Abstract

   Abnormal accumulation of both intracellular and extracellular free
   nucleic acids drives chronic inflammation in intervertebral disc
   degeneration (IVDD). Despite the development of numerous minimally
   invasive treatments for IVDD, systematic approaches targeting the
   chronic inflammation mediated by both nucleic acid types are lacking.
   We propose a dual clearance strategy that inhibits mitochondrial DNA
   release inside nucleus pulposus cells while removing extracellular DNA
   from the disc microenvironment. Using single-cell sequencing and
   clinical samples, we revealed how both nucleic acid types drive
   inflammation. We then developed a targeted nanovesicle system
   delivering a mitochondrial membrane-stabilizing small molecule to block
   DNA leakage and inflammatory signaling, along with a hydrogel that
   captures extracellular DNA to prevent immune activation. In a rat
   model, this approach significantly slowed disease progression. This
   targeted dual nucleic acid clearance strategy provides a approach for
   treating IVDD and offers a theoretical framework for addressing other
   nucleic acid-related inflammatory diseases.

   Subject terms: Nanoparticles, Materials science, Biomaterials
     __________________________________________________________________

   Abnormal accumulation of intra- and extracellular free nucleic acids
   contributes to the chronic inflammation driving intervertebral disc
   degeneration (IVDD). Here, the authors report a nanovesicle system to
   inhibit mitochondrial DNA release in nucleus pulposus cells and remove
   extracellular DNA from the disc microenvironment to block inflammation
   for treatment of IVDD.

Introduction

   Low back pain is a global health issue that severely impacts patients’
   quality of life and imposes a significant burden on society and the
   economy^[44]1. IVDD is a leading cause of low back pain, especially in
   the elderly^[45]2. Current clinical treatments for IVDD—such as
   nonsteroidal anti-inflammatory drugs (NSAIDs), physiotherapy, and
   surgical intervention—are largely palliative and fail to halt or
   reverse disc degeneration^[46]3. Although experimental approaches,
   including stem cell therapy, gene editing, and biomaterial implants,
   have shown promise, they primarily aim to repair damaged tissue or
   suppress inflammation, often overlooking the upstream pathogenic
   drivers^[47]4–[48]6. In particular, the role of senescent nucleus
   pulposus (NP) cells and the inflammation they trigger via nucleic acid
   release remains underexplored. These senescent cells release
   mitochondrial DNA (mtDNA) into the cytosol and extracellular DNA
   (exDNA) into the extracellular matrix, activating the cGAS–STING and
   TLR9 pathways, respectively, to perpetuate sterile
   inflammation^[49]7,[50]8. Targeting these damage-associated nucleic
   acid signaling axes thus represents a mechanistically grounded and
   previously underutilized strategy for treating IVDD.

   Recent advances in biomimetic nanotechnology have highlighted the
   potential of drug-loaded cell membrane-derived vesicles as effective
   platforms for targeted intracellular delivery^[51]9,[52]10. Derived
   from natural cell membranes, these vesicles inherit bioactive
   properties such as immune evasion, prolonged circulation time, and
   homologous cell targeting. Incorporating therapeutic drugs into these
   vesicles improves drug stability and enables efficient delivery to
   targeted disease sites, facilitating precision treatment^[53]11. Their
   inherent biocompatibility and ability to mimic native cell interactions
   make these vesicles ideal platforms for the targeted modulation of
   cytosolic nucleic acid-driven inflammatory responses^[54]12,[55]13.

   Hyaluronic acid (HA) hydrogel, with a composition similar to the
   extracellular matrix (ECM), exhibits good biocompatibility and
   excellent drug-loading capacity, allowing sustained release of
   nanodrugs. It holds great potential for treating IVDD^[56]14,[57]15.
   Additionally, HA can be easily modified, with its surface
   functionalized by specific chemical groups or biological ligands to
   facilitate gel crosslinking reactions^[58]16. Polyamidoamine (PAMAM)
   dendrimers is a macromolecular network that contains numerous primary
   amine groups on its surface^[59]17. At physiological pH, these primary
   amines become protonated, forming positively charged -NH3^+ groups that
   easily capture negatively charged exDNA^[60]17. This functional hybrid
   system provides a means to neutralize exDNA and mitigate downstream
   immune activation.

   Here, we developed a biomimetic nanovesicle system (BNPMT) derived from
   NP cell membranes and loaded with BAI1, a small-molecule inhibitor of
   BAX/BAK-mediated mitochondrial membrane permeabilization (Fig. [61]1).
   To confer mitochondrial targeting capability, TPP^+ was conjugated to
   the vesicle surface via copper-free click chemistry. This
   dual-targeting strategy combines the inherent NP cell-homing ability of
   the membrane with the mitochondrial affinity of TPP^+, enabling
   efficient delivery of BAI1 to the NP cell mitochondria. At the target
   site, BAI1 stabilizes the mitochondrial outer membrane, thereby
   suppressing the aberrant release of mtDNA into the cytosol and
   subsequently blocking the production of senescence-associated secretory
   phenotype (SASP). Metabolomic profiling and mechanistic analyses
   further revealed that BNPMT stabilizes mitochondrial membrane integrity
   and restores mitochondrial function and energy metabolism in senescent
   NP cells. To achieve sustained local delivery and enhance therapeutic
   efficacy within the intervertebral disc, BNPMT was encapsulated in a
   dynamic hydrogel matrix crosslinked via Schiff base bonds between
   oxidized hyaluronic acid and fifth-generation PAMAM dendrimers
   (Fig. [62]1). Beyond its role as a delivery matrix, the PAMAM hydrogel
   also acts as a scavenger for exDNA, attenuating TLR9-mediated
   macrophage activation and downstream inflammatory amplification.
   Together, this dual-modality platform—simultaneously targeting
   intracellular mtDNA release and exDNA accumulation—offers effective
   therapeutic strategy for IVDD. This is the integrated approach that
   intervenes at both intracellular and extracellular levels of nucleic
   acid-mediated inflammation. In a rat model of IVDD, BNPMT treatment
   significantly alleviated disc degeneration and inflammation over an
   8-week period, supporting its translational potential for long-term,
   mechanism-oriented disc repair.

Fig. 1. Schematic overview of the hydrogel-nanocomposite design and its
mechanism in regulating intra-/extracellular nucleic acids to alleviate
intervertebral disc degeneration.

   [63]Fig. 1
   [64]Open in a new tab

   The biomimetic nanovesicles are constructed by encapsulating BAI1 with
   NP cell membranes modified with TPP. Benefiting from the homotypic
   targeting ability of NP cell membranes and the mitochondrial targeting
   of TPP, the nanovesicles are precisely delivered to the mitochondria of
   NP cells. BAI1 inhibits the activation of BAX/BAK, thereby suppressing
   miMOMP, blocking the release of mtDNA into the cytoplasm, and
   subsequently inhibiting the activation of the cGAS-STING signaling
   pathway-mediated secretion of SASP factors. The hydrogel system
   designed for capturing exDNA is cross-linked via Schiff base bonds
   between oxidized hyaluronic acid (OHA) and PAMAM-G5. The primary amine
   groups of PAMAM-G5 become protonated to form positively charged -NH3+
   under physiological pH, enabling the efficient scavenging of exDNA in
   the intervertebral disc microenvironment. This prevents the activation
   of macrophage M1 polarization via the exDNA–TLR9–TRAF6–NF-κB axis and
   helps improve the inflammatory microenvironment within the disc. In
   summary, by eliminating both intracellular and extracellular DNA in NP
   cells, the hydrogel-nanocomposite system effectively remodels the
   inflammatory microenvironment of the intervertebral disc, thereby
   delaying disc degeneration.

Results

NP cell senescence in IVDD: inflammatory activation by cytosolic nucleic
acids and extracellular DNA

