared with WT counterparts of the same age (Figure ##FIG##3##4b##). Similar reductions in mRNA levels were also observed in the WAT, another source of endogenous E2, of KO mice at the 3 and 6 month timepoints (Figure ##FIG##3##4c,d##). The activity of microbial β‐glucuronidase within the gut, which deconjugates estrogen in the intestinal lumen, was reduced at both time points in KO mice (Figure ##FIG##3##4e##). Estrogen could mediate its effects in several ways, one of which is binding to receptors, including ESR1 and ESR2, to mediate a membrane response or genomic response (Heldring et al., ##REF##17615392##2007##). Esr2 was not expressed in the heart (data not shown), whereas Esr1 mRNA was not different between groups compared at both timepoints (Figure ##SUPPL##0##S4a##). Contrarily, ileal and WAT Esr1 mRNA were significantly reduced (p < 0.05) in KO mice at both timepoints (Figure ##FIG##3##4f##; Figure ##SUPPL##0##S4b##), while the reduction in ileal Esr2 mRNA only reached statistical significance in the ileum tissue at the 6 month timepoint (Figure ##FIG##3##4g##). Esr1 was highly expressed at the 6 month timepoint in the bone marrow of female KO, while Esr2 was repressed within the bone marrow at both timepoints in KO mice compared to WT control mice (Figure ##FIG##3##4h,i##).
\",\n \"
To further confirm if IL‐10 is required for estrogen to modulate gut inflammation, lymphocytes from the ileum of 14 month‐old‐female WT and KO mice were isolated and treated with conditioned media with or without E2 (100 nM). E2 treatment lowered lymphocytic Tnf and Il17 mRNA in WT, but not in the KO mice (Figure ##FIG##4##5a,b##). We further assessed the effect of E2 treatment (100 nM) on intestinal crypt‐derived organoids from female KO and WT mice. E2‐treated organoids were faster in developing complex structures (crypt domain) in both WT and KO groups (Figure ##FIG##4##5c##). The mRNA expression of Vil1 and Alpi, which are highly expressed in the intestine, were not affected by E2 treatment in both organoid from WT and KO mice (Figure ##SUPPL##0##S5a,b##). In contrast, E2 treatment increased gene expression of Tjp1 in the KO‐derived organoid (Figure ##FIG##4##5d##), and Ocln in organoid from both WT and KO mice (Figure ##FIG##4##5e##). These data support an estrogenic effect on the enterocytes and not the lymphocytes of old IL‐10 KO mice.
In addition to its role in balancing gut immune reactivity, IL‐10 plays a critical role in interorgan communication (Madsen et al., ##REF##9207273##1997##; Miyoshi et al., ##REF##32835897##2021##; Oshima et al., ##REF##11162203##2001##). The mammalian gut participates in several interorgan communications in the mammalian system (Forkosh & Ilan, ##REF##31168383##2019##; Tripathi et al., ##REF##29748586##2018##; Zaiss et al., ##REF##31305265##2019##). Aging‐associated alterations in these interorgan communications contribute to chronic diseases, including cardiovascular and musculoskeletal disorders. Using the IL‐10 KO frail mouse model (Walston et al., ##REF##18426963##2008##), this study investigated the role of IL‐10 in mediating interplay between the gastrointestinal, skeletal, and cardiovascular systems. We reported a proinflammatory signature in the gut, bone loss and higher bone Fgf23 mRNA, lower serum estrogen, and increased cardiac fibrosis in the absence of IL‐10.
","
The highly expressed Il17a and Tnfa mRNA in the gut of the IL‐10 KO mice is expected, as IL‐10 suppresses both Th1 and Th17 immune responses in regulating immune reactivity (Chaudhry et al., ##REF##21511185##2011##; Couper et al., ##REF##18424693##2008##; Gu et al., ##REF##18506885##2008##). Berg et al. (##REF##8770874##1996##) has also reported a role for Th1 immune response in driving enterocolitis in IL‐10 KO mice. It is noteworthy that the higher gene expression of these proinflammatory cytokines in the female IL‐10 KO mice corresponds with their reduced Tgfb mRNA. TGF‐β impinges on Th1 differentiation by repressing Tbx21 and Stat5a, which encode the master transcription factors for Th1 cells (Gorelik & Flavell, ##REF##10714683##2000##; Lin et al., ##REF##15879087##2005##). The proinflammatory gut signature of the IL‐10 KO mouse could contribute to the loss of epithelial tight junction (Capaldo & Nusrat, ##REF##18952050##2009##; Ma et al., ##REF##14766535##2004##). The repression of Tjp1 and Ocln in male and female IL‐10 KO mice, respectively, at 3 months supports the early onset of inflammaging and further suggests a potential sex‐based difference in the expression of these tight junctions. A compromised gut is critical to the commonly reported increased systemic inflammation in the IL‐10 KO mice (Alake et al., ##REF##36813578##2023##; Kühn et al., ##REF##8402911##1993##).
","
As anticipated, both the female and male IL‐10 KO mice exhibited a bone phenotype characterized by lower cortical and trabecular bone mass. Relative to the WT control mice, alterations in Rankl and Wnt10b in the IL‐10 KO mice suggest greater osteoclastogenesis and reduced osteoblastogenesis contributed to this phenotype. Osteocytes are a major source of IL‐6 in the bone which has been reported to be increased in rodent models of IBD (Metzger et al., ##REF##27796050##2017##). The higher Il6 mRNA noted in the IL‐10 KO mice have the potential to stimulate osteoclast bone resorption (Ishimi et al., ##REF##2121824##1990##; Löwik et al., ##REF##2548501##1989##) and upregulate the expression of the phosphaturic hormone, FGF23 (Durlacher‐Betzer et al., ##REF##29861060##2018##). This response occurs in conjunction with the IL‐10 KO mice's reduced expression of bone Phex and Dmp1, which encode for PHEX and DMP1 and regulate FGF23 (Ling et al., ##REF##16294270##2005##; Martin et al., ##REF##21507898##2011##). PHEX represses Fgf23 gene expression mainly through the FGF receptor signaling (Bär et al., ##REF##31199502##2019##; Lu & Feng, ##REF##21404002##2011##; Martin et al., ##REF##21507898##2011##). More so, PHEX and DMP1 are interaction partners and evidence support the importance of their interaction in lowering plasma FGF23 (Martin et al., ##REF##22930691##2012##). Importantly, the increased expression of osteopontin and osteocalcin in the bone tissue also contributes to the bone phenotype in the IL‐10 KO mice. Osteocalcin appears to facilitate bone resorption (Ducy et al., ##REF##8684484##1996##), whereas osteopontin is a substrate for PHEX and it is known for inhibiting bone mineralization (Barros et al., ##REF##22991293##2013##). The reduced PHEX expression in the IL‐10 KO mice therefore explains the increase in Opn expression and the reduced bone phenotype in IL‐10 KO mice.
","
The increased cardiac fibrosis in IL‐10 KO mice at 6 months could be partly attributed to an increase FGF23 in these mice. Egli‐Spichtig et al. (##REF##31301888##2019##) had previously reported an increased serum iFGF23 in IL‐10 KO mice. Higher serum FGF23 had also been reported to mediate cardiovascular pathologies, including left ventricular hypertrophy, cardiac fibrosis, and vascular calcification (Böckmann et al., ##REF##31540546##2019##; Faul et al., ##REF##21985788##2011##; Hao et al., ##REF##27579618##2016##; Jimbo et al., ##REF##24088960##2014##). Furthermore, we reported a reduced E2 signaling in the cardiac tissue of KO mice, as indicated by a reduced phosphorylation of NOS3 as well as a reduced PI3K expression in the cardiac tissue of the IL10 KO mice. Haynes et al. (##REF##11029403##2000##) had earlier demonstrated that the E2 activation of NOS3 occurs via the PI3K‐Akt pathway and it involves the phosphorylation and activation of Ser1119 residue of NOS3. More so, the anti‐inflammatory potential of E2 was reported to be mediated by an increased IκBα and the prevention of NFκB nuclear translocation or binding (Ghisletti et al., ##REF##15798185##2005##; Xing et al., ##REF##22723832##2012##). In addition, an increased Ser536 phosphorylation of NFκB reportedly inhibits p65 signaling and inflammation (Pradère et al., ##REF##27555662##2016##). Our findings, including the reduced expression of IκBα and phospho Ser536 NFκB in the cardiac tissue of IL‐10 KO mice, therefore support a reduced activation of NOS3 and increased inflammation due to reduced E2 signaling in the cardiac tissue of these mice.
","
In this study, we also demonstrated that IL‐10 KO mice had lower circulating 17‐ β estradiol. 17‐ β estradiol mediates an anti‐inflammatory response in various tissues, including the gut, bone, and vascular endothelium (Goodman et al., ##REF##32632016##2020##; Nilsson, ##REF##17659431##2007##; Weitzmann & Pacifici, ##REF##16670759##2006##). Therefore, the increased gene expression of IL‐6 in the bone and cardiac tissue could be partly attributed to estrogen deficiency in the IL‐10 KO mice. The lowered serum 17‐β estradiol in these mice is explained in part by reduced estrogen synthesis in the ovaries and in adipose tissue as evidenced by a reduced ovarian and WAT Cyp17a1 and Cyp11a1 mRNA. These genes encode for two cytochrome P450 enzymes, both catalyzing important steps in the conversion of cholesterol to estrogens. However, recycling of estrogen was also altered in the KO mice as indicated by reduced activity of microbial β‐glucuronidase. β‐glucuronidase increases serum estrogen by deconjugating the glucuronide‐complex and increasing the bioavailability of estrogen (Baker et al., ##REF##28778332##2017##). Interestingly, Lactobacillus rhamnosus, which has β‐glucuronidase activity (Biernat et al., ##REF##30696850##2019##) had been reported to prevent bone loss and reduce intestinal inflammation and gut permeability in sex‐steroid‐deficient mice (Li et al., ##REF##27111232##2016##). However, our finding contradicted a phylogenetic prediction of an increased β‐glucuronidase in female IL‐10 KO mice that was earlier reported by Son et al. (##REF##31624723##2019##) using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software. This difference might be due to the incomprehensive nature of the 16S sequencing data that was used in those predictions. Moreover, such predictions are gene‐level based suggesting the transcriptional, posttranscriptional and translational regulation of gut bacterial β‐glucuronidase warrants further investigation.
","
The estrogen receptor, ESR2 mediates the anti‐inflammatory role of E2 as well as its role in intestinal organoid regeneration (Goodman et al., ##REF##32632016##2020##; Hases et al., ##REF##32711097##2020##; Lee et al., ##UREF##0##2019##). Our result showed that E2 treatment does not affect the proinflammatory phenotype in isolated lymphocytes from IL‐10 KO mice. However, E2 treatment increased the transcription of tight junctions in intestinal organoids. These findings suggest an interference of E2 signaling by other factors that are present in an inflamed gut. Interestingly, Pierdominici et al. (##REF##26497217##2015##) had previously reported significant repression of Esr2 mRNA in T lymphocytes and intestinal epithelial cells exposed to IL‐6. More so, the role of IL‐10 in suppressing IL‐6 production has been previously reported (Hempel et al., ##REF##7725065##1995##). Therefore, the lack of IL‐10 favored the dominance of proinflammatory molecules that could act partially by suppressing the expression of estrogen receptors to increase proinflammatory response in the gut of IL‐10 KO mice.
","
There are a few limitations in this study. First, the utilization of a global KO model makes it seemingly impossible to fully understand the tissue‐specific role of IL‐10. Future experiments should consider utilizing tissue‐specific KO models to understand the specific roles of IL‐10 in the tissues that were studied and how IL‐10 affects interorgan communication in these tissues. The gene expression of tight junctions does not necessarily determine their localization pattern; however, we are unable to confirm the localization pattern in the tissues that are available. Even though no differences were detected in Tjp1 and Ocln gene expression, future studies should include immunostaining techniques to determine if localization patterns are altered in the IL‐10KO mice since these changes can occur without alterations in overall gene expression. Our supporting in vitro experiments on the effect of E2 in LP lymphocytes and intestinal organoid was limited to female retired breeders. It will be interesting to perform similar experiment at a younger age in both male and female mice. In addition, since lowered E2 was a serendipitous finding at the end of the study and only retired breeders were available, we could not compare the expression level of Cyp2a1 and Cyp2a11 for the age group of mice that were studied. Lastly, whereas it is tempting to assume that E2 was lowered due to the deficiency in IL10, further research is needed to understand the mechanism by which reduced recycling and synthesis of E2 occurs in the IL‐10 KO mice.
","
Based on our findings, including a gut proinflammatory phenotype, an increased bone Fgf23 with corresponding reduced Dmp1 and Phex mRNA, a reduced serum E2 and gut bacterial β‐glucuronidase, as well as a reduced expression of estrogen receptor or downstream target of E2, we conclude that it is likely the combination of reduced IL‐10 and E2 signaling that promote an inflammatory phenotype which increases the secretion of FGF23. As a result, both factors mediate the interorgan communication that leads to gut inflammation and compromised barrier integrity, cardiac dysfunction and decrements in bone mass in the IL‐10 KO mouse.
"],"string":"[\n \"DISCUSSION\",\n \"
In addition to its role in balancing gut immune reactivity, IL‐10 plays a critical role in interorgan communication (Madsen et al., ##REF##9207273##1997##; Miyoshi et al., ##REF##32835897##2021##; Oshima et al., ##REF##11162203##2001##). The mammalian gut participates in several interorgan communications in the mammalian system (Forkosh & Ilan, ##REF##31168383##2019##; Tripathi et al., ##REF##29748586##2018##; Zaiss et al., ##REF##31305265##2019##). Aging‐associated alterations in these interorgan communications contribute to chronic diseases, including cardiovascular and musculoskeletal disorders. Using the IL‐10 KO frail mouse model (Walston et al., ##REF##18426963##2008##), this study investigated the role of IL‐10 in mediating interplay between the gastrointestinal, skeletal, and cardiovascular systems. We reported a proinflammatory signature in the gut, bone loss and higher bone Fgf23 mRNA, lower serum estrogen, and increased cardiac fibrosis in the absence of IL‐10.
\",\n \"
The highly expressed Il17a and Tnfa mRNA in the gut of the IL‐10 KO mice is expected, as IL‐10 suppresses both Th1 and Th17 immune responses in regulating immune reactivity (Chaudhry et al., ##REF##21511185##2011##; Couper et al., ##REF##18424693##2008##; Gu et al., ##REF##18506885##2008##). Berg et al. (##REF##8770874##1996##) has also reported a role for Th1 immune response in driving enterocolitis in IL‐10 KO mice. It is noteworthy that the higher gene expression of these proinflammatory cytokines in the female IL‐10 KO mice corresponds with their reduced Tgfb mRNA. TGF‐β impinges on Th1 differentiation by repressing Tbx21 and Stat5a, which encode the master transcription factors for Th1 cells (Gorelik & Flavell, ##REF##10714683##2000##; Lin et al., ##REF##15879087##2005##). The proinflammatory gut signature of the IL‐10 KO mouse could contribute to the loss of epithelial tight junction (Capaldo & Nusrat, ##REF##18952050##2009##; Ma et al., ##REF##14766535##2004##). The repression of Tjp1 and Ocln in male and female IL‐10 KO mice, respectively, at 3 months supports the early onset of inflammaging and further suggests a potential sex‐based difference in the expression of these tight junctions. A compromised gut is critical to the commonly reported increased systemic inflammation in the IL‐10 KO mice (Alake et al., ##REF##36813578##2023##; Kühn et al., ##REF##8402911##1993##).
\",\n \"
As anticipated, both the female and male IL‐10 KO mice exhibited a bone phenotype characterized by lower cortical and trabecular bone mass. Relative to the WT control mice, alterations in Rankl and Wnt10b in the IL‐10 KO mice suggest greater osteoclastogenesis and reduced osteoblastogenesis contributed to this phenotype. Osteocytes are a major source of IL‐6 in the bone which has been reported to be increased in rodent models of IBD (Metzger et al., ##REF##27796050##2017##). The higher Il6 mRNA noted in the IL‐10 KO mice have the potential to stimulate osteoclast bone resorption (Ishimi et al., ##REF##2121824##1990##; Löwik et al., ##REF##2548501##1989##) and upregulate the expression of the phosphaturic hormone, FGF23 (Durlacher‐Betzer et al., ##REF##29861060##2018##). This response occurs in conjunction with the IL‐10 KO mice's reduced expression of bone Phex and Dmp1, which encode for PHEX and DMP1 and regulate FGF23 (Ling et al., ##REF##16294270##2005##; Martin et al., ##REF##21507898##2011##). PHEX represses Fgf23 gene expression mainly through the FGF receptor signaling (Bär et al., ##REF##31199502##2019##; Lu & Feng, ##REF##21404002##2011##; Martin et al., ##REF##21507898##2011##). More so, PHEX and DMP1 are interaction partners and evidence support the importance of their interaction in lowering plasma FGF23 (Martin et al., ##REF##22930691##2012##). Importantly, the increased expression of osteopontin and osteocalcin in the bone tissue also contributes to the bone phenotype in the IL‐10 KO mice. Osteocalcin appears to facilitate bone resorption (Ducy et al., ##REF##8684484##1996##), whereas osteopontin is a substrate for PHEX and it is known for inhibiting bone mineralization (Barros et al., ##REF##22991293##2013##). The reduced PHEX expression in the IL‐10 KO mice therefore explains the increase in Opn expression and the reduced bone phenotype in IL‐10 KO mice.
