Macrophage membrane-biomimetic nanovehicles delivering amygdalin halt pyroptosis propagation and alleviate endometriosis-associated neuropathic pain

All animal experimental procedures were reviewed and approved by the Animal Ethics Committee of Wannan Medical University (Wuhu, China). The approval number for this study is WNMC-AWE-2024404. All experiments were conducted in accordance with the institutional guidelines and international standards for the care and use of laboratory animals.

Li Wang: Conceptualization, Data curation, Investigation, Project administration, Validation, Writing – original draft. Wenyu Peng: Data curation, Formal analysis, Methodology. Jingyi Wang: Software, Supervision. Jiawen Chen: Validation, Visualization. Mengting Liu: Investigation. Huan Huang: Methodology. Ruizhe Jia: Project administration, Writing – review & editing. Jin Ding: Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing.

To achieve lesion-specific delivery and further enhance the bioavailability of amygdalin (Amy), we designed and constructed a biomimetic nanoparticle system (denoted as Amy@NPs-MM/PL1) by encapsulating Amy within the biodegradable PPNPs, followed by surface camouflage with macrophage membrane that incorporated the PL1 targeting peptide (MM/PL1). We first carried out physicochemical characterization to verify the successful construction of Amy@NPs-MM/PL1. The suspension of Amy@NPs-MM/PL1 displayed a strong Tyndall effect, indicating good colloidal stability and uniform dispersion ( Fig. S1a ). Transmission electron microscopy (TEM) images showed that both the Amy@NPs and Amy@NPs-MM/PL1 exhibited a spherical and homogeneous morphology, with Amy@NPs-MM/PL1 displaying a clear core–shell architecture with an approximately 10 nm membrane layer ( Fig. 1 a). Size analysis result also indicated that membrane coating increased the particle diameter by 10-20 nm ( Fig. 1 b, Fig. S1b ). In addition, we analyzed the zeta potentials of Amy@NPs and Amy@NPs-MM/PL1 using a Zeta potential analyzer. The results in Fig. 3 c showed that the zeta potential of Amy@NPs-MM/PL1 (−30.16 ± 0.12 mV) was significantly lower than that of Amy@NPs (−21.47 ± 0.16 mV), further confirming the successful coating of macrophage membrane. To be noted, the zeta potentials of both Amy@NPs and Amy@NPs-MM/PL1 remained stable for 12 days ( Fig. 1 c), indicating good colloidal stability of the nanoparticles. The EE and DL of Amy@NPs-MM/PL1 were 69.67% and 17.33%, respectively. Amy release from Amy@NPs-MM/PL1 was slow at pH 7.4 but accelerated at pH 5.5, reaching 42.77% at day 6 and 65.26% at day 12 ( Fig. 1 d), supporting pH-triggered release at the acidic lesion site. Next, Coomassie blue staining showed that Amy@NPs-MM/PL1 displayed protein band patterns closely matching those of native macrophage membranes, suggesting successful transfer of the broad membrane protein repertoire ( Fig. 1 e). Western blot results further confirmed the presence of key macrophage markers (i.e., CD11b, CD68, and CD47) on Amy@NPs-MM/PL1, consistent with their high expression in native macrophage membranes ( Fig. 1 f and g). Finally, the intrinsic antioxidant capacity of Amy@NPs-MM/PL1 was evaluated in vitro. The nanoparticles effectively scavenged multiple representative ROS, including H 2 O 2 , O 2 − , ·OH, and DPPH radicals, indicating the remarkable antioxidative potential of Amy@NPs-MM/PL1 ( Fig. 1 h). Overall, these findings collectively confirmed the successful construction of Amy@NPs-MM/PL1, which is a structurally intact, biologically competent nanoplatform with robust physicochemical properties, pH-triggered drug release, preserved membrane functionality, and notable antioxidant capacity. Fig. 1 Physicochemical characterization and antioxidant properties of Amy@NPs-MM/PL1. (a) Transmission electron microscopy (TEM) images showing the morphology of uncoated Amy@NPs and membrane-coated Amy@NPs (i.e., Amy@NPs-MM/PL1). (b) Particle size distribution of Amy@NPs and Amy@NPs-MM/PL1 obtained by statistical analysis of TEM images. (c) Zeta-potentials of Amy@NPs and Amy@NPs-MM/PL1 in 12 consecutive days. (d) Cumulative drug-release profiles of Amy@NPs-MM/PL1 under physiological (pH = 7.4) and acidic (pH = 5.5) conditions. (e) Coomassie brilliant blue staining result displaying the expression of membrane proteins on Amy@NPs-MM/PL1 compared with macrophage membrane (MM). Representative Western blot analysis results of representative membrane-associated proteins (CD11b, CD68, CD47) (f) and the corresponding quantification results (g). (h) Histograms exhibiting the relative levels of hydrogen peroxide (H 2 O 2 ), and the scavenging rates of superoxide anion (O 2 − ), hydroxyl radicals (·OH), and 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radicals after the treatment of PPNPs, Amy@NPs, and Amy@NPs-MM/PL1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Physicochemical characterization and antioxidant properties of Amy@NPs-MM/PL1. (a) Transmission electron microscopy (TEM) images showing the morphology of uncoated Amy@NPs and membrane-coated Amy@NPs (i.e., Amy@NPs-MM/PL1). (b) Particle size distribution of Amy@NPs and Amy@NPs-MM/PL1 obtained by statistical analysis of TEM images. (c) Zeta-potentials of Amy@NPs and Amy@NPs-MM/PL1 in 12 consecutive days. (d) Cumulative drug-release profiles of Amy@NPs-MM/PL1 under physiological (pH = 7.4) and acidic (pH = 5.5) conditions. (e) Coomassie brilliant blue staining result displaying the expression of membrane proteins on Amy@NPs-MM/PL1 compared with macrophage membrane (MM). Representative Western blot analysis results of representative membrane-associated proteins (CD11b, CD68, CD47) (f) and the corresponding quantification results (g). (h) Histograms exhibiting the relative levels of hydrogen peroxide (H 2 O 2 ), and the scavenging rates of superoxide anion (O 2 − ), hydroxyl radicals (·OH), and 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radicals after the treatment of PPNPs, Amy@NPs, and Amy@NPs-MM/PL1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) In