   NP degeneration is a major contributor and primary driver of IVDD.
   Multiple factors—including cellular senescence, apoptosis, mechanical
   overload, inflammation, and oxidative stress—contribute to NP
   degeneration, suggesting that its prevention may offer an effective
   therapeutic strategy for IVDD^[65]18. To further investigate the link
   between nucleus pulposus degeneration and intervertebral disc
   degeneration, this study reanalyzed single-cell transcriptomic data
   from the publicly available [66]GSE165722 database and examined the
   role of nucleus pulposus cells in this process (Fig. [67]2A). Based on
   characteristic gene expression, cells from healthy and degenerated
   intervertebral discs were categorized into 14 groups (Fig. [68]2B, C).
   The 14 cell groups were further classified into four transcriptionally
   distinct clusters (Fig. [69]2D). The volcano plot indicates significant
   upregulation of senescence-related genes (e.g., p21, Tp53) in nucleus
   pulposus cells from degenerated intervertebral discs (Fig. [70]2E).
   Gene Ontology (GO) enrichment analysis of differentially expressed
   genes (DEGs) highlighted significant enrichment in pathways related to
   mitochondrial function, energy metabolism, and inflammation
   (Fig. [71]2F). Notably, mitochondrial function, energy metabolism, and
   inflammation are closely interconnected in senescent cells, and their
   imbalance collectively accelerates cellular senescence^[72]19,[73]20.
   In senescent cells, mitochondrial function declines, energy production
   decreases, and self-repair and stress responses weaken. Meanwhile, ROS
   accumulates, damaging DNA, proteins, and lipids, ultimately impairing
   normal cell function^[74]21,[75]22. Gene set enrichment analysis (GSEA)
   revealed significant upregulation of inflammatory response pathways and
   downregulation of senescence-inhibition pathways in degenerated
   intervertebral discs, suggesting a strong association between
   intervertebral disc degeneration and nucleus pulposus cell senescence
   (Fig. [76]2G, H). To complement these findings, we characterized the
   transcriptomic landscapes of young and senescent NP cells in vitro. Rat
   NP cells were isolated and induced into senescence using doxorubicin
   (Dox), followed by RNA sequencing (Fig. [77]2I). Principal component
   analysis (PCA) demonstrated distinct transcriptomic profiles between NC
   and senescent NP cells (Supplementary Fig. [78]1). Senescent cells
   showed significant upregulation of genes related to the SASP and
   downregulation of ECM synthesis genes (Fig. [79]2J). GO enrichment
   analysis revealed marked enrichment in inflammatory and immune
   activation pathways, alongside suppression of mitochondrial functional
   pathways (Fig. [80]2K). GSEA also confirmed elevated immune and
   inflammatory activity in senescent NP cells compared to normal controls
   (Supplementary Fig. [81]2). Collectively, these results suggest that
   inflammatory senescence pathways are activated in NP cells during IVDD.
   However, the precise mechanisms driving NP cell senescence remain
   incompletely understood. Under stress conditions, mitochondrial
   dysfunction may induce minority mitochondrial outer membrane
   permeabilization (miMOMP), leading to cytosolic leakage of mtDNA and
   subsequent activation of innate immune responses^[82]23. To test this
   hypothesis, we extracted the cytoplasmic fraction of normal and
   senescent NP cells and quantified cytosolic mtDNA via quantitative PCR
   (qPCR). Senescent NP cells exhibited significant mtDNA accumulation in
   the cytoplasm (Fig. [83]2L and Supplementary Fig. [84]3). Moreover,
   histological analyses of human NP tissues from different Pfirrmann
   grades (Fig. [85]2M) and rat disc samples revealed markedly elevated
   expression of STING in degenerated discs (Fig. [86]2N and Supplementary
   Fig. [87]4), implicating activation of the cGAS-STING pathway through
   cytosolic DNA sensing. In addition to intracellular responses,
   senescent cells may release mtDNA into the extracellular
   microenvironment^[88]7, where it can activate macrophages via Toll-like
   receptors (TLRs), thereby amplifying inflammation. While cytosolic DNA
   activates inflammation through the cGAS-STING axis, extracellular
   nucleic acids contribute to immune activation via TLR-mediated
   pathways^[89]24,[90]25. To assess the relevance of extracellular
   nucleic acids in IVDD, we isolated NP tissues from healthy and
   degenerated rats and measured the DNA content in the microenvironment
   following cell isolation. Degenerated discs exhibited significantly
   higher exDNA levels than healthy discs (Fig. [91]2O), further
   supporting the involvement of both intracellular and extracellular
   nucleic acids in IVDD pathology. In summary, these findings uncover a
   pathological axis in IVDD, wherein the aberrant accumulation of nucleic
   acids—both intracellular and extracellular—triggers innate immune
   activation and drives disease progression.

Fig. 2. IVDD is linked to inflammation caused by the accumulation of
intracellular and extracellular nucleic acids.

   [92]Fig. 2
   [93]Open in a new tab

   A Schematic of single-cell RNA-seq in human degenerated disc tissues. B
   UMAP plot of single-cell transcriptomes from human discs. C Bubble plot
   of scaled expression of DEGs across clusters. D UMAP plot of human
   intervertebral disc cells, showing four distinct clusters identified
   via unsupervised clustering. Each dot represents a single cell,
   color-coded by subcluster classification. E Volcano plot showing
   differentially expressed genes in nucleus pulposus cells. F Gene
   Ontology (GO) analysis was performed to investigate the differentially
   expressed genes (DEGs) in nucleus pulposus cells. GO enrichment
   analysis was performed using a hypergeometric distribution algorithm to
   assess the significance of differentially expressed gene enrichment in
   each GO term. Fisher’s exact test was applied separately to the
   Cellular Component. P-values were adjusted for multiple comparisons
   (FDR < 0.05). G, H GSEA plots showing enrichment analysis for nucleus
   pulposus cells. GSEA was performed using the default weighted
   Kolmogorov–Smirnov statistic with FDR < 0.25 as the significance
   threshold. I Schematic of bulk RNA-seq in rat primary NP cells. J
   Heatmap of DEGs in rat NP cells (p value < 0.05, |log2FoldChange| >1).
   K GO enrichment analysis plot for rat nucleus pulposus cells. GO
   enrichment analysis was performed using a hypergeometric distribution
   algorithm to assess the significance of differentially expressed gene
   enrichment in each GO term. Fisher’s exact test was applied separately
   to the Biological Process, Cellular Component, and Molecular Function
   categories. P-values were adjusted for multiple comparisons
   (FDR < 0.05). L Cytoplasmic DNA content in normal vs. senescent rat NP
   cells (n = 3 biological replicates, mean ± s.d.). M MRI scans of human
   intervertebral discs from normal and degenerated groups. N
   Immunohistochemical staining of STING in rat and human NP tissues.
   Scale bar, 50 μm. Representative images are from three independent
   experiments. O exDNA content in control vs. degenerated discs (n = 3
   biological replicates, mean ± s.d.). Created with BioRender.com (A, I).
   Statistical significance was assessed using a two-tailed unpaired
   Student’s t-test. (L, O). Source data are provided as a Source Data
   file.

Development of mitochondrial-targeted nanovesicles (BNPMT) to prevent mtDNA
leakage in NP cells

   As nucleus pulposus cells age, mitochondrial dysfunction occurs,
   potentially causing mtDNA leakage, which activates the innate immune
   pathway and triggers inflammation. Therefore, we initially aimed to
   develop a targeted nanomedicine to prevent mtDNA leakage from
   mitochondria. BAX-mediated miMOMP is a key driver of cytosolic mtDNA
   leakage^[94]26. To target this mechanism, we engineered a biomimetic
   nanovesicle to deliver the BAX inhibitor BAI1, which inhibits miMOMP,
   prevents abnormal cytosolic DNA accumulation in nucleus pulposus cells,
   and suppresses SASP factor release (Fig. [95]3A, B). To achieve
   mitochondrial targeting in nucleus pulposus cells, we modified the
   nucleus pulposus cell membrane surface with the TPP ligand^[96]27.
   First, we co-incubated nucleus pulposus cells with Ac4ManNAz,
   successfully introducing azide groups onto their surface^[97]28. Next,
   we employed copper-free click chemistry to attach the TPP functional
   group to the cell surface. To evaluate the efficiency of the click
   chemistry reaction, we used DBCO-FITC, and fluorescence analysis
   confirmed the successful attachment of FITC to the nucleus pulposus
   cell membrane (Fig. [98]3C).

Fig. 3. Characterization and targeting effect of BNPMT.

   [99]Fig. 3
   [100]Open in a new tab

   A Schematic representation of BNPMT synthesis. B 3D representation of
   BAI1 binding to the BAX protein. C Evaluation of click chemistry
   efficiency on nucleus pulposus cell surfaces. Scale bar, 50 μm.
   Representative images are from three independent experiments. D Western
   blot analysis of Na^+/K^+-ATPase and α-tubulin in nucleus pulposus cell
   membranes and cytoplasm. Representative images are from three
   independent experiments. E Average particle sizes of BNPM and BNPMT
   (n = 6 independent experiments, mean ± s.d.). F Cryo-electron
   microscopy images of BNPMT (Scale bar, 50 nm) and particle size
   distribution of BNPM and BNPMT. G Zeta potentials of BNPM and BNPMT
   (n = 3 independent experiments, mean ± s.d.). H Encapsulation
   efficiency of BNPMT (n = 3 independent experiments, mean ± s.d.). I
   Uptake of FITC, FITC@NPM, and FITC@NPMT by RAW264.7 macrophages and NP
   cells (Scale bar, 50 μm, left). Representative images are from three
   independent experiments. Statistical analysis of FITC fluorescence
   intensity in RAW264.7 and nucleus pulposus cells (n = 3 independent
   experiments, mean ± s.d., right). J Visualization of nanovesicle
   localization after 4h co-incubation of FITC, FITC@NPM, and FITC@NPMT
   with nucleus pulposus cells (Scale bar, 10 μm). Representative images
   are from three independent experiments. K Intensity analysis along the
   selected white line across the entire cell in (J). L Schematic
   representation of BNPMT nanovesicles targeting mitochondria to inhibit
   BAX activation. Created with BioRender.com (L). Statistical analyses
   were performed using one-way ANOVA followed by Tukey’s multiple
   comparison test (I). Source data are provided as a Source Data file.

   We extracted and purified the nucleus pulposus cell membrane, and
   Western blot (WB) analysis confirmed the successful isolation of NP
   cell membranes (NPM) (Fig. [101]3D). We then fabricated engineered
   biomimetic nanovesicles using mechanical extrusion and loaded BAI1 into
   the vesicles via ultrasonic drug loading, yielding BNPM and
   TPP-modified BNPMT^[102]29,[103]30. Cryo-electron microscopy confirmed
   that BNPMT exhibited a typical vesicular morphology. Dynamic light
   scattering (DLS) analysis showed that BNPM had an average hydrodynamic
   diameter of ~150 nm with a zeta potential of −16.7 mV, while BNPMT
   exhibited a larger diameter (~170 nm) and a zeta potential of −14.5 mV
   (Fig. [104]3E–G). The increased vesicle size and altered surface charge
   may result from TPP modification. High-performance liquid
   chromatography (HPLC) analysis determined the drug loading rate of BAI1
   to be 17.8% (Fig. [105]3H). The membrane-derived nanovesicles exhibited
   homotypic targeting ability^[106]31. Incubation of nanovesicles with
   different cell types revealed that those derived from nucleus pulposus
   cell membranes exhibited significantly higher fluorescence in nucleus
   pulposus cells than in macrophages, indicating specific uptake of
   FITC@NPMT and FITC@NPM by nucleus pulposus cells (Fig. [107]3I).
   Furthermore, we analyzed the mitochondrial targeting ability of
   FITC@NPMT. Unlike FITC and FITC@NPM, which primarily localized in the
   cytoplasm of nucleus pulposus cells, TPP-modified FITC@NPMT showed
   strong mitochondrial co-localization, confirming its mitochondrial
   targeting properties (Fig. [108]3J–L).