\",\n \"
The increased cardiac fibrosis in IL‐10 KO mice at 6 months could be partly attributed to an increase FGF23 in these mice. Egli‐Spichtig et al. (##REF##31301888##2019##) had previously reported an increased serum iFGF23 in IL‐10 KO mice. Higher serum FGF23 had also been reported to mediate cardiovascular pathologies, including left ventricular hypertrophy, cardiac fibrosis, and vascular calcification (Böckmann et al., ##REF##31540546##2019##; Faul et al., ##REF##21985788##2011##; Hao et al., ##REF##27579618##2016##; Jimbo et al., ##REF##24088960##2014##). Furthermore, we reported a reduced E2 signaling in the cardiac tissue of KO mice, as indicated by a reduced phosphorylation of NOS3 as well as a reduced PI3K expression in the cardiac tissue of the IL10 KO mice. Haynes et al. (##REF##11029403##2000##) had earlier demonstrated that the E2 activation of NOS3 occurs via the PI3K‐Akt pathway and it involves the phosphorylation and activation of Ser1119 residue of NOS3. More so, the anti‐inflammatory potential of E2 was reported to be mediated by an increased IκBα and the prevention of NFκB nuclear translocation or binding (Ghisletti et al., ##REF##15798185##2005##; Xing et al., ##REF##22723832##2012##). In addition, an increased Ser536 phosphorylation of NFκB reportedly inhibits p65 signaling and inflammation (Pradère et al., ##REF##27555662##2016##). Our findings, including the reduced expression of IκBα and phospho Ser536 NFκB in the cardiac tissue of IL‐10 KO mice, therefore support a reduced activation of NOS3 and increased inflammation due to reduced E2 signaling in the cardiac tissue of these mice.
\",\n \"
In this study, we also demonstrated that IL‐10 KO mice had lower circulating 17‐ β estradiol. 17‐ β estradiol mediates an anti‐inflammatory response in various tissues, including the gut, bone, and vascular endothelium (Goodman et al., ##REF##32632016##2020##; Nilsson, ##REF##17659431##2007##; Weitzmann & Pacifici, ##REF##16670759##2006##). Therefore, the increased gene expression of IL‐6 in the bone and cardiac tissue could be partly attributed to estrogen deficiency in the IL‐10 KO mice. The lowered serum 17‐β estradiol in these mice is explained in part by reduced estrogen synthesis in the ovaries and in adipose tissue as evidenced by a reduced ovarian and WAT Cyp17a1 and Cyp11a1 mRNA. These genes encode for two cytochrome P450 enzymes, both catalyzing important steps in the conversion of cholesterol to estrogens. However, recycling of estrogen was also altered in the KO mice as indicated by reduced activity of microbial β‐glucuronidase. β‐glucuronidase increases serum estrogen by deconjugating the glucuronide‐complex and increasing the bioavailability of estrogen (Baker et al., ##REF##28778332##2017##). Interestingly, Lactobacillus rhamnosus, which has β‐glucuronidase activity (Biernat et al., ##REF##30696850##2019##) had been reported to prevent bone loss and reduce intestinal inflammation and gut permeability in sex‐steroid‐deficient mice (Li et al., ##REF##27111232##2016##). However, our finding contradicted a phylogenetic prediction of an increased β‐glucuronidase in female IL‐10 KO mice that was earlier reported by Son et al. (##REF##31624723##2019##) using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) software. This difference might be due to the incomprehensive nature of the 16S sequencing data that was used in those predictions. Moreover, such predictions are gene‐level based suggesting the transcriptional, posttranscriptional and translational regulation of gut bacterial β‐glucuronidase warrants further investigation.
\",\n \"
The estrogen receptor, ESR2 mediates the anti‐inflammatory role of E2 as well as its role in intestinal organoid regeneration (Goodman et al., ##REF##32632016##2020##; Hases et al., ##REF##32711097##2020##; Lee et al., ##UREF##0##2019##). Our result showed that E2 treatment does not affect the proinflammatory phenotype in isolated lymphocytes from IL‐10 KO mice. However, E2 treatment increased the transcription of tight junctions in intestinal organoids. These findings suggest an interference of E2 signaling by other factors that are present in an inflamed gut. Interestingly, Pierdominici et al. (##REF##26497217##2015##) had previously reported significant repression of Esr2 mRNA in T lymphocytes and intestinal epithelial cells exposed to IL‐6. More so, the role of IL‐10 in suppressing IL‐6 production has been previously reported (Hempel et al., ##REF##7725065##1995##). Therefore, the lack of IL‐10 favored the dominance of proinflammatory molecules that could act partially by suppressing the expression of estrogen receptors to increase proinflammatory response in the gut of IL‐10 KO mice.
\",\n \"
There are a few limitations in this study. First, the utilization of a global KO model makes it seemingly impossible to fully understand the tissue‐specific role of IL‐10. Future experiments should consider utilizing tissue‐specific KO models to understand the specific roles of IL‐10 in the tissues that were studied and how IL‐10 affects interorgan communication in these tissues. The gene expression of tight junctions does not necessarily determine their localization pattern; however, we are unable to confirm the localization pattern in the tissues that are available. Even though no differences were detected in Tjp1 and Ocln gene expression, future studies should include immunostaining techniques to determine if localization patterns are altered in the IL‐10KO mice since these changes can occur without alterations in overall gene expression. Our supporting in vitro experiments on the effect of E2 in LP lymphocytes and intestinal organoid was limited to female retired breeders. It will be interesting to perform similar experiment at a younger age in both male and female mice. In addition, since lowered E2 was a serendipitous finding at the end of the study and only retired breeders were available, we could not compare the expression level of Cyp2a1 and Cyp2a11 for the age group of mice that were studied. Lastly, whereas it is tempting to assume that E2 was lowered due to the deficiency in IL10, further research is needed to understand the mechanism by which reduced recycling and synthesis of E2 occurs in the IL‐10 KO mice.
\",\n \"
Based on our findings, including a gut proinflammatory phenotype, an increased bone Fgf23 with corresponding reduced Dmp1 and Phex mRNA, a reduced serum E2 and gut bacterial β‐glucuronidase, as well as a reduced expression of estrogen receptor or downstream target of E2, we conclude that it is likely the combination of reduced IL‐10 and E2 signaling that promote an inflammatory phenotype which increases the secretion of FGF23. As a result, both factors mediate the interorgan communication that leads to gut inflammation and compromised barrier integrity, cardiac dysfunction and decrements in bone mass in the IL‐10 KO mouse.
Characterization of the interleukin (IL)‐10 knockout (KO) mouse with chronic gut inflammation, cardiovascular dysfunction, and bone loss suggests a critical role for this cytokine in interorgan communication within the gut, bone, and cardiovascular axis. We sought to understand the role of IL‐10 in the cross‐talk between these systems. Six‐week‐old IL‐10 KO mice and their wild type (WT) counterparts were maintained on a standard rodent diet for 3 or 6 months. Gene expression of proinflammatory markers and Fgf23, serum 17β‐estradiol (E2), and cardiac protein expression were assessed. Ileal Il17a and Tnf mRNA increased while Il6 mRNA increased in the bone and heart by at least 2‐fold in IL‐10 KO mice. Bone Dmp1 and Phex mRNA were repressed at 6 months in IL‐10 KO mice, resulting in increased Fgf23 mRNA (~4‐fold) that contributed to increased fibrosis. In the IL‐10 KO mice, gut bacterial β‐glucuronidase activity and ovarian Cyp19a1 mRNA were lower (p < 0.05), consistent with reduced serum E2 and reduced cardiac pNOS3 (Ser1119) in these mice. Treatment of ileal lymphocytes with E2 reduced gut inflammation in WT but not IL‐10 KO mice. In conclusion, our data suggest that diminished estrogen and defective bone mineralization increased FGF23 which contributed to cardiac fibrosis in the IL‐10 KO mouse.
","
Unlike the WT (C57BL6) mouse, IL‐10 KO mouse exhibited tissue (gut, and consequently bone and cardiac) proinflammatory signature and reduced gut bacterial β‐glucuronidase activity. Proinflammatory cytokines favor increased bone production of FGF23, whose action (potentiated by reduced PHEX activity) could increase cardiac fibrosis. The reduced gut bacterial β‐glucuronidase activity decreases conjugated estrogen recycling into its active form, favoring reduced bone mass and cardiac phenotype in the IL‐10 KO mouse.\n
","
\n\n\nAlake, S. E.\n, \nIce, J.\n, \nRobinson, K.\n, \nPrice, P.\n, \nHatter, B.\n, \nWozniak, K.\n, \nLin, D.\n, \nChowanadisai, W.\n, \nSmith, B. J.\n, & \nLucas, E. A.\n (2024). Reduced estrogen signaling contributes to bone loss and cardiac dysfunction in interleukin‐10 knockout mice. Physiological Reports, 12, e15914. 10.14814/phy2.15914\n38217044\n\n
"],"string":"[\n \"Abstract\",\n \"
Characterization of the interleukin (IL)‐10 knockout (KO) mouse with chronic gut inflammation, cardiovascular dysfunction, and bone loss suggests a critical role for this cytokine in interorgan communication within the gut, bone, and cardiovascular axis. We sought to understand the role of IL‐10 in the cross‐talk between these systems. Six‐week‐old IL‐10 KO mice and their wild type (WT) counterparts were maintained on a standard rodent diet for 3 or 6 months. Gene expression of proinflammatory markers and Fgf23, serum 17β‐estradiol (E2), and cardiac protein expression were assessed. Ileal Il17a and Tnf mRNA increased while Il6 mRNA increased in the bone and heart by at least 2‐fold in IL‐10 KO mice. Bone Dmp1 and Phex mRNA were repressed at 6 months in IL‐10 KO mice, resulting in increased Fgf23 mRNA (~4‐fold) that contributed to increased fibrosis. In the IL‐10 KO mice, gut bacterial β‐glucuronidase activity and ovarian Cyp19a1 mRNA were lower (p < 0.05), consistent with reduced serum E2 and reduced cardiac pNOS3 (Ser1119) in these mice. Treatment of ileal lymphocytes with E2 reduced gut inflammation in WT but not IL‐10 KO mice. In conclusion, our data suggest that diminished estrogen and defective bone mineralization increased FGF23 which contributed to cardiac fibrosis in the IL‐10 KO mouse.
\",\n \"
Unlike the WT (C57BL6) mouse, IL‐10 KO mouse exhibited tissue (gut, and consequently bone and cardiac) proinflammatory signature and reduced gut bacterial β‐glucuronidase activity. Proinflammatory cytokines favor increased bone production of FGF23, whose action (potentiated by reduced PHEX activity) could increase cardiac fibrosis. The reduced gut bacterial β‐glucuronidase activity decreases conjugated estrogen recycling into its active form, favoring reduced bone mass and cardiac phenotype in the IL‐10 KO mouse.\\n
\",\n \"
\\n\\n\\nAlake, S. E.\\n, \\nIce, J.\\n, \\nRobinson, K.\\n, \\nPrice, P.\\n, \\nHatter, B.\\n, \\nWozniak, K.\\n, \\nLin, D.\\n, \\nChowanadisai, W.\\n, \\nSmith, B. J.\\n, & \\nLucas, E. A.\\n (2024). Reduced estrogen signaling contributes to bone loss and cardiac dysfunction in interleukin‐10 knockout mice. Physiological Reports, 12, e15914. 10.14814/phy2.15914\\n38217044\\n\\n
Edralin A. Lucas and Brenda J. Smith funding acquisition; Sanmi E. Alake, Karen Wozniak, Dingbo Lin, Winyoo Chowanadisai, Brenda J. Smith, and Edralin A. Lucas designed research; Sanmi E. Alake, John Ice, Kara Robinson, Payton Price, Bethany Hatter, Brenda J. Smith, and Edralin A. Lucas conducted research; Sanmi E. Alake, Brenda J. Smith and Edralin A. Lucas analyzed the data, wrote the paper, and had primary responsibility for the final content. All authors read and approved the final manuscript.
","FUNDING INFORMATION","
This study was funded by the Oklahoma Agricultural Experiment Station (Project # OKL03104 and OKL03105) and the Jim and Lynn Williams Professorship (1‐156560).
","CONFLICT OF INTEREST STATEMENT","
All authors declared no conflicts of interest.
","ETHICS STATEMENT","
All procedures were approved and followed strict guidelines set by the Institution Animal Care of Oklahoma State University (Protocol #IACUC 22‐02‐STW).
Edralin A. Lucas and Brenda J. Smith funding acquisition; Sanmi E. Alake, Karen Wozniak, Dingbo Lin, Winyoo Chowanadisai, Brenda J. Smith, and Edralin A. Lucas designed research; Sanmi E. Alake, John Ice, Kara Robinson, Payton Price, Bethany Hatter, Brenda J. Smith, and Edralin A. Lucas conducted research; Sanmi E. Alake, Brenda J. Smith and Edralin A. Lucas analyzed the data, wrote the paper, and had primary responsibility for the final content. All authors read and approved the final manuscript.
\",\n \"FUNDING INFORMATION\",\n \"
This study was funded by the Oklahoma Agricultural Experiment Station (Project # OKL03104 and OKL03105) and the Jim and Lynn Williams Professorship (1‐156560).
\",\n \"CONFLICT OF INTEREST STATEMENT\",\n \"
All authors declared no conflicts of interest.
\",\n \"ETHICS STATEMENT\",\n \"
All procedures were approved and followed strict guidelines set by the Institution Animal Care of Oklahoma State University (Protocol #IACUC 22‐02‐STW).
Weekly body weight of male (a) and female (b) mice, and ileum gene expression of inflammatory cytokines, Tgfb (c), Il6 (d), Il17a (e), Tnf (f), and tight junctions, Tjp1 (g), and Ocln (h) in WT mice and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months. Values are mean ± SD, n = 12–16 mice/group for a and b, n = 6 mice/group for c and d, n = 12–16 mice/group for e, and n = 6 mice/group for f–h. Based on data analysis using Student's t‐test.
","
Skeletal response of male and female WT and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months: whole body bone mineral density, BMD (a), trabecular bone volume/total volume (BV/TV) of the lumbar vertebra body (b) and distal femur metaphysis (c), femur cortical area (d) and thickness (e), and gene expression of Sost (f), Opn (g), Bglap2 (h) Phex (i) Dmp1 (j), Fgf23 (k), and Il6 (l). Values are mean ± SD, n = 12–16 mice/group for a, n‐8–13 mice/group for b–e, n = 6 mice/group for f–l. Based on data analysis using Student's t‐test.
","
Representative trichrome staining of fixed heart tissue, with blue coloration indicating fibrosis (a), cardiac gene expression of inflammatory markers: Tnf (b), Il6 (c), Il1b (d), and the macrophage marker Adgre1 (e). Representative blots (f), showing cardiac protein expression of: IκB (g), phosphorylated IκB (h), Ratio of pIκB to total IκB (i), phosphorylated NFκB (j), and NFκB (k). Also, representative blots (l) for cardiac protein expression of NOS3 (m), phosphorylated NOS3 (n), and PI3K (o) in WT mice and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months. Values are mean ± SD, n = 6 mice/group for a–e, n = 4–5 mice/group for f–o. Student's t‐test.
","
Serum 17β‐estradiol (a). Gene expression of Cyp19a1 and Cyp11a1 in the ovary (b), and white adipose (WAT; c and d, respectively). β‐glucuronidase enzyme activity in the cecal contents (e), and estrogen receptor gene expression in the ileum, Esr1 (f) and Esr2 (g) and bone marrow (h and i) in WT mice and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months. Values are mean ± SD, n = 8–10 mice/group for a, n = 5 mice/group for b, n = 6 mice/group for c and d, n = 7 mice/group for e, n = 5–6 mice/group for f–i. Based on data analysis using Student's t‐test.
","
Gene expression of proinflammatory cytokines in E2‐treated (100 nM) ileal‐derived lymphocytes isolated from 14 month‐old‐female KO and WT mice: Tnf (a), and Il17 (b). Representative image of intestinal organoids, Day 6, developed from crypt cells derived from 14 month‐old female KO and WT mice, with or without E2 treatment (c). Gene expression of tight junctions in E2‐treated intestinal organoids: Tjp1 (d), and Ocln (e). Values are mean ± SD, n = 3 mice/group for a, b, n = 3 mice/group and replicate = 3/mouse for c–e. Based on data analysis using Student's t‐test.
"],"string":"[\n \"
Weekly body weight of male (a) and female (b) mice, and ileum gene expression of inflammatory cytokines, Tgfb (c), Il6 (d), Il17a (e), Tnf (f), and tight junctions, Tjp1 (g), and Ocln (h) in WT mice and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months. Values are mean ± SD, n = 12–16 mice/group for a and b, n = 6 mice/group for c and d, n = 12–16 mice/group for e, and n = 6 mice/group for f–h. Based on data analysis using Student's t‐test.
\",\n \"
Skeletal response of male and female WT and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months: whole body bone mineral density, BMD (a), trabecular bone volume/total volume (BV/TV) of the lumbar vertebra body (b) and distal femur metaphysis (c), femur cortical area (d) and thickness (e), and gene expression of Sost (f), Opn (g), Bglap2 (h) Phex (i) Dmp1 (j), Fgf23 (k), and Il6 (l). Values are mean ± SD, n = 12–16 mice/group for a, n‐8–13 mice/group for b–e, n = 6 mice/group for f–l. Based on data analysis using Student's t‐test.