a macrophage-hEMSC co-culture model ( Fig. S2a ), Amy@NPs-MM/PL1 showed enhanced cellular entry via membrane fusion, whereas uncoated Amy@NPs were internalized by macrophages through phagocytosis Amy@NPs-MM/PL1 also penetrated the macrophage layer more efficiently and was readily taken up by hEMSCs, indicating that membrane coating promotes trans-barrier delivery. We next evaluated the effects of different treatments on cell viability. Live/dead staining results revealed that Amy@NPs-MM/PL1 enhanced the sustained cytotoxic effects of Amy on hEMSCs, indicating that the membrane coating strategy increased the cytotoxicity of Amy against hEMSCs, thereby enhancing its therapeutic potential. Because oxidative stress is a major trigger of pyroptosis, we then measured the intracellular ROS levels in the differently treated hEMSCs. Both the fluorescence staining ( Fig. 2 a, Fig. S2b ) and ROS (H 2 O 2 , ·OH, DPPH·) detection ( Fig. 2 b) revealed that hEMSCs exposed to lipopolysaccharide (LPS) exhibited marked oxidative stress, consistent with the establishment of a pyroptosis-inducing inflammatory model. Upon subsequent treatment, ROS levels declined to varying degrees across all drug formulations, among which Amy@NPs-MM/PL1 demonstrated the most significant reduction, highlighting its potent antioxidative capability. Fig. 2 Inhibition of pyroptosis and oxidative stress by Amy@NPs-MM/PL1 in vitro. (a) Representative live/dead staining results of hEMSCs after different treatments (Control, PPNPs, Amy, Amy@NPs, and Amy@NPs-MM/PL1) (upper panel), and immunofluorescence results indicating intracellular ROS levels in differently treated hEMSCs (lower panel). (b) Histograms showing the relative levels of H 2 O 2 and the scavenging rates of ·OH and DPPH· radicals in hEMSCs after different treatments. (c) Representative images of hEMSCs: SEM (upper panel), PI/Annexin V-FITC staining showing early (Annexin V + , green) and late (Annexin V + /PI + , orange) pyroptotic cells (second panel), immunofluorescence of C-Caspase-1 (green) and GSDMD-N (red) (third panel), and Ki-67 (lower panel), together with Western blot (d) and ELISA (e) results depicting the expression and secretion changes of pyroptosis-related proteins (CASP-1, C-CASP-1, GSDMD, GSDMD-N, IL-1β, and IL-18) after different treatments. (f) Representative SEM (upper panel), PI/Annexin V staining (second panel), and immunofluorescence images of neurons showing C-Caspase-1 (green)/GSDMD-N (red) (third panel) and BDNF (green)/NGF (red) (lower panel), along with ELISA (g) results illustrating changes in neuro-associated cytokines (BDNF and NGF) under different treatment conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Inhibition of pyroptosis and oxidative stress by Amy@NPs-MM/PL1 in vitro. (a) Representative live/dead staining results of hEMSCs after different treatments (Control, PPNPs, Amy, Amy@NPs, and Amy@NPs-MM/PL1) (upper panel), and immunofluorescence results indicating intracellular ROS levels in differently treated hEMSCs (lower panel). (b) Histograms showing the relative levels of H 2 O 2 and the scavenging rates of ·OH and DPPH· radicals in hEMSCs after different treatments. (c) Representative images of hEMSCs: SEM (upper panel), PI/Annexin V-FITC staining showing early (Annexin V + , green) and late (Annexin V + /PI + , orange) pyroptotic cells (second panel), immunofluorescence of C-Caspase-1 (green) and GSDMD-N (red) (third panel), and Ki-67 (lower panel), together with Western blot (d) and ELISA (e) results depicting the expression and secretion changes of pyroptosis-related proteins (CASP-1, C-CASP-1, GSDMD, GSDMD-N, IL-1β, and IL-18) after different treatments. (f) Representative SEM (upper panel), PI/Annexin V staining (second panel), and immunofluorescence images of neurons showing C-Caspase-1 (green)/GSDMD-N (red) (third panel) and BDNF (green)/NGF (red) (lower panel), along with ELISA (g) results illustrating changes in neuro-associated cytokines (BDNF and NGF) under different treatment conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) To further determine whether Amy@NPs-MM/PL1 could attenuate LPS-induced pyroptotic activation, SEM imaging was performed. LPS-stimulated hEMSCs displayed typical pyroptotic morphology characterized by cell swelling, membrane ballooning, and pore-like ruptures. These structural abnormalities were alleviated following treatment, with Amy@NPs-MM/PL1 showing the most pronounced morphological restoration. PI/Annexin V-FITC staining further confirmed these observations. In LPS-stimulated hEMSCs, the majority of cells exhibited Annexin V + (green) single-positive staining with minimal PI co-localization, indicating an early stage of pyroptosis. Treatment with Amy@NPs-MM/PL1 markedly reduced the number of Annexin V + cells, demonstrating effective suppression of pyroptosis initiation. Moreover, immunofluorescence analysis revealed decreased expression of cleaved Caspase-1 and GSDMD-N, together with reduced Ki-67 levels in Amy@NPs-MM/PL1-treated cells, suggesting effective suppression of pyroptosis and cellular hyperproliferation ( Fig. 2 c). In addition, Western blot analysis results also confirmed the suppression of pyroptosis-related proteins (CASP-1, C-CASP-1, GSDMD, GSDMD-N, IL-1β, IL-18), which was in accordance with the ELISA results ( Fig. 2 d and e). These results fully demonstrated that the Amy@NPs-MM/PL1 treatment can efficiently inhibit the pyroptosis of hEMSCs and alleviate inflammation. To examine whether Amy@NPs-MM/PL1 could block pyroptosis propagation from hEMSCs to neurons, neuronal cells were co-cultured with differently treated hEMSCs. Neurons co-cultured with LPS-stimulated hEMSCs displayed extensive pyroptotic damage, while those co-cultured with hEMSCs from the Amy@NPs-MM/PL1-treated group largely retained intact morphology, indicating effective interruption of pyroptosis propagation. In the co-cultured neurons, PI/Annexin V-FITC staining revealed abundant Annexin V + /PI + double-positive (orange) cells in the LPS group, reflecting late-stage pyroptosis with extensive membrane permeabilization. Following treatment with Amy@NPs-MM/PL1, the proportion of double-positive neurons was significantly reduced, confirming effective blockade of pyroptotic signal transmission. Immunofluorescence results revealed that pyroptosis-related proteins, such as Cleaved-Caspase-1 (green) and GSDMD-N (red), were significantly reduced in the Amy@NPs-MM/PL1 treatment group. Additionally, the expression of nerve growth factor (NGF) (red) was notably decreased, while BDNF (green) was elevated, indicating the reversal of neuroinflammatory and neurodegenerative phenotypes ( Fig. 2 f). Similar results were also observed in Western blot and ELISA assays, confirming the suppression of neuronal pyroptosis and the preservation of neuronal structure and function ( Fig. 2 f, Fig. S2c ). To dissect the mediators of pyroptosis propagation, we first characterized the morphology of EVs derived from pyroptotic hEMSCs using transmission electron microscopy, which revealed typical cup-shaped or spherical vesicles ( Fig. S2d ). We then performed immunofluorescence for NF-κB p65 (green) and GSDMD-N (red) in neurons under different conditions ( Fig. S2e ). Addition of these pyroptotic EVs strongly induced both GSDMD-N expression and p65 nuclear translocation. Inhibition of EV secretion (GW4869) or NLRP3 (MCC950) reduced GSDMD-N but did not notably improve p65 nuclear translocation, indicating that soluble factors also contribute to NF-κB activation. In contrast, Amy@NPs-MM/PL1 treatment simultaneously suppressed GSDMD-N and p65 nuclear translocation, demonstrating its dual ability to block both pyroptosis propagation and upstream inflammatory signaling. Collectively, these findings demonstrate that Amy@NPs-MM/PL1 suppresses hEMSC pyroptosis and blocks its propagation to neurons. Mechanistically, early-pyroptotic hEMSCs release EVs that transmit pyroptotic signals and sustain their own pro-inflammatory state. EVs are key mediators of pyroptosis propagation, while soluble factors contribute to neuronal NF-κB activation. Amy@NPs-MM/PL1 uniquely inhibits both pyroptosis execution and upstream inflammatory signaling, thereby disrupting the “hEMSC pyroptosis–neuroinflammation–pain” cascade in endometriosis. To assess the in vivo therapeutic efficacy of Amy@NPs-MM/PL1, we developed a mouse EM model and administered PBS, GnRH-a, PPNPs, Amy, Amy@NPs, or Amy@NPs-MM/PL1 according to the schedule depicted in Fig. 3 a. There were clear differences in the size and shape of ectopic lesions between the treatment groups ( Fig. 3 b). Amy@NPs-MM/PL1 produced the most significant decrease in both the number and weight of lesions ( Fig. 3 c and d), while body weight remained constant throughout the treatment ( Fig. 3 e). Fig. 3 In vivo therapeutic evaluation of Amy@NPs-MM/PL1 in an endometriosis mouse model. (a) Schematic illustration of the endometriosis mouse model and the treatment schedule for PBS, GnRH-a, PPNPs, Amy, Amy@NPs, and Amy@NPs-MM/PL1. (b–d) Representative images of ectopic lesions from different treatment groups, along with quantitative analysis of lesion number and weight across groups. (e) Body-weight monitoring of mice throughout the treatment period. (f) In vivo fluorescence imaging of Cy5.5 dye alone, Cy5.5-labeled Amy@NPs, and Cy5.5-labeled Amy@NPs-MM/PL1 at 0, 3, and 7 days post-administration. (g) Fluorescence imaging performed ex vivo on excised major organs (heart, liver, spleen, lung, kidney), uterus, and ectopic lesions from the Amy@NPs and Amy@NPs-MM/PL1 groups. (h) Representative H&E and Masson staining images of ectopic lesion tissues from each treatment group. In vivo therapeutic evaluation of Amy@NPs-MM/PL1 in an endometriosis mouse model. (a) Schematic illustration of the endometriosis mouse model and the treatment schedule for PBS, GnRH-a, PPNPs, Amy, Amy@NPs, and Amy@NPs-MM/PL1. (b–d) Representative images of ectopic lesions from different treatment groups, along with quantitative analysis of lesion number and weight across groups. (e) Body-weight monitoring of mice throughout the treatment period. (f) In vivo fluorescence imaging of Cy5.5 dye alone, Cy5.5-labeled Amy@NPs, and Cy5.5-labeled Amy@NPs-MM/PL1 at 0, 3, and 7 days post-administration. (g) Fluorescence imaging performed ex vivo on excised major organs (heart, liver, spleen, lung, kidney), uterus, and ectopic lesions from the Amy@NPs and Amy@NPs-MM/PL1 groups. (h) Representative H&E and Masson staining images of ectopic lesion tissues from each treatment group. To examine the in vivo behavior of the nanoparticles, real-time fluorescence imaging was conducted utilizing Cy5.5-labeled formulations ( Fig. 3 f). The free Cy5.5 dye was quickly removed and was almost impossible to see by day 3. Amy@NPs had a signal that could be seen on day 3 but not on day 7. Amy@NPs-MM/PL1, on the other hand, had strong fluorescence that lasted longer than day 7. This shows that membrane coating allowed for much longer intraperitoneal retention. To further validate PL1-mediated targeting, an in vivo competition assay was performed. Pre-injection of free PL1 peptide significantly reduced the lesion accumulation of Cy5.5-labeled Amy@NPs-MM/PL1 ( Fig. S3 ), providing direct evidence for receptor-mediated specificity. Ex vivo fluorescence imaging further demonstrated the in vivo distribution profiles of the nanoparticles ( Fig. 3 g). Amy@NPs built up not only in ectopic lesions but also showed strong fluorescence in the uterus, which could mean that they were exposed to something else. In contrast, Amy@NPs-MM/PL1 showed very little distribution in the uterus and other major organs. Instead, it only built up strongly and selectively in ectopic lesions. Histological analysis employing H&E and Masson staining demonstrated significant pathological enhancements in Amy@NPs-MM/PL1–treated lesions, characterized by diminished inflammatory infiltration, decreased stromal cell distribution, enhanced extracellular matrix remodeling, and