BNPMT prevents mtDNA leakage, thereby mitigating inflammation in aging NP
cells

   Next, we investigated the intracellular anti-inflammatory effects of
   BNPMT. First, we established an in vitro aging model of nucleus
   pulposus cells using Dox. Following Dox treatment, BAX(6A7) expression
   in aging nucleus pulposus cells significantly increased and
   co-localized with mitochondria, accompanied by a marked rise in
   cytosolic double-stranded DNA (dsDNA) levels (Fig. [109]4A, B). BNPMT
   treatment significantly reduced BAX(6A7) expression and cytosolic dsDNA
   levels in aging nucleus pulposus cells, suggesting that BNPMT
   effectively inhibited BAX activation, suppressed miMOMP, and reduced
   mtDNA leakage into the cytoplasm (Fig. [110]4A, B). Furthermore,
   cytoplasmic fractionation and dsDNA quantification confirmed that BNPMT
   significantly reduced cytoplasmic dsDNA accumulation (Fig. [111]4C). In
   conclusion, these findings demonstrate that BNPMT inhibits miMOMP and
   prevents mtDNA leakage in aging nucleus pulposus cells. The cGAS-STING
   pathway serves as a key cytosolic DNA sensor, recognizing dsDNA and
   activating the downstream NF-κB signaling cascade, which promotes the
   secretion of inflammatory cytokines (e.g., TNF-α and IL-6)^[112]32. To
   determine whether BNPMT suppresses SASP secretion through the
   cGAS–STING pathway following the inhibition of cytosolic mtDNA
   accumulation, we analyzed the expression of key proteins associated
   with the cGAS–STING signaling pathway across different treatment
   groups. WB analysis revealed significantly elevated phosphorylation
   levels of p-STING, p-IRF3, and p-TBK in aging nucleus pulposus cells,
   whereas BNPMT treatment effectively suppressed cGAS-STING pathway
   activation (Fig. [113]4D and Supplementary Fig. [114]5). Additionally,
   qPCR and ELISA analyses demonstrated that BNPMT treatment significantly
   reduced IL-6 and TNF-α mRNA levels in aging nucleus pulposus cells,
   along with a marked decrease in IL-6 secretion (Fig. [115]4E, F). These
   findings suggest that BNPMT mitigates SASP by suppressing the cytosolic
   DNA sensing pathway cGAS-STING.

Fig. 4. BNPMT suppress SASP secretion in NP cells.

   [116]Fig. 4
   [117]Open in a new tab

   A, B Immunofluorescence staining and quantification of BAX6A7 (A) and
   dsDNA (B) in aging NP cells treated with BAI1, BNPM, or BNPMT. Scale
   bars, 10 μm. Representative images are from three independent
   experiments. Quantification of BAX6A7 fluorescence intensity (n = 3
   biological replicates, mean ± s.d.). Quantification of dsDNA
   fluorescence intensity (n = 3 biological replicates, mean ± s.d.). C
   Cytoplasmic dsDNA content in NP cells following different treatments
   (n = 3 biological replicates, mean ± s.d.). D Western blot analysis of
   STING pathway proteins. Representative images are from three
   independent experiments. E mRNA expression levels of IL-6 and TNF-α in
   NP cells from different treatment groups (n = 9 three independent
   biological replicates with three technical replicates each,
   mean ± s.d.). F IL-6 cytokine levels in the supernatants of NP cells
   across different treatment groups (n = 3 biological replicates,
   mean ± s.d.). G Heatmap of differentially expressed genes in SEN vs.
   BNPMT groups. H, I KEGG enrichment analysis of upregulated and
   downregulated gene sets in the BNPMT and SEN groups. KEGG pathway
   enrichment was performed using a hypergeometric test with
   Benjamini–Hochberg correction. Pathways with adjusted P-values
   (FDR) < 0.05 were considered significantly enriched. J GSEA comparing
   the SEN and BNPMT groups for gene sets involved in the NF-κB, JAK-STAT,
   and IL-17 signaling pathways. GSEA was performed using the default
   weighted Kolmogorov–Smirnov statistic with FDR < 0.25 as the
   significance threshold. K Quantitative qPCR analysis of total mtDNA
   levels in senescent NP cells post-treatment with CNPMT or BNPMT (n = 3
   biological replicates, mean ± s.d.). L ELISA quantification of IL-6
   protein secretion in senescent NP cells treated with CNPMT or BNPMT
   (n = 3 biological replicates, mean ± s.d.). M, N Representative
   SA-β-gal staining images of senescent NP cells following treatment with
   CNPMT or BNPMT (N). Scale bar, 100 μm. Quantification of
   SA-β-gal-positive cells (M) (n = 3 biological replicates, mean ± s.d.).
   Statistical analyses were performed using one-way ANOVA followed by
   Tukey’s multiple comparison test (A–C, E, F, K–M). Source data are
   provided as a Source Data file.

   To further investigate the therapeutic effects of BNPMT on aging
   nucleus pulposus cells, we conducted transcriptome sequencing on aging
   nucleus pulposus cells with and without BNPMT treatment. PCA analysis
   demonstrated significant mRNA differences between aging nucleus
   pulposus cells and those treated with BNPMT (Supplementary
   Fig. [118]6). Differential gene expression (DEG) analysis using DESeq2
   revealed that BNPMT treatment significantly downregulated SASP-related
   genes while upregulating ECM synthesis-related genes, consistent with
   previous findings (Fig. [119]4G). Kyoto Encyclopedia of Genes and
   Genomes (KEGG) enrichment analysis indicated that, compared to aging
   nucleus pulposus cells, BNPMT treatment significantly downregulated
   pathways associated with cell aging and inflammation while upregulating
   those related to mitochondrial function (Fig. [120]4H, I). GSEA
   analysis further revealed that BNPMT treatment significantly suppressed
   inflammation-related pathways, including the NF-κB, JAK-STAT, and IL-17
   signaling pathways (Fig. [121]4J). These findings confirm that BNPMT
   exerts an endogenous anti-inflammatory effect in aging nucleus pulposus
   cells by preventing cytosolic DNA accumulation and reversing the
   aging-related inflammatory phenotype. Furthermore, ECM homeostasis,
   primarily regulated by nucleus pulposus cells, is essential for
   maintaining normal disc function, and aging nucleus pulposus cells are
   often linked to an imbalance between ECM synthesis and
   degradation^[122]33. We analyzed ECM-related gene and protein
   expression in aging nucleus pulposus cells following BNPMT treatment
   using WB and qPCR. The results indicated that, compared to untreated
   aging nucleus pulposus cells, BNPMT treatment significantly increased
   the protein and mRNA expression of collagen II (COL2), a key ECM
   component, while also upregulating SOX9 protein expression
   (Supplementary Figs. [123]7 and [124]8). Meanwhile, the protein and
   mRNA expression of the ECM-degrading enzyme matrix metalloproteinase-13
   (MMP13) was significantly downregulated (Supplementary
   Figs. [125]7–[126]9). These data further suggest that BNPMT not only
   suppresses aging-related inflammation but also restores ECM homeostasis
   by promoting functional repair in nucleus pulposus cells.

   To further investigate the differences between BNPMT and conventional
   anti-inflammatory drugs in alleviating NP cell senescence and
   apoptosis, we constructed biomimetic nanovesicles (CNPMT) by
   encapsulating curcumin within TPP-modified NP cell membranes. By
   isolating the cytoplasm of senescent NP cells and quantifying cytosolic
   dsDNA levels, we found that CNPMT exhibited limited ability to prevent
   cytosolic mtDNA accumulation compared to BNPMT (Fig. [127]4K and
   Supplementary Fig. [128]10). Subsequently, we evaluated the
   anti-inflammatory effects of BNPMT and CNPMT using qPCR and ELISA
   assays. The results demonstrated that BNPMT significantly outperformed
   CNPMT in suppressing IL-6 and TNF-α mRNA expression, as well as
   inhibiting IL-6 cytokine secretion in senescent NP cells (Fig. [129]4L
   and Supplementary Fig. [130]11), suggesting a superior
   anti-inflammatory capacity of BNPMT in this context. We then assessed
   the ability of BNPMT and CNPMT to attenuate NP cell senescence and
   apoptosis. SA-β-gal staining revealed that BNPMT more effectively
   reduced NP cell senescence (Fig. [131]4M, N). Furthermore, live/dead
   staining and flow cytometry analysis showed that BNPMT exhibited
   markedly enhanced anti-apoptotic effects compared to CNPMT
   (Supplementary Figs. [132]12 and [133]13). Finally, we investigated ECM
   homeostasis in NP cells after different treatments. qPCR analysis
   indicated that BNPMT significantly upregulated the mRNA expression of
   the ECM component COL2, while effectively downregulating the expression
   of the ECM-degrading enzyme MMP13 (Supplementary Fig. [134]14).
   Collectively, these results highlight that, compared with conventional
   anti-inflammatory drugs, BNPMT demonstrates superior efficacy in
   mitigating NP cell senescence and apoptosis, as well as in modulating
   ECM synthesis and degradation.