\",\n \"
Representative trichrome staining of fixed heart tissue, with blue coloration indicating fibrosis (a), cardiac gene expression of inflammatory markers: Tnf (b), Il6 (c), Il1b (d), and the macrophage marker Adgre1 (e). Representative blots (f), showing cardiac protein expression of: IκB (g), phosphorylated IκB (h), Ratio of pIκB to total IκB (i), phosphorylated NFκB (j), and NFκB (k). Also, representative blots (l) for cardiac protein expression of NOS3 (m), phosphorylated NOS3 (n), and PI3K (o) in WT mice and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months. Values are mean ± SD, n = 6 mice/group for a–e, n = 4–5 mice/group for f–o. Student's t‐test.
\",\n \"
Serum 17β‐estradiol (a). Gene expression of Cyp19a1 and Cyp11a1 in the ovary (b), and white adipose (WAT; c and d, respectively). β‐glucuronidase enzyme activity in the cecal contents (e), and estrogen receptor gene expression in the ileum, Esr1 (f) and Esr2 (g) and bone marrow (h and i) in WT mice and IL‐10 KO (KO) mice fed a semi‐purified diet for 3 or 6 months. Values are mean ± SD, n = 8–10 mice/group for a, n = 5 mice/group for b, n = 6 mice/group for c and d, n = 7 mice/group for e, n = 5–6 mice/group for f–i. Based on data analysis using Student's t‐test.
\",\n \"
Gene expression of proinflammatory cytokines in E2‐treated (100 nM) ileal‐derived lymphocytes isolated from 14 month‐old‐female KO and WT mice: Tnf (a), and Il17 (b). Representative image of intestinal organoids, Day 6, developed from crypt cells derived from 14 month‐old female KO and WT mice, with or without E2 treatment (c). Gene expression of tight junctions in E2‐treated intestinal organoids: Tjp1 (d), and Ocln (e). Values are mean ± SD, n = 3 mice/group for a, b, n = 3 mice/group and replicate = 3/mouse for c–e. Based on data analysis using Student's t‐test.
Sphingolipids are structural molecules of cell membranes that play an important role in maintaining barrier function and fluidity. Sphingolipids were first isolated and identified from brain tissues in the 19th century by German biochemist Johann L. W. Thudichum, who named them after the Sphinx, a creature from Greek mythology, because of their enigmatic nature.[\n##REF##26747648##\n2\n##\n] Sphingolipid metabolites, ceramide (Cer), ceramide‐1‐phosphate (C1P), and sphingosine‐1‐phosphate (S1P) have been recognized as important signaling molecules that regulate cell growth, survival, immune cell trafficking, and vascular and epithelial cell integrity and are particularly important in inflammation and cancer.[\n##REF##35420948##\n1\n##, ##REF##24899305##\n3\n##\n] In addition, sphingosine (Sph), another sphingolipid metabolite, constitutes a class of natural products containing a long aliphatic chain with a polar 2‐amino‐1,3‐diol terminus (2‐amino‐4‐trans‐octadecene‐1,3‐diol), which is generated from ceramides.[\n##REF##16782799##\n4\n##\n] It occurs in the cell membranes of all animals and many plants and plays an important role in a variety of complex biological processes, such as DNA damage,[\n##REF##26943039##\n5\n##\n] apoptosis,[\n##REF##19797232##\n6\n##\n] (including hippocampal neuron and astrocyte apoptosis),[\n##REF##21660956##\n7\n##\n] cell growth,[\n##REF##23221613##\n8\n##\n] differentiation, autophagic processes, and development.[\n##REF##25697338##\n9\n##\n] Significantly, abnormal sphingosine metabolism can induce various diseases, such as cancers and neurodegenerative diseases, including NPC (Niemann‐Pick disease type C), AD (Alzheimer's disease),[\n##REF##18547682##\n10\n##\n] and others.[\n##REF##27830727##\n11\n##\n] Based on the above, developing suitable probes or chemical tools to study sphingolipids and their metabolites and decipher their roles in cellular biology is urgently needed.
","
A plethora of approaches have been developed to detect cellular sphingolipids and metabolites. With respect to instrumental development, liquid chromatography‐mass spectrometry (LC‐MS), mass spectrometry (MS) and imaging mass spectrometry (IMS) techniques have become the method of choice for the detection and quantification of sphingolipid metabolites.[\n##REF##23359681##\n12\n##\n] Alternatively, sphingomyelin can be stained by fluorescent protein conjugates such as recombinant lysenin and equinatoxin.[\n##REF##25389132##\n13\n##\n] More interestingly and importantly, based on the widespread applications of fluorescence imaging technology in chemical biology, numerous methods for fluorescently labeling sphingolipids and metabolites have been developed and extensively used as sphingolipid probes to study the subcellular localization and metabolism of sphingolipids using fluorescence microscopy.[\n##REF##33496698##\n14\n##\n] Although there are many ways to label sphingolipids and metabolites, such as sphingosine, few methods have been developed for the direct detection of sphingolipids and metabolites, especially sphingosine.[\n##REF##23359681##\n12\n##, ##REF##25389132##\n13\n##, ##REF##34509716##\n15\n##\n] In 2020, Devaraj et al. first developed an ingenious fluorescently labeled aldehyde probe that chemoselectively reacts with terminal 1,2‐amino alcohol to detect endogenous Sph.[\n##REF##33044062##\n16\n##\n] Hence, the development of a simple and efficient novel method for highly selective and ultrasensitive detection of sphingosine in living cells remains highly useful and desirable.
","
In contrast to other sphingolipids, sphingosine has a unique terminal amino alcohol structure similar to that of norepinephrine (NE). Notably, Yin et al. pioneered the highly powerful “protect‐deprotect” strategy for NE detection.[\n##REF##33000941##\n17\n##\n\n]\n Herein, a series of fluorogenic probes, DMS‐X (X = 2F, F, Cl, Br, I), with thiocarbonate protecting groups were developed (Scheme ##FIG##0##\n1\n##). Different halo substituents were introduced to control the reactivity between the probes and Sph. The results indicated that the 2,6‐difluoro‐substituted probe DMS‐2F can specifically and selectively detect sphingosine in a short time with good response sensitivity compared to other probes. Interestingly, neurotransmitters (NE, EP, and DA) did not interfere with the response of DMS‐2F to Sph. Furthermore, the results of intracellular studies suggest that DMS‐2F allows fluorescent imaging of exogenous and endogenous Sph, especially can monitor the Aβ42 oligomers‐induced Sph variation in live neutral cells and zebrafish in vivo. Based on the characteristics of reactive fluorescence probes, which largely avoid the background interference of probe molecules, this new NIR fluorogenic probe provides a new research idea for the selective and sensitive detection of sphingosine in vitro and in vivo.
"],"string":"[\n \"Introduction\",\n \"
Sphingolipids are structural molecules of cell membranes that play an important role in maintaining barrier function and fluidity. Sphingolipids were first isolated and identified from brain tissues in the 19th century by German biochemist Johann L. W. Thudichum, who named them after the Sphinx, a creature from Greek mythology, because of their enigmatic nature.[\\n##REF##26747648##\\n2\\n##\\n] Sphingolipid metabolites, ceramide (Cer), ceramide‐1‐phosphate (C1P), and sphingosine‐1‐phosphate (S1P) have been recognized as important signaling molecules that regulate cell growth, survival, immune cell trafficking, and vascular and epithelial cell integrity and are particularly important in inflammation and cancer.[\\n##REF##35420948##\\n1\\n##, ##REF##24899305##\\n3\\n##\\n] In addition, sphingosine (Sph), another sphingolipid metabolite, constitutes a class of natural products containing a long aliphatic chain with a polar 2‐amino‐1,3‐diol terminus (2‐amino‐4‐trans‐octadecene‐1,3‐diol), which is generated from ceramides.[\\n##REF##16782799##\\n4\\n##\\n] It occurs in the cell membranes of all animals and many plants and plays an important role in a variety of complex biological processes, such as DNA damage,[\\n##REF##26943039##\\n5\\n##\\n] apoptosis,[\\n##REF##19797232##\\n6\\n##\\n] (including hippocampal neuron and astrocyte apoptosis),[\\n##REF##21660956##\\n7\\n##\\n] cell growth,[\\n##REF##23221613##\\n8\\n##\\n] differentiation, autophagic processes, and development.[\\n##REF##25697338##\\n9\\n##\\n] Significantly, abnormal sphingosine metabolism can induce various diseases, such as cancers and neurodegenerative diseases, including NPC (Niemann‐Pick disease type C), AD (Alzheimer's disease),[\\n##REF##18547682##\\n10\\n##\\n] and others.[\\n##REF##27830727##\\n11\\n##\\n] Based on the above, developing suitable probes or chemical tools to study sphingolipids and their metabolites and decipher their roles in cellular biology is urgently needed.
\",\n \"
A plethora of approaches have been developed to detect cellular sphingolipids and metabolites. With respect to instrumental development, liquid chromatography‐mass spectrometry (LC‐MS), mass spectrometry (MS) and imaging mass spectrometry (IMS) techniques have become the method of choice for the detection and quantification of sphingolipid metabolites.[\\n##REF##23359681##\\n12\\n##\\n] Alternatively, sphingomyelin can be stained by fluorescent protein conjugates such as recombinant lysenin and equinatoxin.[\\n##REF##25389132##\\n13\\n##\\n] More interestingly and importantly, based on the widespread applications of fluorescence imaging technology in chemical biology, numerous methods for fluorescently labeling sphingolipids and metabolites have been developed and extensively used as sphingolipid probes to study the subcellular localization and metabolism of sphingolipids using fluorescence microscopy.[\\n##REF##33496698##\\n14\\n##\\n] Although there are many ways to label sphingolipids and metabolites, such as sphingosine, few methods have been developed for the direct detection of sphingolipids and metabolites, especially sphingosine.[\\n##REF##23359681##\\n12\\n##, ##REF##25389132##\\n13\\n##, ##REF##34509716##\\n15\\n##\\n] In 2020, Devaraj et al. first developed an ingenious fluorescently labeled aldehyde probe that chemoselectively reacts with terminal 1,2‐amino alcohol to detect endogenous Sph.[\\n##REF##33044062##\\n16\\n##\\n] Hence, the development of a simple and efficient novel method for highly selective and ultrasensitive detection of sphingosine in living cells remains highly useful and desirable.
\",\n \"
In contrast to other sphingolipids, sphingosine has a unique terminal amino alcohol structure similar to that of norepinephrine (NE). Notably, Yin et al. pioneered the highly powerful “protect‐deprotect” strategy for NE detection.[\\n##REF##33000941##\\n17\\n##\\n\\n]\\n Herein, a series of fluorogenic probes, DMS‐X (X = 2F, F, Cl, Br, I), with thiocarbonate protecting groups were developed (Scheme ##FIG##0##\\n1\\n##). Different halo substituents were introduced to control the reactivity between the probes and Sph. The results indicated that the 2,6‐difluoro‐substituted probe DMS‐2F can specifically and selectively detect sphingosine in a short time with good response sensitivity compared to other probes. Interestingly, neurotransmitters (NE, EP, and DA) did not interfere with the response of DMS‐2F to Sph. Furthermore, the results of intracellular studies suggest that DMS‐2F allows fluorescent imaging of exogenous and endogenous Sph, especially can monitor the Aβ42 oligomers‐induced Sph variation in live neutral cells and zebrafish in vivo. Based on the characteristics of reactive fluorescence probes, which largely avoid the background interference of probe molecules, this new NIR fluorogenic probe provides a new research idea for the selective and sensitive detection of sphingosine in vitro and in vivo.
\"\n]"},"methods":{"kind":"list like","value":[],"string":"[]"},"results":{"kind":"list like","value":["Results and Discussion","
The detailed synthesis procedure (Scheme ##SUPPL##0##S1##, Supporting Information) and structural characterization (Figures ##SUPPL##0##S1–S30##, Supporting Information) of fluorogenic probes DMS‐X are provided in the Supporting Information.
","
To evaluate the stability of DMS‐X (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I), solutions of DMS‐X in PBS buffer were first exposed to a UV lamp (365 nm), and the change in fluorescence intensities of DMS‐X was monitored separately at different times. As shown in Figure ##SUPPL##0##S31a## (Supporting Information), the fluorescence intensities of DMS‐X were almost unchanged after 52 h of exposure to a UV lamp. The investigation of fluorescent changes of DMS‐X in different pH buffers at different times also showed excellent stability (Figure ##SUPPL##0##S31b–f##, Supporting Information). These results indicate that the DMS‐X probes have good photostability and acid‐base resistance properties.
","
Next, the ability of DMS‐X probes (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to react with Sph under physiological conditions (pH 7.4, 37 °C) was tested using the vesicles of DMPC to mimic biological membranes. After reacting with Sph, the fluorescent emission results showed that only DMS‐2F had significant fluorescence enhancement at 660 nm (Figure ##FIG##1##\n1a##), while the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) exhibited only slight fluorescence changes (Figure ##SUPPL##0##S32a–h##, Supporting Information). To further validate the superiority of DMS‐2F in detecting sphingosine, the fluorescence changes of DMS‐X were examined when incubated with Sph for different times in the DMPC system (with or without Sph). As shown in Figure ##FIG##1##1b##, an obvious fluorescence enhancement (I/I0 = 21.08) was detected in the DMS‐2F and Sph system when incubated at 37 °C for 10 min. With increasing incubation time, the fluorescence intensity of DMS‐2F gradually increased, reaching the highest intensity at 4 h (I/I0 = 57.60) (Figure ##FIG##1##1c##), which was fivefold faster than that reported for the Sph probe.[\n##REF##33044062##\n16\n##\n] However, the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) did not show significant fluorescence changes even after 6 h of incubation with Sph (Figure ##FIG##1##1b##; Figure ##SUPPL##0##S33##, Supporting Information). These findings suggest that DMS‐2F can be used as a highly efficient fluorogenic probe in response to Sph.
","
To evaluate the appropriate pH range for the Sph recognition process of DMS‐2F, pH control experiments were carefully carried out in PBS‐buffered solutions (pH 3 to 9). After incubation with 200 µm Sph in buffer solutions of different pH values for 30 min at 37 °C, the fluorescence of DMS‐2F at 660 nm was significantly enhanced when the pH ranged from 6 to 9 (Figure ##SUPPL##0##S34##, Supporting Information), indicating that DMS‐2F could efficiently fluorogenically detect Sph in the physiological pH range.
","
Concentration‐dependent fluorescence experiments were subsequently performed by incubating DMS‐2F (5 µm) with various concentrations of Sph (0–200 µm) in DMPC vesicles for 30 min at 37 °C. The results showed that the fluorescence intensity of DMS‐2F at 660 nm gradually increased with increasing Sph concentrations (Figure ##FIG##1##1d##). Moreover, an excellent linear relationship between the emission intensities was observed in the range of 0–110 µm (Figure ##FIG##1##1e##). The limit of detection (LOD) of DMS‐2F for Sph was calculated as 9.33 ± 0.41 nm according to the 3σ/m method. These findings suggest that DMS‐2F has the capability for ultrasensitive detection of Sph.
","
To further determine whether DMS‐2F could selectively detect Sph in living cells, DMS‐2F (5 εm) was incubated in DMPC vesicles composed of several naturally abundant lipid species and other possible interferents (including amino acids with structures similar to Sph, such as cysteine (Cys), lysine (Lys), serine (Ser), threonine (Thr), tyrosine (Tyr) (260 εm) and glutathione (GSH) (2 mm); naturally abundant lipid species, sphingomyelin (SM, 200 εm) and ceramide (Cer, 200 εm); and neurotransmitters: NE (norepinephrine, 200 εm), DA (dopamine, 200 εm), and EP (epinephrine, 200 εm) for 30 min at 37 °C. The results showed that a significant fluorescence turn‐on was observed when DMS‐2F (5 εm) was incubated with Sph (200 εm). However, when DMS‐2F was incubated with another analyte, there was a weak change in fluorescence intensity compared to the untreated DMS‐2F probe (Figure ##FIG##1##1f##). These results are consistent with the design of the DMS‐2F probe to selectively detect Sph in the DMPC system.
","
The specific response mechanism of DMS‐2F to Sph was confirmed by liquid chromatograph mass spectrometer (LC‐MS), high‐resolution mass spectrometry (HR‐MS), fluorescence spectroscopy, UV‐Visible spectroscopy, and theoretical calculation. DMS‐2F was exposed to Sph for 6 h at 37 °C, followed by LC−MS analysis and the reaction products of a five‐membered cyclic oxazolidinone‐based Sph compound (0.83 min) and the fluorophore DM‐2F (3.57 min) were successfully detected (Figure ##FIG##2##\n2a–c##). In addition, HR‐MS experiments again confirmed that the fluorophore was released after DMS‐2F reacted with Sph, producing a five‐membered cyclic oxazolidinone‐based Sph compound (Figure ##SUPPL##0##S35##, Supporting Information). Subsequently, the UV−vis and fluorescence response of DMS‐2F to Sph were determined by adding 200 εm Sph to 1 mL of PBS buffer containing 5 εm\nDMS‐2F and incubating at 37 °C for 6 h. As illustrated in Figure ##FIG##2##2d##, incubation with Sph resulted in a decrease in the UV‒vis absorption of DMS‐2F at 408 nm and an increase in absorption at 463 nm. The spectral properties of the system after reaction with Sph were in good agreement with the fluorophore DM‐2F. These results indicated that the response mechanism of DMS‐2F to Sph should be attributed to the nucleophilic addition and elimination reaction between ─NH2 of Sph and the thiocarbonate‐ester group of DMS‐2F. When the reaction is triggered, the fluorophore DM‐2F is released and emits significant fluorescence. The probable response mechanism of DMS‐2F to Sph is shown in Scheme ##FIG##0##1##. According to the proposed mechanism above, the bond cleavage between the carbon atom of the carbonyl group (C20) and the aryl oxide (O21) in probes is the key factor for the fluorogenic response of DMS‐X to Sph. Therefore, the bond energy of C20─O21 was a determining factor for screening the optimal probe for the response of Sph. To further understand the response priority of DMS‐2F and other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to Sph, theoretical calculations were carried out. The structures of DMS‐X were optimized by density functional theory (DFT) studies using the B3LYP 6–31G (d, p) levels (Figure ##FIG##2##2f,g##: Figure ##SUPPL##0##S36##, Supporting Information), and the bond lengths of C20─O21 in DMS‐X were evaluated. As shown in Figure ##FIG##2##2e##, the bond length of DMS‐2F is significantly longer than that of the other probes, which further indicates that DMS‐2F has a better response effect than the other probes. Additionally, the ground‐state geometry structures of Sph and NE were optimized by DFT at the B3LYP/6‐31G levels to investigate the selective response mechanism of DMS‐2F toward Sph. The calculation results indicate that there are hydrogen bonding interactions between the amino group of NE and its neighboring hydroxyl group (Figure ##SUPPL##0##S37a##, Supporting Information), which was detrimental to the DMS‐2F response to NE, whereas such interactions are absent in the structure of Sph (Figure ##SUPPL##0##S37b##, Supporting Information). Additionally, the electrostatic surface potential (ESP) maps reveal that the amino moieties of Sph possess higher electronegativity compared to those of NE (Figures ##SUPPL##0##S37c,d##, Supporting Information). These findings suggest that Sph can readily react with DMS‐2F, leading to its selective response.