reduced fibrosis. These results show that Amy@NPs-MM/PL1 not only stops lesions from growing, but it also stays in the body for a long time, targets lesions more effectively, and doesn't build up in other places. This biomimetic nanosystem effectively reshapes the pathological microenvironment of ectopic lesions by combining membrane-mediated immune evasion with targeted delivery of Amy. This supports its potential as a safer and more effective treatment for EM. Amy@NPs-MM/PL1 treatment altered the inflammatory microenvironment of endometriotic lesions by concurrently inhibiting pyroptotic signaling and restricting subsequent cytokine release. Immunofluorescence analysis demonstrated a comprehensive reduction in proliferative, angiogenic, and invasive activities within the lesions, characterized by significantly diminished expression of Ki-67/CD31 and Vimentin/MMP-9 ( Fig. 4 a). Ectopic lesions showed more inflammatory activation than eutopic endometrium. Following treatment with Amy@NPs-MM/PL1, inflammasome activation was markedly inhibited, as evidenced by decreased levels of Cleaved-Caspase-1, GSDMD-N, IL-1β, and IL-18, signifying effective suppression of pyroptosis and inflammatory signaling. Amy@NPs-MM/PL1 also blocked the TNF-α/TNFR1/NF-κB pathway and turned off genes that respond to NF-κB, such as COX-2, MMP-2, MMP-9, and VEGF ( Fig. 4 a–c). This coordinated regulation not only reduced inflammation in the area, but it also slowed down the remodeling of the extracellular matrix and fibrosis in ectopic lesions. Fig. 4 Expression profiling of pyroptosis-related and inflammatory markers in endometriotic lesions. (a–d) Representative immunofluorescence, Western blot, heatmap, and ELISA results showing the expression patterns of pyroptosis-related proteins, inflammatory cytokines, and angiogenesis or fibrosis markers (Ki-67/CD31, Vimentin/MMP-9, C-CASP-1/GSDMD-N, IL-1β/IL-18, TNF-α, TNFR1, NF-κB, COX-2, MMP-2, MMP-9, VEGF, and PGE 2 ) in eutopic and ectopic endometrial tissues as well as in different treatment groups (PBS, PPNPs, Amy, Amy@NPs, and Amy@NPs-MM/PL1). Expression profiling of pyroptosis-related and inflammatory markers in endometriotic lesions. (a–d) Representative immunofluorescence, Western blot, heatmap, and ELISA results showing the expression patterns of pyroptosis-related proteins, inflammatory cytokines, and angiogenesis or fibrosis markers (Ki-67/CD31, Vimentin/MMP-9, C-CASP-1/GSDMD-N, IL-1β/IL-18, TNF-α, TNFR1, NF-κB, COX-2, MMP-2, MMP-9, VEGF, and PGE 2 ) in eutopic and ectopic endometrial tissues as well as in different treatment groups (PBS, PPNPs, Amy, Amy@NPs, and Amy@NPs-MM/PL1). A systemic cytokine assessment showed that the levels of IL-1β, IL-18, TNF-α, and PGE 2 in the serum were much lower in mice treated with Amy@NPs-MM/PL1. This suggests that the nanoplatform effectively reduced systemic inflammation ( Fig. 4 d). In summary, these results show that Amy@NPs-MM/PL1 breaks the pyroptosis–inflammation feedback loop and brings the immune system back into balance. This stops the growth of endometriotic lesions at both the tissue and systemic levels. In our study, we observed that the activity levels of EM model mice were significantly reduced compared to the healthy control group. Based on in vitro experiments, we hypothesize that this change is closely related to pyroptosis propagation from the EM lesion microenvironment and the subsequent neuroinflammation. This process likely leads to severe pain and, through chronic pain and hyperalgesia, results in the development of anxiety and depressive-like behaviors in these mice. To further explore these mechanisms and assess whether the Amy@NPs-MM/PL1 nanoparticle system can improve these conditions, we performed a comprehensive series of behavioral assessments, immunohistochemical analyses, metabolomic profiling, and other biochemical and molecular studies on three groups: healthy mice, PBS-treated EM model mice (control group), and Amy@NPs-MM/PL1-treated EM model mice (Amy@NPs-MM/PL1 treatment group). In the open field test, control mice showed significantly reduced locomotor activity compared to Amy@NPs-MM/PL1-treated mice, indicating that the treatment mitigated EM-related neuroinflammation and pain. In forced swim and tail suspension tests, control mice exhibited increased immobility time, a classic sign of depressive-like behavior, whereas treated mice showed markedly reduced immobility. The sucrose preference test revealed anhedonia in control mice (reduced sucrose preference), which was restored by Amy@NPs-MM/PL1. Von Frey testing showed that control mice had a lower paw withdrawal threshold (heightened pain sensitivity), while treatment significantly elevated the threshold, indicating reduced hyperalgesia ( Fig. 5 a). Collectively, Amy@NPs-MM/PL1 effectively alleviated EM-induced pain, depressive-like behaviors, and reduced activity. Fig. 5 Comprehensive Analysis of the Effect of Amy@NPs-MM/PL1 Treatment on Neuroinflammation and Metabolism in Endometriosis Mice. (a) Schematic diagrams (left) and statistical histograms (right) of behavioral tests in mice from different treatment groups, including the open field test, swimming test, tail suspension test, sucrose preference test, and pain sensitivity test. (b) Immunohistochemical staining results of IBA-1 and Ki67 in hippocampal sections of mice from different groups at the end of treatment. (c) Bubble plot showing the levels of TNF-α, IL-1β, 3-NT, and NSE in the cerebrospinal fluid of sacrificed mice after different treatments. (d) Principal component analysis (PCA) results of serum samples from mice in different groups after treatment. Control group: Endometriosis mice treated with PBS; QC group: Quality control samples. (e) Heatmap displaying the relative abundance of 14 representative differential metabolites in serum samples from different groups. (f) Histograms showing the relative levels of four representative metabolites (Indole, Linoleoylcarnitine, Taurine, and N-Arachidonylglycine) in serum samples