BNPMT-induced metabolic reprogramming in senescent NP cells

   To further explore the mechanism of BNPMT intervention in senescent
   nucleus pulposus cells, we conducted metabolomics analysis and examined
   the metabolic profiles of the NC, SEN, and BNPMT groups using Partial
   Least Squares Discriminant Analysis. PCA revealed significant metabolic
   differences among the NC, SEN, and BNPMT groups (Fig. [135]5A). The
   metabolic profile of BNPMT-treated senescent NP cells closely resembled
   that of young cells, further confirming that BNPMT partially reverses
   the senescent phenotype of nucleus pulposus cells, suggesting that
   metabolic reprogramming may contribute to the functional restoration of
   senescent NP cells (Fig. [136]5B). BNPMT intervention induced
   significant metabolic reprogramming in nucleus pulposus cells compared
   to the SEN group, affecting metabolites such as pyruvate, alanine,
   glutamate, guanosine, inosine, and uracil (Fig. [137]5B). Pyruvate lies
   at the intersection of glycolysis and mitochondrial metabolism, and can
   promote NAD^+ regeneration through lactate production, thereby
   maintaining mitochondrial function and delaying cellular
   senescence^[138]34. Glutamate is a precursor of α-ketoglutarate and can
   enter the TCA cycle to support mitochondrial respiration; it also
   serves as a substrate for glutathione (GSH) synthesis, contributing to
   antioxidant defense^[139]35. KEGG pathway enrichment analysis of
   differentially expressed metabolites revealed that BNPMT primarily
   reverses metabolic alterations in senescent nucleus pulposus cells
   through multiple pathways, including alanine, aspartate, and glutamate
   metabolism; purine metabolism; pyrimidine metabolism; and the
   biosynthesis of pantothenate and Coenzyme A (Fig. [140]5C).
   Furthermore, metabolomics analysis allowed us to establish the
   metabolic profiles of nucleus pulposus cells under different
   interventions (Fig. [141]5D, E). The biosynthesis of pantothenate and
   Coenzyme A, along with purine and pyrimidine metabolism, plays a
   crucial role in regulating cellular energy metabolism, ensuring
   sufficient energy for basic metabolic and repair functions^[142]36. The
   biosynthesis of pantothenate and Coenzyme A, glutathione synthesis, and
   β-alanine metabolism are involved in cellular antioxidant and stress
   responses, aiding in the elimination of excess free radicals, reduction
   of oxidative damage, and deceleration of cellular
   aging^[143]37,[144]38. Additionally, pyrimidine and purine metabolism
   contribute to ECM synthesis and stability by supplying essential
   molecules for gene replication, transcription, and repair, thereby
   supporting the structural and functional integrity of the
   intervertebral disc^[145]39. To further verify the functional relevance
   of these metabolic alterations, we examined ATP levels in NP cells
   (Fig. [146]5F). BNPMT treatment significantly increased ATP production
   in senescent NP cells, indicating improved energy metabolism. We also
   assessed mitochondrial membrane potential and mitochondrial Reactive
   Oxygen Species (ROS) levels. BNPMT-treated NP cells showed restoration
   of mitochondrial membrane potential and a marked reduction in
   mitochondrial ROS generation, suggesting enhanced mitochondrial
   function (Fig. [147]5G and Supplementary Fig. [148]15). Collectively,
   these results, together with the metabolomic findings, suggest that
   BNPMT promotes metabolic reprogramming in senescent NP cells, leading
   to the restoration of mitochondrial function and attenuation of
   senescence-associated phenotypes. These findings underscore a potential
   mechanistic link between metabolic regulation and cellular aging in NP
   cells.

Fig. 5. Metabolic alterations in nucleus pulposus cells following BNPMT
treatment.

   [149]Fig. 5
   [150]Open in a new tab

   A PCA plot showing the metabolic profiles of nucleus pulposus cells
   from the SEN, NC, and BNPMT groups (n = 6 biological replicates). B
   Heatmap illustrating the differential metabolites in nucleus pulposus
   cells among the SEN, NC, and BNPMT groups (n = 6 biological
   replicates). C KEGG pathway enrichment bubble plot depicting metabolite
   distribution in nucleus pulposus cells from the SEN, NC, and BNPMT
   groups. KEGG pathway enrichment was performed using a hypergeometric
   test with Benjamini–Hochberg correction. Pathways with adjusted
   P-values (FDR) < 0.05 were considered significantly enriched. D
   Metabolomics network analysis of the SEN, NC, and BNPMT groups. Yellow,
   blue, and black rectangular borders represent metabolites that
   increased, decreased, or remained unchanged in BNPMT compared to the
   SEN group. Brown, blue, and white-filled rectangles indicate
   metabolites that increased, decreased, or remained unchanged in the SEN
   group compared to the NC group. Dashed rectangles indicate undetected
   metabolites. E Schematic diagram illustrating differential metabolic
   pathways in nucleus pulposus cells between the SEN and BNPMT groups. F
   Quantification of intracellular ATP levels in NP cells following
   different treatments (n = 6 biological replicates, mean ± s.d.). G
   Representative TMRE staining images (Scale bar, 50 μm) and
   corresponding fluorescence intensity analysis (n = 3 biological
   replicates, mean ± s.d.) in NP cells from each group. Created with
   BioRender.com (E). Statistical analyses were performed using one-way
   ANOVA followed by Tukey’s multiple comparison test (F, G). Source data
   are provided as a Source Data file.

Synthesis and characterization of H-P gel for capturing exDNA

   Next, we aim to develop a hydrogel system capable of capturing exDNA
   (Fig. [151]6A). The hydrogel is crosslinked via Schiff base chemistry
   between oxidized hyaluronic acid and PAMAM, where the positively
   charged PAMAM effectively captures the negatively charged exDNA. We
   evaluated the biocompatibility and gelation time of PAMAM dendrimers
   with different generations (Supplementary Figs. [152]16 and [153]17),
   and ultimately selected PAMAM-G5 due to its superior stability and
   biocompatibility, making it a suitable component for constructing the
   exDNA-capturing hydrogel system. As shown in Fig. [154]6B, when equal
   volumes of OHA aqueous solution and PAMAM solution are mixed at room
   temperature, a hydrogel (H-P gel) rapidly forms. Scanning electron
   microscopy (SEM) imaging of freeze-dried H-P gel reveals a typical
   porous microstructure, which is conducive to vesicle loading
   (Fig. [155]6C). Next, we evaluated the injectability and self-healing
   properties of the H-P gel. The hydrogel was continuously extruded
   through a 26-gauge needle, successfully forming the letters “NP”
   without blockage (Fig. [156]6D). Meanwhile, rheological analysis
   demonstrated shear-thinning behavior, as the viscosity of the H-P gel
   decreased sharply when the shear rate increased from 0.1 to 1000 s^−1,
   confirming its injectability (Fig. [157]6E). The self-healing ability
   of the H-P gel was further examined. In macroscopic tests, two
   separated pieces of H-P gel fused into a single, continuous gel after
   10 min of contact (Fig. [158]6F). Additionally, strain sweep
   experiments indicated that the storage modulus (G′) and loss modulus
   (G″) remained nearly constant up to 30% strain, suggesting that the H-P
   gel can withstand relatively large elastic deformations. However, at
   80% strain, the intersection of G′ and G″ marked the critical point at
   which the hydrogel network collapsed (Fig. [159]6G). To further assess
   self-healing behavior, continuous alternating strain scanning was
   conducted. Under a high dynamic strain (200%), the G′ value decreased
   below G″, indicating network disruption. When the strain was reduced to
   1%, the hydrogel fully recovered its G′ and G″ values within seconds,
   maintaining this recovery over three alternating cycles, thus
   demonstrating excellent self-healing capability (Fig. [160]6H). The
   dynamic Schiff base reactions in the H-P gel occur rapidly at room or
   physiological temperatures. The aldehyde groups in OHA react with the
   amino groups in PAMAM to form Schiff base bonds, achieving a dynamic
   equilibrium that imparts both injectability and self-healing properties
   to the hydrogel. To enable in vivo tracking, indocyanine green (ICG)
   was covalently conjugated to the hydrogel using ICG-NHS, which forms
   stable amide bonds with the abundant primary amine groups (–NH[2]) on
   PAMAM dendrimers. In vivo fluorescence imaging was performed on days 0,
   3, 7, 14, and 21 post injection to dynamically monitor the persistence
   and degradation of the hydrogel within the intervertebral disc space
   (Supplementary Fig. [161]18). The results demonstrated that the H-P gel
   exhibited a slow degradation profile, with detectable fluorescence
   signals remaining up to 21 days after injection.

Fig. 6. Characterization and Function of H-P Hydrogel.

   [162]Fig. 6
   [163]Open in a new tab

   A, B Schematic (A) and image (B) of H-P GEL formation from 1% PAMAM-G5
   and 5% HA-CHO (1:1). Representative images are from three independent
   experiments. C SEM image of H-P GEL. Scale bar, 400 μm. Representative
   images are from three independent experiments. D Injectability of H-P
   GEL. E Viscosity curve demonstrating the shear-thinning behavior of the
   hydrogel as shear rates increase (0.1–1000 s^−1). F Images showing the
   self-healing ability of the hydrogel. G Storage (G′) and loss modulus
   (G′′) of the hydrogel in a strain amplitude sweep (γ = 1–1000%) at a
   fixed frequency. H Rheological behavior of the hydrogel under
   alternating strains (1–200%). I Schematic of RAW264.7 cell co-culture.
   J Flow cytometry of CpG uptake (left) and corresponding statistical
   graph (right) (n = 4 biological replicates, mean ± s.d.). K Flow
   cytometry of iNOS expression (left) and corresponding statistical graph
   (right) (n = 3 biological replicates, mean ± s.d.). L Expression levels
   of TLR9, TNF-α, and IL-6 mRNA in RAW264.7 cells (n = 9 three
   independent biological replicates with three technical replicates each,
   mean ± s.d.). M TNF-α and IL-6 (n = 5 biological replicates,
   mean ± s.d.) cytokine levels in RAW264.7 cell culture supernatant. N
   Diagram of conditioned medium preparation. O mRNA levels of COL2, ACAN
   in NP cells after treatment with different conditioned media (n = 9
   three independent biological replicates with three technical replicates
   each, mean ± s.d.). P mRNA levels of MMP3 and MMP13 in NP cells after
   treatment with different conditioned media (n = 9, three independent
   biological replicates with three technical replicates each,
   mean ± s.d.). Q Western blot for COL2 and MMP3. Representative images
   are from three independent experiments. R, S Immunofluorescence
   staining of COL2 and MMP3 in NP cells. Scale bars, 100 μm. along with
   fluorescence intensity statistical graph (n = 4 biological replicates,
   mean ± s.d.). Created with BioRender.com (I, N). Statistical analyses
   were performed using one-way ANOVA followed by Tukey’s multiple
   comparison test (J–M, O, P, R, S). Source data are provided as a Source
   Data file.