","
These promising in vitro results prompted us to explore the imaging effect of DMS‐X in living cells. Using MTT assays, it was confirmed that DMS‐X was non‐toxic to different cell lines (Figures ##SUPPL##0##S38## and ##SUPPL##0##S39##, Supporting Information). First, to determine whether DMS‐2F could react with Sph in live cells, A549 cells were incubated with DMS‐2F (10 εm) for 30 min, and then the cell medium was exchanged with a new medium containing 40 εm Sph and incubated for different amounts of time (30–150 min). As shown in Figure ##SUPPL##0##S40## (Supporting Information), as the Sph incubation time increased, the cells showed a gradual enhancement in red fluorescence emission within 1.5 h. To assess whether the response of DMS‐2F to Sph is concentration‐dependent in living cells, DMS‐2F‐pretreated A549 and HepG2 cells were incubated with different concentrations of Sph (0, 5, 10, and 20 εM). After 2 h of incubation, cells treated with exogenous Sph showed a dose‐dependent fluorescence enhancement signal (Figure ##SUPPL##0##S41##, Supporting Information). Furthermore, to confirm that the observed increase in cellular fluorescence was a result of the release of the fluorophore DM‐2F after the reaction between DMS‐2F and Sph, A549 cells were further treated with DM‐2F (10 εm) (Figure ##SUPPL##0##S42##, Supporting Information). Cells treated with DM‐2F exhibited a fluorescent imaging pattern consistent with that observed in cells treated with DMS‐2F and Sph. These results indicate that DMS‐2F could react with Sph in living cells within a short period of time and release an enhanced fluorescence signal by generating the fluorophore DM‐2F in a dose‐dependent manner.
","
Since elevated sphingosine in mammalian cells is closely associated with apoptosis, to evaluate the response ability of DMS‐2F to endogenous Sph levels in live cells, cancer cells (rat adrenal pheochromocytoma PC12 cells, human non‐small cell lung cancer A549 cells, and brain glioma U87 cell lines) and normal lung fibroblast MRC‐5 cells were exposed to serum‐free medium (opti‐MEM containing 10 εm\nDMS‐2F), which could result in elevated Sph levels. As shown in Figure ##SUPPL##0##S43a,b## (Supporting Information), after 2 h of incubation, the fluorescence of live cells treated with opti‐MEM all showed a significant increase. Taken together, these results suggest that DMS‐2F is sensitive enough to probe native sphingosine in various live cells, and can be used for imaging differences in sphingosine levels in living cells.
","
Abnormal sphingolipid metabolism has been reported to induce Alzheimer's disease (AD), and substantial evidence also support that amyloid‐β (Aβ42) plays an important role in AD. In vitro, Aβ42 has been shown to induce apoptosis via the sphingomyelin pathway in various brain cells, including PC12 cells, etc.[\n##REF##18547682##\n10\n##, ##REF##14748735##\n18\n##\n] Accordingly, we attempted to assess whether the level of Sph will be changed in PC12 cells after treatment with Aβ42 peptide. We first treated PC12 cells with 10 εm Aβ42 oligomers for different times and then incubated them with DMS‐2F, as shown in Figure ##SUPPL##0##S44## (Supporting Information), the fluorescence of PC12 cells treated with Aβ42 oligomers was significantly increased. These observations undoubtedly revealed that intracellular Sph was up‐regulated when neuronal cells were challenged by Aβ42 oligomers. To further correlate Sph generation with Aβ42 oligomers, PC12 cells were treated with different concentrations of Aβ42 oligomers and Aβ42 monomers. After 12 h of incubation, a gradual increase in fluorescence was observed in PC12 cells treated with Aβ42 oligomers, however, weak fluorescence changes were observed in Aβ42 monomers treated cells (Figure ##FIG##3##\n3a,b,d,e##). Together, these findings suggested that neuronal cells experienced overexpression of Sph during Aβ42 administration and that the up‐regulated Sph levels correlated with both the incubation time and dosage of Aβ42 oligomers, indicating that the abnormal sphingolipid metabolism in neuronal cells is tightly related to the stimulation of Aβ42 oligomers.
","
Since the overexpression of Aβ protein induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade, and previous studies have illustrated that H2O2 could induce neurotoxicity in PC12 cells,[\n##REF##14748735##\n18\n##, ##REF##22133762##\n19\n##\n] we further investigated the Sph variations induced by H2O2. PC12 cells were treated with different concentrations of H2O2, after 4 h of incubation, a gradual increase in fluorescence was detected in PC12 cells treated with H2O2 (Figure ##FIG##3##3c,f##), suggesting that the Sph variation occurred during the oxidative stress process. In addition, the changes of Sph levels after being treated with Aβ42 oligomers and H2O2 in other cell lines were also assessed. U87, A549, HepG2, and MRC5 cells were treated with Aβ42 oligomers and H2O2 for 12 and 4 h, respectively, and then incubated with DMS‐2F for 2 h. As shown in Figure ##SUPPL##0##S45## (Supporting Information), significant fluorescence enhancement was exhibited in PC12 and U87 cells, while the fluorescence in other cell lines was not evident. These results indicate that the fluorogenic probe DMS‐2F can successfully monitor the level changes of Sph during Aβ42 oligomers and H2O2‐induced apoptosis in neuronal cells.
","
Since changes in Sph levels are associated with the abnormal sphingolipid metabolism in cells, to investigate the possible mechanisms of Aβ42 oligomers and H2O2‐induced changes in Sph levels, we further investigated intracellular sphingomyelinase and ceramidase activities in Aβ42 oligomers and H2O2 treated cells. PC12 cells were preincubated for 2 h in the presence of 10 µm amitriptyline hydrochloride (AMI, an acid sphingomyelinase inhibitor[\n##UREF##0##\n20\n##\n]), 20 µm GW4869 (a neutral sphingomyelinase inhibitor[\n##REF##20629193##\n21\n##\n]), 10 µm LCL‐521 (an acid ceramidase inhibitor[\n##REF##25456083##\n22\n##\n]), 500 µm N‐acetyl‐cysteine (NAC, an efficient antioxidant[\n##UREF##1##\n23\n##\n]) and 1 mm Trolox (a reactive oxygen scavenger [\n##REF##17013382##\n24\n##\n]), and then treated with Aβ42 oligomers (10 µm, 12 h) or H2O2 (50 µm, 4 h), respectively. As shown in Figure ##FIG##4##\n4a,c##, the fluorescence of the cells preincubated with the inhibitors prior to the treatment with Aβ42 oligomers was lower than that of the control group. This suggests that the Aβ42 oligomers increase Sph levels by elevating intracellular ROS and enhancing intracellular acidic and neutral sphingomyelinase and acidic ceramidase activities. However, the increase in intracellular Sph levels in H2O2‐treated cells appeared to be more correlated with neutral sphingomyelinase activity. This is supported by the reduced imaging intensity observed in cells pretreated with the nerve sphingomyelinase inhibitor, GW4869, as well as the reactive oxygen scavengers, NAC and Trolox (Figure ##FIG##4##4b,d##). Therefore, it is hypothesized that the possible mechanism of Aβ42 oligomers and H2O2‐induced up‐regulation of intracellular Sph levels is as follows: Aβ42 oligomers induced an increase in intracellular ROS levels, which then activated neutral sphingomyelinase (NSMase), leading to the hydrolysis of sphingomyelin (SM) and the production of ceramides (Cer). This process ultimately results in an increase in intracellular Sph levels. Moreover, Aβ42 oligomers can activate acidic sphingomyelinase (ASMase) and acidic ceramidase (ACDase), which also contribute to the hydrolysis of SM and Cer, thereby increasing Sph levels (Figure ##FIG##4##4e##).
","
To further validate the possible mechanisms proposed above, changes in ROS levels after different times of treatment of PC12 cells with Aβ42 oligomers were monitored using a ROS specific probe. We found that changes of ROS levels in cellular mitochondria upon Aβ42 oligomers treatment showed a gradual increase and reached the peak at 1 h. With prolonged treatment time, the ROS levels gradually decreased and eventually returned to the initial level (Figure ##FIG##5##\n5a–c##; Figure ##SUPPL##0##S46##, Supporting Information). The activations of SMase and CDase were then investigated. The treatment of PC12 cells with 1 µm Aβ42 oligomers and 50 µm H2O2 induced a time‐dependent increase in SMase activity and reached maximum levels at 6 and 3 h, respectively (Figure ##FIG##5##5d,e##; Figure ##SUPPL##0##S47##, Supporting Information). Notably, the widely used ASMase and NSMase inhibitors AMI and GW4869 strongly inhibited Aβ42 oligomers‐induced SMase activation, respectively, while only GW4869 inhibited H2O2‐induced SMase activation, and the ROS scavenger NAC and Trolox inhibited both the Aβ42 oligomers‐ and H2O2‐induced SMase activation (Figure ##FIG##5##5f,g##). Immunoblotting and immunofluorescence experiments provided additional support for the aforementioned experimental results (Figure ##FIG##5##5h–j##; Figure ##SUPPL##0##S48##, Supporting Information). This suggests that Aβ42 oligomers can induce the activation of ASMase and NSMase, respectively, whereas H2O2 can only induce the activation of NSMase. In addition, the ELISA test results for CDase indicated that the intracellular ceramidase level reached a maximum after 4 h of treatment with Aβ42 oligomers. It was observed that H2O2 did not activate intracellular CDase, but had an inhibitory effect, which aligns with the findings reported in the literature,[\n##REF##34459070##\n25\n##\n] (Figure ##FIG##5##5l,m##; Figure ##SUPPL##0##S49##, Supporting Information). Moreover, the activation effect of ROS scavenger to Aβ42 oligomers induced CDase activity and immunoblotting experiments on ACDase further supported that Aβ42 oligomers can activate ACDase, while H2O2 inhibits its activity (Figure ##FIG##5##5h,k##; Figure ##SUPPL##0##S50##, Supporting Information). Taken together, these results suggest that Aβ42 oligomers directly activate ASMase and ACDase, and also increase ROS levels, which activate NSMase and ultimately accelerate sphingolipid metabolism and increase the level of Sph.
","
Encouraged by the excellent results of live cell imaging, we further investigated the in vivo detection in a living animal model. The transparent zebrafish larva was selected as a model because zebrafish are optically transparent and have a high degree of homology with mammals.[\n##REF##21240407##\n26\n##\n] Zebrafish larvae (3 days old) were incubated with Aβ42 oligomers (10 εm) for 12 h and then treated with DMS‐2F (10 εm) for 2 h. As shown in Figure ##FIG##6##\n6\n##, zebrafish treated with DMS‐2F without Aβ42 oligomers exhibited weak fluorescence, and notably, the Aβ42 oligomers treated zebrafish showed bright red fluorescence in the brain, yolk sac, and intestine. However, the fluorescence intensity of zebrafish pretreated with inhibitors (AMI, GW4869, LCL‐521, and Trolox) and further treated with Aβ42 oligomers and DMS‐2F was significantly weaker than that of zebrafish treated without inhibitors. Based on these results, it is shown that in vivo, Aβ42 oligomers can still induce the upregulation of Sph levels by the same mechanism as in neuronal cells. Hence, this new probe DMS‐2F successfully realized the detection of Sph in vivo.
"],"string":"[\n \"Results and Discussion\",\n \"
The detailed synthesis procedure (Scheme ##SUPPL##0##S1##, Supporting Information) and structural characterization (Figures ##SUPPL##0##S1–S30##, Supporting Information) of fluorogenic probes DMS‐X are provided in the Supporting Information.
\",\n \"
To evaluate the stability of DMS‐X (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I), solutions of DMS‐X in PBS buffer were first exposed to a UV lamp (365 nm), and the change in fluorescence intensities of DMS‐X was monitored separately at different times. As shown in Figure ##SUPPL##0##S31a## (Supporting Information), the fluorescence intensities of DMS‐X were almost unchanged after 52 h of exposure to a UV lamp. The investigation of fluorescent changes of DMS‐X in different pH buffers at different times also showed excellent stability (Figure ##SUPPL##0##S31b–f##, Supporting Information). These results indicate that the DMS‐X probes have good photostability and acid‐base resistance properties.
\",\n \"
Next, the ability of DMS‐X probes (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to react with Sph under physiological conditions (pH 7.4, 37 °C) was tested using the vesicles of DMPC to mimic biological membranes. After reacting with Sph, the fluorescent emission results showed that only DMS‐2F had significant fluorescence enhancement at 660 nm (Figure ##FIG##1##\\n1a##), while the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) exhibited only slight fluorescence changes (Figure ##SUPPL##0##S32a–h##, Supporting Information). To further validate the superiority of DMS‐2F in detecting sphingosine, the fluorescence changes of DMS‐X were examined when incubated with Sph for different times in the DMPC system (with or without Sph). As shown in Figure ##FIG##1##1b##, an obvious fluorescence enhancement (I/I0 = 21.08) was detected in the DMS‐2F and Sph system when incubated at 37 °C for 10 min. With increasing incubation time, the fluorescence intensity of DMS‐2F gradually increased, reaching the highest intensity at 4 h (I/I0 = 57.60) (Figure ##FIG##1##1c##), which was fivefold faster than that reported for the Sph probe.[\\n##REF##33044062##\\n16\\n##\\n] However, the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) did not show significant fluorescence changes even after 6 h of incubation with Sph (Figure ##FIG##1##1b##; Figure ##SUPPL##0##S33##, Supporting Information). These findings suggest that DMS‐2F can be used as a highly efficient fluorogenic probe in response to Sph.
\",\n \"
To evaluate the appropriate pH range for the Sph recognition process of DMS‐2F, pH control experiments were carefully carried out in PBS‐buffered solutions (pH 3 to 9). After incubation with 200 µm Sph in buffer solutions of different pH values for 30 min at 37 °C, the fluorescence of DMS‐2F at 660 nm was significantly enhanced when the pH ranged from 6 to 9 (Figure ##SUPPL##0##S34##, Supporting Information), indicating that DMS‐2F could efficiently fluorogenically detect Sph in the physiological pH range.
\",\n \"
Concentration‐dependent fluorescence experiments were subsequently performed by incubating DMS‐2F (5 µm) with various concentrations of Sph (0–200 µm) in DMPC vesicles for 30 min at 37 °C. The results showed that the fluorescence intensity of DMS‐2F at 660 nm gradually increased with increasing Sph concentrations (Figure ##FIG##1##1d##). Moreover, an excellent linear relationship between the emission intensities was observed in the range of 0–110 µm (Figure ##FIG##1##1e##). The limit of detection (LOD) of DMS‐2F for Sph was calculated as 9.33 ± 0.41 nm according to the 3σ/m method. These findings suggest that DMS‐2F has the capability for ultrasensitive detection of Sph.
\",\n \"
To further determine whether DMS‐2F could selectively detect Sph in living cells, DMS‐2F (5 εm) was incubated in DMPC vesicles composed of several naturally abundant lipid species and other possible interferents (including amino acids with structures similar to Sph, such as cysteine (Cys), lysine (Lys), serine (Ser), threonine (Thr), tyrosine (Tyr) (260 εm) and glutathione (GSH) (2 mm); naturally abundant lipid species, sphingomyelin (SM, 200 εm) and ceramide (Cer, 200 εm); and neurotransmitters: NE (norepinephrine, 200 εm), DA (dopamine, 200 εm), and EP (epinephrine, 200 εm) for 30 min at 37 °C. The results showed that a significant fluorescence turn‐on was observed when DMS‐2F (5 εm) was incubated with Sph (200 εm). However, when DMS‐2F was incubated with another analyte, there was a weak change in fluorescence intensity compared to the untreated DMS‐2F probe (Figure ##FIG##1##1f##). These results are consistent with the design of the DMS‐2F probe to selectively detect Sph in the DMPC system.