from different groups of mice. (g) WB bands and heatmaps of key proteins in metabolic pathways related to the four representative metabolites (AHR, AMPKα1, NLRP3, and GPR18). Comprehensive Analysis of the Effect of Amy@NPs-MM/PL1 Treatment on Neuroinflammation and Metabolism in Endometriosis Mice. (a) Schematic diagrams (left) and statistical histograms (right) of behavioral tests in mice from different treatment groups, including the open field test, swimming test, tail suspension test, sucrose preference test, and pain sensitivity test. (b) Immunohistochemical staining results of IBA-1 and Ki67 in hippocampal sections of mice from different groups at the end of treatment. (c) Bubble plot showing the levels of TNF-α, IL-1β, 3-NT, and NSE in the cerebrospinal fluid of sacrificed mice after different treatments. (d) Principal component analysis (PCA) results of serum samples from mice in different groups after treatment. Control group: Endometriosis mice treated with PBS; QC group: Quality control samples. (e) Heatmap displaying the relative abundance of 14 representative differential metabolites in serum samples from different groups. (f) Histograms showing the relative levels of four representative metabolites (Indole, Linoleoylcarnitine, Taurine, and N-Arachidonylglycine) in serum samples from different groups of mice. (g) WB bands and heatmaps of key proteins in metabolic pathways related to the four representative metabolites (AHR, AMPKα1, NLRP3, and GPR18). Immunohistochemical staining of the hippocampus ( Fig. 5 b) revealed that in the control group, microglia exhibited an amoeboid morphology with enlarged cell bodies and reduced processes, indicating activation, while KI-67 levels were decreased. In contrast, Amy@NPs-MM/PL1 treatment restored normal microglial morphology and KI-67 levels, indicating reduced neuroinflammation and improved neurogenesis. To dynamically assess central sensitization, we performed a time-course analysis of glial activation in the spinal cord dorsal horn at 0, 1, 2, and 3 weeks post-treatment ( Fig. S4a ). At week 0, ionized calcium-binding adapter molecule 1 (IBA-1)-positive microglia displayed an amoeboid morphology and glial fibrillary acidic protein (GFAP) fluorescence was markedly elevated. Over time, microglia progressively transitioned to a ramified phenotype and GFAP signal gradually decreased, reaching healthy levels by week 3. This dynamic recovery correlated with the progressive improvement in pain behavior ( Fig. 5 a). ELISA of cerebrospinal fluid further confirmed lower levels of TNF-α, IL-1β, 3-nitrotyrosine (3-NT) , and neuron-specific enolase (NSE) in the treatment group ( Fig. 5 c). Together, these data demonstrate that Amy@NPs-MM/PL1 alleviates neuroinflammation and central sensitization in a time-dependent manner. Untargeted metabolomics revealed distinct metabolic profiles among healthy, control, and Amy@NPs-MM/PL1 groups (PCA, Fig. 5 d). A heatmap of 14 differential metabolites associated with EM progression, pain, and neuroinflammation is shown in Fig. 5 e. Compared to controls, Amy@NPs-MM/PL1 increased beneficial metabolites (e.g., thiamine) and decreased harmful ones (e.g., oleylcarnitine). KEGG pathway enrichment ( Fig. S4b -c) further supported the observed changes. Indole reduction aligned with 'Tryptophan metabolism' and aryl hydrocarbon receptor (AHR)-related pathways, suggesting AHR-mediated suppression of inflammation. Linoleoylcarnitine reduction correlated with 'Metabolic pathways' and energy homeostasis networks, linking to AMP-activated protein kinase α1 subunit (AMPKa1) activation and metabolic restoration. Taurine elevation was supported by 'Primary bile acid biosynthesis' and 'Bile secretion' pathways, likely enhancing NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome regulation and reducing neuroinflammation. N-arachidonylglycine reduction matched 'Steroid hormone biosynthesis', accompanied by changes in its receptor G protein-coupled receptor 18 (GPR18), reflecting reduced inflammation and enhanced neurorepair. These findings were corroborated by Western blot analysis of neuroinflammation-related proteins. Together, these data demonstrate that Amy@NPs-MM/PL1 alleviates EM-associated pain and neuroinflammation via comprehensive metabolic reprogramming. To verify the systemic biosafety of Amy@NPs-MM/PL1, we compared healthy mice treated with the formulation against untreated controls through histopathological and biochemical evaluations. H&E staining revealed no noticeable tissue damage, necrosis, or inflammatory infiltration in major organs, including the uterus, heart, liver, spleen, lung, and kidney ( Fig. 6 a), indicating the absence of histological toxicity. Fig. 6 Biosafety evaluation of Amy@NPs-MM/PL1 in vivo. (a) Representative H&E-stained images of major organs (uterus, heart, liver, spleen, lung, and kidney) from control and Amy@NPs-MM/PL1–treated mice. (b) Quantitative analyses of hematological, biochemical, and hormonal parameters, including AST, ALP, BUN, Cr, ALT, WBC, RBC, PLT, E2, and PG, comparing control and Amy@NPs-MM/PL1 groups. Biosafety evaluation of Amy@NPs-MM/PL1 in vivo. (a) Representative H&E-stained images of major organs (uterus, heart, liver, spleen, lung, and kidney) from control and Amy@NPs-MM/PL1–treated mice. (b) Quantitative analyses of hematological, biochemical, and hormonal parameters, including AST, ALP, BUN, Cr, ALT, WBC, RBC, PLT, E2, and PG, comparing control and Amy@NPs-MM/PL1 groups. Additionally, hematological and biochemical parameters, including AST, ALP, BUN, Cr, ALT, WBC, RBC, and PLT, remained within normal physiological limits, exhibiting no significant differences between the control and Amy@NPs-MM/PL1 groups. Likewise, serum hormone concentrations of estradiol (E2) and progesterone (PG) exhibited no significant changes ( Fig. 6 b). These findings collectively indicate that Amy@NPs-MM/PL1 exhibits superior biocompatibility and systemic safety, thereby endorsing its potential for prolonged therapeutic use in EM.