   exDNA in the intervertebral disc environment activates macrophage TLRs,
   triggering local inflammation, accelerating nucleus pulposus cell
   aging, and disrupting the metabolic balance between ECM synthesis and
   degradation^[164]40. The primary amine groups on the surface of PAMAM
   can be protonated at physiological pH (pH = 7.4), forming positively
   charged-NH[3]^+ groups that effectively capture negatively charged
   exDNA (Fig. [165]6I). Flow cytometry analysis demonstrated that after
   co-incubation with H-P gel, the uptake of exDNA mimic CpG-FITC by
   macrophages was significantly reduced, confirming that H-P gel can
   sequester exDNA from the environment and inhibit CpG-FITC
   internalization by macrophages (Fig. [166]6J). Upon activation of TLRs
   on the macrophage surface, M1 polarization occurs, leading to the
   release of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β,
   thereby initiating and exacerbating local inflammation. Flow cytometry
   results showed that after H-P gel captured environmental CpG, it
   significantly downregulated the expression of the M1 macrophage marker
   inducible nitric oxide synthase (iNOS) (Fig. [167]6K). Furthermore,
   qPCR and ELISA analyses demonstrated that co-incubation with H-P gel
   significantly reduced the mRNA expression levels of TLR9, IL-6, and
   TNF-α, as well as the secretion of the inflammatory cytokines IL-6 and
   TNF-α in macrophages (Fig. [168]6L, M). These findings indicate that
   H-P gel effectively suppresses exogenous exDNA-induced inflammation in
   the intervertebral disc. Chronic exposure of nucleus pulposus cells to
   an inflammatory microenvironment suppresses their function, disrupts
   normal metabolic processes, accelerates ECM degradation, and promotes
   disc degeneration. To investigate whether mitigating exogenous
   exDNA-induced inflammation could restore ECM homeostasis in nucleus
   pulposus cells, we collected conditioned media from CpG-treated
   macrophages and macrophages co-incubated with H-P gel. Nucleus pulposus
   cells were then cultured in these conditioned media for 24 h to assess
   ECM-related markers and determine whether alleviating macrophage
   inflammation could restore the balance between ECM synthesis and
   degradation (Fig. [169]6N). qPCR analysis revealed that the mRNA
   expression levels of key ECM components, COL2 and aggrecan (ACAN), were
   significantly upregulated, whereas the expression levels of ECM
   degradation enzymes matrix MMP13 and MMP3 were markedly downregulated
   (Fig. [170]6O, P). WB and immunofluorescence staining results also
   confirmed this (Fig. [171]6Q–S). Collectively, these results
   demonstrate that H-P gel effectively mitigates macrophage-driven
   inflammation, thereby restoring ECM synthesis and degradation
   homeostasis in nucleus pulposus cells.

Therapeutic effect of BNPMT @ H-P in a puncture-induced rat intervertebral
disc degeneration model

   To minimize potential side effects caused by off-target effects of
   BNPMT in vivo, we adopted a localized delivery strategy. BNPMT was
   encapsulated within an H-P gel, which confined its bioactivity to the
   lesion site and reduced systemic exposure. Moreover, the H-P gel
   enables the sustained release of BNPMT within the intervertebral disc,
   thereby enhancing its therapeutic efficacy. Subsequently, the
   therapeutic potential of BNPMT@H-P was evaluated using a
   puncture-induced rat model of IVDD (Fig. [172]7A). After 8 weeks of
   treatment, major organs and serum samples were collected for
   histological and biochemical analysis to assess the biosafety of
   BNPMT@H-P (Supplementary Figs. [173]19 and [174]20). H&E staining
   revealed no evident pathological changes in major organs, and serum
   biochemical parameters showed no significant differences compared with
   controls. These results indicate that BNPMT@H-P does not elicit
   systemic immune responses and possesses favorable biocompatibility.
   Given the near-infrared fluorescence properties of ICG, which offer
   superior tissue penetration and reduced background autofluorescence in
   vivo^[175]41, ICG enables more reliable and sensitive imaging outcomes.
   To simulate the in vivo release behavior of BNPMT, we encapsulated ICG
   within NP cell membrane–coated nanovesicles and further embedded these
   nanovesicles into H-P gel for subsequent tracking and imaging. ICG@NPMT
   and ICG@NPMT@H-P were administered into the rat IVD to mimic the in
   vivo release profile of BNPMT@H-P. In vivo imaging revealed that
   ICG@NPMT rapidly diffused and was cleared from the IVD site by day 7,
   whereas ICG@NPMT encapsulated in H-P gel persisted at the site for up
   to 21 days. This suggests that H-P gel can sustain a higher local drug
   concentration within the IVD for an extended duration (Fig. [176]7B,
   C). BNPMT, H-P gel, and BNPMT@H-P were injected into the rat IVD using
   a 26G needle. The control group underwent no puncture, while the injury
   group received no injection. The disc height index (DHI) of the five
   groups was assessed using radiographic analysis. At four weeks
   post-surgery, the DHI value in the BNPMT@H-P group was comparable to
   that of the control group but significantly higher than in the BNPMT,
   H-P, and non-injection groups. At eight weeks post-surgery, BNPMT@H-P
   significantly attenuated the puncture-induced reduction in IVD height,
   whereas BNPMT or H-P treatment alone had a minimal effect
   (Fig. [177]7D, E). MRI analysis, which assesses IVD water content and
   serves as a key diagnostic indicator of IVD degeneration, revealed
   uneven disc signals in the IVDD, BNPMT groups, and H-P groups,
   suggesting reduced water content^[178]42. These findings were
   consistent with radiographic assessments, as DHI and MRI results were
   aligned, further supporting the potential of BNPMT@H-P in delaying IVD
   degeneration in vivo (Fig. [179]7F, G). The therapeutic efficacy of
   minimally invasive BNPMT@H-P hydrogel injection in inhibiting IVD
   degeneration was validated through histological and immunohistochemical
   analyses at 4 and 8 weeks post-surgery. H&E staining was performed to
   evaluate the morphology of the NP, fibrous tissue, and their
   boundaries^[180]43. During the 4- to 8-week observation period, the
   BNPMT@H-P group exhibited a reduction in NP tissue area; however, the
   tissue loss was significantly less than that observed in the BNPMT,
   H-P, and non-injection groups. Furthermore, in the BNPMT, H-P, and
   non-injection groups, the boundary between the NP and annulus fibrosus
   (AF) appeared blurred, and the NP region exhibited significant atrophy,
   indicating IVD fibrosis and the loss of the organized NP-AF structure
   (Fig. [181]7H). Safranin O-Fast Green staining was performed to assess
   proteoglycan content in the NP. In the NC group, the NP region was rich
   in proteoglycans, whereas proteoglycan levels were markedly reduced in
   the BNPMT, H-P, and non-injection groups. However, proteoglycan
   depletion was less pronounced in the BNPMT@H-P gel group compared to
   the other puncture groups (Fig. [182]7I). Histological grading revealed
   that the BNPMT@H-P group had significantly lower degeneration scores at
   both 4 and 8 weeks compared to the BNPMT, H-P, and non-injection groups
   (Fig. [183]7J).

Fig. 7. Significant delay of intervertebral disc degeneration progression by
BNPMT@H-P in a puncture-induced rat IVD degeneration model.

   [184]Fig. 7
   [185]Open in a new tab

   A Schematic illustration of puncture-induced IVDD and therapeutic
   intervention in rat model. B In vivo IVIS imaging at 0, 3, 7, 14, and
   21 days post-injection showing dynamic changes in fluorescence
   intensity at the rat intervertebral disc site (Color scale: blue [low]
   to red [high]). C Quantification of fluorescence signal intensity in
   experimental groups over 8 weeks (n = 3 independent rats, mean ± s.d.).
   D Representative μCT reconstructions of rat tail intervertebral discs
   across treatment groups. E Quantitative analysis of intervertebral DHI
   from radiographic measurements (n = 3 independent rats, mean  ± s.d.).
   F Comparative MRI cross-sections of rat tail intervertebral discs in
   experimental cohorts. G NP T2-signal intensity quantification at 4- and
   8-week timepoints (n = 3 independent rats, mean ± s.d.). H Temporal
   progression of intervertebral disc morphology demonstrated by H&E
   staining (Multiple timepoints/treatment groups). I Extracellular matrix
   remodeling visualized through Safranin O/Fast Green staining
   (Representative timepoints/groups). J Histopathological grading system
   scores following surgical intervention (4/8-week post-operation, n = 3
   independent rats, mean ± s.d.). Statistical significance between groups
   at each time point was assessed using an unpaired two-tailed Student’s
   t-test (C). Created with BioRender.com (A). Statistical analyses were
   performed using one-way ANOVA followed by Tukey’s multiple comparison
   test (E, G, J). Source data are provided as a Source Data file.

   Next, we evaluated the in vivo capability of the BNPMT@H-P dual-mode
   system to eliminate exDNA. Initially, we performed immunofluorescence
   staining for dsDNA on intervertebral disc sections (Fig. [186]8A and
   Supplementary Fig. [187]21), and the results showed a significant
   reduction in dsDNA fluorescence intensity in NP tissues following
   BNPMT@H-P treatment. To further quantitatively assess the hydrogel
   system’s ability to eliminate exDNA from the tissue microenvironment,
   we conducted a quantitative analysis at the tissue level. We collected
   NP tissues and isolated NP cells, then extracted the extracellular
   matrix from the nucleus pulposus to quantitatively analyze the exDNA
   levels in the NP microenvironment (Fig. [188]8B). The results showed a
   significant reduction in exDNA levels after BNPMT@H-P administration,
   consistent with the immunofluorescence findings, suggesting effective
   exDNA clearance by BNPMT@H-P within the disc microenvironment. In
   addition, to evaluate the in vivo efficacy of the hydrogel system in
   suppressing the cytoplasmic release of mtDNA, we conducted qPCR
   analysis. qPCR analysis of cytoplasmic DNA from isolated NP cells
   revealed a significant reduction in mtDNA levels compared to the IVDD
   group (Fig. [189]8C). Collectively, these results indicate that
   BNPMT@H-P effectively clears exDNA from the NP microenvironment and
   inhibits the release of mtDNA into the cytoplasm of NP cells. To
   determine whether the removal of both intracellular and extracellular
   dsDNA by BNPMT@H-P could suppress STING expression in NP cells and
   reduce inflammation within the disc, we performed immunohistochemical
   staining for STING in the intervertebral discs (Fig. [190]8D, E and
   Supplementary Fig. [191]22), and immunofluorescence staining for IL-6
   and IL-1β (Fig. [192]8F, G). The results showed that BNPMT@H-P
   significantly downregulated STING expression and reduced inflammatory
   cytokine levels, suggesting that BNPMT@H-P may suppress the release of
   SASP factors from NP cells via inhibition of the STING pathway.
   Meanwhile, immunofluorescence analysis of the senescence markers p16
   and p21 (Fig. [193]8H, I and Supplementary Figs. [194]23 and [195]24)
   was performed to assess whether BNPMT@H-P could reverse NP cell
   senescence. Post-injection, a marked reduction in the average
   fluorescence intensity of p16 and p21 was observed, indicating that
   BNPMT@H-P can alleviate cellular senescence in NP cells, thereby
   contributing to IVDD treatment. Furthermore, IHC analysis revealed a
   significant increase in the expression of the key ECM marker COL2 and a
   decrease in the ECM degradation marker MMP3 in the BNPMT@H-P group
   compared with other puncture groups (Fig. [196]8J, K and Supplementary
   Figs. [197]25 and [198]26). These findings suggest that BNPMT@H-P
   promotes intervertebral disc regeneration by remodeling the disc
   microenvironment and mitigating NP cell senescence.

