Cartilage targeting hydrogel nanoplatform degrades BRD4 to alleviate osteoarthritis via Nav1.7 axis

Domain: biology
Published: 2026-03-28
Editorial outcome: minor_revision

Abstract
Osteoarthritis (OA) is a common degenerative joint disease with limited disease-modifying therapies. Emerging evidence suggests that epigenetic dysregulation contributes to cartilage degeneration, but effective strategies to selectively target these pathways remain lacking. Here we show that the BRD4/Nav1.7 axis drives inflammatory and metabolic dysfunction in OA. Integrated single-cell and transcriptomic analyses identify BRD4 as a key regulator that enhances Nav1.7 transcription, promoting mitochondrial impairment and catabolic activation in chondrocytes. To therapeutically target this pathway, we develop a biomimetic hydrogel system incorporating chondrocyte membrane-coated nanoparticles for cartilage-specific delivery of a BRD4 proteolysis-targeting chimera (PROTAC), a molecule designed to induce selective protein degradation. This nanoplatform enables efficient intra-articular delivery, immune evasion and targeted retention in cartilage. Treatment suppresses inflammatory responses, restores mitochondrial function and reduces cartilage degeneration and pain behaviors in two mouse models of OA. These findings establish targeted BRD4 degradation as a disease-modifying strategy and provide a precision nanotherapeutic platform for OA.

Introduction
Osteoarthritis (OA) is the most prevalent degenerative joint disorder and severely impairs the quality of life in middle-aged and elderly populations 1. It is characterized by cartilage destruction, synovial inflammation, osteophyte formation, and chronic pain, leading to restricted mobility and, in advanced cases, functional disability 2, 3. With global population aging, the incidence and disability rates of OA have continued to rise, posing a significant public health burden 4, 5. Current clinical management strategies for OA primarily aim to alleviate symptoms and delay structural deterioration. These approaches include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular hyaluronic acid injections, and joint replacement surgery 6. However, these approaches largely remain palliative, lacking precision in targeting underlying disease mechanisms, especially during the early and middle stages when cartilage deterioration may still be reversible 7. Therefore, there is an urgent need to investigate OA pathogenesis at the molecular level to facilitate the development of critical targeted therapies.

In recent years, the role of epigenetic regulator BRD4 (Bromodomain-containing protein 4) in OA has garnered increasing attention 8, 9. BRD4 recognizes histone acetylation marks and regulates the transcription of genes involved in inflammatory responses and cellular metabolism, thereby promoting inflammatory activation and extracellular matrix (ECM) degradation in chondrocytes 10, 11. Studies have shown that BRD4 is highly expressed in OA cartilage, with its elevated activity closely associated with cartilage deterioration 12, 13. Meanwhile, the voltage-gated sodium channel Nav1.7, traditionally recognized for its role in nociceptive signaling in neurons, has been found to be aberrantly expressed in chondrocytes and implicated in both cartilage dysfunction and pain perception 14-16. Emerging evidence suggests that BRD4 may contribute to OA progression by modulating Nav1.7 transcriptional activity 14. However, whether BRD4 activates Nav1.7 expression through epigenetic mechanisms to establish a pathogenic signaling axis remains unclear and requires further investigation.

From a therapeutic perspective, the emergence of Proteolysis Targeting Chimera (PROTAC) technology has offered a critical approach for targeting "undruggable" proteins 17. Unlike traditional small-molecule inhibitors, PROTACs induce selective protein degradation by recruiting target proteins to E3 ubiquitin ligases, thereby enabling efficient and sustained elimination of disease-associated proteins 18, 19. As a nuclear protein, BRD4 is inherently challenging to inhibit efficiently through conventional methods, whereas PROTACs provide a feasible means for its precise modulation 20, 21. Despite their promise, PROTACs face notable limitations, including high molecular weight, poor membrane permeability, and insufficient tissue specificity, which restrict their performance in complex biological environments 22. In recent years, stimulus-responsive nano-PROTAC technology has emerged as an innovative therapeutic strategy. By incorporating PROTACs into nanocarriers and integrating internal or external stimuli such as redox conditions, enzymatic activity, or light, this strategy enables spatiotemporal control of protein degradation, thereby enhancing targeting precision, reducing systemic toxicity, and facilitating the clinical translation of PROTAC-based therapies 23. To further improve therapeutic efficacy, the development of nanocarrier platforms with tissue-targeting capabilities has become a critical breakthrough 24. Cartilage tissue is characterized by poor vascularization and strong barrier properties, making effective drug penetration and sustained retention highly dependent on intelligent delivery systems. In recent years, multifunctional nanomaterials have been extensively validated for precise drug delivery across complex biological barriers, including the blood-brain barrier and the cartilage barrier, by rational modulation of their physicochemical properties. These advances provide a strong theoretical basis for the design of the drug delivery platform used in the present study 25. Moreover, highly programmable delivery systems represented by DNA nanostructures have attracted increasing attention as an emerging frontier in biomedicine. Owing to their excellent biocompatibility and structural design flexibility, DNA-based nanostructures exhibit distinct advantages for the precise delivery of diverse bioactive cargos, including proteins and nucleic acids 26.

Based on these considerations, the present study aimed to elucidate the key mechanism by which BRD4 activates Nav1.7 expression through histone acetylation, thereby promoting cartilage degeneration and OA progression. The study further defined the pathogenic role of the BRD4/Nav1.7 axis in the OA disease process. On this foundation, a cartilage-targeted PROTAC nanodelivery system was developed to achieve precise BRD4 degradation at the epigenetic level, with the objective of intervening at the upstream origin of disease progression. This work not only advances the mechanistic understanding of OA pathogenesis by revealing the noncanonical role of Nav1.7 and its epigenetic regulation, but also provides technical validation for the integration of PROTAC-based protein degradation with targeted nanodelivery strategies in OA. From a clinical perspective, the findings identify critical therapeutic targets and intervention strategies for early cartilage protection, disease modification, and pain alleviation, thereby offering a conceptual and translational framework for precision medicine approaches in degenerative joint diseases.

Results
Identification of Degeneration-Associated Chondrocyte Populations via Single-Cell Sequencing
To systematically identify chondrocyte subpopulations closely associated with OA progression, articular cartilage tissues were harvested from the knee joints of mice in the normal group (Sham surgery) and the OA model group (8 weeks post-destabilization of the medial meniscus [DMM] surgery). scRNA-seq was performed using the 10x Genomics platform (Figure 1A). After stringent quality control, 14,743 cells were retained for downstream analysis, including 7,870 cells from the Sham group and 6,873 cells from the OA group. Data were normalized, followed by PCA for dimensionality reduction and t-distributed stochastic neighbor embedding (t-SNE) for cell clustering. Nineteen distinct cell clusters (Clusters 0–18) were identified (Figure 1B). Cluster annotation was performed based on established marker gene expression patterns and prior reports 27. Using Adamts5 and Mmp13 as markers of degenerative chondrocytes, Cluster 4 was annotated as the degenerative chondrocyte population (Figure 1C-D; Supplementary Table 1). Cell proportion analysis revealed a marked increase in both chondrocytes and degenerative chondrocytes, with the latter showing the most pronounced elevation (Figure 1E-F).

Identification of the Key Regulator BRD4 via Transcriptomic Profiling and Marker Gene Integration
To further identify key regulators governing the function of degeneration-associated chondrocytes (Cluster 4), bulk RNA sequencing was performed on cartilage tissues from Sham and OA mice (8 weeks post-DMM surgery) (Supplementary Fig. 1A), with six biological replicates per group (n = 6). RNA integrity was confirmed (RIN > 8.0), followed by standard quality control. Differential expression analysis was conducted using the limma package (v3.56.2), with thresholds of log FC > 0.5 or < -0.5 and p < 0.05. A total of 689 upregulated and 714 downregulated genes were identified (Supplementary Fig. 1B). Sample clustering results are shown in Supplementary Fig. 1C. GO enrichment analysis revealed that the upregulated genes were significantly associated with pathways such as the negative regulation of cartilage development (Supplementary Fig. 1D-E), suggesting epigenetic and transcriptional dysregulation in OA-associated cartilage degeneration.

To precisely pinpoint key regulators associated with degenerative chondrocytes (Cluster 4), an intersection analysis was conducted between the upregulated genes identified in the degenerative chondrocyte cluster from scRNA-seq and those obtained from bulk RNA-seq. This intersection yielded 18 candidate genes (Supplementary Fig. 1F). PPI network analysis using STRING (v12.0) indicated that BRD4 occupied a central hub position within the network (degree = 15) (Supplementary Fig. 1G) and exhibit strong interactions with epigenetic regulators such as EP300. Previous studies have implicated BRD4 in OA progression 12. Notably, BRD4 was significantly upregulated in both datasets, with log FC values of 1.14 in scRNA-seq and 2.02 in bulk RNA-seq, both reaching statistical significance (p < 0.05) (Supplementary Fig. 1H).

Silencing BRD4 Alleviates Mitochondrial Dysfunction in TNF-α-Treated ATDC5 Cells
To evaluate the role of BRD4 in oxidative stress and mitochondrial injury, ATDC5 cells were divided into four groups: Ctrl, TNF-α, TNF-α + sh-NC, and TNF-α + sh-BRD4 (workflow shown in Supplementary Fig. 2A). BRD4 knockdown efficiency was first confirmed. RT-qPCR and WB analyses showed that, compared with the Ctrl group, BRD4 mRNA (P<0.001, 95.00% CI=-1.646 to -1.061) and protein (P<0.001, 95.00% CI=-3.824 to -3.549) levels were significantly upregulated in the TNF-α group. In contrast, BRD4 mRNA (P<0.001, 95.00% CI=0.3041 to 0.8892) and protein (P<0.001, 95.00% CI=4.079 to 4.354) expression was markedly reduced in the TNF-α + sh-BRD4 group compared with the TNF-α + sh-NC group (Supplementary Fig. 2B-C).

Reactive oxygen species (ROS) levels were measured using the DCFH-DA probe. ROS production was significantly elevated in the TNF-α group relative to the Ctrl group (P<0.001, 95.00% CI=-1.343 to -0.8898), whereas it was markedly reduced in the TNF-α + sh-BRD4 group compared to the TNF-α + sh-NC group (P<0.001, 95.00% CI=0.8165 to 1.270) (Supplementary Fig. 2D). TEM revealed pronounced mitochondrial swelling and cristae disruption in the TNF-α group, whereas mitochondrial ultrastructure was notably improved in the TNF-α + sh-BRD4 group, with more preserved cristae compared with the TNF-α + sh-NC group (Supplementary Fig. 2E).

Subsequently, JC-1 staining was used to assess changes in ΔΨm. The TNF-α group showed a significant decrease in the JC-1 A/M ratio relative to the Ctrl group (P<0.001, 95.00% CI=0.5565 to 1.084). This reduction was reversed by BRD4 knockdown (P<0.001, 95.00% CI=-1.000 to -0.4731), as evidenced by a significantly increased A/M ratio in the TNF-α + sh-BRD4 group relative to the TNF-α + sh-NC group (Supplementary Fig. 2F-G). Consistently, WB analysis showed that TNF-α markedly reduced the expression of mitochondrial functional proteins TFAM (P<0.001, 95.00% CI=0.8323 to 1.008) and ATP synthase subunit beta (ATPB) (P<0.001, 95.00% CI=0.3754 to 0.6646). In contrast, both TFAM (P<0.001, 95.00% CI=-0.4877 to -0.3123) and ATPB (P=0.001, 95.00% CI=-0.4179 to -0.1287) expression were markedly upregulated in the TNF-α + sh-BRD4 group relative to the TNF-α + sh-NC group (Supplementary Fig. 2H). Immunofluorescence staining produced consistent results, further confirming the improvement in mitochondrial function (Supplementary Fig. 2I-J).

To assess cellular energy metabolism, intracellular ATP levels were quantified. ATP content was significantly reduced in the TNF-α group compared with the Ctrl group (P=0.003, 95.00% CI=0.6229 to 2.384), while BRD4 knockdown notably restored ATP levels in the TNF-α + sh-BRD4 group compared to the TNF-α + sh-NC group (P=0.009, 95.00% CI=-2.100 to -0.3396) (Supplementary Fig. 2K).

Collectively, these data demonstrate that BRD4 silencing effectively suppresses TNF-α-induced ROS accumulation, rescues mitochondrial ultrastructure, stabilizes ΔΨm, and enhances ATP synthesis, indicating a pivotal regulatory role of BRD4 in chondrocyte mitochondrial dysfunction (Supplementary Fig. 2L).

Silencing BRD4 Promotes Anabolic Metabolism and Suppresses Catabolic Activity in TNF-α-Treated ATDC5 Cells
To examine the role of BRD4 in TNF-α-induced metabolic imbalance, ATDC5 cells were assigned to four groups: Ctrl, TNF-α, TNF-α + sh-NC, and TNF-α + sh-BRD4 (workflow shown in Supplementary Fig. 3A). RT-qPCR analysis demonstrated that inflammatory cytokine expression was markedly elevated after TNF-α stimulation, with significant increases in IL-6 (P<0.001, 95.00% CI=-4.755 to -4.331) and IL-1β (P<0.001, 95.00% CI=-10.11 to -8.839) compared with the Ctrl group. However, their expression was markedly reduced in the TNF-α + sh-BRD4 group relative to the TNF-α + sh-NC group (IL-6: P<0.001, 95.00% CI=1.095 to 1.519; IL-1β: P<0.001, 95.00% CI=1.479 to 2.747) (Supplementary Fig. 3B). WB analysis further confirmed that IL-6 protein levels were significantly increased in the TNF-α group (P<0.001, 95.00% CI=-2.632 to -2.381) and significantly decreased (P<0.001, 95.00% CI=3.511 to 3.762) following BRD4 knockdown (Supplementary Fig. 3C). Consistent results from immunofluorescence staining further validated the inhibitory effect of BRD4 silencing on TNF-α-induced inflammation (Supplementary Fig. 3D).