Amy was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (China). Poly(lactic-co-glycolic acid) (PLGA, 50:50, molecular weight: 30,000–60,000 Da) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG) were obtained from Xi'an Ruixi Biological Technology Co., Ltd. (China). Cyanine5.5 N-hydroxysuccinimide ester (Cy5.5) was purchased from Lumiprobe (USA). All other chemicals and reagents were of analytical grade and used as received. Primary human EM-associated stromal cells (hEMSCs; SUNNCELL, China) were used and cultured in stromal cell–specific complete medium (BaiDi Biotechnology Co., Ltd, China). RAW264.7 murine macrophages (Pricella, China) were maintained in RAW264.7 cell–specific medium (BDBIO, China). All cultures were kept in a humidified incubator at 37 °C with 5% CO 2 . For in vitro studies, Amy@NPs-MM/PL1 was used at 100 μg/mL (nanoparticle total mass). Free Amy (20 μg/mL) and empty PPNPs (100 μg/mL) served as controls. To induce pyroptosis, hEMSCs were stimulated with 1 μg/mL lipopolysaccharide (LPS; Sigma, USA) for 24 h. To establish the macrophage-hEMSC co-culture model, RAW264.7 macrophages were seeded on the upper chamber of 24-well transwell inserts (0.4 μm pore size, Corning, USA) at a density of 5 × 10 4  cells/insert, while hEMSCs were seeded in the lower chamber at a density of 2 × 10 4  cells/well. After 24 h co-culture for barrier formation, Cy5.5-labeled formulations were added to the upper chamber. Following another 12 h, cells from both chambers were collected for uptake analysis. Amy-loaded PEG-PLGA nanoparticles (Amy@NPs) were prepared using a microfluidic method. Briefly, Amy, PLGA, and Cy5.5 dye were dissolved in acetone to form the organic phase, which was mixed with a 2% (w/v) PVA aqueous solution in a staggered herringbone micromixer (SHM) chip with 200 μm × 100 μm microchannels at a flow rate ratio of 3:9 (organic: aqueous, mL/min) and a total flow rate of 12 mL/min. The resulting emulsion was dialyzed against deionized water using a 300 kDa molecular weight cut-off (MWCO) membrane to remove the organic solvent, filtered through a 0.22 μm syringe filter, and concentrated using a 100 kDa ultrafiltration tube to obtain Amy@NPs. To prepare Amy@NPs-MM/PL1, RAW264.7 cell membranes were extracted by repeated freeze–thaw cycles and differential centrifugation. PL1 lipid-targeting peptides (PPRRRGLIKLKTS) were then inserted into the isolated macrophage membranes. Briefly, PL1 peptides were dissolved in a small volume of dimethyl sulfoxide (DMSO) and added to the extracted macrophage membranes (resuspended in PBS, pH 7.4, at 1 mg/mL protein concentration) at a final concentration of 50 μg/mL (peptide: membrane protein mass ratio of 1:20). The peptide insertion was achieved by incubating the mixture at 37 °C for 1 h with gentle agitation, allowing efficient fusion of PL1 peptides with the macrophage membranes. After the peptide insertion, Amy@NPs were mixed with the modified membranes at a 1:1 protein-to-nanoparticle mass ratio, followed by probe sonication (30% amplitude, 5 min, ice bath) and extrusion through a 200 nm polycarbonate membrane using a mini-extruder for membrane coating. The resulting membrane-coated nanoparticles were then used for the preparation of Amy@NPs-MM/PL1. Nanoparticle size and zeta potential were measured using dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern, UK). Morphological observation was performed using transmission electron microscopy (TEM; HT7700, Hitachi, Japan). The drug loading capacity (DL) and encapsulation efficiency (EE) were determined using a high-performance liquid chromatography (HPLC) system (Elite, China). Drug release profiles were evaluated by a dialysis method using PBS (pH 7.4) and acetate buffer (pH 5.5) at 37 °C. Samples were collected at predetermined time points and analyzed by UV–Vis spectrophotometry at 269 nm. Fluorescence spectra of Cy5.5-loaded nanoparticles were recorded using a microplate reader (λ_ex = 675 nm, λ_em = 700–750 nm). The levels of hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 − ), hydroxyl radicals (·OH), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were measured using specific assay kits from Abbkine. For hydrogen peroxide, the concentration was determined using a dedicated kit. For superoxide anion, hydroxyl radicals, and DPPH radicals, the scavenging rates were assessed based on the reduction in absorbance following the reaction with specific reagents. The assays were performed according to the manufacturer's instructions, and the results were expressed as the percentage of radicals scavenged or the concentration of hydrogen peroxide. A murine model of EM was established using an allogeneic transplantation method. Female BALB/c mice (6–8 weeks old, 18–22 g, SPF grade, purchased from Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., China) were housed under standard laboratory conditions (22 ± 2 °C, 12 h light/dark cycle, ad libitum access to food and water). All experimental procedures were approved by the Animal Ethics Committee of Wannan Medical College. Donor mice received subcutaneous injections of estradiol benzoate (0.2 mg/kg) once every other day for 7 consecutive days to induce a proliferative endometrial state. On day 8, uteri were excised under sterile conditions, and endometrial tissues were minced into fragments (<1 mm in diameter) using ophthalmic scissors. These tissue fragments were suspended in sterile PBS and injected intraperitoneally (200 μL per mouse) into recipient mice to induce ectopic lesion formation. One week after transplantation, a subset of mice was euthanized to confirm model establishment via histological analysis. The remaining mice were randomly assigned into six groups (n = 6 per group): (1) PBS, (2) Gonadotropin-releasing hormone analogue (GnRH-a) (3) PPNPs, (4) Amy, (5) Amy@NPs, and (6) Amy@NPs-MM/PL1. For in vivo treatment, free Amy was administered via intraperitoneal injection at a dose of 5 mg/kg body weight. Amy@NPs-MM/PL1 was administered at an equivalent amygdalin dose of 5 mg/kg, corresponding to a nanoparticle dose of approximately 28.9 mg/kg based on a drug loading capacity of 17.33%. PPNPs were administered at the same nanoparticle dose (28.9 mg/kg) to serve as a vehicle control. The GnRH-a (Leuprorelin, 1 mg/kg) was used as a positive control. All treatments were administered three times per week for 3 consecutive weeks, with a total of 9 injections per mouse. The control group received an equal volume of sterile PBS. Behavioral assessments were conducted to evaluate anxiety, depression-like behavior, and pain sensitivity in mice. The open field test (10 min) was used to measure general movement and anxiety. Spending more time in the center of the field meant less anxiety. The swimming test (5 min) was used to assess depressive-like behavior, with the immobility time