\",\n \"
The specific response mechanism of DMS‐2F to Sph was confirmed by liquid chromatograph mass spectrometer (LC‐MS), high‐resolution mass spectrometry (HR‐MS), fluorescence spectroscopy, UV‐Visible spectroscopy, and theoretical calculation. DMS‐2F was exposed to Sph for 6 h at 37 °C, followed by LC−MS analysis and the reaction products of a five‐membered cyclic oxazolidinone‐based Sph compound (0.83 min) and the fluorophore DM‐2F (3.57 min) were successfully detected (Figure ##FIG##2##\\n2a–c##). In addition, HR‐MS experiments again confirmed that the fluorophore was released after DMS‐2F reacted with Sph, producing a five‐membered cyclic oxazolidinone‐based Sph compound (Figure ##SUPPL##0##S35##, Supporting Information). Subsequently, the UV−vis and fluorescence response of DMS‐2F to Sph were determined by adding 200 εm Sph to 1 mL of PBS buffer containing 5 εm\\nDMS‐2F and incubating at 37 °C for 6 h. As illustrated in Figure ##FIG##2##2d##, incubation with Sph resulted in a decrease in the UV‒vis absorption of DMS‐2F at 408 nm and an increase in absorption at 463 nm. The spectral properties of the system after reaction with Sph were in good agreement with the fluorophore DM‐2F. These results indicated that the response mechanism of DMS‐2F to Sph should be attributed to the nucleophilic addition and elimination reaction between ─NH2 of Sph and the thiocarbonate‐ester group of DMS‐2F. When the reaction is triggered, the fluorophore DM‐2F is released and emits significant fluorescence. The probable response mechanism of DMS‐2F to Sph is shown in Scheme ##FIG##0##1##. According to the proposed mechanism above, the bond cleavage between the carbon atom of the carbonyl group (C20) and the aryl oxide (O21) in probes is the key factor for the fluorogenic response of DMS‐X to Sph. Therefore, the bond energy of C20─O21 was a determining factor for screening the optimal probe for the response of Sph. To further understand the response priority of DMS‐2F and other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to Sph, theoretical calculations were carried out. The structures of DMS‐X were optimized by density functional theory (DFT) studies using the B3LYP 6–31G (d, p) levels (Figure ##FIG##2##2f,g##: Figure ##SUPPL##0##S36##, Supporting Information), and the bond lengths of C20─O21 in DMS‐X were evaluated. As shown in Figure ##FIG##2##2e##, the bond length of DMS‐2F is significantly longer than that of the other probes, which further indicates that DMS‐2F has a better response effect than the other probes. Additionally, the ground‐state geometry structures of Sph and NE were optimized by DFT at the B3LYP/6‐31G levels to investigate the selective response mechanism of DMS‐2F toward Sph. The calculation results indicate that there are hydrogen bonding interactions between the amino group of NE and its neighboring hydroxyl group (Figure ##SUPPL##0##S37a##, Supporting Information), which was detrimental to the DMS‐2F response to NE, whereas such interactions are absent in the structure of Sph (Figure ##SUPPL##0##S37b##, Supporting Information). Additionally, the electrostatic surface potential (ESP) maps reveal that the amino moieties of Sph possess higher electronegativity compared to those of NE (Figures ##SUPPL##0##S37c,d##, Supporting Information). These findings suggest that Sph can readily react with DMS‐2F, leading to its selective response.
\",\n \"
These promising in vitro results prompted us to explore the imaging effect of DMS‐X in living cells. Using MTT assays, it was confirmed that DMS‐X was non‐toxic to different cell lines (Figures ##SUPPL##0##S38## and ##SUPPL##0##S39##, Supporting Information). First, to determine whether DMS‐2F could react with Sph in live cells, A549 cells were incubated with DMS‐2F (10 εm) for 30 min, and then the cell medium was exchanged with a new medium containing 40 εm Sph and incubated for different amounts of time (30–150 min). As shown in Figure ##SUPPL##0##S40## (Supporting Information), as the Sph incubation time increased, the cells showed a gradual enhancement in red fluorescence emission within 1.5 h. To assess whether the response of DMS‐2F to Sph is concentration‐dependent in living cells, DMS‐2F‐pretreated A549 and HepG2 cells were incubated with different concentrations of Sph (0, 5, 10, and 20 εM). After 2 h of incubation, cells treated with exogenous Sph showed a dose‐dependent fluorescence enhancement signal (Figure ##SUPPL##0##S41##, Supporting Information). Furthermore, to confirm that the observed increase in cellular fluorescence was a result of the release of the fluorophore DM‐2F after the reaction between DMS‐2F and Sph, A549 cells were further treated with DM‐2F (10 εm) (Figure ##SUPPL##0##S42##, Supporting Information). Cells treated with DM‐2F exhibited a fluorescent imaging pattern consistent with that observed in cells treated with DMS‐2F and Sph. These results indicate that DMS‐2F could react with Sph in living cells within a short period of time and release an enhanced fluorescence signal by generating the fluorophore DM‐2F in a dose‐dependent manner.
\",\n \"
Since elevated sphingosine in mammalian cells is closely associated with apoptosis, to evaluate the response ability of DMS‐2F to endogenous Sph levels in live cells, cancer cells (rat adrenal pheochromocytoma PC12 cells, human non‐small cell lung cancer A549 cells, and brain glioma U87 cell lines) and normal lung fibroblast MRC‐5 cells were exposed to serum‐free medium (opti‐MEM containing 10 εm\\nDMS‐2F), which could result in elevated Sph levels. As shown in Figure ##SUPPL##0##S43a,b## (Supporting Information), after 2 h of incubation, the fluorescence of live cells treated with opti‐MEM all showed a significant increase. Taken together, these results suggest that DMS‐2F is sensitive enough to probe native sphingosine in various live cells, and can be used for imaging differences in sphingosine levels in living cells.
\",\n \"
Abnormal sphingolipid metabolism has been reported to induce Alzheimer's disease (AD), and substantial evidence also support that amyloid‐β (Aβ42) plays an important role in AD. In vitro, Aβ42 has been shown to induce apoptosis via the sphingomyelin pathway in various brain cells, including PC12 cells, etc.[\\n##REF##18547682##\\n10\\n##, ##REF##14748735##\\n18\\n##\\n] Accordingly, we attempted to assess whether the level of Sph will be changed in PC12 cells after treatment with Aβ42 peptide. We first treated PC12 cells with 10 εm Aβ42 oligomers for different times and then incubated them with DMS‐2F, as shown in Figure ##SUPPL##0##S44## (Supporting Information), the fluorescence of PC12 cells treated with Aβ42 oligomers was significantly increased. These observations undoubtedly revealed that intracellular Sph was up‐regulated when neuronal cells were challenged by Aβ42 oligomers. To further correlate Sph generation with Aβ42 oligomers, PC12 cells were treated with different concentrations of Aβ42 oligomers and Aβ42 monomers. After 12 h of incubation, a gradual increase in fluorescence was observed in PC12 cells treated with Aβ42 oligomers, however, weak fluorescence changes were observed in Aβ42 monomers treated cells (Figure ##FIG##3##\\n3a,b,d,e##). Together, these findings suggested that neuronal cells experienced overexpression of Sph during Aβ42 administration and that the up‐regulated Sph levels correlated with both the incubation time and dosage of Aβ42 oligomers, indicating that the abnormal sphingolipid metabolism in neuronal cells is tightly related to the stimulation of Aβ42 oligomers.
\",\n \"
Since the overexpression of Aβ protein induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade, and previous studies have illustrated that H2O2 could induce neurotoxicity in PC12 cells,[\\n##REF##14748735##\\n18\\n##, ##REF##22133762##\\n19\\n##\\n] we further investigated the Sph variations induced by H2O2. PC12 cells were treated with different concentrations of H2O2, after 4 h of incubation, a gradual increase in fluorescence was detected in PC12 cells treated with H2O2 (Figure ##FIG##3##3c,f##), suggesting that the Sph variation occurred during the oxidative stress process. In addition, the changes of Sph levels after being treated with Aβ42 oligomers and H2O2 in other cell lines were also assessed. U87, A549, HepG2, and MRC5 cells were treated with Aβ42 oligomers and H2O2 for 12 and 4 h, respectively, and then incubated with DMS‐2F for 2 h. As shown in Figure ##SUPPL##0##S45## (Supporting Information), significant fluorescence enhancement was exhibited in PC12 and U87 cells, while the fluorescence in other cell lines was not evident. These results indicate that the fluorogenic probe DMS‐2F can successfully monitor the level changes of Sph during Aβ42 oligomers and H2O2‐induced apoptosis in neuronal cells.
\",\n \"
Since changes in Sph levels are associated with the abnormal sphingolipid metabolism in cells, to investigate the possible mechanisms of Aβ42 oligomers and H2O2‐induced changes in Sph levels, we further investigated intracellular sphingomyelinase and ceramidase activities in Aβ42 oligomers and H2O2 treated cells. PC12 cells were preincubated for 2 h in the presence of 10 µm amitriptyline hydrochloride (AMI, an acid sphingomyelinase inhibitor[\\n##UREF##0##\\n20\\n##\\n]), 20 µm GW4869 (a neutral sphingomyelinase inhibitor[\\n##REF##20629193##\\n21\\n##\\n]), 10 µm LCL‐521 (an acid ceramidase inhibitor[\\n##REF##25456083##\\n22\\n##\\n]), 500 µm N‐acetyl‐cysteine (NAC, an efficient antioxidant[\\n##UREF##1##\\n23\\n##\\n]) and 1 mm Trolox (a reactive oxygen scavenger [\\n##REF##17013382##\\n24\\n##\\n]), and then treated with Aβ42 oligomers (10 µm, 12 h) or H2O2 (50 µm, 4 h), respectively. As shown in Figure ##FIG##4##\\n4a,c##, the fluorescence of the cells preincubated with the inhibitors prior to the treatment with Aβ42 oligomers was lower than that of the control group. This suggests that the Aβ42 oligomers increase Sph levels by elevating intracellular ROS and enhancing intracellular acidic and neutral sphingomyelinase and acidic ceramidase activities. However, the increase in intracellular Sph levels in H2O2‐treated cells appeared to be more correlated with neutral sphingomyelinase activity. This is supported by the reduced imaging intensity observed in cells pretreated with the nerve sphingomyelinase inhibitor, GW4869, as well as the reactive oxygen scavengers, NAC and Trolox (Figure ##FIG##4##4b,d##). Therefore, it is hypothesized that the possible mechanism of Aβ42 oligomers and H2O2‐induced up‐regulation of intracellular Sph levels is as follows: Aβ42 oligomers induced an increase in intracellular ROS levels, which then activated neutral sphingomyelinase (NSMase), leading to the hydrolysis of sphingomyelin (SM) and the production of ceramides (Cer). This process ultimately results in an increase in intracellular Sph levels. Moreover, Aβ42 oligomers can activate acidic sphingomyelinase (ASMase) and acidic ceramidase (ACDase), which also contribute to the hydrolysis of SM and Cer, thereby increasing Sph levels (Figure ##FIG##4##4e##).
\",\n \"
To further validate the possible mechanisms proposed above, changes in ROS levels after different times of treatment of PC12 cells with Aβ42 oligomers were monitored using a ROS specific probe. We found that changes of ROS levels in cellular mitochondria upon Aβ42 oligomers treatment showed a gradual increase and reached the peak at 1 h. With prolonged treatment time, the ROS levels gradually decreased and eventually returned to the initial level (Figure ##FIG##5##\\n5a–c##; Figure ##SUPPL##0##S46##, Supporting Information). The activations of SMase and CDase were then investigated. The treatment of PC12 cells with 1 µm Aβ42 oligomers and 50 µm H2O2 induced a time‐dependent increase in SMase activity and reached maximum levels at 6 and 3 h, respectively (Figure ##FIG##5##5d,e##; Figure ##SUPPL##0##S47##, Supporting Information). Notably, the widely used ASMase and NSMase inhibitors AMI and GW4869 strongly inhibited Aβ42 oligomers‐induced SMase activation, respectively, while only GW4869 inhibited H2O2‐induced SMase activation, and the ROS scavenger NAC and Trolox inhibited both the Aβ42 oligomers‐ and H2O2‐induced SMase activation (Figure ##FIG##5##5f,g##). Immunoblotting and immunofluorescence experiments provided additional support for the aforementioned experimental results (Figure ##FIG##5##5h–j##; Figure ##SUPPL##0##S48##, Supporting Information). This suggests that Aβ42 oligomers can induce the activation of ASMase and NSMase, respectively, whereas H2O2 can only induce the activation of NSMase. In addition, the ELISA test results for CDase indicated that the intracellular ceramidase level reached a maximum after 4 h of treatment with Aβ42 oligomers. It was observed that H2O2 did not activate intracellular CDase, but had an inhibitory effect, which aligns with the findings reported in the literature,[\\n##REF##34459070##\\n25\\n##\\n] (Figure ##FIG##5##5l,m##; Figure ##SUPPL##0##S49##, Supporting Information). Moreover, the activation effect of ROS scavenger to Aβ42 oligomers induced CDase activity and immunoblotting experiments on ACDase further supported that Aβ42 oligomers can activate ACDase, while H2O2 inhibits its activity (Figure ##FIG##5##5h,k##; Figure ##SUPPL##0##S50##, Supporting Information). Taken together, these results suggest that Aβ42 oligomers directly activate ASMase and ACDase, and also increase ROS levels, which activate NSMase and ultimately accelerate sphingolipid metabolism and increase the level of Sph.
\",\n \"
Encouraged by the excellent results of live cell imaging, we further investigated the in vivo detection in a living animal model. The transparent zebrafish larva was selected as a model because zebrafish are optically transparent and have a high degree of homology with mammals.[\\n##REF##21240407##\\n26\\n##\\n] Zebrafish larvae (3 days old) were incubated with Aβ42 oligomers (10 εm) for 12 h and then treated with DMS‐2F (10 εm) for 2 h. As shown in Figure ##FIG##6##\\n6\\n##, zebrafish treated with DMS‐2F without Aβ42 oligomers exhibited weak fluorescence, and notably, the Aβ42 oligomers treated zebrafish showed bright red fluorescence in the brain, yolk sac, and intestine. However, the fluorescence intensity of zebrafish pretreated with inhibitors (AMI, GW4869, LCL‐521, and Trolox) and further treated with Aβ42 oligomers and DMS‐2F was significantly weaker than that of zebrafish treated without inhibitors. Based on these results, it is shown that in vivo, Aβ42 oligomers can still induce the upregulation of Sph levels by the same mechanism as in neuronal cells. Hence, this new probe DMS‐2F successfully realized the detection of Sph in vivo.
\"\n]"},"discussion":{"kind":"list like","value":["Results and Discussion","
The detailed synthesis procedure (Scheme ##SUPPL##0##S1##, Supporting Information) and structural characterization (Figures ##SUPPL##0##S1–S30##, Supporting Information) of fluorogenic probes DMS‐X are provided in the Supporting Information.
","
To evaluate the stability of DMS‐X (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I), solutions of DMS‐X in PBS buffer were first exposed to a UV lamp (365 nm), and the change in fluorescence intensities of DMS‐X was monitored separately at different times. As shown in Figure ##SUPPL##0##S31a## (Supporting Information), the fluorescence intensities of DMS‐X were almost unchanged after 52 h of exposure to a UV lamp. The investigation of fluorescent changes of DMS‐X in different pH buffers at different times also showed excellent stability (Figure ##SUPPL##0##S31b–f##, Supporting Information). These results indicate that the DMS‐X probes have good photostability and acid‐base resistance properties.
","
Next, the ability of DMS‐X probes (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to react with Sph under physiological conditions (pH 7.4, 37 °C) was tested using the vesicles of DMPC to mimic biological membranes. After reacting with Sph, the fluorescent emission results showed that only DMS‐2F had significant fluorescence enhancement at 660 nm (Figure ##FIG##1##\n1a##), while the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) exhibited only slight fluorescence changes (Figure ##SUPPL##0##S32a–h##, Supporting Information). To further validate the superiority of DMS‐2F in detecting sphingosine, the fluorescence changes of DMS‐X were examined when incubated with Sph for different times in the DMPC system (with or without Sph). As shown in Figure ##FIG##1##1b##, an obvious fluorescence enhancement (I/I0 = 21.08) was detected in the DMS‐2F and Sph system when incubated at 37 °C for 10 min. With increasing incubation time, the fluorescence intensity of DMS‐2F gradually increased, reaching the highest intensity at 4 h (I/I0 = 57.60) (Figure ##FIG##1##1c##), which was fivefold faster than that reported for the Sph probe.[\n##REF##33044062##\n16\n##\n] However, the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) did not show significant fluorescence changes even after 6 h of incubation with Sph (Figure ##FIG##1##1b##; Figure ##SUPPL##0##S33##, Supporting Information). These findings suggest that DMS‐2F can be used as a highly efficient fluorogenic probe in response to Sph.
","
To evaluate the appropriate pH range for the Sph recognition process of DMS‐2F, pH control experiments were carefully carried out in PBS‐buffered solutions (pH 3 to 9). After incubation with 200 µm Sph in buffer solutions of different pH values for 30 min at 37 °C, the fluorescence of DMS‐2F at 660 nm was significantly enhanced when the pH ranged from 6 to 9 (Figure ##SUPPL##0##S34##, Supporting Information), indicating that DMS‐2F could efficiently fluorogenically detect Sph in the physiological pH range.
","
Concentration‐dependent fluorescence experiments were subsequently performed by incubating DMS‐2F (5 µm) with various concentrations of Sph (0–200 µm) in DMPC vesicles for 30 min at 37 °C. The results showed that the fluorescence intensity of DMS‐2F at 660 nm gradually increased with increasing Sph concentrations (Figure ##FIG##1##1d##). Moreover, an excellent linear relationship between the emission intensities was observed in the range of 0–110 µm (Figure ##FIG##1##1e##). The limit of detection (LOD) of DMS‐2F for Sph was calculated as 9.33 ± 0.41 nm according to the 3σ/m method. These findings suggest that DMS‐2F has the capability for ultrasensitive detection of Sph.