The impact of BRD4 silencing on anabolic metabolism was then evaluated. RT-qPCR showed that Sox9 (P<0.001, 95.00% CI=0.2529 to 0.5204) and Col2a1 (P<0.001, 95.00% CI=0.4580 to 0.5486) mRNA levels were significantly downregulated in the TNF-α group compared with the Ctrl group, whereas both transcripts were significantly restored in the TNF-α + sh-BRD4 group relative to the TNF-α + sh-NC group (Sox9: P<0.001, 95.00% CI=-0.4304 to -0.1629; Col2a1: P<0.001, 95.00% CI=-0.4420 to -0.3514) (Supplementary Fig. 3E). WB analysis yielded consistent results, showing that SOX9 (P<0.001, 95.00% CI=0.3291 to 0.5509) and COL II (P<0.001, 95.00% CI=0.3889 to 0.7045) protein levels were markedly decreased in the TNF-α group and significantly increased after BRD4 silencing (SOX9: P<0.001, 95.00% CI=-0.7343 to -0.5124; COL II: P<0.001, 95.00% CI=-0.6478 to -0.3322) (Supplementary Fig. 3F). Immunofluorescence staining further demonstrated enhanced SOX9 and COL II signals in the TNF-α + sh-BRD4 group (Supplementary Fig. 3G), indicating restoration of anabolic activity.

To evaluate cartilage matrix degradation, the expression of matrix-degrading enzymes Mmp13, Adamts4, and Adamts5 was assessed. RT-qPCR results showed that mRNA levels of all three genes were significantly downregulated in the TNF-α + sh-BRD4 group (Mmp13: P<0.001, 95.00% CI=2.438 to 3.269; Adamts4: P<0.001, 95.00% CI=1.831 to 2.602; Adamts5: P<0.001, 95.00% CI=0.9654 to 1.201) (Supplementary Fig. 3H). Consistently, WB analysis showed that MMP13 protein expression was markedly increased in the TNF-α group compared with the Ctrl group (P<0.001, 95.00% CI=-1.538 to -1.342), and significantly suppressed by BRD4 knockdown (P<0.001, 95.00% CI=2.039 to 2.235) (Supplementary Fig. 3I). This expression pattern was further validated by immunofluorescence staining (Supplementary Fig. 3J).

Collectively, these results demonstrate that BRD4 silencing attenuates TNF-α-induced inflammatory responses, restores anabolic metabolism, and suppresses catabolic enzyme expression, thereby contributing to the maintenance of cartilage metabolic homeostasis.

Transcriptomic Profiling Identifies Nav1.7 as a Downstream Target of BRD4
To systematically identify key downstream effectors regulated by BRD4, transcriptome sequencing was performed on ATDC5 cells from the sh-NC and sh-BRD4 groups (workflow shown in Supplementary Fig. 4A). Differential expression analysis was conducted using the limma package (v3.56.2) with a threshold of |log FC| > 0.5 and p < 0.05, resulting in 184 upregulated and 392 downregulated genes (Supplementary Fig. 4B-C). GO enrichment analysis revealed that downregulated genes were predominantly associated with “ion transmembrane transport” and “sodium channel activity,” indicating that BRD4 knockdown markedly affects ion channel–related functions in chondrocytes (Supplementary Fig. 4D).

Based on the global expression matrix, WGCNA was performed (soft-threshold power = 6; scale-free R2 > 0.85) (Supplementary Fig. 4E). Module-trait relationship analysis identified the blue module as being most strongly correlated with BRD4 knockdown (R = -0.99, p < 0.001). Genes within this module were filtered using MM > 0.8 and GS > 0.6 and intersected with DEGs, yielding 534 candidate genes (Supplementary Fig. 4F).

To further refine potential target genes, the LASSO regression was applied to the 534 candidates, identifying seven feature genes (Supplementary Fig. 4G). Among them, Scn9a, which encodes the voltage-gated sodium channel Nav1.7, emerged as the most significantly upregulated gene (log FC = 1.81, p < 0.05) (Supplementary Fig. 4H). Through the integrated analysis of transcriptomic profiling, WGCNA, and machine learning-based feature selection, Scn9a was identified as a critical downstream effector regulated by BRD4.

Additionally, GSEA of the whole-transcriptome dataset revealed significant downregulation of pathways related to potassium ion channels, the tricarboxylic acid (TCA) cycle and respiratory chain, and ECM metabolism, including collagen synthesis and degradation, following BRD4 depletion (Supplementary Fig. 4I). These results suggest that BRD4 contributes to cartilage degeneration by coordinately regulating ion channel activity, cellular energy metabolism, and ECM remodeling.

Nav1.7 Expression Is Elevated in OA Chondrocytes and Contributes to Sodium Current Formation
To evaluate the expression pattern of Nav1.7 (encoded by Scn9a) under osteoarthritic conditions, an OA-like cellular model was established by TNF-α treatment of ATDC5 cells. (workflow shown in Figure 2A). RT-qPCR analysis demonstrated a significant upregulation of Scn9a mRNA in the TNF-α group compared with the Ctrl group (P<0.001, 95.00% CI=0.425 to 0.788) (Figure 2B). Consistently, WB analysis revealed a marked increase in Nav1.7 protein expression following TNF-α stimulation (P<0.001, 95.00% CI=7.89 to 8.16) (Figure 2C). Immunofluorescence staining further showed enhanced Nav1.7 fluorescence intensity in TNF-α-treated cells, with prominent localization to the plasma membrane (Figure 2D). To confirm subcellular distribution, membrane and cytoplasmic fractions were isolated for WB analysis. Nav1.7 was predominantly detected in the membrane fraction, and membrane-associated Nav1.7 expression was significantly increased in the TNF-α group (P<0.001, 95.00% CI=2.85 to 3.13) (Figure 2E). These results indicate that Nav1.7 is transcriptionally and translationally upregulated under OA-like conditions and exhibits enhanced membrane localization, consistent with its function as a voltage-gated sodium channel.

Whole-cell patch-clamp recordings were then performed to assess Nav1.7 functional activity. Depolarizing voltage steps from -60 mV to +50 mV elicited robust inward sodium currents (Figure 2F). Compared to the Ctrl group, the TNF-α-treated group exhibited a significantly increased sodium current amplitude (Figure 2G). Application of 1 μM TTX markedly suppressed the inward current, indicating a TTX-sensitive (TTX-S) sodium current consistent with Nav1.7 electrophysiological properties (Figure 2H).

To further verify the specific contribution of Nav1.7, ATDC5 cells were treated with either the non-selective sodium channel blocker TTX (1 μM) or the Nav1.7-selective inhibitor ProTx II (25 nM). Both treatments significantly reduced sodium current amplitudes relative to the TNF-α group (Figure 2I). Normalized and overlaid current traces under TTX and ProTx II conditions displayed highly similar activation and inactivation kinetics (Figure 2J). Kinetic analysis revealed no significant differences between TTX-S and ProTx II-sensitive currents in inactivation time constant (1.00 ± 0.05 ms vs. 1.06 ± 0.08 ms) or time to peak (1.44 ± 0.08 ms vs. 1.52 ± 0.05 ms) (Figure 2K). Together, these findings indicate that the TNF-α-induced sodium current in ATDC5 cells is primarily mediated by Nav1.7, supporting its potential role as a key contributor to OA progression.

Nav1.7 Deletion Alleviates Cartilage Degeneration and Pain-Related Behaviors in OA
To elucidate the role of Nav1.7 in chondrocytes during OA progression, cartilage-specific Nav1.7 knockout mice (Nav1.7chondrocyte) and control littermates (Nav1.7flox) were generated and subjected to DMM and MIA models to mimic traumatic and chemically induced OA, respectively (workflow shown in Supplementary Fig. 5A). H&E (P<0.001, 95.00% CI=-3.06 to -1.27) and Safranin O staining (P<0.001, 95.00% CI=-2.81 to -1.19) revealed that, in both DMM and MIA models, Nav1.7chondrocyte mice exhibited markedly attenuated cartilage degeneration, improved matrix staining, and significantly lower OARSI scores compared to Nav1.7flox controls (Supplementary Fig. 5B-D). Furthermore, in the DMM model, Nav1.7chondrocyte mice showed reduced osteophyte formation, decreased subchondral bone plate thickening, and significantly lower synovial hyperplasia scores (Supplementary Fig. 5E), indicating that Nav1.7 deletion effectively mitigates OA-associated histopathological changes.

To assess the impact of Nav1.7 deletion on OA-associated pain behaviors, open field tests and von Frey mechanical stimulation assays were conducted in both DMM and MIA models. In both models, Nav1.7chondrocyte mice displayed significantly increased locomotor activity and higher mechanical withdrawal thresholds compared with Nav1.7flox controls (Supplementary Fig. 5F-G). These findings indicate that the loss of Nav1.7 in chondrocytes exerts a protective effect by alleviating both structural pathology and pain perception in OA.

To further investigate the underlying mechanisms, the expression of key metabolic markers in cartilage tissue was evaluated in both models. Immunohistochemical staining revealed that, relative to Nav1.7flox mice, Nav1.7chondrocyte mice showed significantly increased expression of COL II and ACAN, indicating enhanced anabolic metabolism. Conversely, MMP13 and COMP fragment levels were markedly reduced, reflecting suppression of matrix degradation (Supplementary Fig. 5H-J). These findings indicate that chondrocyte-specific Nav1.7 deletion preserves cartilage homeostasis by promoting matrix synthesis and inhibiting catabolic processes.

Finally, we examined whether Nav1.7 deficiency affected BRD4 expression. The results showed that, compared with the Nav1.7flox group, there was no significant change in BRD4 expression levels in the articular cartilage of the Nav1.7chondrocyte group (Supplementary Fig. 5K-L). These findings indicate that the regulatory role of BRD4 in chondrocytes and OA is independent of Nav1.7.

BRD4 Promotes Nav1.7 Transcriptional Activation Through Histone Acetylation
Based on the preceding bioinformatics analysis, we investigated whether BRD4 regulates Scn9a transcription. ATDC5 cells were divided into four groups: Ctrl, TNF-α, TNF-α + sh-NC, and TNF-α + sh-BRD4 (workflow shown in Figure 3A). RT-qPCR analysis showed that Scn9a mRNA expression was significantly increased in the TNF-α group compared with the Ctrl group (P<0.001, 95.00% CI=-1.019 to -0.6073), while BRD4 knockdown significantly reduced Scn9a expression relative to the TNF-α + sh-NC group (P<0.001, 95.00% CI=0.8440 to 1.256) (Figure 3B). Consistently, WB analysis revealed that Nav1.7 protein levels were significantly elevated following TNF-α stimulation (P<0.001, 95.00% CI=-0.8013 to -0.5987) and were substantially suppressed by BRD4 silencing (P<0.001, 95.00% CI=1.339 to 1.541) (Figure 3C).

To further examine the functional association between BRD4 and Nav1.7, immunofluorescence co-staining was performed. Compared with the Ctrl group, the TNF-α group exhibited a significant increase in Nav1.7-positive cells (P<0.001, 95.00% CI=-1.863 to -1.250) as well as Nav1.7/BRD4 double-positive cells (P<0.001, 95.00% CI=-1.591 to -0.9419). In contrast, both populations were significantly reduced in the TNF-α + sh-BRD4 group relative to the TNF-α + sh-NC group (P<0.001, 95.00% CI=0.5371 to 1.363) (Figure 3D). These findings suggest that BRD4 knockdown not only inhibits Nav1.7 protein expression but also reduces its co-localization with BRD4, indicating a functional regulatory relationship.

Mechanistically, ChIP-qPCR analysis demonstrated that histone H3 acetylation (H3ac) at the Scn9a promoter was significantly increased in the TNF-α group relative to the Ctrl group, whereas BRD4 knockdown markedly reduced H3ac enrichment compared with the TNF-α + sh-NC group (Figure 3E). Consistent with these findings, dual-luciferase reporter assays showed that Scn9a promoter activity was significantly enhanced by TNF-α treatment (P<0.001, 95.00% CI=-0.533 to -0.234) and significantly suppressed following BRD4 silencing (P<0.001, 95.00% CI=0.197 to 0.496) (Figure 3F).

These results collectively indicate that BRD4 enhances Nav1.7 expression by promoting localized histone acetylation at the Scn9a promoter, thereby increasing its transcriptional activity.

BRD4-Mediated Activation of Nav1.7 Exacerbates Mitochondrial Dysfunction in TNF-α-Treated ATDC5 Cells
To clarify the interplay between BRD4 and Nav1.7 in oxidative stress regulation, TNF-α-treated ATDC5 cells were assigned to four groups: sh-NC, sh-BRD4, OD1 (Nav1.7 activator), and sh-BRD4 + OD1 (workflow shown in Supplementary Fig. 6A). Intracellular ROS levels were assessed using the DCFH-DA fluorescent probe. Compared with the sh-NC group, ROS levels were significantly reduced in the sh-BRD4 group (P<0.001, 95.00% CI=0.3529 to 0.8204), but markedly elevated in the OD1 group (P<0.001, 95.00% CI=-2.264 to -1.796). Notably, ROS levels were also significantly elevated in the sh-BRD4 + OD1 group compared with sh-BRD4 alone (P<0.001, 95.00% CI=-0.9137 to -0.4463) (Supplementary Fig. 6B-C). These findings suggest that activation of Nav1.7 can reverse the inhibitory effect of BRD4 knockdown on ROS production.