Fig. 8. Analysis of treatment effects after injection of BNPMT@H-P.

   [199]Fig. 8
   [200]Open in a new tab

   A Representative immunofluorescence images of dsDNA staining in IVD
   tissues at 8 weeks post-treatment. Scale bar, 100 μm. Representative
   images are from three independent experiments. B Quantification of
   exDNA levels in the IVD microenvironment across different treatment
   groups (n = 3 independent rats, mean ± s.d.). C Cytoplasmic mtDNA
   levels in NP cells isolated from IVD tissues of different treatment
   groups (n = 3 biological replicates, mean ± s.d.). D Representative
   Immunohistochemistry images of STING expression in IVD tissues at 8
   weeks post-treatment across different experimental groups. Scale bar,
   100 μm. E Quantitative analysis of STING expression levels at week 8.
   (n = 3 independent rats, mean ± s.d.). F Representative
   immunofluorescence images of IL-6, IL-1β staining in IVD tissues at 8
   weeks post-treatment. Scale bar, 100 μm. G Quantitative analysis of
   fluorescence intensities for IL-6, IL-1β at week 8. (n = 3 independent
   rats, mean ± s.d.). H Representative immunofluorescence images of p16,
   p21 staining in IVD tissues at 8 weeks post-treatment. Scale bar,
   100 μm. I Quantitative analysis of fluorescence intensities for p16,
   p21 at week 8. (n = 3 independent rats, mean ± s.d.). J Representative
   Immunohistochemistry images of COL2 and MMP3 expression in IVD tissues
   at 8 weeks post-treatment across different experimental groups. Scale
   bar, 100 μm. K Quantitative analysis of COL2 and MMP3 expression levels
   at 8-week. (n = 3 independent rats, mean ± s.d.). Statistical analyses
   were performed using one-way ANOVA followed by Tukey’s multiple
   comparison test (B, C, E, G, I, K). Source data are provided as a
   Source Data file.

Discussion

   In this study, we developed a hydrogel-based nanocomposite system
   (BNPMT@H-P) that implements an organelle–environment synergistic
   strategy for the treatment of IVDD. By simultaneously targeting
   intracellular mtDNA release and capturing exDNA, this platform
   interrupts the self-amplifying inflammatory loop driven by pathogenic
   nucleic acids. The biomimetic nanovesicles derived from NP cell
   membranes enabled homologous targeting and mitochondrial delivery of
   BAI1, restoring mitochondrial integrity, suppressing SASP factor
   release, and improving metabolic function in senescent NP cells.
   Meanwhile, the PAMAM-based hydrogel matrix not only enabled sustained
   local drug release but also neutralized pro-inflammatory exDNA through
   electrostatic binding, thereby modulating the extracellular immune
   microenvironment. Importantly, this dual-modality approach not only
   achieved robust anti-inflammatory effects but also enhanced the disc’s
   intrinsic capacity for self-repair by regulating NP cell fate and
   function. The therapeutic benefits were sustained over an 8-week course
   in a rat model of trauma-induced IVDD, demonstrating the translational
   potential of this organelle–environment synergistic strategy as a
   paradigm for nucleic acid-targeted IVDD therapy.

   Although our model was based on injury-induced disc degeneration, which
   replicates key pathological features such as mitochondrial dysfunction,
   sterile inflammation, and ECM breakdown, the mechanisms targeted by our
   therapy are broadly relevant to other clinically prevalent IVDD
   subtypes^[201]44,[202]45. In age-related IVDD, mitochondrial
   instability and progressive accumulation of senescent NP cells are
   well-established hallmarks^[203]18,[204]46,[205]47. Similarly, in
   mechanical stress-induced degeneration—common in physically demanding
   occupations or spinal deformities—chronic compression accelerates NP
   cell senescence, elevates mtDNA and exDNA levels, and drives
   pro-inflammatory cascades^[206]48. Given that BNPMT@H-P system
   specifically targets these conserved degenerative mechanisms, we
   believe it holds promise beyond acute injury contexts and may offer
   broader applicability across the heterogeneous clinical spectrum of
   IVDD.

   Despite encouraging preclinical results, several challenges remain
   before clinical translation can be realized. The injectability and
   mechanical compatibility of the hydrogel in human intervertebral discs
   need thorough evaluation, as human discs are larger and endure greater
   mechanical loads than rodent models^[207]49; thus, the hydrogel must
   sustain structural integrity and controlled release under physiological
   stress without compromising disc biomechanics. Optimizing viscoelastic
   properties and enabling minimally invasive delivery via fine-gauge
   needles are critical^[208]50. Long-term biocompatibility and safety
   must also be established, since repeated injections and potential
   immune responses over extended periods in human discs remain
   uncharacterized despite generally favorable safety profiles of PAMAM
   dendrimers and hyaluronic acid derivatives^[209]51. Moreover, given the
   multifactorial nature of human IVDD—including age-related degeneration,
   mechanical overload, metabolic and genetic factors—it is essential to
   evaluate efficacy across diverse, clinically relevant models such as
   age-associated and mechanically induced degeneration^[210]52. Scalable
   production of biomimetic nanovesicles with consistent quality and
   stability is also vital for regulatory approval and
   commercialization^[211]29. Importantly, our platform leverages
   biocompatible, tunable components—cell membrane-derived vesicles for
   homotypic targeting, BAI1 as a mitochondrial stabilizer, and PAMAM/HA
   hydrogels for localized, sustained drug release and exDNA
   scavenging—offering adaptability to different disease stages and
   administration routes (e.g., image-guided injection). Future work
   should assess long-term safety, biodistribution, and regenerative
   outcomes in spontaneous or age-related IVDD models, while optimizing
   vesicle formulations and dosing regimens tailored to patient-specific
   factors to enhance therapeutic precision and clinical feasibility.

   Together, our work provides a mechanistically informed and clinically
   adaptable framework for treating IVDD by modulating nucleic acid–driven
   inflammation. This strategy opens new avenues for developing
   non-surgical, cell-free therapies that preserve disc function and delay
   disease progression in diverse patient populations.

Methods

Data collection and reanalysis

   Single-cell RNA sequencing (scRNA-seq) data from degenerated and
   non-degenerated intervertebral discs of a single individual were
   obtained from the Gene Expression Omnibus database ([212]GSE165722).
   The scRNA-seq analysis was conducted using the 10× Genomics platform,
   and data processing was performed with the Seurat package. Low-quality
   cells were filtered out based on the following criteria: cells
   expressing fewer than 200 or more than 6000 genes were excluded, as
   abnormal gene counts may indicate dying cells, cell membrane damage, or
   doublets. Additionally, cells with mitochondrial gene content exceeding
   40% were removed, as this suggests excessive cytoplasmic leakage and
   mRNA escape. After quality control, high-quality cells were normalized
   and scaled using the “NormalizeData” and “ScaleData” functions to
   achieve linear transformation. PCA was conducted on the top 2000
   variable genes, and the 30 most significant principal components were
   selected for clustering analysis. To correct for batch effects, the
   “RunHarmony” function from the Harmony package was applied to integrate
   scRNA-seq data across multiple samples. Clustering results were
   visualized using Uniform Manifold Approximation and Projection (UMAP).
   Finally, cells were classified based on characteristic markers, and
   DEGs were identified. GO enrichment analyses were performed for each
   cell group.

mRNA sequencing experimental method and analysis

   Total RNA was extracted using the TRIzol reagent (Invitrogen, USA)
   following the manufacturer’s protocol. RNA purity and concentration
   were measured with a NanoDrop 2000 spectrophotometer (Thermo
   Scientific, USA), while RNA integrity was assessed using an Agilent
   2100 Bioanalyzer (Agilent Technologies, USA). Library preparation was
   performed using the VAHTS Universal V6 RNA-seq Library Prep Kit
   according to the manufacturer’s instructions. Transcriptome sequencing
   and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).
   Bioinformatic analysis was performed using the OECloud tools at
   ([213]https://cloud.oebiotech.com/task/). The volcano map (Or other
   graphics) was drawn based on the [214]R on the OECloud platform
   ([215]https://cloud.oebiotech.com/task/).