","
To further determine whether DMS‐2F could selectively detect Sph in living cells, DMS‐2F (5 εm) was incubated in DMPC vesicles composed of several naturally abundant lipid species and other possible interferents (including amino acids with structures similar to Sph, such as cysteine (Cys), lysine (Lys), serine (Ser), threonine (Thr), tyrosine (Tyr) (260 εm) and glutathione (GSH) (2 mm); naturally abundant lipid species, sphingomyelin (SM, 200 εm) and ceramide (Cer, 200 εm); and neurotransmitters: NE (norepinephrine, 200 εm), DA (dopamine, 200 εm), and EP (epinephrine, 200 εm) for 30 min at 37 °C. The results showed that a significant fluorescence turn‐on was observed when DMS‐2F (5 εm) was incubated with Sph (200 εm). However, when DMS‐2F was incubated with another analyte, there was a weak change in fluorescence intensity compared to the untreated DMS‐2F probe (Figure ##FIG##1##1f##). These results are consistent with the design of the DMS‐2F probe to selectively detect Sph in the DMPC system.
","
The specific response mechanism of DMS‐2F to Sph was confirmed by liquid chromatograph mass spectrometer (LC‐MS), high‐resolution mass spectrometry (HR‐MS), fluorescence spectroscopy, UV‐Visible spectroscopy, and theoretical calculation. DMS‐2F was exposed to Sph for 6 h at 37 °C, followed by LC−MS analysis and the reaction products of a five‐membered cyclic oxazolidinone‐based Sph compound (0.83 min) and the fluorophore DM‐2F (3.57 min) were successfully detected (Figure ##FIG##2##\n2a–c##). In addition, HR‐MS experiments again confirmed that the fluorophore was released after DMS‐2F reacted with Sph, producing a five‐membered cyclic oxazolidinone‐based Sph compound (Figure ##SUPPL##0##S35##, Supporting Information). Subsequently, the UV−vis and fluorescence response of DMS‐2F to Sph were determined by adding 200 εm Sph to 1 mL of PBS buffer containing 5 εm\nDMS‐2F and incubating at 37 °C for 6 h. As illustrated in Figure ##FIG##2##2d##, incubation with Sph resulted in a decrease in the UV‒vis absorption of DMS‐2F at 408 nm and an increase in absorption at 463 nm. The spectral properties of the system after reaction with Sph were in good agreement with the fluorophore DM‐2F. These results indicated that the response mechanism of DMS‐2F to Sph should be attributed to the nucleophilic addition and elimination reaction between ─NH2 of Sph and the thiocarbonate‐ester group of DMS‐2F. When the reaction is triggered, the fluorophore DM‐2F is released and emits significant fluorescence. The probable response mechanism of DMS‐2F to Sph is shown in Scheme ##FIG##0##1##. According to the proposed mechanism above, the bond cleavage between the carbon atom of the carbonyl group (C20) and the aryl oxide (O21) in probes is the key factor for the fluorogenic response of DMS‐X to Sph. Therefore, the bond energy of C20─O21 was a determining factor for screening the optimal probe for the response of Sph. To further understand the response priority of DMS‐2F and other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to Sph, theoretical calculations were carried out. The structures of DMS‐X were optimized by density functional theory (DFT) studies using the B3LYP 6–31G (d, p) levels (Figure ##FIG##2##2f,g##: Figure ##SUPPL##0##S36##, Supporting Information), and the bond lengths of C20─O21 in DMS‐X were evaluated. As shown in Figure ##FIG##2##2e##, the bond length of DMS‐2F is significantly longer than that of the other probes, which further indicates that DMS‐2F has a better response effect than the other probes. Additionally, the ground‐state geometry structures of Sph and NE were optimized by DFT at the B3LYP/6‐31G levels to investigate the selective response mechanism of DMS‐2F toward Sph. The calculation results indicate that there are hydrogen bonding interactions between the amino group of NE and its neighboring hydroxyl group (Figure ##SUPPL##0##S37a##, Supporting Information), which was detrimental to the DMS‐2F response to NE, whereas such interactions are absent in the structure of Sph (Figure ##SUPPL##0##S37b##, Supporting Information). Additionally, the electrostatic surface potential (ESP) maps reveal that the amino moieties of Sph possess higher electronegativity compared to those of NE (Figures ##SUPPL##0##S37c,d##, Supporting Information). These findings suggest that Sph can readily react with DMS‐2F, leading to its selective response.
","
These promising in vitro results prompted us to explore the imaging effect of DMS‐X in living cells. Using MTT assays, it was confirmed that DMS‐X was non‐toxic to different cell lines (Figures ##SUPPL##0##S38## and ##SUPPL##0##S39##, Supporting Information). First, to determine whether DMS‐2F could react with Sph in live cells, A549 cells were incubated with DMS‐2F (10 εm) for 30 min, and then the cell medium was exchanged with a new medium containing 40 εm Sph and incubated for different amounts of time (30–150 min). As shown in Figure ##SUPPL##0##S40## (Supporting Information), as the Sph incubation time increased, the cells showed a gradual enhancement in red fluorescence emission within 1.5 h. To assess whether the response of DMS‐2F to Sph is concentration‐dependent in living cells, DMS‐2F‐pretreated A549 and HepG2 cells were incubated with different concentrations of Sph (0, 5, 10, and 20 εM). After 2 h of incubation, cells treated with exogenous Sph showed a dose‐dependent fluorescence enhancement signal (Figure ##SUPPL##0##S41##, Supporting Information). Furthermore, to confirm that the observed increase in cellular fluorescence was a result of the release of the fluorophore DM‐2F after the reaction between DMS‐2F and Sph, A549 cells were further treated with DM‐2F (10 εm) (Figure ##SUPPL##0##S42##, Supporting Information). Cells treated with DM‐2F exhibited a fluorescent imaging pattern consistent with that observed in cells treated with DMS‐2F and Sph. These results indicate that DMS‐2F could react with Sph in living cells within a short period of time and release an enhanced fluorescence signal by generating the fluorophore DM‐2F in a dose‐dependent manner.
","
Since elevated sphingosine in mammalian cells is closely associated with apoptosis, to evaluate the response ability of DMS‐2F to endogenous Sph levels in live cells, cancer cells (rat adrenal pheochromocytoma PC12 cells, human non‐small cell lung cancer A549 cells, and brain glioma U87 cell lines) and normal lung fibroblast MRC‐5 cells were exposed to serum‐free medium (opti‐MEM containing 10 εm\nDMS‐2F), which could result in elevated Sph levels. As shown in Figure ##SUPPL##0##S43a,b## (Supporting Information), after 2 h of incubation, the fluorescence of live cells treated with opti‐MEM all showed a significant increase. Taken together, these results suggest that DMS‐2F is sensitive enough to probe native sphingosine in various live cells, and can be used for imaging differences in sphingosine levels in living cells.
","
Abnormal sphingolipid metabolism has been reported to induce Alzheimer's disease (AD), and substantial evidence also support that amyloid‐β (Aβ42) plays an important role in AD. In vitro, Aβ42 has been shown to induce apoptosis via the sphingomyelin pathway in various brain cells, including PC12 cells, etc.[\n##REF##18547682##\n10\n##, ##REF##14748735##\n18\n##\n] Accordingly, we attempted to assess whether the level of Sph will be changed in PC12 cells after treatment with Aβ42 peptide. We first treated PC12 cells with 10 εm Aβ42 oligomers for different times and then incubated them with DMS‐2F, as shown in Figure ##SUPPL##0##S44## (Supporting Information), the fluorescence of PC12 cells treated with Aβ42 oligomers was significantly increased. These observations undoubtedly revealed that intracellular Sph was up‐regulated when neuronal cells were challenged by Aβ42 oligomers. To further correlate Sph generation with Aβ42 oligomers, PC12 cells were treated with different concentrations of Aβ42 oligomers and Aβ42 monomers. After 12 h of incubation, a gradual increase in fluorescence was observed in PC12 cells treated with Aβ42 oligomers, however, weak fluorescence changes were observed in Aβ42 monomers treated cells (Figure ##FIG##3##\n3a,b,d,e##). Together, these findings suggested that neuronal cells experienced overexpression of Sph during Aβ42 administration and that the up‐regulated Sph levels correlated with both the incubation time and dosage of Aβ42 oligomers, indicating that the abnormal sphingolipid metabolism in neuronal cells is tightly related to the stimulation of Aβ42 oligomers.
","
Since the overexpression of Aβ protein induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade, and previous studies have illustrated that H2O2 could induce neurotoxicity in PC12 cells,[\n##REF##14748735##\n18\n##, ##REF##22133762##\n19\n##\n] we further investigated the Sph variations induced by H2O2. PC12 cells were treated with different concentrations of H2O2, after 4 h of incubation, a gradual increase in fluorescence was detected in PC12 cells treated with H2O2 (Figure ##FIG##3##3c,f##), suggesting that the Sph variation occurred during the oxidative stress process. In addition, the changes of Sph levels after being treated with Aβ42 oligomers and H2O2 in other cell lines were also assessed. U87, A549, HepG2, and MRC5 cells were treated with Aβ42 oligomers and H2O2 for 12 and 4 h, respectively, and then incubated with DMS‐2F for 2 h. As shown in Figure ##SUPPL##0##S45## (Supporting Information), significant fluorescence enhancement was exhibited in PC12 and U87 cells, while the fluorescence in other cell lines was not evident. These results indicate that the fluorogenic probe DMS‐2F can successfully monitor the level changes of Sph during Aβ42 oligomers and H2O2‐induced apoptosis in neuronal cells.
","
Since changes in Sph levels are associated with the abnormal sphingolipid metabolism in cells, to investigate the possible mechanisms of Aβ42 oligomers and H2O2‐induced changes in Sph levels, we further investigated intracellular sphingomyelinase and ceramidase activities in Aβ42 oligomers and H2O2 treated cells. PC12 cells were preincubated for 2 h in the presence of 10 µm amitriptyline hydrochloride (AMI, an acid sphingomyelinase inhibitor[\n##UREF##0##\n20\n##\n]), 20 µm GW4869 (a neutral sphingomyelinase inhibitor[\n##REF##20629193##\n21\n##\n]), 10 µm LCL‐521 (an acid ceramidase inhibitor[\n##REF##25456083##\n22\n##\n]), 500 µm N‐acetyl‐cysteine (NAC, an efficient antioxidant[\n##UREF##1##\n23\n##\n]) and 1 mm Trolox (a reactive oxygen scavenger [\n##REF##17013382##\n24\n##\n]), and then treated with Aβ42 oligomers (10 µm, 12 h) or H2O2 (50 µm, 4 h), respectively. As shown in Figure ##FIG##4##\n4a,c##, the fluorescence of the cells preincubated with the inhibitors prior to the treatment with Aβ42 oligomers was lower than that of the control group. This suggests that the Aβ42 oligomers increase Sph levels by elevating intracellular ROS and enhancing intracellular acidic and neutral sphingomyelinase and acidic ceramidase activities. However, the increase in intracellular Sph levels in H2O2‐treated cells appeared to be more correlated with neutral sphingomyelinase activity. This is supported by the reduced imaging intensity observed in cells pretreated with the nerve sphingomyelinase inhibitor, GW4869, as well as the reactive oxygen scavengers, NAC and Trolox (Figure ##FIG##4##4b,d##). Therefore, it is hypothesized that the possible mechanism of Aβ42 oligomers and H2O2‐induced up‐regulation of intracellular Sph levels is as follows: Aβ42 oligomers induced an increase in intracellular ROS levels, which then activated neutral sphingomyelinase (NSMase), leading to the hydrolysis of sphingomyelin (SM) and the production of ceramides (Cer). This process ultimately results in an increase in intracellular Sph levels. Moreover, Aβ42 oligomers can activate acidic sphingomyelinase (ASMase) and acidic ceramidase (ACDase), which also contribute to the hydrolysis of SM and Cer, thereby increasing Sph levels (Figure ##FIG##4##4e##).
","
To further validate the possible mechanisms proposed above, changes in ROS levels after different times of treatment of PC12 cells with Aβ42 oligomers were monitored using a ROS specific probe. We found that changes of ROS levels in cellular mitochondria upon Aβ42 oligomers treatment showed a gradual increase and reached the peak at 1 h. With prolonged treatment time, the ROS levels gradually decreased and eventually returned to the initial level (Figure ##FIG##5##\n5a–c##; Figure ##SUPPL##0##S46##, Supporting Information). The activations of SMase and CDase were then investigated. The treatment of PC12 cells with 1 µm Aβ42 oligomers and 50 µm H2O2 induced a time‐dependent increase in SMase activity and reached maximum levels at 6 and 3 h, respectively (Figure ##FIG##5##5d,e##; Figure ##SUPPL##0##S47##, Supporting Information). Notably, the widely used ASMase and NSMase inhibitors AMI and GW4869 strongly inhibited Aβ42 oligomers‐induced SMase activation, respectively, while only GW4869 inhibited H2O2‐induced SMase activation, and the ROS scavenger NAC and Trolox inhibited both the Aβ42 oligomers‐ and H2O2‐induced SMase activation (Figure ##FIG##5##5f,g##). Immunoblotting and immunofluorescence experiments provided additional support for the aforementioned experimental results (Figure ##FIG##5##5h–j##; Figure ##SUPPL##0##S48##, Supporting Information). This suggests that Aβ42 oligomers can induce the activation of ASMase and NSMase, respectively, whereas H2O2 can only induce the activation of NSMase. In addition, the ELISA test results for CDase indicated that the intracellular ceramidase level reached a maximum after 4 h of treatment with Aβ42 oligomers. It was observed that H2O2 did not activate intracellular CDase, but had an inhibitory effect, which aligns with the findings reported in the literature,[\n##REF##34459070##\n25\n##\n] (Figure ##FIG##5##5l,m##; Figure ##SUPPL##0##S49##, Supporting Information). Moreover, the activation effect of ROS scavenger to Aβ42 oligomers induced CDase activity and immunoblotting experiments on ACDase further supported that Aβ42 oligomers can activate ACDase, while H2O2 inhibits its activity (Figure ##FIG##5##5h,k##; Figure ##SUPPL##0##S50##, Supporting Information). Taken together, these results suggest that Aβ42 oligomers directly activate ASMase and ACDase, and also increase ROS levels, which activate NSMase and ultimately accelerate sphingolipid metabolism and increase the level of Sph.
","
Encouraged by the excellent results of live cell imaging, we further investigated the in vivo detection in a living animal model. The transparent zebrafish larva was selected as a model because zebrafish are optically transparent and have a high degree of homology with mammals.[\n##REF##21240407##\n26\n##\n] Zebrafish larvae (3 days old) were incubated with Aβ42 oligomers (10 εm) for 12 h and then treated with DMS‐2F (10 εm) for 2 h. As shown in Figure ##FIG##6##\n6\n##, zebrafish treated with DMS‐2F without Aβ42 oligomers exhibited weak fluorescence, and notably, the Aβ42 oligomers treated zebrafish showed bright red fluorescence in the brain, yolk sac, and intestine. However, the fluorescence intensity of zebrafish pretreated with inhibitors (AMI, GW4869, LCL‐521, and Trolox) and further treated with Aβ42 oligomers and DMS‐2F was significantly weaker than that of zebrafish treated without inhibitors. Based on these results, it is shown that in vivo, Aβ42 oligomers can still induce the upregulation of Sph levels by the same mechanism as in neuronal cells. Hence, this new probe DMS‐2F successfully realized the detection of Sph in vivo.
"],"string":"[\n \"Results and Discussion\",\n \"
The detailed synthesis procedure (Scheme ##SUPPL##0##S1##, Supporting Information) and structural characterization (Figures ##SUPPL##0##S1–S30##, Supporting Information) of fluorogenic probes DMS‐X are provided in the Supporting Information.
\",\n \"
To evaluate the stability of DMS‐X (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I), solutions of DMS‐X in PBS buffer were first exposed to a UV lamp (365 nm), and the change in fluorescence intensities of DMS‐X was monitored separately at different times. As shown in Figure ##SUPPL##0##S31a## (Supporting Information), the fluorescence intensities of DMS‐X were almost unchanged after 52 h of exposure to a UV lamp. The investigation of fluorescent changes of DMS‐X in different pH buffers at different times also showed excellent stability (Figure ##SUPPL##0##S31b–f##, Supporting Information). These results indicate that the DMS‐X probes have good photostability and acid‐base resistance properties.
\",\n \"
Next, the ability of DMS‐X probes (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to react with Sph under physiological conditions (pH 7.4, 37 °C) was tested using the vesicles of DMPC to mimic biological membranes. After reacting with Sph, the fluorescent emission results showed that only DMS‐2F had significant fluorescence enhancement at 660 nm (Figure ##FIG##1##\\n1a##), while the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) exhibited only slight fluorescence changes (Figure ##SUPPL##0##S32a–h##, Supporting Information). To further validate the superiority of DMS‐2F in detecting sphingosine, the fluorescence changes of DMS‐X were examined when incubated with Sph for different times in the DMPC system (with or without Sph). As shown in Figure ##FIG##1##1b##, an obvious fluorescence enhancement (I/I0 = 21.08) was detected in the DMS‐2F and Sph system when incubated at 37 °C for 10 min. With increasing incubation time, the fluorescence intensity of DMS‐2F gradually increased, reaching the highest intensity at 4 h (I/I0 = 57.60) (Figure ##FIG##1##1c##), which was fivefold faster than that reported for the Sph probe.[\\n##REF##33044062##\\n16\\n##\\n] However, the other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) did not show significant fluorescence changes even after 6 h of incubation with Sph (Figure ##FIG##1##1b##; Figure ##SUPPL##0##S33##, Supporting Information). These findings suggest that DMS‐2F can be used as a highly efficient fluorogenic probe in response to Sph.