Subsequently, ΔΨm was evaluated using JC-1 staining. The JC-1 A/M ratio was significantly increased in the sh-BRD4 group relative to sh-NC (P=0.002, 95.00% CI=0.2543 to 0.8990). In contrast, the A/M ratio was significantly decreased in both the OD1 (P<0.001, 95.00% CI=-1.239 to -0.5943) and sh-BRD4 + OD1 groups (P=0.002, 95.00% CI=-0.8890 to -0.2443) relative to their respective controls (Supplementary Fig. 6D-E). TEM further supported these findings: mitochondria in the sh-BRD4 group displayed more intact cristae and reduced swelling, whereas OD1 treatment induced pronounced cristae disruption, which was further aggravated in the sh-BRD4 + OD1 group (Supplementary Fig. 6F).

To further assess the status of cellular energy metabolism, mitochondrial function-related proteins and intracellular ATP levels were measured. WB analysis revealed that TFAM (P<0.001, 95.00% CI=-0.8297 to -0.6436) and ATPB (P<0.001, 95.00% CI=-1.247 to -1.059) protein levels were significantly increased in the sh-BRD4 group compared with sh-NC. In contrast, both TFAM (P<0.001, 95.00% CI=0.4869 to 0.6731) and ATPB (P<0.001, 95.00% CI=0.1494 to 0.3373) expression were markedly reduced in the OD1 group relative to sh-NC, and in the sh-BRD4 + OD1 group compared to sh-BRD4 alone (TFAM: P<0.001, 95.00% CI=0.9703 to 1.156; ATPB: P<0.001, 95.00% CI=1.039 to 1.227) (Supplementary Fig. 6G-H). Consistently, ATP quantification revealed a significant increase in the sh-BRD4 group compared with sh-NC (P<0.001, 95.00% CI=-2.850 to -1.497), while ATP levels were significantly decreased in the OD1 (P=0.01, 95.00% CI=0.1869 to 1.540) and sh-BRD4 + OD1 groups (P=0.01, 95.00% CI=-1.573 to -0.2202) compared to their respective controls (Supplementary Fig. 6I). Taken together, these results indicate that BRD4 exacerbates TNF-α-induced ROS accumulation, mitochondrial dysfunction, and energy metabolism disturbances through Nav1.7 activation.

BRD4 Activation of Nav1.7 Suppresses Anabolic Metabolism and Promotes Catabolic Activity in TNF-α-Treated ATDC5 Cells
To delineate the roles of BRD4 and Nav1.7 in inflammatory and metabolic regulation, TNF-α-treated ATDC5 cells were assigned to four groups: sh-NC, sh-BRD4, OD1 (Nav1.7 activator), and sh-BRD4 + OD1 (workflow shown in Supplementary Fig. 7A). RT-qPCR analysis revealed that IL-6 (P<0.001, 95.00% CI=0.6360 to 1.257) and IL-1β (P<0.001, 95.00% CI=0.5842 to 1.449) mRNA levels were significantly reduced in the sh-BRD4 group compared to the sh-NC group. In contrast, both cytokines were significantly upregulated in the OD1 group relative to sh-NC (IL-6: P<0.001, 95.00% CI=-9.284 to -8.663; IL-1β: P<0.001, 95.00% CI=-8.072 to -7.208) and in the sh-BRD4 + OD1 group compared to sh-BRD4 alone (IL-6: P<0.001, 95.00% CI=1.257 to -0.6360; IL-1β: P<0.001, 95.00% CI=-1.389 to -0.5242) (Supplementary Fig. 7B). WB and immunofluorescence analyses corroborated these findings, indicating that Nav1.7 activation counteracts the anti-inflammatory effects of BRD4 knockdown (Supplementary Fig. 7C-D).

The impact on anabolic metabolism was next evaluated. RT-qPCR revealed that Sox9 and Col2a1 were significantly upregulated in the sh-BRD4 group compared with sh-NC (Sox9: P<0.001, 95.00% CI=-1.546 to -0.6272; Col2a1: P<0.001, 95.00% CI=-1.170 to -0.8100), but significantly downregulated in the OD1 group (Sox9: P<0.001, 95.00% CI=0.1005 to 1.020; Col2a1: P<0.001, 95.00% CI=0.3533 to 0.7134) and the sh-BRD4 + OD1 group (Sox9: P<0.001, 95.00% CI=0.6272 to 1.546; Col2a1: P<0.001, 95.00% CI=0.9133 to 1.273) (Supplementary Fig. 7E). WB analysis further confirmed these findings: SOX9 (P<0.001, 95.00% CI=-0.5841 to -0.3492) and COL II (P<0.001, 95.00% CI=0.7126 to 0.9474) protein levels were elevated in the sh-BRD4 group but markedly reduced in the OD1 (Sox9: P<0.001, 95.00% CI=0.7126 to 0.9474; Col2a1: P<0.001, 95.00% CI=0.6473 to 0.8527) and sh-BRD4 + OD1 groups (Sox9: P<0.001, 95.00% CI=0.7326 to 0.9674; Col2a1: P<0.001, 95.00% CI=0.9073 to 1.113) (Supplementary Fig. 7F). Immunofluorescence staining revealed enhanced fluorescence intensity of SOX9 and COL II in the sh-BRD4 group, which was markedly reduced following OD1 treatment (Supplementary Fig. 7G), indicating that BRD4 suppresses cartilage anabolic metabolism through Nav1.7 signaling.

To further define the cooperative role of BRD4 and Nav1.7 in catabolic regulation, the expression of matrix-degrading enzymes was examined. RT-qPCR analysis demonstrated that Mmp13, Adamts4, and Adamts5 were significantly downregulated in the sh-BRD4 group compared with the sh-NC group (Mmp13: P=0.007, 95.00% CI=0.2505 to 1.389; Adamts4: P=0.02, 95.00% CI=0.1315 to 1.495; Adamts5: P<0.001, 95.00% CI=0.5507 to 1.049). In contrast, their expression was significantly elevated in both the OD1 group (relative to the sh-NC group) (Mmp13: P<0.001, 95.00% CI=-9.519 to -8.381; Adamts4: P<0.001, 95.00% CI=-4.478 to -3.115; Adamts5: P<0.001, 95.00% CI=-1.026 to -0.5274) and the sh-BRD4 + OD1 group (relative to the sh-BRD4 group) (Mmp13: P<0.002, 95.00% CI=-1.599 to -0.4605; Adamts4: P=0.02, 95.00% CI=-1.528 to -0.1648; Adamts5: P<0.001, 95.00% CI=-1.093 to -0.5940) (Supplementary Fig. 7H). Consistently, WB analysis revealed that BRD4 silencing significantly suppressed MMP13 protein expression, whereas OD1-mediated Nav1.7 activation robustly restored MMP13 levels (all P<0.001) (Supplementary Fig. 7I). Immunofluorescence staining further confirmed that MMP13-positive signals were strongly enhanced following OD1 stimulation. Although BRD4 knockdown inhibited this upregulation, OD1 treatment partially reversed the effect (Supplementary Fig. 7J).

Collectively, these results indicate that Nav1.7 not only mediates BRD4-driven suppression of anabolic metabolism but also contributes to its promotion of cartilage matrix degradation.

The BRD4-Targeting PROTAC dBET1 Exhibits Low Cytotoxicity and Effectively Induces BRD4 Degradation
To assess the feasibility of pharmacologically targeting BRD4 in chondrocytes, the effects of the PROTAC dBET1 on BRD4 expression and cell viability were evaluated in ATDC5 cells. WB analysis showed that dBET1 markedly reduced BRD4 protein levels at concentrations ≥0.5 μM, with degradation reaching a plateau beyond 1 μM (Supplementary Fig. 8A and Supplementary Fig. 8C). Time-course experiments further demonstrated that 1 μM dBET1 induced a progressive decrease in BRD4 expression, achieving maximal degradation at 24 hours (Supplementary Fig. 8B and S8D). These findings indicate that dBET1 effectively promotes BRD4 degradation in ATDC5 cells in a dose- and time-dependent manner.

Cytotoxicity was then assessed using a CCK-8 assay under two conditions: direct exposure to dBET1 (0.1–10 μM) for 24 hours, and 2-hour dBET1 pretreatment followed by TNF-α (1 μg/mL) co-treatment for 18 hours. Cell viability was significantly reduced only at 10 μM, whereas concentrations between 0.1 and 5 μM showed no significant cytotoxicity (Supplementary Fig. 8E-F). Long-term toxicity assessment further revealed that treatment with 1 μM dBET1 did not induce cytotoxic effects in ATDC5 cells or MSCs over extended culture periods (Supplementary Fig. 8G–H). Collectively, these results demonstrate that dBET1 efficiently induces BRD4 degradation while maintaining a favorable cytotoxicity profile, supporting the selection of 1 μM as the optimal concentration for subsequent experiments.

Preparation and Characterization of CCM@PM
To develop a cartilage-targeted delivery platform for BRD4 PROTAC, plasma membranes were isolated from P1 primary chondrocytes and used to coat functionalized MSNs (workflow shown in Supplementary Fig. 10A). RT-qPCR analysis showed a progressive reduction in Col2a1 and Acan expression with increasing passage number, whereas Col1a expression remained relatively stable, indicating chondrocyte dedifferentiation and supporting the selection of P1 cells for membrane extraction (Supplementary Fig. 9A). Morphologically, P1 chondrocytes displayed a polygonal shape (Supplementary Fig. 9B). Alcian blue staining confirmed abundant glycosaminoglycan production, and COL II immunofluorescence further verified preservation of the chondrocyte phenotype (Supplementary Fig. 9C). Immunofluorescence analysis demonstrated that P1-derived membranes were enriched in adhesion molecules, including integrin β1, α3, αV, CD44, N-cadherin, and E-cadherin, supporting their suitability for targeted delivery (Supplementary Fig. 9D).

Successful conjugation of the cartilage-targeting peptide to mesoporous silica nanoparticles (PepM) was verified by FTIR analysis. The FTIR spectra of PepM exhibited characteristic absorption peaks corresponding to C=O, C–N, and C–H functional groups, indicating effective peptide modification (Supplementary Fig. 10B). To further evaluate whether PepM mesoporous silica nanoparticles possessed pore structure features suitable for dBET1 loading, nitrogen adsorption-desorption isotherms and pore size distribution analyses were performed. The nitrogen adsorption-desorption isotherms demonstrated that PepM exhibited favorable adsorption capacity across the entire relative pressure range, displaying a typical mesoporous material isotherm, from which the specific surface area parameters were obtained (Supplementary Fig. 11A). Pore size distribution analysis based on DFT revealed a narrowly distributed pore size range, with a dominant pore diameter in the nanometer scale (Supplementary Fig. 11B), which meets the requirements for dBET1 loading and diffusion. Elution experiments showed that both PM and CCM@PM exhibited slow and reversible release behavior without resistance to elution characteristic of covalent binding, indicating that dBET1 was encapsulated within the nanoparticles via non-covalent interactions (Supplementary Fig. 12). In addition, zeta potential measurements further confirmed alterations in the surface charge properties following modification (Supplementary Fig. 10C).

TEM combined with EDS analysis revealed that PME nanoparticles were spherical with an average diameter of approximately 194 nm, and exhibited nitrogen and sulfur signals, confirming successful organic functionalization (Supplementary Fig. 10D). Fluorescence spectroscopy of FITC-labeled PROTAC-loaded PM showed a characteristic absorption peak at 516 nm, indicating effective drug encapsulation (Supplementary Fig. 10E).

Following incubation of PMI with chondrocyte cell membranes, CCM@PM nanoparticles displayed a slightly increased diameter and a dense peripheral coating by TEM. EDS analysis detected uniform phosphorus signals in addition to nitrogen and sulfur, confirming retention of membrane phospholipid components (Supplementary Fig. 10F). Dynamic light scattering (DLS) measurements further confirmed the increase in particle size, supporting successful membrane encapsulation (Supplementary Fig. 10G). Confocal fluorescence imaging demonstrated strong colocalization between FITC-labeled PM (green) and DiI-labeled CCM (red), indicating efficient fusion of the chondrocyte membrane with the nanoparticle surface (Supplementary Fig. 10H). WB analysis confirmed that CCM@PM retained key chondrocyte membrane proteins, including CD44, integrin β1, and N-cadherin (Supplementary Fig. 10I), supporting preservation of biomimetic adhesion and recognition functions critical for targeted cartilage delivery.

CCM@PM Mediates Cartilage-Specific Cellular Uptake via Dual Clathrin-Dependent and Macropinocytic Pathways while Preserving PROTAC Activity
To characterize the cellular uptake of CCM@PM in chondrocytes, Cy5.5-labeled PM, RM@PM, and CCM@PM were incubated with chondrocytes for 2 hours. Confocal microscopy showed markedly stronger intracellular red fluorescence in the CCM@PM group than in the PM or RM@PM groups (Supplementary Fig. 13A). Flow cytometry confirmed a significantly higher mean fluorescence intensity for CCM@PM, indicating that chondrocyte membrane coating substantially enhanced uptake efficiency (Supplementary Fig. 13B-C). In contrast, uptake levels across BMSCs and RAW264.7 macrophages were comparable among all three formulations, suggesting that the CCM coating confers cartilage-specific targeting (Supplementary Fig. 13D).