Metabolomics sequencing and analysis

   NP cell samples from each experimental group were collected and washed
   2–3 times with pre-cooled PBS at 4 °C. After removing the PBS, the
   cells were immediately snap-frozen in liquid nitrogen. For metabolite
   extraction, each sample was treated with a total of 1 mL pre-chilled
   methanol:water (v:v = 4:1), added in two aliquots into 1.5 mL EP tubes.
   The samples were thoroughly dispersed using a pipette and subjected to
   ultrasonication in an ice bath (1620 W, 10 min, 6 s on / 4 s off
   cycles). This was followed by an additional 20 min of ultrasonic
   extraction in an ice-water bath and incubation at –40 °C overnight.
   Subsequently, the samples were centrifuged at 12,000 × g for 10 min at
   4 °C. A total of 800 μL of supernatant was collected (in two 400 μL
   portions), transferred to LC-MS vials, and evaporated to dryness. The
   dried residue was reconstituted in 300 μL of methanol:water (v:v = 1:4)
   containing internal standards (4 μg/mL), vortexed for 1 min,
   ultrasonicated for 10 min, and incubated again at –40 °C overnight.
   After a second centrifugation at 12,000 × g for 20 min at 4 °C, 150 μL
   of the supernatant was transferred to LC-MS vials equipped with inserts
   for analysis. All extraction reagents were pre-cooled to −20 °C prior
   to use. Quality control (QC) samples were prepared by pooling equal
   volumes of extracts from all samples. Untargeted metabolomics analysis
   was performed using a Waters ACQUITY UPLC I-Class Plus system coupled
   with a Thermo Scientific Q Exactive high-resolution mass spectrometer.
   Chromatographic separation was carried out on an ACQUITY UPLC HSS T3
   column (100 mm × 2.1 mm, 1.8 μm) maintained at 45 °C. The mobile phases
   consisted of solvent A (water with 0.1% formic acid) and solvent B
   (acetonitrile), delivered at a flow rate of 0.35 mL/min. The injection
   volume was 5 μL. Raw data were processed and analyzed by OE Biotech
   Co., Ltd. (Shanghai, China). Bioinformatic analysis was performed using
   OECloud tools ([216]https://cloud.oebiotech.com/task/). Volcano plots
   and other visualizations were generated using R software
   ([217]https://www.r-project.org/) via the OECloud platform.

Cell culture

   Four-week-old male Sprague-Dawley rats were obtained from the
   Experimental Animal Center of Zhejiang Chinese Medical University,
   China. NP tissue was isolated from the rat tail intervertebral discs,
   minced, and digested with type II collagenase at 37 °C for 4 h. The
   resulting cell suspension was filtered twice through a 70 μm
   single-cell strainer to obtain a single-cell suspension, followed by
   centrifugation at 1000 × g for 5 min to collect primary NP cells. The
   collected NPCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
   supplemented with 10% fetal bovine serum (Sigma-Aldrich, USA) and 1%
   streptomycin-penicillin solution (Gibco, USA). NP cells were cultured
   at 37 °C and 5% CO[2]. The culture medium was refreshed the following
   day, and cells were passaged upon reaching 80% confluence. NP cells in
   the second passage were regarded as normal state and used to perform
   experiments.

   RAW264.7 cell lines were purchased from CCTCC (Wuhan, China). The
   macrophage was cultured in DMEM containing 10% FBS.

Membrane surface modification of nucleus pulposus cells with TPP

   The isolated nucleus pulposus cells were seeded in 10 cm culture dishes
   and cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1%
   penicillin/streptomycin (PS), and 10 μM non-natural mannose derivative
   (Ac4ManNAz) for 48 h. After incubation, the cells were washed twice
   with PBS, and the medium was replaced with serum-free medium containing
   10 μM DBCO-TPP for a 2-h treatment. In the experiment characterizing
   the efficiency of the click chemistry reaction, DBCO-TPP was replaced
   with equal concentration of DBCO-FITC.

Preparation and characterization of biomimetic nanovesicles

   NP cell membranes and TPP-modified NP cell membranes were extracted
   using a Membrane and Cytosol Protein Extraction Kit (Beyotime, China)
   according to the manufacturer’s instructions. The membrane protein
   concentration was adjusted to 1 mg/mL using a BCA Protein Assay Kit
   (Beyotime, China). The isolated membranes were then extruded through a
   220 nm polycarbonate membrane at least five times to ensure uniform
   vesicle dispersion. BAI1 (HY-103269, MCE, USA) was dissolved in DMSO at
   a concentration of 10 mM and mixed with an equal volume of PBS
   containing either unmodified NP membranes nanovesicles (NPM) or
   TPP-modified NP membranes nanovesicles (NPMT). The resulting mixture
   was sonicated on ice using an ultrasonic processor (10 s on, 5 s off,
   repeated for 10 cycles) to obtain BAI1-loaded nanovesicles, termed BNPM
   and BNPMT. The nanovesicles were subsequently collected by
   ultracentrifugation at 20,000 × g for 1 h at 4 °C. The BAI1 loading
   efficiency was determined by HPLC (Agilent 1260, USA). The morphology
   of the nanovesicles was characterized using cryogenic transmission
   electron microscopy (Cryo-EM), and their hydrodynamic diameter and zeta
   potential were measured using a Zetasizer Nano ZS90 DLS instrument
   (Malvern Panalytical, UK).

BNPMT targeting nucleus pulposus cells mitochondria

   Confocal microscopy was used to assess the targeting efficiency of
   nanovesicles. FITC-encapsulated nanovesicles were prepared using the
   same method as BNPM and BNPMT to evaluate their targeting capabilities.
   For cellular uptake experiments, equal numbers of NP cells and RAW264.7
   macrophages were incubated with free FITC, FITC@NPM, or FITC@NPMT (with
   the equal FITC concentration of 10 µg/mL) for 4 h. After incubation,
   nuclei were stained with Hoechst dye (Yeasen, China). For mitochondrial
   targeting experiments, MitoTracker (Beyotime, China) was used to label
   mitochondria in NP cells during BNPM and BNPMT uptake assays. Live-cell
   imaging was performed using a Nikon confocal laser scanning microscope
   to observe nanovesicle uptake under different treatment conditions.

Cells treatment

   To induce senescence in primary NPCs, cells were treated with 200 nM
   Dox for 24 h, followed by culture in fresh medium for an additional 4
   days. In the NC group, NPCs were treated with an equal volume of PBS
   under the same conditions. In the experimental group, BAI1, BNPM, BNPMT
   (2.5 μM based on BAI1) was co-cultured with NPCs for 24 h.

Cytoplasmic DNA isolation

   After collecting the NP cells with different treatments, they were
   placed in 500 μL of buffer (Containing 150 mM NaCl, 50 mM HEPES, pH
   7.4, and 25 μg/ml digitonin (Sigma-Aldrich, USA) and incubated at room
   temperature for 10 min. The cells were then centrifuged at 150 × g for
   5 min at 4 °C, and the supernatant was collected. This supernatant was
   further centrifuged twice at 150 × g at 4 °C, followed by a final
   centrifugation at 17,000 × g for 10 min to obtain the cytoplasmic
   fraction. DNA was extracted from the cytoplasmic fraction using the
   DNeasy Blood & Tissue Kit (Qiagen, 69504) according to the
   manufacturer’s instructions. Finally, the concentration of the
   extracted DNA was measured using the Qubit™ dsDNA Assay Kit
   (Invitrogen, USA).

Co-culture of macrophages with H-P gel

   RAW264.7 macrophages were seeded into the lower chambers of 12-well
   Transwell plates for co-culture with H-P hydrogel. The experimental
   groups were designed as follows: (1) NC group, with no hydrogel added;
   (2) CpG group, with CpG added to the lower chamber at a final
   concentration of 1 μg/mL in DMEM; and (3) CpG + H-P group, with H-P
   hydrogel placed in the upper chamber and CpG (1 μg/mL) added to the
   lower chamber medium. After 24 h of incubation, macrophages and their
   supernatants were collected for flow cytometry, qPCR, and ELISA
   analyses. Preparation of Conditioned Medium: The macrophage
   supernatants were centrifuged at 1000 × g for 5 min, and the resulting
   supernatants were mixed with an equal volume of fresh complete DMEM to
   generate conditioned media for each group. NP cells were then cultured
   in the respective conditioned media for 24 h, followed by
   immunofluorescence staining, ELISA, WB, and qPCR analysis.

Immunocytochemistry

   After washing the cells three times with PBS, we added 4%
   paraformaldehyde to completely cover and fix the cells at room
   temperature for 15 min. Next, the cells were washed three times with
   PBST, and then 0.1% Triton ×-100 was added to permeabilize the cells,
   followed by a 20-min incubation at room temperature and three
   additional PBST washes. The cells were then blocked with 5% BSA at room
   temperature for 60 min. After blocking, primary antibodies (BAX6A7
   antibody, Santa Cruz, USA; TOMM20 antibody, Abcam, UK; dsDNA marker,
   Santa Cruz, USA; Collagen Type II Polyclonal antibody, Proteintech,
   China; MMP3 antibody, Proteintech, China) were added, and the cells
   were incubated overnight at 4 °C. Following the removal of primary
   antibodies, the cells were washed three times with PBST and incubated
   with secondary antibodies Alexa Fluor® 488 (Abcam, UK) or Alexa Fluor®
   594 (Abcam, UK) at room temperature for 1 h. After this, the cells were
   washed three times with PBST and stained with DAPI (Solarbio, China) to
   visualize the nuclei. Finally, cell images were captured using a
   confocal microscope to assess the localization and expression of the
   target proteins.