\",\n \"
To evaluate the appropriate pH range for the Sph recognition process of DMS‐2F, pH control experiments were carefully carried out in PBS‐buffered solutions (pH 3 to 9). After incubation with 200 µm Sph in buffer solutions of different pH values for 30 min at 37 °C, the fluorescence of DMS‐2F at 660 nm was significantly enhanced when the pH ranged from 6 to 9 (Figure ##SUPPL##0##S34##, Supporting Information), indicating that DMS‐2F could efficiently fluorogenically detect Sph in the physiological pH range.
\",\n \"
Concentration‐dependent fluorescence experiments were subsequently performed by incubating DMS‐2F (5 µm) with various concentrations of Sph (0–200 µm) in DMPC vesicles for 30 min at 37 °C. The results showed that the fluorescence intensity of DMS‐2F at 660 nm gradually increased with increasing Sph concentrations (Figure ##FIG##1##1d##). Moreover, an excellent linear relationship between the emission intensities was observed in the range of 0–110 µm (Figure ##FIG##1##1e##). The limit of detection (LOD) of DMS‐2F for Sph was calculated as 9.33 ± 0.41 nm according to the 3σ/m method. These findings suggest that DMS‐2F has the capability for ultrasensitive detection of Sph.
\",\n \"
To further determine whether DMS‐2F could selectively detect Sph in living cells, DMS‐2F (5 εm) was incubated in DMPC vesicles composed of several naturally abundant lipid species and other possible interferents (including amino acids with structures similar to Sph, such as cysteine (Cys), lysine (Lys), serine (Ser), threonine (Thr), tyrosine (Tyr) (260 εm) and glutathione (GSH) (2 mm); naturally abundant lipid species, sphingomyelin (SM, 200 εm) and ceramide (Cer, 200 εm); and neurotransmitters: NE (norepinephrine, 200 εm), DA (dopamine, 200 εm), and EP (epinephrine, 200 εm) for 30 min at 37 °C. The results showed that a significant fluorescence turn‐on was observed when DMS‐2F (5 εm) was incubated with Sph (200 εm). However, when DMS‐2F was incubated with another analyte, there was a weak change in fluorescence intensity compared to the untreated DMS‐2F probe (Figure ##FIG##1##1f##). These results are consistent with the design of the DMS‐2F probe to selectively detect Sph in the DMPC system.
\",\n \"
The specific response mechanism of DMS‐2F to Sph was confirmed by liquid chromatograph mass spectrometer (LC‐MS), high‐resolution mass spectrometry (HR‐MS), fluorescence spectroscopy, UV‐Visible spectroscopy, and theoretical calculation. DMS‐2F was exposed to Sph for 6 h at 37 °C, followed by LC−MS analysis and the reaction products of a five‐membered cyclic oxazolidinone‐based Sph compound (0.83 min) and the fluorophore DM‐2F (3.57 min) were successfully detected (Figure ##FIG##2##\\n2a–c##). In addition, HR‐MS experiments again confirmed that the fluorophore was released after DMS‐2F reacted with Sph, producing a five‐membered cyclic oxazolidinone‐based Sph compound (Figure ##SUPPL##0##S35##, Supporting Information). Subsequently, the UV−vis and fluorescence response of DMS‐2F to Sph were determined by adding 200 εm Sph to 1 mL of PBS buffer containing 5 εm\\nDMS‐2F and incubating at 37 °C for 6 h. As illustrated in Figure ##FIG##2##2d##, incubation with Sph resulted in a decrease in the UV‒vis absorption of DMS‐2F at 408 nm and an increase in absorption at 463 nm. The spectral properties of the system after reaction with Sph were in good agreement with the fluorophore DM‐2F. These results indicated that the response mechanism of DMS‐2F to Sph should be attributed to the nucleophilic addition and elimination reaction between ─NH2 of Sph and the thiocarbonate‐ester group of DMS‐2F. When the reaction is triggered, the fluorophore DM‐2F is released and emits significant fluorescence. The probable response mechanism of DMS‐2F to Sph is shown in Scheme ##FIG##0##1##. According to the proposed mechanism above, the bond cleavage between the carbon atom of the carbonyl group (C20) and the aryl oxide (O21) in probes is the key factor for the fluorogenic response of DMS‐X to Sph. Therefore, the bond energy of C20─O21 was a determining factor for screening the optimal probe for the response of Sph. To further understand the response priority of DMS‐2F and other probes (DMS‐F, DMS‐Cl, DMS‐Br, and DMS‐I) to Sph, theoretical calculations were carried out. The structures of DMS‐X were optimized by density functional theory (DFT) studies using the B3LYP 6–31G (d, p) levels (Figure ##FIG##2##2f,g##: Figure ##SUPPL##0##S36##, Supporting Information), and the bond lengths of C20─O21 in DMS‐X were evaluated. As shown in Figure ##FIG##2##2e##, the bond length of DMS‐2F is significantly longer than that of the other probes, which further indicates that DMS‐2F has a better response effect than the other probes. Additionally, the ground‐state geometry structures of Sph and NE were optimized by DFT at the B3LYP/6‐31G levels to investigate the selective response mechanism of DMS‐2F toward Sph. The calculation results indicate that there are hydrogen bonding interactions between the amino group of NE and its neighboring hydroxyl group (Figure ##SUPPL##0##S37a##, Supporting Information), which was detrimental to the DMS‐2F response to NE, whereas such interactions are absent in the structure of Sph (Figure ##SUPPL##0##S37b##, Supporting Information). Additionally, the electrostatic surface potential (ESP) maps reveal that the amino moieties of Sph possess higher electronegativity compared to those of NE (Figures ##SUPPL##0##S37c,d##, Supporting Information). These findings suggest that Sph can readily react with DMS‐2F, leading to its selective response.
\",\n \"
These promising in vitro results prompted us to explore the imaging effect of DMS‐X in living cells. Using MTT assays, it was confirmed that DMS‐X was non‐toxic to different cell lines (Figures ##SUPPL##0##S38## and ##SUPPL##0##S39##, Supporting Information). First, to determine whether DMS‐2F could react with Sph in live cells, A549 cells were incubated with DMS‐2F (10 εm) for 30 min, and then the cell medium was exchanged with a new medium containing 40 εm Sph and incubated for different amounts of time (30–150 min). As shown in Figure ##SUPPL##0##S40## (Supporting Information), as the Sph incubation time increased, the cells showed a gradual enhancement in red fluorescence emission within 1.5 h. To assess whether the response of DMS‐2F to Sph is concentration‐dependent in living cells, DMS‐2F‐pretreated A549 and HepG2 cells were incubated with different concentrations of Sph (0, 5, 10, and 20 εM). After 2 h of incubation, cells treated with exogenous Sph showed a dose‐dependent fluorescence enhancement signal (Figure ##SUPPL##0##S41##, Supporting Information). Furthermore, to confirm that the observed increase in cellular fluorescence was a result of the release of the fluorophore DM‐2F after the reaction between DMS‐2F and Sph, A549 cells were further treated with DM‐2F (10 εm) (Figure ##SUPPL##0##S42##, Supporting Information). Cells treated with DM‐2F exhibited a fluorescent imaging pattern consistent with that observed in cells treated with DMS‐2F and Sph. These results indicate that DMS‐2F could react with Sph in living cells within a short period of time and release an enhanced fluorescence signal by generating the fluorophore DM‐2F in a dose‐dependent manner.
\",\n \"
Since elevated sphingosine in mammalian cells is closely associated with apoptosis, to evaluate the response ability of DMS‐2F to endogenous Sph levels in live cells, cancer cells (rat adrenal pheochromocytoma PC12 cells, human non‐small cell lung cancer A549 cells, and brain glioma U87 cell lines) and normal lung fibroblast MRC‐5 cells were exposed to serum‐free medium (opti‐MEM containing 10 εm\\nDMS‐2F), which could result in elevated Sph levels. As shown in Figure ##SUPPL##0##S43a,b## (Supporting Information), after 2 h of incubation, the fluorescence of live cells treated with opti‐MEM all showed a significant increase. Taken together, these results suggest that DMS‐2F is sensitive enough to probe native sphingosine in various live cells, and can be used for imaging differences in sphingosine levels in living cells.
\",\n \"
Abnormal sphingolipid metabolism has been reported to induce Alzheimer's disease (AD), and substantial evidence also support that amyloid‐β (Aβ42) plays an important role in AD. In vitro, Aβ42 has been shown to induce apoptosis via the sphingomyelin pathway in various brain cells, including PC12 cells, etc.[\\n##REF##18547682##\\n10\\n##, ##REF##14748735##\\n18\\n##\\n] Accordingly, we attempted to assess whether the level of Sph will be changed in PC12 cells after treatment with Aβ42 peptide. We first treated PC12 cells with 10 εm Aβ42 oligomers for different times and then incubated them with DMS‐2F, as shown in Figure ##SUPPL##0##S44## (Supporting Information), the fluorescence of PC12 cells treated with Aβ42 oligomers was significantly increased. These observations undoubtedly revealed that intracellular Sph was up‐regulated when neuronal cells were challenged by Aβ42 oligomers. To further correlate Sph generation with Aβ42 oligomers, PC12 cells were treated with different concentrations of Aβ42 oligomers and Aβ42 monomers. After 12 h of incubation, a gradual increase in fluorescence was observed in PC12 cells treated with Aβ42 oligomers, however, weak fluorescence changes were observed in Aβ42 monomers treated cells (Figure ##FIG##3##\\n3a,b,d,e##). Together, these findings suggested that neuronal cells experienced overexpression of Sph during Aβ42 administration and that the up‐regulated Sph levels correlated with both the incubation time and dosage of Aβ42 oligomers, indicating that the abnormal sphingolipid metabolism in neuronal cells is tightly related to the stimulation of Aβ42 oligomers.
\",\n \"
Since the overexpression of Aβ protein induces mitochondrial oxidative stress and activates the intrinsic apoptotic cascade, and previous studies have illustrated that H2O2 could induce neurotoxicity in PC12 cells,[\\n##REF##14748735##\\n18\\n##, ##REF##22133762##\\n19\\n##\\n] we further investigated the Sph variations induced by H2O2. PC12 cells were treated with different concentrations of H2O2, after 4 h of incubation, a gradual increase in fluorescence was detected in PC12 cells treated with H2O2 (Figure ##FIG##3##3c,f##), suggesting that the Sph variation occurred during the oxidative stress process. In addition, the changes of Sph levels after being treated with Aβ42 oligomers and H2O2 in other cell lines were also assessed. U87, A549, HepG2, and MRC5 cells were treated with Aβ42 oligomers and H2O2 for 12 and 4 h, respectively, and then incubated with DMS‐2F for 2 h. As shown in Figure ##SUPPL##0##S45## (Supporting Information), significant fluorescence enhancement was exhibited in PC12 and U87 cells, while the fluorescence in other cell lines was not evident. These results indicate that the fluorogenic probe DMS‐2F can successfully monitor the level changes of Sph during Aβ42 oligomers and H2O2‐induced apoptosis in neuronal cells.
\",\n \"
Since changes in Sph levels are associated with the abnormal sphingolipid metabolism in cells, to investigate the possible mechanisms of Aβ42 oligomers and H2O2‐induced changes in Sph levels, we further investigated intracellular sphingomyelinase and ceramidase activities in Aβ42 oligomers and H2O2 treated cells. PC12 cells were preincubated for 2 h in the presence of 10 µm amitriptyline hydrochloride (AMI, an acid sphingomyelinase inhibitor[\\n##UREF##0##\\n20\\n##\\n]), 20 µm GW4869 (a neutral sphingomyelinase inhibitor[\\n##REF##20629193##\\n21\\n##\\n]), 10 µm LCL‐521 (an acid ceramidase inhibitor[\\n##REF##25456083##\\n22\\n##\\n]), 500 µm N‐acetyl‐cysteine (NAC, an efficient antioxidant[\\n##UREF##1##\\n23\\n##\\n]) and 1 mm Trolox (a reactive oxygen scavenger [\\n##REF##17013382##\\n24\\n##\\n]), and then treated with Aβ42 oligomers (10 µm, 12 h) or H2O2 (50 µm, 4 h), respectively. As shown in Figure ##FIG##4##\\n4a,c##, the fluorescence of the cells preincubated with the inhibitors prior to the treatment with Aβ42 oligomers was lower than that of the control group. This suggests that the Aβ42 oligomers increase Sph levels by elevating intracellular ROS and enhancing intracellular acidic and neutral sphingomyelinase and acidic ceramidase activities. However, the increase in intracellular Sph levels in H2O2‐treated cells appeared to be more correlated with neutral sphingomyelinase activity. This is supported by the reduced imaging intensity observed in cells pretreated with the nerve sphingomyelinase inhibitor, GW4869, as well as the reactive oxygen scavengers, NAC and Trolox (Figure ##FIG##4##4b,d##). Therefore, it is hypothesized that the possible mechanism of Aβ42 oligomers and H2O2‐induced up‐regulation of intracellular Sph levels is as follows: Aβ42 oligomers induced an increase in intracellular ROS levels, which then activated neutral sphingomyelinase (NSMase), leading to the hydrolysis of sphingomyelin (SM) and the production of ceramides (Cer). This process ultimately results in an increase in intracellular Sph levels. Moreover, Aβ42 oligomers can activate acidic sphingomyelinase (ASMase) and acidic ceramidase (ACDase), which also contribute to the hydrolysis of SM and Cer, thereby increasing Sph levels (Figure ##FIG##4##4e##).
\",\n \"
To further validate the possible mechanisms proposed above, changes in ROS levels after different times of treatment of PC12 cells with Aβ42 oligomers were monitored using a ROS specific probe. We found that changes of ROS levels in cellular mitochondria upon Aβ42 oligomers treatment showed a gradual increase and reached the peak at 1 h. With prolonged treatment time, the ROS levels gradually decreased and eventually returned to the initial level (Figure ##FIG##5##\\n5a–c##; Figure ##SUPPL##0##S46##, Supporting Information). The activations of SMase and CDase were then investigated. The treatment of PC12 cells with 1 µm Aβ42 oligomers and 50 µm H2O2 induced a time‐dependent increase in SMase activity and reached maximum levels at 6 and 3 h, respectively (Figure ##FIG##5##5d,e##; Figure ##SUPPL##0##S47##, Supporting Information). Notably, the widely used ASMase and NSMase inhibitors AMI and GW4869 strongly inhibited Aβ42 oligomers‐induced SMase activation, respectively, while only GW4869 inhibited H2O2‐induced SMase activation, and the ROS scavenger NAC and Trolox inhibited both the Aβ42 oligomers‐ and H2O2‐induced SMase activation (Figure ##FIG##5##5f,g##). Immunoblotting and immunofluorescence experiments provided additional support for the aforementioned experimental results (Figure ##FIG##5##5h–j##; Figure ##SUPPL##0##S48##, Supporting Information). This suggests that Aβ42 oligomers can induce the activation of ASMase and NSMase, respectively, whereas H2O2 can only induce the activation of NSMase. In addition, the ELISA test results for CDase indicated that the intracellular ceramidase level reached a maximum after 4 h of treatment with Aβ42 oligomers. It was observed that H2O2 did not activate intracellular CDase, but had an inhibitory effect, which aligns with the findings reported in the literature,[\\n##REF##34459070##\\n25\\n##\\n] (Figure ##FIG##5##5l,m##; Figure ##SUPPL##0##S49##, Supporting Information). Moreover, the activation effect of ROS scavenger to Aβ42 oligomers induced CDase activity and immunoblotting experiments on ACDase further supported that Aβ42 oligomers can activate ACDase, while H2O2 inhibits its activity (Figure ##FIG##5##5h,k##; Figure ##SUPPL##0##S50##, Supporting Information). Taken together, these results suggest that Aβ42 oligomers directly activate ASMase and ACDase, and also increase ROS levels, which activate NSMase and ultimately accelerate sphingolipid metabolism and increase the level of Sph.
\",\n \"
Encouraged by the excellent results of live cell imaging, we further investigated the in vivo detection in a living animal model. The transparent zebrafish larva was selected as a model because zebrafish are optically transparent and have a high degree of homology with mammals.[\\n##REF##21240407##\\n26\\n##\\n] Zebrafish larvae (3 days old) were incubated with Aβ42 oligomers (10 εm) for 12 h and then treated with DMS‐2F (10 εm) for 2 h. As shown in Figure ##FIG##6##\\n6\\n##, zebrafish treated with DMS‐2F without Aβ42 oligomers exhibited weak fluorescence, and notably, the Aβ42 oligomers treated zebrafish showed bright red fluorescence in the brain, yolk sac, and intestine. However, the fluorescence intensity of zebrafish pretreated with inhibitors (AMI, GW4869, LCL‐521, and Trolox) and further treated with Aβ42 oligomers and DMS‐2F was significantly weaker than that of zebrafish treated without inhibitors. Based on these results, it is shown that in vivo, Aβ42 oligomers can still induce the upregulation of Sph levels by the same mechanism as in neuronal cells. Hence, this new probe DMS‐2F successfully realized the detection of Sph in vivo.