Endocytic mechanisms underlying CCM@PM internalization were then examined. Cy5.5-labeled CCM@PM was incubated with chondrocytes, followed by addition of transferrin (clathrin-mediated), dextran (macropinocytosis), or cholera toxin (caveolae-mediated) during the final 30 minutes. Confocal imaging revealed strong colocalization of CCM@PM with transferrin and dextran, but minimal overlap with cholera toxin (Supplementary Fig. 13E). Pearson correlation analysis confirmed that CCM@PM was predominantly internalized via clathrin-mediated endocytosis and macropinocytosis (Supplementary Fig. 13F). Consistently, pretreatment with Dynasore (clathrin inhibitor) or Rottlerin (macropinocytosis inhibitor) significantly reduced CCM@PM uptake, whereas other inhibitors had limited effects (Supplementary Fig. 13G).

To determine the contribution of membrane proteins to targeted uptake, CCM@PM was subjected to functional blocking with antibodies against CD44, integrin β1, α1, α3, αV, E-cadherin, and N-cadherin, with trypsin digestion as a positive control. All blocking conditions significantly reduced CCM@PM internalization, with E-cadherin inhibition producing the most pronounced reduction, approaching the effect of trypsin treatment (Supplementary Fig. 13H).

To compare the extracellular release behavior of PROTACs delivered by different carrier systems, the in vitro release kinetics of dBET1 from PM and CCM@PM were first evaluated. The results showed that both formulations exhibited a time-dependent cumulative release profile. However, PM displayed a relatively faster release rate, whereas CCM@PM showed a lower release fraction within the first 12 h (Supplementary Fig. 14A), indicating that cell membrane coating can partially retard premature drug release. To investigate the intracellular trafficking and subcellular localization dynamics of BRD4-PROTAC following cellular internalization, FITC-labeled BRD4-PROTAC was subjected to confocal imaging in conjunction with Rab5 (an early endosome marker) or LAMP1 (a lysosomal marker) staining at different time points. The fluorescence signal of BRD4-PROTAC was predominantly distributed in a punctate pattern at the cell periphery and exhibited pronounced colocalization with Rab5-positive vesicles, suggesting that the PROTAC initially enters cells via classical endocytic pathways and localizes to early endosome-associated compartments (Supplementary Fig. 14B-C). With prolonged incubation, the BRD4-PROTAC fluorescence gradually transitioned from a highly punctate, vesicular distribution to a more diffuse cytoplasmic pattern, accompanied by a reduced degree of overlap with LAMP1-labeled lysosomes and the appearance of fluorescence signals dissociated from lysosomal compartments (Supplementary Fig. 14D-E). These time-resolved imaging results indicate that, following endosomal/lysosomal accumulation, BRD4-PROTAC can undergo partial lysosomal escape and progressively accumulate within the cytosolic space.

The intracellular functional activity of PROTACs following release from the nanoscale delivery systems was further evaluated. WB analysis revealed that treatment with free dBET1 led to a progressive reduction in BRD4 protein levels beginning at 6 h, with near-complete depletion observed at 24-48 h. In comparison, cells treated with PM or CCM@PM also exhibited time-dependent BRD4 downregulation; however, the degradation kinetics were relatively slower and more gradual (Supplementary Fig. 14F). Quantitative analysis further demonstrated that, at equivalent incubation times, free dBET1 induced the greatest extent of BRD4 degradation, followed by PM and then CCM@PM (Supplementary Fig. 14G). These results indicate that dBET1 retains its BRD4-targeting degradation activity after being released from the nanocarrier systems.

In summary, the cartilage cell membrane coating endows the nanodelivery system with specific cellular recognition and enhanced uptake efficiency, with internalization mediated by membrane protein–dependent clathrin-mediated endocytosis and macropinocytosis. Following cellular entry, the encapsulated PROTAC is capable of escaping lysosomal sequestration, being released into the cytosol, and maintaining its BRD4-degrading activity. Together, these findings demonstrate that CCM@PM offers a significant advantage in achieving precise cellular delivery while preserving the functional efficacy of PROTACs.

Preparation and Characterization of Rh-Gel@CCM@PM
To develop an injectable nanoparticle delivery system with favorable physicochemical properties, this study utilized the natural herbal molecule Rh to form a three-dimensional network hydrogel (Rh-Gel) via non-covalent self-assembly (Figure 4A). Rh was dissolved in sodium bicarbonate buffer at elevated temperature and allowed to cool, yielding a stable red gel. UV-visible spectroscopy showed characteristic absorption peaks at 260 nm and 435 nm, consistent with stable π-π stacking interactions (Figure 4B). Fluorescence analysis showed a prominent emission peak at 592 nm, suggesting densely packed fluorescent aggregates within the hydrogel (Figure 4C). Zeta potential analysis demonstrated that Rh-Gel possessed a moderate surface charge suitable for stable nanoparticle incorporation (Figure 4D). SEM imaging showed a porous three-dimensional network composed of interwoven nanofibers (Figure 4E). Furthermore, Rh-Gel exhibited temperature-dependent degradation behavior: it gradually degraded over three days at physiological temperature (37°C) but remained stable at 25°C, highlighting its suitability for in vivo applications (Figure 4F).

CCM@PM nanoparticles were encapsulated within Rh-Gel to fabricate the Rh-Gel@CCM@PM composite system. UV-visible absorption spectra revealed characteristic peaks corresponding to both Rh and FITC-labeled CCM@PM, indicating successful integration of the two components (Figure 4G). The Zeta potential of the composite shifted toward the characteristic value of CCM, further confirming the successful embedding of CCM@PM into the hydrogel matrix (Figure 4H). Confocal microscopy demonstrated a strong colocalization of the green FITC signal with the red fluorescence of the hydrogel, suggesting uniform distribution and efficient encapsulation of the nanoparticles within the gel network (Figure 4I).

The pH-responsive release behavior of Rh-Gel@CCM@PM was then evaluated. CCM@PM release was markedly accelerated at pH 5.5 compared with pH 7.4, while release under neutral conditions remained relatively slow (Figure 4J). TEM analysis showed that released CCM@PM maintained consistent particle size and structural integrity without aggregation throughout the release process (Figure 4K). WB analysis further confirmed that released nanoparticles retained high levels of key chondrocyte membrane proteins, including CD44, Integrin β1, and N-cadherin, demonstrating preservation of membrane protein structure and function following hydrogel encapsulation (Figure 4L).

To systematically evaluate the mechanical responsiveness and injectability of the Rh-Gel@CCM@PM hydrogel, rheological characterization was first performed. During amplitude sweep measurements, the hydrogel exhibited a typical viscoelastic response in the low-strain region; with increasing strain, the storage modulus gradually decreased and eventually reached a plateau, indicating structural yielding (Supplementary Fig. 15A). Cyclic shear tests demonstrated that under alternating low- and high-shear conditions, both the storage modulus and loss modulus displayed reproducible and periodic variations, reflecting the reversible mechanical behavior of the hydrogel (Supplementary Fig. 15B). Frequency sweep analysis showed that across the frequency range of 0.1-100 Hz, the storage modulus consistently exceeded the loss modulus, indicating stable frequency-dependent viscoelastic properties (Supplementary Fig. 15C). Time sweep measurements further revealed that under constant strain, both moduli remained relatively stable over time, suggesting good structural integrity (Supplementary Fig. 15D). In addition, viscosity-shear rate analysis indicated pronounced shear-thinning behavior, with viscosity continuously decreasing as the shear rate increased (Supplementary Fig. 15E), supporting the suitability of the hydrogel for injectable applications.

With respect to injectability, the hydrogel could be smoothly extruded through syringes equipped with needles of different gauges. Continuous filamentous or droplet-like gel structures were readily formed using 23G, 25G, and 27G needles (Supplementary Fig. 15F). Photographs of the injection process further illustrated the flow behavior and shape retention of the hydrogel under applied mechanical force (Supplementary Fig. 15G). To evaluate stability under simulated joint cavity conditions, the hydrogel was incubated in synovial fluid–mimicking solution and monitored over time. Macroscopic observation revealed progressive swelling and volumetric changes, accompanied by a gradual color transition of the surrounding medium from transparent to yellow (Supplementary Fig. 15H).

To assess long-term storage stability and suitability for repeated administration, samples stored under a nitrogen atmosphere at 4 °C for 0, 1, 2, 4, and 8 weeks were systematically evaluated for appearance, injectability, and rheological properties. Throughout the 0–8 week storage period, the hydrogel maintained a uniform brown appearance without visible macroscopic alterations (Supplementary Fig. 16A). Samples from all storage time points could be smoothly extruded through a 27G needle, exhibiting consistent filament formation and droplet behavior (Supplementary Fig. 16B). Shear rheological analysis further demonstrated that all samples retained stable shear-thinning characteristics, with viscosity decreasing as shear rate increased and no noticeable shifts in the viscosity profiles across different storage durations (Supplementary Fig. 16C–G).

In summary, Rh-Gel@CCM@PM exhibits robust structural integrity, acid-responsive behavior, sustained membrane protein retention, and reliable injectability, even after prolonged storage.

Adhesion, Penetration, and Retention Properties of Rh-Gel@CCM@PM in Cartilage Tissue
To assess the contribution of CCM coating to cartilage targeting, the adhesion and distribution of Cy5.5-labeled PM, RM@PM, and CCM@PM were compared in murine cartilage tissue. Fresh cartilage samples were incubated with each formulation for 12 hours, followed by PBS washing and SEM analysis. CCM@PM displayed markedly stronger adhesion to cartilage fibers than PM or RM@PM. Notably, trypsin pretreatment significantly reduced CCM@PM binding, indicating that membrane proteins mediate its high-affinity interaction with cartilage tissue (Supplementary Fig. 17A).

Three-dimensional penetration was further evaluated by confocal imaging of cartilage sections. Both CCM@PM and Rh-Gel@CCM@PM penetrated deeply into the cartilage matrix, whereas PM and RM@PM were largely restricted to superficial layers (Supplementary Fig. 17B). These findings suggest that the CCM structure enhances tissue penetration. For in vivo evaluation, a murine knee joint intra-articular injection model was established, and local fluorescence signals were monitored on days 1, 10, 28, and 35 post-injection. Rh-Gel@CCM@PM exhibited the strongest fluorescence intensity, slowest signal decay, and longest intra-articular retention among all groups. Kinetic analysis revealed a significantly prolonged half-life for Rh-Gel@CCM@PM (Supplementary Fig. 17C-D). Importantly, no detectable fluorescence was observed in major organs for any formulation on days 1 or 10, indicating favorable tissue specificity (Supplementary Fig. 17E).

In summary, Rh-Gel@CCM@PM exhibits good cartilage adhesion, enhanced tissue penetration, and prolonged in vivo retention within the joint cavity.

Rh-Gel@CCM@PM Exhibits Favorable Biocompatibility
To systematically evaluate the in vivo biocompatibility of Rh-Gel@CCM@PM, mice were subjected to intra-articular injections once weekly for eight consecutive weeks. Treatment groups included Rh-Gel@CCM@PM, Rh-Gel, Rh solution (Rh Sol), and PBS. At the end of the treatment period, blood samples were collected for hematological and serum biochemical analyses. All groups exhibited normal levels of white blood cells (WBC), red blood cells (RBC), and platelets (PLT), as well as liver and kidney function markers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE). No significant differences were detected between the Rh-Gel@CCM@PM group and the PBS control group (Supplementary Fig. 18A-B).

Histological evaluation of major organs was subsequently performed. Heart, liver, spleen, lungs, kidneys, and joint tissues were harvested, paraffin-embedded, and stained with H&E. No signs of inflammation, necrosis, or cellular degeneration were observed in any organs from the Rh-Gel@CCM@PM group. Moreover, no pathological differences were detected compared with the Rh-Gel, Rh Sol, or PBS groups (Supplementary Fig. 18C).

Rh-Gel@CCM@PM Hydrogel Attenuates Mitochondrial Dysfunction in TNF-α-Treated ATDC5 Cells
A TNF-α-induced ATDC5 cell model was established, and cells were assigned to four groups: TNF-α, Rh-Gel, CCM@PM, and Rh-Gel@CCM@PM (workflow shown in Figure 5A). WB analysis demonstrated that, compared with the TNF-α group, both CCM@PM and Rh-Gel@CCM@PM significantly reduced BRD4 protein levels (CCM@PM: P<0.001, 95.00% CI=0.5857 to 0.7877; Rh-Gel@CCM@PM: P<0.001, 95.00% CI=0.6323 to 0.8343) and Nav1.7 (CCM@PM: P<0.001, 95.00% CI=0.4406 to 0.6927; Rh-Gel@CCM@PM: P<0.001, 95.00% CI=0.4939 to 0.7461). RT-qPCR analysis showed that Nav1.7 mRNA expression mirrored its protein changes, whereas BRD4 mRNA levels remained unchanged among groups, indicating that BRD4 suppression primarily occurred through post-translational regulation (Figure 5B-D). These findings indicate that Rh-Gel@CCM@PM may exert mitochondrial protective effects by inhibiting the BRD4/Nav1.7 pathway.