in the water indicating the level of depression. The tail suspension test (6 min) assessed depression-like behavior by recording immobility time. The sucrose preference test (24 h) evaluated anhedonia, a symptom of depression, by measuring the preference for a sucrose solution. The Von Frey test was used to assess mechanical pain sensitivity by applying calibrated filaments to the hind paw and recording the withdrawal threshold. These tests provided a comprehensive analysis of the animals’ emotional and sensory responses. All behavioral assessments were conducted by experimenters blinded to the treatment group allocation. Proteins were extracted with RIPA buffer (Beyotime, China) containing protease/phosphatase inhibitors and quantified by BCA (Beyotime). Equal amounts were resolved by SDS–PAGE, transferred to PVDF membranes (Millipore, USA), blocked with rapid blocking buffer (e.g., QuickBlock, Beyotime), and incubated overnight at 4 °C with primary antibodies against TNF-α, TNFR1, NF-κB p65, Caspase-1, Cleaved-Caspase-1, GSDMD, GSDMD-N, IL-1β, IL-18, COX-2, MMP2, MMP9, VEGF, BDNF, NGF, β-III-tubulin, and GAPDH. These antibodies were purchased from HUABIO (China) and Abcam (UK). HRP-conjugated secondary antibodies were applied for 1 h at room temperature, signals were developed with ECL (Thermo Fisher, USA), and quantified using ImageJ. Tissue sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0) at 95 °C for 15 min. Sections were permeabilized with 0.3% Triton X-100 and blocked with 5% BSA for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against Ki-67, CD31, MMP9, NGF, cleaved caspase-1, GSDMD-N, IL-1β, and IL-18 (Immunoway, USA). After washing, fluorescence labeling was performed using a mouse/rabbit triple-color fluorescence detection kit (Immunoway, USA) according to the manufacturer's instructions. Nuclei were counterstained with DAPI, slides were mounted with antifade medium, and images were acquired on a confocal laser scanning microscope (Leica, Germany). The levels of IL-1β, IL-18, BDNF, NGF, PGE2, TNF-α, 3-NT, and NSE in the cell supernatant, serum, or cerebrospinal fluid were measured using ELISA kits (ELK Biotechnology, China). The assay was performed according to the manufacturer's instructions. Briefly, 96-well plates were pre-coated with specific capture antibodies for each target analyte. After blocking with 5% BSA, cell supernatant, serum, or cerebrospinal fluid samples were added to the wells and incubated at 37 °C for 80 min. After washing the plates, biotinylated detection antibodies were added and incubated for 50 min at 37 °C, followed by the addition of Streptavidin-HRP and a second incubation. The color was developed using TMB substrate solution, and the reaction was terminated by adding Stop Solution. Absorbance was measured at 450 nm using a microplate reader. The cytokine levels were quantified by comparison with standard curves provided in the kits. At the end of the treatment, mice were sacrificed, and their tissues were collected for histological analysis. Brain tissues and endometrial lesions were fixed in 4% formaldehyde and embedded in paraffin. Sections (5 μm) were cut and stained with hematoxylin and eosin (H&E) or immunohistochemically for IBA-1 and KI-67 using specific antibodies from Abcam. The staining results were evaluated under a 40 × microscope. At the end of the treatment, serum samples were collected from each group of mice. Non-targeted serum metabolomics analysis was performed using liquid chromatography coupled with mass spectrometry (LC-MS) to explore the metabolic differences between the different groups. Principal component analysis (PCA) was used to assess the metabolic variation between the groups. Data were log-transformed and normalized using MetaX software for further analysis. Data are presented as the means ± SD (n = 3). Significant differences between two groups were analyzed using unpaired two-tailed Student's t-tests. No significant difference was denoted as "ns," and statistical significance was denoted as P < 0.05, ∗P < 0.01, and ∗∗∗P < 0.001. Analyses were performed in GraphPad Prism (10.1.2).

Endometriosis remains therapeutically challenging because existing therapies inadequately address both ectopic lesion survival and chronic pelvic pain [ 24 , 25 ]. We identified a novel “lesion microenvironment – pyroptosis propagation – neuroinflammation” axis and developed a dual-targeted biomimetic nanotherapeutic, Amy@NPs-MM/PL1, to disrupt it. Our principal findings are: (1) pyroptotic endometrial stromal cells drive neuronal activation; (2) macrophage membrane coating and PL1 peptide enable enhanced lesion targeting; (3) the nanoplatform inhibits stromal cell pyroptosis and blocks intercellular propagation to neurons; (4) it reduces lesion burden, reverses pain and depressive-like behaviors, and restores metabolic homeostasis. The persistence of pain in EM, often disproportionate to lesion size, suggests mechanisms beyond local inflammation [ 26 ]. In the lesion microenvironment, oxidative stress induces pyroptosis. Pyroptotic cells release extracellular vesicles (EVs) that propagate pyroptotic signals to neighboring neurons, triggering neuroinflammation and central sensitization. Using a transwell co-culture system, we demonstrated that EVs from pyroptotic hEMSCs are both necessary and sufficient for this propagation, while soluble factors also contribute to neuronal NF-κB activation. Notably, Amy@NPs-MM/PL1 simultaneously suppressed EV-mediated pyroptosis transmission and upstream NF-κB signaling, distinguishing it from a conventional NLRP3 inhibitor (MCC950) that only reduced GSDMD-N without blocking p65 nuclear translocation. These data establish EVs as key mediators of pyroptosis propagation and highlight the dual mechanism of Amy@NPs-MM/PL1. The macrophage membrane coating enables immune evasion and innate tropism for inflammatory sites [ 27 , 28 ]. The selection of amygdalin (Amy) was based on its anti-inflammatory properties [ 29 ]. Mechanistically, its broad-spectrum ROS scavenging activity derives from phenolic hydroxyl groups (direct radical neutralization) and nitrile group-mediated metal chelation (inhibiting Fenton reaction) [ [30] , [31] , [32] ]. Additionally, Amy activates the Nrf2 pathway, upregulating endogenous antioxidant enzymes (SOD, CAT, GPx) [ 33 ]. This dual mechanism underlies the potent ROS scavenging observed in our study. Amy@NPs-MM/PL1 markedly inhibits NLRP3 inflammasome activation and GSDMD cleavage, effectively terminating the pyroptotic cascade. In vivo, reduced hippocampal IBA-1 and KI-67 levels correlated with relief of mechanical allodynia and depressive-like behaviors [ 34 ], confirming that peripheral lesion targeting modulates central nervous system responses. Beyond direct cellular effects, untargeted metabolomics revealed that Amy@NPs-MM/PL1 