In summary, we have developed a series of easily synthesized fluorogenic probes, named DMS‐X, by introducing different halogen substituents and thiocarbonate protecting groups. DMS‐2F can specifically detect sphingosine in a short period of time (within 4 h) and has excellent response sensitivity (LOD, 9.33 nm). DMS‐2F is capable of fluorescence tracing and imaging of exogenous and endogenous sphingosine in living cells. Importantly, we, for the first time, evaluated the Sph levels during Aβ42 oligomers and H2O2‐induced apoptosis of PC12 cells by fluorescent imaging approach and further verified that Aβ42 oligomers can induce abnormal sphingolipid metabolism by increasing the intracellular ROS levels and inducing the activation of sphingomyelinase and ceramidase during sphingolipid metabolism, which ultimately led to the increase of Sph level. Finally, we successfully realized the in vivo detection of Aβ42 oligomers‐induced Sph in a living zebrafish model. Based on the properties of reactive fluorogenic probes, this new probe greatly avoids the background interference of probe molecules and provides a novel strategy for monitoring sphingosine in living cells and in vivo.
"],"string":"[\n \"Conclusion\",\n \"
In summary, we have developed a series of easily synthesized fluorogenic probes, named DMS‐X, by introducing different halogen substituents and thiocarbonate protecting groups. DMS‐2F can specifically detect sphingosine in a short period of time (within 4 h) and has excellent response sensitivity (LOD, 9.33 nm). DMS‐2F is capable of fluorescence tracing and imaging of exogenous and endogenous sphingosine in living cells. Importantly, we, for the first time, evaluated the Sph levels during Aβ42 oligomers and H2O2‐induced apoptosis of PC12 cells by fluorescent imaging approach and further verified that Aβ42 oligomers can induce abnormal sphingolipid metabolism by increasing the intracellular ROS levels and inducing the activation of sphingomyelinase and ceramidase during sphingolipid metabolism, which ultimately led to the increase of Sph level. Finally, we successfully realized the in vivo detection of Aβ42 oligomers‐induced Sph in a living zebrafish model. Based on the properties of reactive fluorogenic probes, this new probe greatly avoids the background interference of probe molecules and provides a novel strategy for monitoring sphingosine in living cells and in vivo.
Sphingosine (Sph) plays important roles in various complex biological processes. Abnormalities in Sph metabolism can result in various diseases, including neurodegenerative disorders. However, due to the lack of rapid and accurate detection methods, understanding sph metabolic in related diseases is limited. Herein, a series of near‐infrared fluorogenic probes DMS‐X (X = 2F, F, Cl, Br, and I) are designed and synthesized. The fast oxazolidinone ring formation enables the DMS‐2F to detect Sph selectively and ultrasensitively, and the detection limit reaches 9.33 ± 0.41 nm. Moreover, it is demonstrated that DMS‐2F exhibited a dose‐ and time‐dependent response to Sph and can detect sph in living cells. Importantly, for the first time, the changes in Sph levels induced by Aβ42 oligomers and H2O2 are assessed through a fluorescent imaging approach, and further validated the physiological processes by which Aβ42 oligomers and reactive oxygen species (ROS)‐induce changes in intracellular Sph levels. Additionally, the distribution of Sph in living zebrafish is successfully mapped by in vivo imaging of a zebrafish model. This work provides a simple and efficient method for probing Sph in living cells and in vivo, which will facilitate investigation into the metabolic process of Sph and the connection between Sph and disease pathologies.
","
A novel fluorogenic probe for specifical sphingosine detecting is constructed via a rational molecular design strategy. The probe could image Aβ42 oligomers‐induced Sph in neural cells and in vivo.\n\n
","
\n\n\nY.\nChen\n, \nT.\nHao\n, \nJ.\nWang\n, \nY.\nChen\n, \nX.\nWang\n, \nW.\nWei\n, \nJ.\nZhao\n, \nY.\nQian\n, A Near‐Infrared Fluorogenic Probe for Rapid, Specific, and Ultrasensitive Detection of Sphingosine in Living Cells and In Vivo. Adv. Sci.\n2024, 11, 2307598. 10.1002/advs.202307598\n\n
"],"string":"[\n \"Abstract\",\n \"
Sphingosine (Sph) plays important roles in various complex biological processes. Abnormalities in Sph metabolism can result in various diseases, including neurodegenerative disorders. However, due to the lack of rapid and accurate detection methods, understanding sph metabolic in related diseases is limited. Herein, a series of near‐infrared fluorogenic probes DMS‐X (X = 2F, F, Cl, Br, and I) are designed and synthesized. The fast oxazolidinone ring formation enables the DMS‐2F to detect Sph selectively and ultrasensitively, and the detection limit reaches 9.33 ± 0.41 nm. Moreover, it is demonstrated that DMS‐2F exhibited a dose‐ and time‐dependent response to Sph and can detect sph in living cells. Importantly, for the first time, the changes in Sph levels induced by Aβ42 oligomers and H2O2 are assessed through a fluorescent imaging approach, and further validated the physiological processes by which Aβ42 oligomers and reactive oxygen species (ROS)‐induce changes in intracellular Sph levels. Additionally, the distribution of Sph in living zebrafish is successfully mapped by in vivo imaging of a zebrafish model. This work provides a simple and efficient method for probing Sph in living cells and in vivo, which will facilitate investigation into the metabolic process of Sph and the connection between Sph and disease pathologies.
\",\n \"
A novel fluorogenic probe for specifical sphingosine detecting is constructed via a rational molecular design strategy. The probe could image Aβ42 oligomers‐induced Sph in neural cells and in vivo.\\n\\n
\",\n \"
\\n\\n\\nY.\\nChen\\n, \\nT.\\nHao\\n, \\nJ.\\nWang\\n, \\nY.\\nChen\\n, \\nX.\\nWang\\n, \\nW.\\nWei\\n, \\nJ.\\nZhao\\n, \\nY.\\nQian\\n, A Near‐Infrared Fluorogenic Probe for Rapid, Specific, and Ultrasensitive Detection of Sphingosine in Living Cells and In Vivo. Adv. Sci.\\n2024, 11, 2307598. 10.1002/advs.202307598\\n\\n
\"\n]"},"body":{"kind":"list like","value":["Conflict of Interest","
The authors declare no conflict of interest.
","Supporting information"],"string":"[\n \"Conflict of Interest\",\n \"
This work was supported by the National Key Research Program 2019YFA0905801, the National Natural Science Foundation of China (22025701, 22207053, 22177048 and 22074065), the National Science Fund for Excellent Young Scholars (22222704), the Natural Science Foundation of Jiangsu Province (BK20202004 and BK20220764), Shenzhen Basic Research Program (JCYJ20170413150538897, JCYJ20180508182240106), the National Key Research and Development Program of China (2017YFA0208200, 2016YFB0700600, 2015CB659300), The Fundamental Research Funds for the Central Universities, 2021 Strategic Research Project of the Science and Technology Commission of the Ministry of Education of China.
","Data Availability Statement","
The data that support the findings of this study are available in the supplementary material of this article.
"],"string":"[\n \"Acknowledgements\",\n \"
This work was supported by the National Key Research Program 2019YFA0905801, the National Natural Science Foundation of China (22025701, 22207053, 22177048 and 22074065), the National Science Fund for Excellent Young Scholars (22222704), the Natural Science Foundation of Jiangsu Province (BK20202004 and BK20220764), Shenzhen Basic Research Program (JCYJ20170413150538897, JCYJ20180508182240106), the National Key Research and Development Program of China (2017YFA0208200, 2016YFB0700600, 2015CB659300), The Fundamental Research Funds for the Central Universities, 2021 Strategic Research Project of the Science and Technology Commission of the Ministry of Education of China.
\",\n \"Data Availability Statement\",\n \"
The data that support the findings of this study are available in the supplementary material of this article.
\"\n]"},"figure":{"kind":"list like","value":["
a) The response process of DMS‐2F with Sph; b) Designed structure of the fluorogenic probes DMS‐X and c) Structure of Sph and oxazolidinone‐based Sph after reacting with DMS‐2F.
","
a) Fluorescence spectra of DMS‐2F in PBS and DMPC vesicles (with/without Sph), inset: histograms for fluorescent intensity at 660 nm; b,c) Fluorescence results of DMS‐X (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br and DMS‐I) for Sph under physiological conditions and incubated for different times: b) 10 min, c) 0–360 min, respectively; d) Concentration response of DMS‐2F to Sph from 0 to 200 µm; e) Fluorescence spectrum linear range for Sph from 0 to 110 µm; f) Fluorescence intensity ratios of DMS‐2F and DMS‐2F toward various analytes (λ\nex = 540 nm, λ\nem = 660 nm, slit = 10/10 nm, pH 7.4, 37 °C.
","
a,b,c) The LC‐MS of DMS‐2F (5 mm) in MeOH after reacting with Sph (100 mm) for 6 h at 37 °C; d) UV‒vis absorption spectra of DM‐2F (5 µm), DMS‐2F (5 µm) and DMS‐2F (5 µm) reacted with Sph (200 µm) in PBS buffer; e) Bond length between C21─O20 in DMS‐X evaluated by theoretical calculations, inset: the chemical structure of DMS‐X; f) View of the energy‐optimized structure and g) The molecular electrostatic potential (ESP) surface for DMS‐2F from Gauss View.
","
Fluorescence images of PC12 cells treated with different concentrations of a) Aβ42 monomers and b) Aβ42 oligomers for 12 h and then incubated with 10 εm\nDMS‐2F for 2 h. c) Fluorescence images of PC12 cells treated with different concentrations of H2O2 for 4 h and then incubated with 10 µm\nDMS‐2F for 2 h. d,e,f) Mean fluorescent intensities of PC12 cells in panels (a), (b), and (c), respectively. Scale bar: 20 µm. The data are expressed as the mean ± SD. One‐way ANOVA was used to compare multiple groups: *\nP ≤0.05, **\nP ≤0.01, ***\nP ≤0.001, ****\nP ≤0.0001.
","
Fluorescence images of PC12 cells preincubated with different inhibitors and then treated with a) 10 εm Aβ42 oligomers for 12 h and b) 50 εm H2O2 for 4 h, and then incubated with 10 εm\nDMS‐2F for 2 h, scale bar: 10 µm; c,d) Mean fluorescent intensities of PC12 cells in panels (a) and (b), respectively; e) Schematic pathway proposed for the Aβ42 oligomers, ROS, and sphingolipid changes in the PC12 cells. All values are expressed as the mean ± SD of triplicates. One‐way ANOVA was used to compare multiple groups: ns P> 0.05, *\nP ≤0.05, **\nP ≤0.01, ***\nP ≤0.001, ****\nP ≤0.0001.
","
a) Fluorescence image of ROS in PC12 cells induced by Aβ42 oligomers at different time, scale bar: 5 µm; b,c) Mean fluorescent intensities of PC12 cells in panels (a); d,e). The level of SMase released in the supernatant of PC12 cells treated with Aβ42 oligomers and H2O2 for different durations measured by ELISA; f,g). The level of SMase released in the supernatant of PC12 cells pretreated with different inhibitors and then incubated by Aβ42 oligomers and H2O2 measured by ELISA; h) WB blot analysis of the expression of ASMase and ACDase in PC12 cells after incubation with Aβ42 oligomers and H2O2 at different times; i) Immunofluorescence analysis of ASMase in Aβ42 oligomers‐incubated PC12 cells pretreated or not pretreated with inhibitors AMI and NAC, scale bar: 10 µm; j) Mean fluorescent intensities of PC12 cells in panels Figure ##FIG##5##5(h)##; k) The level of CDase released in the supernatant of PC12 cells pretreated with different inhibitors and then incubated by Aβ42 oligomers measured by ELISA; l,m). The level of CDase released in the supernatant of PC12 cells treated with Aβ42 oligomers and H2O2 for different durations measured by ELISA; All values are expressed as the mean ± SD of triplicates. One‐way ANOVA was used to compare multiple groups: *\nP ≤0.05, **\nP ≤0.01, ***\nP ≤0.001, ****\nP ≤0.0001.
","
a) Fluorescence images of zebrafish preincubated with/without different inhibitors (20 µm AMI, 40 µm GW4869, 20 µm LCL‐521, and 1mm Trolox) and treated with 10 εm Aβ42 oligomers for 12 h and then incubated with 10 εm\nDMS‐2F for 2 h, scale bar: 200 µm; Mean fluorescent intensities of zebrafish position b) brain, c) yolk sac, and d) intestine, respectively. All values are expressed as the mean ± SD of triplicates. One‐way ANOVA was used to compare multiple groups: ns P> 0.05, *\nP ≤0.05, **\nP ≤0.01, ***\nP ≤0.001, ****\nP ≤0.0001.
"],"string":"[\n \"
a) The response process of DMS‐2F with Sph; b) Designed structure of the fluorogenic probes DMS‐X and c) Structure of Sph and oxazolidinone‐based Sph after reacting with DMS‐2F.
\",\n \"
a) Fluorescence spectra of DMS‐2F in PBS and DMPC vesicles (with/without Sph), inset: histograms for fluorescent intensity at 660 nm; b,c) Fluorescence results of DMS‐X (DMS‐2F, DMS‐F, DMS‐Cl, DMS‐Br and DMS‐I) for Sph under physiological conditions and incubated for different times: b) 10 min, c) 0–360 min, respectively; d) Concentration response of DMS‐2F to Sph from 0 to 200 µm; e) Fluorescence spectrum linear range for Sph from 0 to 110 µm; f) Fluorescence intensity ratios of DMS‐2F and DMS‐2F toward various analytes (λ\\nex = 540 nm, λ\\nem = 660 nm, slit = 10/10 nm, pH 7.4, 37 °C.
\",\n \"
a,b,c) The LC‐MS of DMS‐2F (5 mm) in MeOH after reacting with Sph (100 mm) for 6 h at 37 °C; d) UV‒vis absorption spectra of DM‐2F (5 µm), DMS‐2F (5 µm) and DMS‐2F (5 µm) reacted with Sph (200 µm) in PBS buffer; e) Bond length between C21─O20 in DMS‐X evaluated by theoretical calculations, inset: the chemical structure of DMS‐X; f) View of the energy‐optimized structure and g) The molecular electrostatic potential (ESP) surface for DMS‐2F from Gauss View.
\",\n \"
Fluorescence images of PC12 cells treated with different concentrations of a) Aβ42 monomers and b) Aβ42 oligomers for 12 h and then incubated with 10 εm\\nDMS‐2F for 2 h. c) Fluorescence images of PC12 cells treated with different concentrations of H2O2 for 4 h and then incubated with 10 µm\\nDMS‐2F for 2 h. d,e,f) Mean fluorescent intensities of PC12 cells in panels (a), (b), and (c), respectively. Scale bar: 20 µm. The data are expressed as the mean ± SD. One‐way ANOVA was used to compare multiple groups: *\\nP ≤0.05, **\\nP ≤0.01, ***\\nP ≤0.001, ****\\nP ≤0.0001.
\",\n \"
Fluorescence images of PC12 cells preincubated with different inhibitors and then treated with a) 10 εm Aβ42 oligomers for 12 h and b) 50 εm H2O2 for 4 h, and then incubated with 10 εm\\nDMS‐2F for 2 h, scale bar: 10 µm; c,d) Mean fluorescent intensities of PC12 cells in panels (a) and (b), respectively; e) Schematic pathway proposed for the Aβ42 oligomers, ROS, and sphingolipid changes in the PC12 cells. All values are expressed as the mean ± SD of triplicates. One‐way ANOVA was used to compare multiple groups: ns P> 0.05, *\\nP ≤0.05, **\\nP ≤0.01, ***\\nP ≤0.001, ****\\nP ≤0.0001.
\",\n \"
a) Fluorescence image of ROS in PC12 cells induced by Aβ42 oligomers at different time, scale bar: 5 µm; b,c) Mean fluorescent intensities of PC12 cells in panels (a); d,e). The level of SMase released in the supernatant of PC12 cells treated with Aβ42 oligomers and H2O2 for different durations measured by ELISA; f,g). The level of SMase released in the supernatant of PC12 cells pretreated with different inhibitors and then incubated by Aβ42 oligomers and H2O2 measured by ELISA; h) WB blot analysis of the expression of ASMase and ACDase in PC12 cells after incubation with Aβ42 oligomers and H2O2 at different times; i) Immunofluorescence analysis of ASMase in Aβ42 oligomers‐incubated PC12 cells pretreated or not pretreated with inhibitors AMI and NAC, scale bar: 10 µm; j) Mean fluorescent intensities of PC12 cells in panels Figure ##FIG##5##5(h)##; k) The level of CDase released in the supernatant of PC12 cells pretreated with different inhibitors and then incubated by Aβ42 oligomers measured by ELISA; l,m). The level of CDase released in the supernatant of PC12 cells treated with Aβ42 oligomers and H2O2 for different durations measured by ELISA; All values are expressed as the mean ± SD of triplicates. One‐way ANOVA was used to compare multiple groups: *\\nP ≤0.05, **\\nP ≤0.01, ***\\nP ≤0.001, ****\\nP ≤0.0001.
\",\n \"
a) Fluorescence images of zebrafish preincubated with/without different inhibitors (20 µm AMI, 40 µm GW4869, 20 µm LCL‐521, and 1mm Trolox) and treated with 10 εm Aβ42 oligomers for 12 h and then incubated with 10 εm\\nDMS‐2F for 2 h, scale bar: 200 µm; Mean fluorescent intensities of zebrafish position b) brain, c) yolk sac, and d) intestine, respectively. All values are expressed as the mean ± SD of triplicates. One‐way ANOVA was used to compare multiple groups: ns P> 0.05, *\\nP ≤0.05, **\\nP ≤0.01, ***\\nP ≤0.001, ****\\nP ≤0.0001.