To assess intracellular oxidative stress, DCFH-DA fluorescence probes were used to measure ROS accumulation. Compared with the TNF-α group, both the Rh-Gel (P<0.001, 95.00% CI=0.3484 to 0.8716) and CCM@PM (P<0.001, 95.00% CI=0.6251 to 1.148) groups showed significantly decreased ROS levels. Furthermore, ROS reduction was even more pronounced in the Rh-Gel@CCM@PM group relative to the individual treatment groups (P=0.04, 95.00% CI=0.01509 to 0.5382) (Figure 5E). ΔΨm was then evaluated using JC-1 staining. The results demonstrated that, compared with the TNF-α group, both the Rh-Gel (P=0.004, 95.00% CI=-0.9097 to -0.2103) and CCM@PM (P<0.001, 95.00% CI=-1.446 to -0.7470) groups exhibited significantly increased JC-1 A/M ratios. Importantly, the Rh-Gel@CCM@PM group showed a further significant elevation in A/M ratio compared to the Rh-Gel and CCM@PM groups (P=0.005, 95.00% CI=-0.8863 to -0.1870) (Figure 5F-G), suggesting superior preservation of mitochondrial integrity. TEM revealed that, compared with the TNF-α group, both the Rh-Gel and CCM@PM groups exhibited more intact mitochondrial cristae structures. Notably, cristae morphology was more clearly restored in the Rh-Gel@CCM@PM group, indicating superior mitochondrial preservation (Figure 5H).

To further investigate changes in cellular energy metabolism, we assessed the expression of mitochondrial function-related proteins TFAM and ATPB, along with intracellular ATP levels. WB analysis showed that, relative to the TNF-α group, both Rh-Gel and CCM@PM treatments significantly upregulated TFAM (Rh-Gel: P<0.001, 95.00% CI=-0.6200 to -0.4867; CCM@PM: P<0.001, 95.00% CI=-2.123 to -1.990) and ATPB (Rh-Gel: P<0.001, 95.00% CI=-3.863 to -3.657; CCM@PM: P<0.001, 95.00% CI=-13.40 to -13.19) expression. Notably, Rh-Gel@CCM@PM induced a further and more pronounced upregulation of both TFAM and ATPB compared with either treatment alone (Figure 5I). Consistently with these findings, ATP quantification revealed the highest ATP content in the Rh-Gel@CCM@PM group among all groups tested (Figure 5J).

In addition, we further validated the effects of Rh-Gel@CCM@PM hydrogel on mitochondrial function in TNF-treated HACs. The overall response patterns closely mirrored those observed in ATDC5 cells, confirming that Rh-Gel@CCM@PM effectively alleviates TNF-induced mitochondrial dysfunction in human primary chondrocytes (Supplementary Fig. 19).

Rh-Gel@CCM@PM Hydrogel promotes Anabolic Activity and suppresses Catabolic Processes in Chondrocytes
A TNF-α-stimulated ATDC5 cell model was established to assess anabolic and catabolic regulation under inflammatory conditions. Cells were divided into four groups: TNF-α, Rh-Gel, CCM@PM, and Rh-Gel@CCM@PM (workflow shown in Figure 6A). RT-qPCR analysis revealed that mRNA levels of inflammatory cytokines IL-6 (Rh-Gel: P<0.001, 95.00% CI=0.1245 to 0.3489; CCM@PM: P<0.001, 95.00% CI=0.1411 to 0.3655) and IL-1β (Rh-Gel: P<0.001, 95.00% CI=0.2884 to 0.7049; CCM@PM: P<0.001, 95.00% CI=0.5918 to 1.008) were significantly downregulated in both the Rh-Gel and CCM@PM groups compared with the TNF-α group. Notably, the Rh-Gel@CCM@PM group exhibited a further and more pronounced reduction in these cytokines (Figure 6B). WB analysis confirmed that IL-6 protein levels were lowest in the Rh-Gel@CCM@PM group (Figure 6C), which was further supported by immunofluorescence staining (Figure 6D). Although a quantitative release profile of FITC-dBET1 was not independently presented, the prolonged BRD4 degradation and Nav1.7 inhibition in the Rh-Gel@CCM@PM group compared with free dBET1 indicate effective intracellular release and sustained cytosolic bioavailability of the PROTAC.

Subsequently, key markers of anabolic metabolism were evaluated. RT-qPCR results demonstrated that, compared with the TNF-α group, both Rh-Gel and CCM@PM treatments significantly upregulated mRNA expression of Sox9 (Rh-Gel: P=0.004, 95.00% CI=-0.7089 to -0.1644; CCM@PM: P<0.001, 95.00% CI=-2.136 to -1.591) and Col2a1 (Rh-Gel: P=0.002, 95.00% CI=-0.4608 to -0.1325; CCM@PM: P<0.001, 95.00% CI=-1.454 to -1.126). Importantly, Rh-Gel@CCM@PM treatment led to an even greater increase in these transcripts (Figure 6E). WB analysis revealed corresponding increases in SOX9 and COL II protein expression, again most prominent in the Rh-Gel@CCM@PM group (Figure 6F). Immunofluorescence staining further demonstrated the strongest SOX9 and COL II signals following Rh-Gel@CCM@PM treatment (Figure 6G). These results collectively indicate that Rh-Gel@CCM@PM markedly enhances the anabolic capacity of ATDC5 cells under inflammatory stress.

Catabolic activity was subsequently assessed. RT-qPCR analysis showed that Mmp13, Adamts4, and Adamts5 mRNA levels were significantly reduced in both Rh-Gel and CCM@PM groups compared with TNF-α alone, with further suppression in the Rh-Gel@CCM@PM group (Figure 6H). Consistent with these findings, WB analysis revealed that MMP13 protein levels were lowest in the Rh-Gel@CCM@PM group (Figure 6I). Immunofluorescence staining further confirmed this trend, showing the weakest MMP13-positive signal in the Rh-Gel@CCM@PM group (Figure 6J-K). These results indicate that Rh-Gel@CCM@PM effectively suppresses the expression of cartilage matrix degradation-associated enzymes, thereby exerting a pronounced anti-catabolic effect. Compared with free dBET1, the Rh-Gel@CCM@PM group exhibited a more sustained therapeutic window for both BRD4 degradation and Nav1.7 inhibition, which was maintained throughout the ≤24 h observation period. This prolonged activity is consistent with the proposed triggered-release mechanism involving gradual release from the Rh-Gel matrix combined with endosomal acidification and intracellular reductive conditions-induced drug release from CCM@PM. These findings indicate that the PROTAC undergoes effective intracellular dissociation and achieves sustained cytosolic availability.

In parallel, we further examined the effects of Rh-Gel@CCM@PM hydrogel on cartilage anabolic and catabolic metabolism in TNF-treated HACs. The overall experimental trends were largely consistent with those observed in ATDC5 cells, demonstrating that Rh-Gel@CCM@PM hydrogel promotes anabolic activity while suppressing catabolic responses in TNF-stimulated human primary chondrocytes (Supplementary Fig. 20).

Rh-Gel@CCM@PM Hydrogel Alleviates Cartilage Damage and Pain-Related Behaviors in DMM-Induced OA Mice
A DMM-induced OA mouse model was established and mice were assigned to five groups: Sham, DMM, Rh-Gel, CCM@PM, and Rh-Gel@CCM@PM (schematic in Figure 7A). To verify the regulatory effect of Rh-Gel@CCM@PM on the BRD4/Nav1.7 axis in vivo, WB was performed to assess BRD4 and Nav1.7 expression in knee cartilage tissue. Compared with the Sham group, the DMM group showed significant upregulation of both BRD4 (P<0.001, 95.00% CI=-2.359 to -2.188) and Nav1.7 (P<0.001, 95.00% CI=-6.820 to -6.666); both were markedly reduced in the Rh-Gel and CCM@PM groups. Notably, the Rh-Gel@CCM@PM group exhibited the most pronounced suppression of BRD4 and Nav1.7 expression (Figure 7B).

Behavioral assessments demonstrated that DMM mice exhibited significantly reduced locomotor distance and decreased mechanical pain thresholds relative to Sham controls. Both parameters were partially improved by Rh-Gel and CCM@PM, whereas Rh-Gel@CCM@PM treatment produced the greatest recovery of locomotor activity and the most pronounced attenuation of pain sensitivity (Figure 7C-D).

Micro-CT analysis revealed severe subchondral bone remodeling in the DMM group, characterized by disorganized trabecular architecture and significantly increased BV/TV (P<0.001, 95.00% CI=-32.35 to -17.58), BMD (P<0.001, 95.00% CI=-25.78 to -14.13), and Tb.Th indices (P<0.001, 95.00% CI=-0.06715 to -0.03619). These abnormalities were partially alleviated in the Rh-Gel and CCM@PM group, while the Rh-Gel@CCM@PM group showed well-aligned trabecular bone with significantly reduced bone morphometric parameters (Figure 7E-F). H&E and SO/FG staining further demonstrated cartilage deterioration in the DMM group, including reduced cartilage thickness, surface irregularity, and diminished staining intensity. Both Rh-Gel and CCM@PM groups showed partial morphological restoration, whereas the Rh-Gel@CCM@PM group exhibited improved cartilage integrity, with a continuous surface and enhanced staining (Figure 7G). Consistent with these findings, the OARSI score was significantly reduced in the Rh-Gel@CCM@PM group (Figure 7H). Immunofluorescence staining showed that COL II and ACAN levels were increased in the Rh-Gel@CCM@PM group, while IL-6 expression was significantly suppressed, indicating enhanced matrix preservation and reduced inflammation (Figure 7I).

Rh-Gel@CCM@PM Hydrogel Alleviates Cartilage Damage and Pain-Related Behaviors in MIA-Induced OA Mice
A mouse model of OA was established using MIA, and animals were assigned to five groups: Sham, MIA, Rh-Gel, CCM@PM, and Rh-Gel@CCM@PM to evaluate the therapeutic effects of each intervention on OA progression (schematic in Figure 8A). Behavioral assessments showed that, compared with the Sham group, MIA mice exhibited markedly reduced locomotor activity and significantly lower mechanical pain thresholds. Both Rh-Gel and CCM@PM partially improved these deficits, whereas Rh-Gel@CCM@PM produced the most pronounced recovery, with behavioral outcomes approaching those of the Sham group (Figure 8B-C). These findings suggest that Rh-Gel@CCM@PM effectively mitigates MIA-induced behavioral abnormalities and chronic pain.

Micro-CT analysis revealed severe subchondral bone remodeling in the MIA group, characterized by disorganized trabeculae and significantly increased BV/TV (P<0.001, 95.00% CI=-31.56 to -14.40), BMD (P<0.001, 95.00% CI=-22.41 to -15.83), and Tb.Th indices (P<0.001, 95.00% CI=-0.07014 to -0.03320). These abnormalities were moderately alleviated by Rh-Gel or CCM@PM, while Rh-Gel@CCM@PM restored near-normal trabecular architecture with significant reductions in all bone morphometric parameters (Figure 8D-E). Histological staining further confirmed cartilage deterioration in the MIA group, characterized by reduced cartilage thickness, surface roughness, and diminished staining intensity. Rh-Gel and CCM@PM treatments attenuated cartilage damage to varying degrees. In contrast, the Rh-Gel@CCM@PM group exhibited markedly improved cartilage morphology, including a smoother surface, greater thickness, and uniform staining (Figure 8F). Quantitative analyses demonstrated that the Rh-Gel@CCM@PM group had the lowest OARSI score and the highest GAG content and cartilage thickness among all groups (Figure 8G-I).

To validate the joint-protective mechanism, the expression levels of BRD4 and Nav1.7 proteins in joint tissues were examined. WB analysis revealed that BRD4 (P<0.001, 95.00% CI=-2.807 to -2.693) and Nav1.7 (P<0.001, 95.00% CI=-6.803 to -6.674) expression were significantly elevated in the MIA group compared to the Sham group. Treatment with either Rh-Gel or CCM@PM significantly reduced the expression of both proteins, while Rh-Gel@CCM@PM led to an even more pronounced downregulation (Figure 8J). Immunofluorescence staining further demonstrated that Rh-Gel@CCM@PM treatment markedly enhanced the expression of COL II and ACAN while significantly suppressing IL-6 levels, indicating its superior regulatory effects on anabolic metabolism and inflammatory responses (Figure 8K). Collectively, these findings suggest that Rh-Gel@CCM@PM delays MIA-induced OA progression by suppressing the BRD4/Nav1.7 axis, preserving cartilage matrix homeostasis, and alleviating subchondral bone remodeling disorders.

Methods
Ethical Statement
All animal experiments were approved by the Animal Ethics Committee of The First Affiliated Hospital of Henan University (No. 2025-03-069). All procedures complied with the Guide for the Care and Use of Laboratory Animals.

Single-Cell RNA Sequencing (scRNA-seq)
Cells were isolated from the knee cartilage tissue of C57BL/6J mice in the sham-operated (control) group and the OA model group (eight weeks post-destabilization of the medial meniscus (DMM) surgery) (n=1 per group). Single-cell suspensions were prepared using a three-step digestion protocol with collagenase II (Sigma, CN6885) and collagenase D (Roche, 11088858001). Single-cell capture and cDNA library construction were performed on the 10x Genomics Chromium platform (Single Cell 3' Reagent Kits v3.1). Raw sequencing data were aligned to the reference genome (GRCm38 mm10) using Cell Ranger (v6.1.2). Stringent quality control was applied to retain cells with 200–4000 detected genes, while cells with more than 10% mitochondrial gene content were excluded.