Western blotting

   Cells were lysed using a lysis buffer (Fude, China) supplemented with
   phosphatase inhibitors (Beyotime, China) and protease inhibitors
   (Beyotime, China). The protein concentration of the lysate was
   determined using the BCA protein assay kit (Beyotime, China). Next,
   protein samples were mixed with protein loading buffer (Fude, China)
   and boiled in a metal bath at 100 °C for 5 min. Proteins were then
   separated by SDS-PAGE and transferred onto a PVDF membrane (Millipore,
   USA). To prevent nonspecific binding, the membrane was blocked with 5%
   BSA at room temperature for 1 h. After blocking, primary antibodies
   (ATP1A3 Polyclonal antibody, Proteintech, China; Alpha Tubulin
   Polyclonal antibody, Proteintech, China; Phospho-TMEM173/STING (Ser366)
   Antibody, Affinity, USA; TMEM173/STING Antibody, Affinity, USA;
   Phospho-IRF3 (Ser396) Antibody, Affinity, USA; IRF3 Antibody, Affinity,
   USA; Phospho-TBK1 (Ser172) Antibody, Affinity, USA; TBK1 Antibody,
   Affinity, USA; GAPDH Polyclonal antibody, Proteintech, China; Collagen
   II Antibody, Affinity, USA; MMP3 Antibody, Affinity, USA) were added,
   and the membrane was incubated overnight at 4 °C. The membrane was then
   washed three times with TBST to remove any unbound primary antibodies,
   followed by incubation with secondary antibodies (HRP-conjugated Goat
   Anti-Rabbit IgG(H + L), Proteintech, China; HRP-conjugated Goat
   Anti-mouse IgG(H + L), Proteintech, China) at room temperature for 1 h.
   A final wash with TBST was performed, and target protein signals were
   detected using the ECL chemiluminescence method (Fude, China). Protein
   band intensities were semi-quantified using ImageJ software.

qRT-PCR and ELISA analyses

   Total RNA from cells was extracted using an RNA extraction kit
   (Accurate Biology, China). RNA concentration was measured using the
   NanoDrop ND-1000 spectrophotometer. Subsequently, RNA was
   reverse-transcribed into cDNA using the Evo M-MLV reverse transcription
   kit (Accurate Biology, China). Real-time qPCR was performed using qPCR
   SYBR Green Master Mix (Yeasen, China), with specific primer sequences
   provided in Supplementary Table [218]1. For cytosolic DNA analysis,
   20 ng of dsDNA was used for qPCR. Cell supernatants were collected, and
   cytokine concentrations were determined using ELISA kits (Boster,
   China).

Flow cytometry

   Macrophages were co-incubated with CpG-FITC and HP hydrogel for 24 h.
   After incubation, the cells were collected and analyzed by flow
   cytometry (Beckman, USA) to assess the FITC fluorescence intensity,
   which reflects their uptake of CpG-FITC. To evaluate macrophage M1
   polarization, a similar co-incubation was performed, after which the
   cells were collected and resuspended in 0.1% Triton ×-100, then
   incubated at room temperature for 10 min. Following centrifugation at
   1000 × g for 5 min to discard the supernatant, the cells were
   resuspended in 5% BSA and incubated at room temperature for 30 min.
   After another centrifugation at 1000 × g for 5 min, PE-anti-iNOS
   antibody (BioLegend, USA) was added and the cells were incubated for
   30 min. Finally, the cells were analyzed by flow cytometry to determine
   their fluorescence, indicating the level of M1 polarization. After
   treatment, NP cells from different groups were collected and stained
   using an Annexin V-FITC apoptosis detection kit (Beyotime, China)
   according to the manufacturer’s instructions. The stained cells were
   then analyzed by flow cytometry.

Preparation and characterization of H-P Gel/BNPMT-loaded H-P Gel

   HA-CHO was synthesized via the sodium periodate oxidation
   method^[219]53. To form the H-P hydrogel, 5 wt% lyophilized HA-CHO and
   1 wt% PAMAM solution were each dissolved in equal volumes of PBS and
   mixed at room temperature. For the preparation of BNPMT-loaded H-P
   hydrogel, HA-CHO was first dissolved in PBS containing BNPMT, followed
   by mixing with an equal volume of PAMAM solution. Gel formation was
   visually confirmed by observing the vial for signs of gelation. To
   enhance visualization, a non-reactive dye was added to the hydrogel,
   and its injectability and self-healing properties were documented using
   photography. The dynamic rheological properties of the H-P hydrogel
   were evaluated using a strain-controlled rheometer (Anton Paar MCR302,
   Austria).

Rat IVDD model construction

   Eight-week-old male Sprague-Dawley (SD) rats were obtained from the
   Animal Experiment Center of Zhejiang Chinese Medical University
   (China). All animal experiments were approved by the Animal Experiment
   Ethics Committee of Zhejiang Chinese Medical University. The rats were
   housed in a pathogen-free environment under a 12-h light/dark cycle,
   with ad libitum access to food and water. They were randomly assigned
   to different groups for in vivo experiments. The rats were anesthetized
   by intraperitoneal injection of sodium pentobarbital (45 mg/kg) and
   placed in a prone position. A 20-gauge needle was used to puncture the
   Co6/7 intervertebral disc from the dorsal side; the needle was inserted
   from the center of the disc to the opposite side, rotated 180°, and
   held for 10 s. The Co5/6 or Co7/8 intervertebral discs were preserved
   intact as internal controls. Evaluations were performed at 4 and 8
   weeks post-treatment. The control group received no puncture or
   injection. The BNPMT group was injected with 20 μL of BNPMT
   nanovesicles (Containing 0.5 mM BAI1). The H-P group received 20 μL of
   H-P hydrogel alone. The BNPMT@H-P group was administered 20 μL of
   BNPMT-loaded H-P hydrogel (Containing 0.5 mM BAI1).

Radiological evaluation analysis

   Rats were euthanized at 4 and 8 weeks after treatment, and their tail
   intervertebral disc specimens were collected and fixed in formaldehyde.
   μCT (SkyScan, Belgium) and MRI (Universal Corporation, USA) were used
   to evaluate intervertebral disc degeneration in the rat tail, focusing
   on changes in nucleus pulposus signal intensity and intervertebral
   space height. X-ray images were analyzed using ImageJ software to
   calculate the Disc Height Index (DHI%). T2-weighted images were also
   obtained through coronal scans. Pathological damage to the tail
   intervertebral discs was assessed by three experienced radiologists and
   three orthopedic surgeons based on the modified Thomson classification.

In vivo imaging of rats

   ICG was encapsulated into nanovesicles derived from nucleus pulposus
   cell membranes (ICG@NPMT with a final ICG concentration of 10 µg/mL)
   using an ultrasonic co-extrusion method to evaluate their in vivo
   release behavior. The ICG@NPMT nanovesicles were subsequently
   encapsulated within the H-P hydrogel and injected into the rat
   coccygeal intervertebral disc at day 0. This approach enabled real-time
   tracking of the nanovesicles’ release profile in vivo.In parallel, to
   evaluate the in vivo degradation behavior of the H-P hydrogel itself,
   ICG was covalently conjugated to the hydrogel using ICG-NHS, which
   forms stable amide bonds with the abundant primary amine groups
   (–NH[2]) on PAMAM dendrimers. The ICG-labeled hydrogel was similarly
   injected into the rat intervertebral disc at day 0. Fluorescence
   signals were captured at predetermined time points (Day 0, 3, 7, 14,
   and 21) using an in vivo imaging system (IVIS Lumina III, PerkinElmer,
   USA) to monitor the dynamic release of nanovesicles and the degradation
   of the hydrogel matrix, respectively.

Histological evaluation, immunohistochemistry, and immunofluorescence

   At 4 and 8 weeks after model induction, rats in each group were
   sacrificed, and caudal intervertebral disc specimens were collected and
   fixed in 4% paraformaldehyde. The specimens were then decalcified in
   15% EDTA for at least 4 weeks and embedded in paraffin to prepare 5 μm
   continuous sections. H&E and Safranin O/Fast Green staining were
   performed to observe changes in the intervertebral disc tissue
   structure and collagen content. To further assess ECM synthesis and
   degradation in the disc, as well as the expression of inflammatory
   factors, immunohistochemistry and immunofluorescence staining were
   performed. The sections were deparaffinized with xylene and a gradient
   of ethanol (100–80%) and underwent antigen retrieval using citrate
   buffer. Blocking was carried out with a 5% BSA solution, followed by
   overnight incubation with primary antibodies at 4 °C. Secondary
   antibodies (HRP or fluorescence-labeled) were incubated at 37 °C for
   1 h. DAB was used for color development, followed by counterstaining
   with hematoxylin. Finally, sections were mounted with neutral resin or
   anti-fade mounting medium and observed using a scanning system.

Statistical analysis

   All experiments were repeated at least three times. Semi-quantitative
   analysis was performed on at least three samples, and the results are
   expressed as the mean ± standard deviation (s.d.). Statistical analysis
   was conducted using GraphPad Prism 10 software (GraphPad Software
   Inc.). Comparisons between two groups were made using an unpaired
   two-tailed Student’s t-test, and for multiple group comparisons,
   one-way ANOVA followed by Tukey’s post hoc test was applied. A P-value
   of less than 0.05 was considered statistically significant.

Ethics approval

   All ethical aspects of the human-related components of this study,
   including the consultation of patient medical data and the collection
   and use of human nucleus pulposus (NP) tissue samples, were approved by
   the Ethics Committee of Hangzhou Hospital of Traditional Chinese
   Medicine (Grant number: 2022KY039). Normal human NP tissue samples were
   collected from patients aged 27–31 years with burst fractures. Based on
   MRI T2-weighted imaging and the Pfirrmann grading system, the
   non-degenerated intervertebral discs were classified as grade I.
   Degenerated human NP tissue samples were collected from patients aged
   56–62 years with lumbar disc herniation, with the corresponding discs
   graded as Pfirrmann grade IV. All selected patients had no history of
   cardiovascular or cerebrovascular diseases, cancer, infections, immune
   or endocrine disorders, or organ dysfunction. In total, three pairs of
   samples (three healthy and three degenerated) were used for
   immunohistochemical analysis.

   Written informed consent was obtained from all patients and volunteers
   for the collection and use of their tissue samples in scientific
   research. In addition, separate written informed consent was obtained
   to publish information that could potentially identify individuals
   (e.g., age, sex).

   All relevant approvals from China’s Ministry of Science and Technology
   regarding the use and export of human genetic resources relevant to
   this study have also been obtained.

   All animal-related procedures, including animal acquisition, surgical
   operations, and sample collection, were approved by the Animal Ethics
   Committee of Zhejiang Chinese Medical University (Grant number:
   20250331-08).

Reporting summary

   Further information on research design is available in the [220]Nature
   Portfolio Reporting Summary linked to this article.

Supplementary information

   [221]Supplementary information^ (2.8MB, pdf)
   [222]41467_2025_63194_MOESM2_ESM.pdf^ (4.7KB, pdf)

   Description of Additional Supplementary Files
   [223]Supplementary Data 1^ (163.8KB, xlsx)
   [224]Reporting Summary^ (124.6KB, pdf)
   [225]Transparent Peer Review file^ (3MB, pdf)

Source data

   [226]Source Data^ (1MB, xlsx)

Acknowledgements