restores systemic metabolic homeostasis. KEGG pathway enrichment showed significant alterations in tryptophan metabolism, primary bile acid biosynthesis, and steroid hormone biosynthesis, which support the observed changes in indole, taurine, and N-arachidonylglycine. The restored equilibrium in pathways like glutathione metabolism and taurine/hypotaurine metabolism signifies improved antioxidant capacity, whereas alterations in arachidonic acid metabolism suggest a mitigated inflammatory condition [ [35] , [36] , [37] , [38] ]. This metabolic reprogramming likely contributes to the broad therapeutic benefits, including improved behavioral outcomes. The Amy@NPs-MM/PL1 system exhibits distinct translational advantages. Unlike GnRH agonists that cause hypoestrogenic side effects, our nanoplatform achieves anti-lesion and analgesic efficacy without perturbing physiological hormone levels, offering a fertility-sparing alternative. Compared to conventional non-targeted nanoparticles, our dual-targeted design facilitates immune evasion and precise lesion homing. We acknowledge that this is an initial proof-of-concept study. The intraperitoneal route may challenge patient compliance; alternative strategies (intravenous or local delivery during laparoscopy) merit consideration. Lesion heterogeneity in Tenascin-C expression could be addressed by patient stratification using PL1-conjugated imaging probes, with the macrophage membrane providing complementary homing. Long-term safety is critical; while acute toxicity data are reassuring, repeated dosing may elicit immune responses. Preservation of CD47 “self” markers and use of autologous membranes could minimize immunogenicity, but extended toxicology studies remain essential. In conclusion, this study identifies a novel pyroptosis-mediated communication axis in endometriosis and introduces a strategically formulated nanotherapeutic to disrupt it. Amy@NPs-MM/PL1 integrates biomimetic targeting, mechanism-driven action, and multi-modal validation. Future work should clarify the contribution of other cell types (e.g., macrophages) to this axis and explore combination with hormonal therapies. By shifting focus from symptom relief to mechanistic disruption of a key pathology-pain pathway, this work offers an innovative, translatable framework for more effective endometriosis therapy.

Endometriosis (EM) is a chronic gynecological disorder characterized by the extrauterine growth of endometrial-like tissue, affecting millions of women of reproductive age [ 1 , 2 ]. Beyond infertility, over 60% of patients suffer from debilitating pelvic and extrapelvic pain (e.g., chronic back pain, fibromyalgia, migraines), which significantly impairs their quality of life [ 3 , 4 ]. Current treatment options, such as hormonal suppression and surgery, provide only temporary symptom relief and are associated with high recurrence rates and side effects [ 5 , 6 ]. These limitations underscore an urgent need to elucidate the biological mechanisms perpetuating lesion survival and pain, facilitating the development of targeted, mechanism-based therapies. Recent studies revealed that the aberrant inflammation and oxidative stress are central drivers of EM progression [ [7] , [8] , [9] ]. In the ectopic microenvironment, excessive reactive oxygen species (ROS) can lead to disrupted redox balance, further leading to inflammasome activation and subsequent pyroptosis, which is defined as the inflammation-programmed cell death resulting from caspase-1- and gasdermin D (GSDMD)-mediated pore formation [ 10 , 11 ]. Notably, pyroptosis is not a cell-autonomous process. Pyroptotic cells release IL-1β, IL-18, and damage-associated molecular patterns (DAMPs), which propagate inflammatory signals to neighboring cells via extracellular vesicles, amplifying tissue-level inflammation [ [12] , [13] , [14] ]. Based on this, we hypothesize that pyroptotic endometrial stromal cells may initiate pyroptosis propagation to adjacent sensory neurons, triggering neuroinflammation. This "pyroptosis-neuroinflammation" axis may represent a fundamental pathological mechanism underlying EM-associated chronic pain. Natural compounds exhibiting multi-target regulatory activity have garnered heightened interest due to their capacity to modulate intricate inflammatory networks [ 15 ]. Amygdalin (Amy), a naturally occurring cyanogenic glycoside derived from bitter apricot kernels [ 16 ], exhibits antioxidant, anti-inflammatory, and antifibrotic properties in various disease models [ 17 , 18 ]. Previous studies showed that Amy reduces oxidative stress and inhibits key pyroptosis-related molecules, including NLRP3, ASC, and IL-1β [ 19 ]. These findings position Amy as a promising candidate for targeting the pyroptosis-neuroinflammation axis in EM. However, its clinical translation is hampered by cyanide-related toxicity, poor bioavailability, and rapid metabolic degradation, necessitating a safer and more efficient delivery system [ 20 ]. In this research, we developed a multifunctional nanodrug platform encapsulating Amy for targeted intervention in EM. The resulting nanosystem, termed Amy@NPs-MM/PL1, consists of a poly(ethylene glycol)–poly(lactic-co-glycolic acid) (PEG–PLGA) core loaded with Amy, cloaked with a biomimetic macrophage membrane (MM) , and decorated with PL1 peptides (PPRRRGLIKLKTS) for dual-targeting capability. The PEG–PLGA matrix ensures controlled drug release and stabilizes the inherently low-bioavailability payload [ 21 ]. In endometriotic lesions, inflammatory cell encapsulation inhibits drug penetration; however, macrophage membrane cloaking imparts nanoparticles with inflammatory homing, membrane fusion capability, and immune evasion, facilitating their traversal of the inflammatory barrier and entry into the lesion without phagocytosis [ 22 ]. Additionally, the PL1 peptide enhances targeting specificity by binding to the C-terminal domain of Tenascin-C (TNC-C), which is highly overexpressed in EM lesions compared to other peritoneal inflammatory sites [ 23 ]. Our findings demonstrate that Amy@NPs-MM/PL1 effectively disrupts the pathological axis by simultaneously scavenging lesion-derived ROS, blocking the caspase-1/GSDMD pyroptotic cascade in stromal cells, and attenuating secondary neuronal inflammatory injury. Consequently, this biomimetic nanoplatform significantly restrains lesion progression and ameliorates pain outcomes, presenting an innovative, microenvironment-targeted approach for comprehensive EM management.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jin Ding reports financial support was provided by National Natural Science Foundation of China. Jin Ding reports financial support was provided by Clinical Medicine Transformation Project of Anhui Provincial Department of Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.