Single-Cell Data Analysis
Data analysis was conducted using the Seurat package (v5.1.0) in R (v4.3.0). Quality control filters were set to retain cells with 200-6000 detected genes and mitochondrial gene content below 10%. Gene expression data were normalized using the LogNormalize method with a scaling factor of 10,000, and the top 2000 highly variable genes were identified using the vst method. Dimensionality reduction was performed by principal component analysis (PCA) based on 50 principal components (PCs), with the top 30 PCs selected via ElbowPlot. Batch effects associated with sample origin (orig.ident) were corrected using the Harmony algorithm. Clustering was conducted based on the top 30 Harmony-corrected PCs, and the optimal resolution parameter (0.4) was determined through multiresolution comparison using the clustree method. Marker gene identification and differential expression analysis were performed with thresholds set at min.pct = 0.25 and logfc.threshold = 0.25, retaining only significantly upregulated genes. All analyses were performed using R (v4.3.0). Because the sample size for each group was n = 1, single-cell sequencing was used solely for cell type identification and cell state characterization, rather than for cross-group statistical inference. The Harmony algorithm (v1.2.0) was applied solely to remove technical batch effects associated with sample origin and to enable joint visualization in a unified low-dimensional space. Key differential conclusions were derived from bulk RNA sequencing data with biological replicates and were further supported by subsequent functional validation experiments.

Transcriptome Sequencing and Analysis
Full-thickness cartilage was collected from the knee joints of mice in the sham group and the OA group (eight weeks post-DMM surgery) (n=6 per group). Total RNA was extracted using TRIzol reagent (Invitrogen), and RNA integrity was verified with an Agilent 2100 Bioanalyzer, ensuring a RIN > 8.0. RNA libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit and sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end reads, targeting a depth of 40 million reads per sample. Raw data were quality-checked using FastQC (v0.11.9), aligned to the mm10 reference genome with Hisat2 (v2.2.1), and quantified using featureCounts (v2.0.3). Differential expression analysis was performed with the limma package (v3.56.2) in R (v4.3.0), applying the voom method for normalization. Genes with |log fold change| > 0.5 and p < 0.05 were considered significantly differentially expressed.

Gene Ontology (GO) Enrichment Analysis
GO enrichment analysis was conducted on the differentially expressed genes (DEGs) identified from bulk RNA-seq using the clusterProfiler package (v4.6.2) in R (v4.3.0). Gene annotation was based on the org.Mm.eg.db database (v3.16.0). Statistical significance of enrichment was assessed using the hypergeometric test, with p-values adjusted by the Benjamini-Hochberg method (adjusted p < 0.05). Visualization was carried out using enrichplot (v1.18.4), with the top 20 most significantly enriched pathways displayed (showCategory = 20).

Visualization of Gene Overlap
To illustrate the overlap between genes specifically upregulated in degeneration-associated chondrocyte clusters identified by single-cell analysis (min.pct = 0.25, logfc.threshold = 0.25) and DEGs from bulk RNA-seq (|log FC| > 0.5, p-value < 0.05), a Venn diagram was generated using the Microbioinformatics online analysis platform (http://www.bioinformatics.com.cn). Two gene lists derived from the single-cell and transcriptome datasets were used as input, and the diagram was produced with default parameters.

Protein-Protein Interaction (PPI) Network Analysis
The PPI network of the intersecting genes was constructed using the STRING database (v12.0), with a confidence score threshold of 0.400. The resulting network file was imported into Cytoscape (v3.9.1) for topological analysis. Node degree and betweenness centrality were calculated using the CytoHubba plugin (v0.1). Core nodes were defined as those within the top 10% ranked by degree.

Weighted Gene Co-Expression Network Analysis (WGCNA)
RNA sequencing was performed on ATDC5 cells treated with sh-NC and sh-BRD4, with five biological replicates per group. A weighted gene co-expression network was constructed using the WGCNA package (v1.73) in R (v4.3.0). Low-expression genes were first filtered using a threshold of CPM > 1. The soft-thresholding power was set to 6 to satisfy the scale-free topology criterion (R2 > 0.85). Co-expression modules were identified using the dynamic tree cut algorithm with parameters deepSplit = 2 and minModuleSize = 30. Module eigengenes were then correlated with treatment groups. Candidate genes were selected based on module membership (MM > 0.8) and gene significance (GS > 0.6).

Least Absolute Shrinkage and Selection Operator (LASSO) Regression Analysis
Based on the gene expression matrix derived from WGCNA screening, LASSO regression was performed using the glmnet package (v4.1-6) in R (v4.3.0). L1 regularization was applied with the parameter α = 1, and the optimal λ value was determined by 10-fold cross-validation using the λ.1se criterion. Feature genes were defined as those with non-zero coefficients following cross-validation.

Gene set enrichment analysis (GSEA)
GSEA was performed using R (v4.2.1) with the clusterProfiler package (v4.4.4). Gene sets were obtained from the MSigDB Collections database (c2.cp.all.v2022.1.Hs.symbols.gmt), which contains 3,050 curated canonical pathway gene sets, and gene set information was retrieved using the msigdbr package (v7.5.1). Prior to analysis, genes identified from differential expression analysis were ranked, and gene identifiers were unified using the org.Mm.eg.db package. GSEA was then conducted with clusterProfiler to calculate the normalized enrichment score (NES) and multiple-testing–adjusted p values for each gene set. The analysis was performed for mouse (Mus musculus). Statistical significance was defined as a false discovery rate (FDR) q value < 0.25, with key results highlighted at q < 0.05.

dBET1 Treatment and BRD4 Degradation Assessment in ATDC5 Cells
To evaluate the effect of dBET1 on BRD4 protein degradation, ATDC5 cells were treated with a gradient of dBET1 concentrations (0.1, 0.5, 1, 5, and 10 μM) for 24 hours. Additionally, cells were treated with 1 μM dBET1 for varying durations (2, 6, 12, and 24 hours) to assess time-dependent effects. dBET1 was purchased from Selleck (Houston, TX, USA), dissolved in DMSO to prepare a stock solution, and stored at -20 °C. Working concentrations were freshly diluted before use. Following treatment, total protein was extracted using RIPA lysis buffer (Beyotime), quantified via the BCA method, and subjected to Western blot (WB) to determine BRD4 protein expression levels.

Cytotoxicity Evaluation of dBET1
The cytotoxicity of dBET1 in ATDC5 cells was assessed using a CCK-8 assay kit (Beyotime, Shanghai, China). Cells were seeded in 96-well plates at a density of 2 × 104 cells/mL and incubated overnight. Two experimental conditions were applied: (1) cells were directly treated with dBET1 at various concentrations (0.1, 0.5, 1, 5, and 10 μM) for 24 hours without inflammatory stimulation; (2) cells were pretreated with dBET1 at the same concentrations for 2 hours, followed by co-incubation with 1 μg/mL recombinant mouse TNF-α (Peprotech, USA) for an additional 18 hours. To evaluate long-term cytotoxicity, ATDC5 cells and mesenchymal stem cells (MSCs) were treated with 1 μM dBET1 for 1, 3, 5, and 7 days. Subsequently, 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 1 hour. Absorbance was recorded at 450 nm, and cell viability was calculated accordingly.

Chondrocyte Isolation and Culture
Articular cartilage was harvested from the tibial plateaus and femoral condyles of male mice aged 3-4 days. Under sterile conditions, the tissue was cut into small fragments (2-5 mm3) and incubated in DMEM containing 0.2% type II collagenase (17101-015, Gibco, USA) at 37 °C with shaking at 150 rpm for 5 hours. The digested suspension was filtered through a 70 μm cell strainer, centrifuged at 800 × g for 5 minutes, and washed three times with PBS. The resulting cell pellet was resuspended in DMEM supplemented with 10% fetal bovine serum (FBS; 10099-141, Gibco, USA) and penicillin-streptomycin (15140122, Gibco, USA; 10,000 U/mL). Cells were cultured in a 37 °C incubator with 5% CO2 and passaged upon reaching approximately 80% confluence. Passage 1 (P1) chondrocytes were used for subsequent plasma membrane extraction.

Isolation of Chondrocyte Plasma Membranes
Primary P1 mouse chondrocytes were used for plasma membrane extraction. After washing with PBS, the cells were gently scraped using a cell scraper (Corning, USA) and collected by centrifugation at 800 × g for 5 minutes at 25 °C. The cell pellet was resuspended in 3 mL of ice-cold plasma membrane isolation buffer containing 75 mM sucrose, 225 mM mannitol, 30 mM Tris-HCl (pH 7.4), 0.5% (w/v) bovine serum albumin (BSA; A7030, Sigma-Aldrich, USA), and 0.5 mM EGTA, supplemented with a phosphatase/protease inhibitor cocktail (P1265, Beyotime, China). To achieve complete cell disruption, the suspension was subjected to probe sonication on ice at 30 W (2 s on/3 s off) for 5 minutes. The lysate was centrifuged at 3000 × g for 5 minutes at 4 °C to remove nuclei and intact cells, and the resulting supernatant was collected. This supernatant was then centrifuged at 10,000 × g for 10 minutes at 4 °C to eliminate mitochondria; the pellet was discarded, and the supernatant retained. Finally, the supernatant was ultracentrifuged at 100,000 × g for 2 hours at 4 °C to isolate the plasma membrane fraction. The membrane pellet was resuspended in 0.2 mM EDTA solution, and the membrane protein concentration was quantified using a BCA protein assay kit (P0012, Beyotime, China). The isolated plasma membrane samples were aliquoted and stored at -80 °C to avoid repeated freeze-thaw cycles. Primary chondrocytes at passage 1 (P1) were selected as the membrane source primarily to ensure batch-to-batch consistency and membrane integrity, thereby preserving a stable adhesion and receptor phenotype and minimizing preparation-related variability. The resulting chondrocyte cell membranes (CCM) retained key membrane proteins and adhesion molecules after isolation, such as receptors, integrins, and glycosaminoglycan-binding sites, providing the structural and functional basis for constructing the subsequent membrane-camouflaged delivery system.

Isolation of Mouse Erythrocyte Plasma Membranes
Fresh whole blood was collected from adult male C57BL/6 mice via retro-orbital venous plexus puncture. The blood samples were centrifuged at 3000 × g for 5 minutes to remove serum and then washed twice with PBS buffer (pH 7.4). The washed erythrocytes were resuspended in hypotonic PBS (0.2× PBS: 20 mM NaCl, 4 mM Na2HPO4, 0.8 mM KH2PO4) under ice-bath conditions to induce membrane lysis. The lysate was centrifuged at 10,000 × g for 10 minutes at 4 °C to remove free hemoglobin (HGB), and the pellet was collected. The lysis and centrifugation steps were repeated until the supernatant became clear and colorless, indicating complete removal of HGB. The final erythrocyte plasma membrane fraction was resuspended in PBS and stored short-term at 4 °C or aliquoted and preserved long-term at -80 °C to avoid repeated freeze-thaw cycles.

Preparation of CCM-Coated Peptide-Modified Mesoporous Silica Nanoparticles (CCM@PM)
Deionized water (1000 mL) was adjusted to pH ≈ 11 using NH4OH solution (25-28 wt% NH3·H2O). Under vigorous stirring at 50 °C, CTAB (1.12 g) was dissolved, followed by slow dropwise addition of TEOS (52.8 mL). The reaction was stirred for 2 hours and allowed to stand overnight. The precipitate was collected by centrifugation at 5,400 × g for 10 minutes, washed alternately with deionized water and ethanol, and ultrasonically dispersed in ethanol for 30 minutes. The dispersion was mixed with a water:toluene (1:1, v/v) solution (20 mL) and transferred into a PTFE-lined autoclave for hydrothermal treatment at 140 °C for 4 days, yielding mesoporous silica nanoparticles (MSNs). To remove the CTAB template, the MSNs were refluxed in 9 mL HCl/400 mL methanol at 70 °C for 36 hours, washed to neutral pH, and vacuum-dried for 20 hours.

Subsequently, 1 g of MSNs was dispersed in 50 mL anhydrous toluene, and 1 mL 3-mercaptopropyltrimethoxysilane (MPTMS) was added under nitrogen protection. The mixture was stirred at 110 °C for 8 hours, centrifuged, washed with ethanol, and collected as thiol-functionalized MSNs (T-M). T-M (10 mg) was dissolved in 2 mL methanol and reacted with 5 mg 2,2'-dipyridyl disulfide at ambient temperature for 12 hours. After washing and drying, pyridine-functionalized MSNs (PyM) were obtained. PyM was then reacted with 2 mg cartilage-targeting peptide (DWRVIIPPRPSA, C-terminal cysteine-modified, synthesized by Chutai Bio) at ambient temperature for 12 hours. Following centrifugation (16,000 × g, 10 minutes) and methanol washing, peptide-modified MSNs (PM) were obtained.

For drug loading, PM (10 mg) was mixed with 2 mg FITC-labeled BRD4-PROTAC in anhydrous ethanol and processed using a negative-pressure drying and rehydration method. Drug encapsulation efficiency was determined using a UV-Vis spectrophotometer (UV-2600, Shimadzu, Japan) at 516 nm based on a standard calibration curve. Finally, mouse P1 CCM and PM nanoparticles were mixed at a mass ratio of 2:1 and subjected to ultrasonic water bath treatment (37 kHz) for 1 minute at ambient temperature. The mixture was then centrifuged at 16,000 × g for 15 minutes at 4 °C to remove unbound membrane fragments, yielding CCM@PM. PROTACs were loaded via hydrophobic interactions and pore adsorption, representing a physical encapsulation strategy that does not involve covalent bond formation.

Nitrogen Adsorption-Desorption Measurement and Pore Structure Analysis
PepM powder was vacuum-dried at 80 °C for 12 hours and analyzed using a specific surface area analyzer (ASAP 2020, Micromeritics, USA). Nitrogen adsorption–desorption measurements were conducted over the full relative pressure range at 77 K. The specific surface area was calculated using the Brunauer–Emmett–Teller (BET) model within a relative pressure (P/P₀) range of 0.05–0.30. Pore size distribution was determined by fitting the adsorption data using the density functional theory (DFT) method, and pore structure curves were generated accordingly.

dBET1 Elution Assay
To evaluate dBET1 elutability, suspensions of dBET1-loaded PM or CCM@PM were placed in glass beakers containing prewarmed PBS buffer (pH 7.4) at a suspension-to-medium volume ratio of 1:20. Samples were maintained at 37 °C with constant mixing at 150 rpm to ensure uniform dispersion. At predetermined time points, aliquots were collected and centrifuged at 10,000 × g for 10 minutes to separate nanoparticles. The supernatant was collected for drug quantification, and an equal volume of PBS was replenished to maintain a constant total volume. dBET1 concentration was measured using a UV-visible spectrophotometer, and cumulative elution profiles were recorded over time. Low-adsorption centrifuge tubes were used throughout, and all procedures were conducted under constant temperature conditions to minimize external variability.

Characterization of PM and CCM@PM Nanoparticles
The incorporation of functional groups on the nanoparticles was assessed using Fourier-transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Fisher Scientific, USA) within the 4000-500 cm⁻¹ range. Characteristic peaks corresponding to thiol, pyridine groups, cell membrane proteins (e.g., C=O, C-N, S-H), and drug-loading signatures were identified. The particle morphology was examined by transmission electron microscopy (TEM, JEM-2100, JEOL, Japan). For TEM analysis, 10 μL of aqueous dispersion was deposited onto carbon-coated copper grids, air-dried, and imaged without staining. Elemental mapping of N, S, and P was performed using energy-dispersive spectroscopy (EDS) coupled with TEM to evaluate the homogeneity of cell membrane components and organic ligands. Hydrodynamic diameter and zeta potential were measured using a Delsa Nano C particle analyzer (Beckman Coulter, USA) equipped with a 633 nm He-Ne laser (4 mW) at 25 °C, and Delsa Nano software was used to assess size and surface charge variations associated with membrane coating. Ultraviolet-visible (UV-Vis) absorption spectra were acquired using a spectrophotometer (U-2910, Hitachi, Japan) over the 200–800 nm range in a 2 mm quartz cuvette, with the characteristic absorption peak of FITC-labeled BRD4-PROTAC detected at 516 nm. Elemental composition was further analyzed by field-emission scanning electron microscopy (FE-SEM, S-4800, Hitachi, Japan) combined with EDS to confirm membrane-coating element distribution. The encapsulation efficiency of CCM@PM, calculated using a standard calibration curve, remained stable at 82.43 ± 2.16% across three independent experiments, with a drug loading capacity of 7.52%.

In Vitro Release Kinetics Assay
dBET1-loaded PM or CCM@PM nanoparticles were loaded into dialysis bags (molecular weight cutoff, 3.5 kDa) and immersed in PBS (pH 7.4). Samples were incubated at 37 °C on a horizontal shaker at 100 rpm. At predetermined time points, aliquots of the external medium were collected and replaced with equal volumes of fresh PBS. dBET1 concentration was quantified using a UV-visible spectrophotometer, and cumulative release profiles were recorded over time.

Cellular Targeting and Uptake Analysis
To evaluate the targeting ability and endocytosis efficiency of different nanoparticle platforms in chondrocytes, Cy5.5-labeled unmodified mesoporous silica nanoparticles, red blood cell membrane-coated nanoparticles (RM@PM), and CCM@PM were incubated with primary mouse chondrocytes for 2 hours. For confocal imaging, cells were washed three times with PBS, stained with DAPI (1 μg/mL, D9542, Sigma-Aldrich, USA) for 5 minutes, and imaged using a laser scanning confocal microscope (LSM 710, Zeiss Microsystems, Germany). For flow cytometry analysis, cells were washed with PBS, centrifuged at 400 × g for 3 minutes, and resuspended in FACS buffer (PBS containing 2% FBS and 1 mM EDTA). Data acquisition was performed on a BD Accuri C6 flow cytometer (BD Biosciences, USA) using BD Accuri C6 software (v.1.0.264.21). A minimum of 10,000 events were recorded per sample. Debris and doublets were excluded based on forward scatter (FSC) and side scatter (SSC) properties. The gating strategy used to identify the main cell population and determine Cy5.5 fluorescence intensity is outlined in Supplementary Figure 21. To further assess the selective uptake capacity of CCM@PM in heterologous cell types, the three types of nanoparticles were separately incubated with bone marrow mesenchymal stem cells (BMSCs, CP-M131, Procell, China) and macrophages (RAW264.7, CL-0190, Procell, China). Uptake efficiency in each cell type was evaluated using the same flow cytometry procedure and gating parameters. Final data analysis was conducted using FlowJo software (v10.8, Tree Star, USA).

Endocytic Mechanism of Chondrocyte Uptake for Membrane-Coated Nanoparticles
To investigate endocytic pathways, Cy5.5-labeled PM, RM@PM, and CCM@PM were incubated with primary mouse chondrocytes. Cells were seeded in 20 mm glass-bottom confocal dishes and cultured overnight to approximately 50% confluence, then incubated in serum-free DMEM containing nanoparticles (200 μg/mL) for 4 hours. During the final 30 minutes, Alexa Fluor 488-labeled pathway markers were added: cholera toxin (5 μg/mL, caveolae-mediated, C34775, Invitrogen, USA), transferrin (50 μg/mL, clathrin-mediated, T13342, Invitrogen, USA), and dextran (500 μg/mL, macropinocytosis, D22910, Invitrogen, USA). Cells were fixed with 4% paraformaldehyde (PFA; P0099, Beyotime, China) for 15 minutes, washed with PBS, counterstained with DAPI (1 μg/mL) for 5 minutes, and imaged by confocal microscopy to assess fluorescence colocalization.

For inhibitor-based validation, chondrocytes were seeded in six-well plates at 2 × 105 cells/well and cultured overnight, then pretreated at 37 °C for 30 minutes with Dynasore (40 μM, clathrin-mediated inhibition), Rottlerin (2 μM, macropinocytosis inhibition), Nystatin (54 μM, caveolae-mediated inhibition), or Cytochalasin D (10 μM, phagocytosis inhibition) (all from Sigma-Aldrich, USA). Cells were subsequently incubated with Cy5.5-labeled PM, RM@PM, or CCM@PM (200 μg/mL) for 2 hours. Parallel incubation at 4 °C was performed to assess energy dependence, and cells incubated at 37 °C without inhibitors served as positive controls. After washing with PBS, 1 × 104 cells were collected and analyzed using the BD Accuri C6 (BD Biosciences, USA), with data processed using FlowJo (v10.5, BD Biosciences, USA).

Confocal Imaging of Subcellular Localization of BRD4-PROTAC
Chondrocytes were seeded on glass coverslips or confocal-compatible culture dishes and incubated with FITC-labeled BRD4-PROTAC after attachment. At designated time points, culture medium was replaced with medium containing antibodies against Rab5 or LAMP1 for immunofluorescence labeling. Cells were washed with PBS, fixed with 4% PFA for 10 minutes, permeabilized with 0.1% Triton X-100, and incubated with appropriate primary and fluorescent secondary antibodies. Nuclei were counterstained with DAPI. Multichannel fluorescence images were acquired using a laser scanning confocal microscope (Leica or Zeiss) and saved for subsequent image analysis.

Scanning Electron Microscopy (SEM) Observation
To examine the adsorption capacity of surface-modified nanoparticles on mouse cartilage tissue, fresh femoral head cartilage was harvested from 8-week-old male C57BL/6J mice and pre-equilibrated for 2 hours in phenol red-free DMEM supplemented with 10% FBS (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) at 37 °C in a 5% CO2 incubator. Cy5.5-labeled PM, RM@PM, and CCM@PM were then added at a final concentration of 200 μg/mL and incubated for an additional 12 hours. After incubation, the cartilage tissues were thoroughly rinsed with PBS and immediately fixed in 2.5% glutaraldehyde for 12 hours. The fixed tissues were cryoprotected with 15% sucrose, followed by graded ethanol dehydration (30%, 50%, 70%, 90%, and 100%), freeze-dried, and sputter-coated with gold. The distribution and surface adsorption of nanoparticles on the cartilage were subsequently observed using an SEM (S-4800, Hitachi, Japan).

Cartilage Tissue Penetration Assay
Fresh femoral head cartilage from 8-week-old male C57BL/6J mice was treated under conditions identical to the SEM experiment. Cartilage samples were incubated with Cy5.5-labeled PM, RM@PM, and CCM@PM at 200 μg/mL for 3 days, washed with PBS, and embedded in Tissue-Tek O.C.T. compound. Frozen sections (10 μm) were prepared using a cryostat (Leica Biosystems, Germany), stained with DAPI (1 μg/mL) for 5 minutes, washed, and mounted with Fluoromount-G (SouthernBiotech, USA). Penetration depth and fluorescence distribution were examined using a confocal laser scanning microscope.

Intra-articular Retention and In Vivo Distribution
An intra-articular injection model was established to evaluate joint retention of nanoparticles. Cy5.5-labeled PM, RM@PM, and CCM@PM were prepared, along with a free Cy5.5 control. Each formulation was prepared at 5 mg/mL (based on total nanoparticle mass), and 50 μL diluted in sterile 0.9% NaCl was injected into the right knee joint cavity. All formulations were injectable in situ gelling systems. Specifically, Rh-Gel@CCM@PM remained in a flowable sol state at ambient temperature and was injected through a 27 G needle, rapidly forming a gel in response to the local microenvironment without implantation of a preformed hydrogel. On days 1, 10, 28, and 35 post-injection, fluorescence signals from the joint region were captured using an in vivo imaging system (IVIS Spectrum, PerkinElmer, USA). The region of interest (ROI) was defined as the knee joint area. Image analysis was conducted using Living Image software (v4.5, PerkinElmer, USA), with excitation/emission wavelengths set to 675/720 nm for Cy5.5. In addition, at days 1 and 10 post-injection, major organs, including the heart, spleen, liver, lungs, and kidneys, were harvested to assess the biodistribution and tissue accumulation of fluorescence signals.

Preparation and Characterization of Hydrogel
The rhein-based hydrogel (Rh-Gel) was formed through the self-assembly of the herbal compound rhein (Rh, R726100, Aladdin, China). Rh was dissolved in 0.1 M NaHCO3 buffer (pH 8.3) at 4 mg/mL, heated to 60 °C until fully dissolved, and slowly cooled to ambient temperature to form a uniform red, transparent gel. Rh-Gel was stored at 4 °C in the dark until use. UV-Vis absorption spectra were recorded from 200–800 nm using a spectrophotometer (UV-2600, Shimadzu, Japan), with characteristic peaks at 260 nm and 435 nm. Fluorescence spectra were acquired using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) at an excitation wavelength of 435 nm and an emission range of 500–700 nm, with an emission peak at 592 nm. Zeta potential was measured at 25 °C using a Zetasizer Nano ZS90 (Malvern Instruments, UK), with triplicate measurements averaged. Lyophilized Rh-Gel samples were sputter-coated with gold and examined by FE-SEM (SU8020, Hitachi, Japan) at 5 kV to visualize the three-dimensional nanofiber microstructure.

Construction and Characterization of Rhein-Based Hydrogel Loaded with Chondrocyte Cell Membrane-Coated Peptide-Modified Mesoporous Silica Nanoparticles (Rh-Gel@CCM@PM)
Rh-Gel precursor sol was prepared as above and maintained in a flowable state. CCM@PM dispersion was added to achieve a final nanoparticle concentration of 20 mg/mL, yielding an injectable Rh-Gel@CCM@PM composite sol with an embedded CCM@PM content of 2 mg/mL gel. For in vitro characterization, the sol was injected into molds (~5 mm diameter) and allowed to gel for 30–60 minutes. PBS (pH 7.4) was then added, and samples were equilibrated for 1 h to obtain composite hydrogels.

UV-Vis spectra were collected over 300–700 nm (1 nm step size) in a 1 cm quartz cuvette using the UV-2600, with absorption peaks at 554 nm for Rh and 495 nm for FITC-labeled CCM@PM. Zeta potential was measured at 25 °C using the Zetasizer Nano ZS90 (Malvern Instruments, UK), with results expressed as mean ± SD. Confocal laser scanning microscopy was used to visualize nanoparticle distribution within the hydrogel using a 40× water-immersion objective, with excitation at 488 nm for FITC and 561 nm for Rh. Z-stack imaging was performed at 1 μm step size to assess spatial localization within hydrogel pores.

Rheological Characterization
Rheological measurements of Rh-Gel@CCM@PM hydrogels were performed using a parallel-plate rheometer (MCR 302, Anton Paar, Austria). Freshly prepared hydrogels were equilibrated at ambient temperature for 30 min and loaded onto a 25 mm parallel-plate geometry with a 1.0 mm gap. Excess material was trimmed to ensure uniform stress distribution. All measurements were conducted at 25°C under solvent trap protection to prevent dehydration. Amplitude sweep tests were performed in oscillatory mode over a strain range of 0.01%-500% at a fixed frequency of 1 Hz to obtain strain-dependent storage modulus (G′) and loss modulus (G″). Cyclic shear tests were conducted by alternately applying 1% and 300% strain with a cycle duration of 30 seconds. Frequency sweep measurements were carried out within the linear viscoelastic region at 1% strain over a frequency range of 0.1-100 Hz. Time sweep tests were performed at a constant strain of 1% and frequency of 1 Hz for 10-30 minutes. Viscosity-shear rate profiles were obtained under steady-state flow by increasing the shear rate from 0.1 to 1000 s-1. All data were collected and exported using RheoCompass for subsequent analysis.

Hydrogel Storage Stability and Injectability Assessment
Freshly prepared brown hydrogels were sealed in nitrogen-filled centrifuge tubes and stored at 4°C for 0, 1, 2, 4, and 8 weeks. At each time point, samples were photographed to document macroscopic appearance under identical conditions. Hydrogels were then loaded into 1 mL syringes fitted with 27 G needles for injectability testing. Injection was performed under consistent applied pressure and experimental conditions, and extrusion behavior, including flow continuity and shape retention, was recorded in real time. Hydrogels stored for 0, 1, 2, 4, and 8 weeks were further subjected to shear rheological testing to evaluate shear-thinning behavior. Viscosity-shear rate curves were generated, with each storage time point measured in triplicate to ensure reproducibility.

pH-Responsive Drug Release Assay
To evaluate the pH-responsive release behavior of Rh-Gel@CCM@PM, hydrogel samples (4 mg/mL) were sealed in dialysis bags (molecular weight cut-off, 3.5 kDa; Solarbio, China) and immersed in 100 mL PBS buffer at pH 7.4 or pH 5.5, simulating the physiological joint environment and the mildly acidic inflammatory microenvironment, respectively. The dialysis systems were incubated at 37 °C on a shaker at 60 rpm. At predetermined time points (0.5, 1, 2, 3, 4, 5, 6, and 7 days), 10 mL of release medium was collected and replaced with an equal volume of fresh PBS to maintain a constant system volume. The absorbance of the collected samples was measured at 452 nm using a UV spectrophotometer (UV-2600, Shimadzu, Japan), and the cumulative release percentage was calculated to generate the drug release profile. To monitor the release of FITC-labeled CCM@PM nanoparticles, fluorescence intensity of the collected medium was measured using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) with excitation at 488 nm and emission at 516 nm. Samples collected at each time point were examined by TEM (JEM-2100, JEOL, Japan) to evaluate particle size and structural integrity of CCM@PM nanoparticles.

ATDC5 Cell Culture and Experimental Grouping
ATDC5 mouse chondrogenic progenitor cells were obtained from Wuhan Procell Life Science & Technology Co., Ltd. (CL-0856, Procell, China). Human articular chondrocytes (HAC) and mouse synovial cells (MSC) were purchased from the same supplier (CP-H096 and CP-M083, respectively). ATDC5 cells were cultured as adherent monolayers in DMEM/F12 medium (11320-033, Gibco, USA) supplemented with 10% FBS (10099-141, Gibco, USA) and 1% penicillin-streptomycin (15140122, Gibco, USA), and maintained in a humidified incubator at 37 °C with 5% CO2. The culture medium was refreshed every 2-3 days. HACs were cultured in human articular chondrocyte complete medium (CM-H096, Procell, China) with medium changes every 3-4 days, while MSCs were maintained in mouse synovial cell complete medium (CM-M083, Procell, China) with medium replaced every 2-3 days. Cells within two passages were used for all experiments 40.

When ATDC5 cells reached approximately 70% confluence, they were randomly assigned to the following groups: Ctrl group (untreated control); TNF-α group (10 ng/mL recombinant mouse TNF-α, 315-01A, PeproTech, USA, 24 hours); TNF-α + sh-NC group (sh-NC lentiviral transduction followed by TNF-α treatment); TNF-α + sh-BRD4 group (sh-BRD4 lentiviral transduction followed by TNF-α treatment); sh-NC group; sh-BRD4 group; OD1 group (Nav1.7 agonist OD1, 100 nM; 5941, Tocris Bioscience, USA, 4 hours); and sh-BRD4 + OD1 group (sh-BRD4 transduction followed by OD1 treatment). For patch-clamp electrophysiology, two additional groups were included: TTX group (TTX, 1 μM, Tocris Bioscience, UK, 48 hours following TNF-α priming at 10 ng/mL for 24 hours) and ProTx II group (Nav1.7-specific blocker ProTx II, 25 nM; HY-P1221, MedChemExpress, China, 48 hours following TNF-α priming) 41, 42. When HACs reached approximately 70% confluence, they were randomly assigned to a TNF-α group (10 ng/mL recombinant human TNF-α, SRP3177, Sigma-Aldrich, USA, 24 hours), an Rh-Gel group, a CCM@PM group, and an Rh-Gel@CCM@PM group.

shRNA Transfection Assay
To achieve stable BRD4 knockdown in ATDC5 cells, two BRD4-specific short hairpin RNA sequences (sh-BRD4) as well as a non-targeting sequence (a negative control [sh-NC]) were designed and synthesized (Supplementary Table 2).

All sequences were subcloned into the lentiviral vector GV493 (U6-MCS-Ubiquitin-EGFP-IRES-puromycin, GeneChem). Lentiviruses were produced in HEK293T cells (CL-0005, Procell, China), yielding a viral titer of 2 × 108 TU/mL. The viral supernatant was purified by ultrafiltration for subsequent infection. ATDC5 cells were seeded in six-well plates at 2 × 10⁵ cells/well and cultured to 40-50% confluence. For transduction, 500 μL of transfection mixture containing lentivirus (MOI = 50), 5 μg/mL PolyJet (SL100688, SignaGen, USA), and 1 μg/mL Polybrene (TR-1003, Sigma-Aldrich, USA) was added to each well and incubated for 6 hours. Subsequently, 1 mL of complete medium was added. At 48 hours post-infection, cells were selected with 2 μg/mL puromycin (A1113803, Gibco, USA) for 72 hours to establish stable cell lines. Infection efficiency was evaluated by GFP fluorescence using a fluorescence microscope (Leica DMi8, Leica Microsystems, Germany), and only cultures with ≥80% GFP positivity were used for subsequent experiments. On day 5 post-infection, cells were harvested for total RNA and protein extraction. BRD4 knockdown efficiency was evaluated by RT-qPCR and WB analysis.

Membrane and Cytoplasmic Protein Isolation
Membrane and cytoplasmic proteins were isolated using a membrane protein extraction kit (P0033, Beyotime, China). The separated fractions were subjected to WB analysis to assess Nav1.7 expression in the membrane compartment.

Whole-Cell Patch-Clamp Recording of Sodium Currents
Whole-cell patch-clamp recordings were performed at ambient temperature using the Axon Patch-Clamp 700B system (Molecular Devices, USA). The extracellular solution contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 5 mM CsCl, and 20 mM tetraethylammonium chloride (TEA-Cl), adjusted to pH 7.3 with CsOH. The pipette (intracellular) solution consisted of 110 mM CsF, 25 mM NaCl, 1 mM CaCl2, 10 mM EGTA, 20 mM TEA, 10 mM glucose, and 10 mM HEPES, also adjusted to pH 7.3 with CsOH. Patch pipettes had a resistance of 3-5 MΩ. Recordings were conducted with a holding potential of -60 mV. A series of depolarizing voltage steps from -60 mV to +50 mV, in 10 mV increments and each lasting 50 ms, was applied to evoke inward sodium currents. Electrical signals were amplified using an Axopatch 200B amplifier and digitized via a Digidata 1322A interface at a sampling rate of 5-10 kHz. Data were acquired using pCLAMP 9.2 software and analyzed with Clampfit 9.2 (Molecular Devices, USA). Nav1.7 channel functionality was verified by repeating the recording protocol following treatment with either 1 μM TTX (Tocris Bioscience, UK) or 25 nM ProTx II (HY-P1221, MCE, China) 41, 42.

Detection of Intracellular ROS Levels Using DCFH-DA Probe
ATDC5 cells were seeded in six-well plates and, after designated treatments, washed twice with PBS. Cells were incubated with 10 μM DCFH-DA probe (50101ES01, YEASEN, China) for 30 min at 37 °C in the dark, washed three times with PBS, and counterstained with DAPI (1 μg/mL, D9542, Sigma-Aldrich, USA) for 5 minutes. Coverslips were mounted after a final PBS rinse. Green fluorescence was imaged using a laser scanning confocal microscope (TCS SP8, Leica Microsystems, Germany) with excitation/emission wavelengths of 488 nm/500-530 nm for DCFH-DA and 405 nm/420-480 nm for DAPI. Intracellular ROS levels were quantified using ImageJ (v1.53c, NIH, USA) based on fluorescence intensity.

TEM for Mitochondrial Morphology
Following treatment, cells were collected and immediately fixed in 2.5% glutaraldehyde solution (P1126, Solarbio, China) at 4 °C for 12 h. Post-fixation was carried out using 1% osmium tetroxide (P1125, Solarbio, China) for 2 h, followed by PBS washing. Samples were dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%) and embedded in epoxy resin. Ultrathin sections (~70 nm) were obtained using an ultramicrotome and stained with uranyl acetate and lead citrate. Mitochondrial morphology and cristae structure were examined using a TEM at an accelerating voltage of 80 kV, and representative images were captured for further analysis.

JC-1 Dye Detection of Mitochondrial Membrane Potential (ΔΨm)
Following treatment, cells were incubated with JC-1 working solution (C2006, Beyotime, China) for 20 minutes at 37 °C in the dark, then washed three times with PBS. Red fluorescence (JC-1 aggregates, high membrane potential) and green fluorescence (JC-1 monomers, low membrane potential) were imaged using an inverted fluorescence microscope (DMi8, Leica Microsystems, Germany). The red-to-green fluorescence intensity ratio (Aggregate/Monomer, A/M ratio) was calculated using ImageJ to evaluate changes in ΔΨm.

ATP Content Measurement
Following treatment, ATP levels were quantified using an ATP assay kit (S0026, Beyotime, China). Cell lysates were mixed with the ATP detection reagent, and luminescence intensity (RLU) was measured using a Luminoskan Ascent microplate luminometer (Thermo Fisher Scientific, USA). ATP standard solutions (1-10 μmol/L) were used to generate a standard curve, and ATP concentrations were calculated accordingly. Results were expressed as ATP content (μmol/L) to evaluate cellular energy metabolism.

ChIP-qPCR Detection of Histone H3 Acetylation (H3ac) Modification at the Scn9a Promoter
Chromatin immunoprecipitation was performed using the ChIP Assay Kit (17-295, Millipore, USA). ATDC5 cells were crosslinked with 1% formaldehyde for 10 minutes, quenched with 125 mM glycine, lysed, and sonicated to 200-600 bp fragments using a Bioruptor Plus (Diagenode, Belgium). Equal amounts of chromatin were incubated overnight at 4 °C with magnetic Protein A/G beads pre-coated with either an anti-H3ac antibody (06-599, Millipore, USA, 1:100) or rabbit IgG isotype control (ab172730, Abcam, UK, 1:100). Following elution and proteinase K digestion, DNA was purified and subjected to qPCR targeting the promoter region of Scn9a (Nav1.7). Relative enrichment was quantified using the 2-ΔCt method, with the IgG group serving as the negative control.

Dual-Luciferase Reporter Assay for Scn9a Promoter Activity
The mouse Scn9a promoter was cloned into the pGL3-Basic vector (E1751, Promega, USA). ATDC5 cells were co-transfected with pGL3-Scn9a (500 ng/well) and Renilla luciferase control pGM-T (50 ng/well, Invitrogen, USA) using Lipofectamine 2000 (11668500, Invitrogen, USA). After 24 h of transfection, cells were treated with TNF-α and cultured for an additional 24 h. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (E1910, Promega, USA) on a GloMax luminometer (Promega, USA). Promoter activity was calculated as the Firefly to Renilla luciferase ratio and expressed as a fold change relative to the NC group.

Establishment and Grouping of Animal Models
Eight-week-old male C57BL/6J mice (20-22 g; Slaccas, Shanghai, China) were housed under SPF conditions (22 ± 2 °C, 50 ± 10% humidity, 12-h light/dark cycle). Chondrocyte-specific Nav1.7 knockout mice (Nav1.7chondrocyte) and their littermate controls (Nav1.7flox) were generated by crossing Agc1-creERT2 mice (Jackson, 019148) with Scn9a flox mice (S-CKO-04938, Cyagen, China). At 10 weeks, CreERT2 was induced by intraperitoneal tamoxifen (150 mg/kg; T5648, Sigma-Aldrich, USA) in sunflower oil (S5007, Sigma-Aldrich, USA) once daily for five days. Littermate controls were used throughout.

DMM Model: Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The right knee underwent medial parapatellar incision and transection of the medial meniscotibial ligament; Sham mice received capsule incision only. Animals were randomized into Sham, DMM, Rh-Gel, CCM@PM, and Rh-Gel@CCM@PM groups. Intra-articular injections (10 μL PBS or treatment) were administered weekly for eight weeks. CCM@PM was dosed at 2.0 mg/mL (20 μg per joint). For Rh-Gel@CCM@PM, CCM@PM was embedded at 2 mg/mL gel, delivering an equivalent 20 μg per joint. Behavioral testing followed the final injection, and knees were harvested for analysis.

Monosodium Iodoacetate (MIA) Model: Anesthesia was performed as described above. On day 3, 10 μL of MIA (2 mg/mL, 12513, Sigma-Aldrich, USA) was injected intra-articularly into the knee joint to establish the model. Mice were randomly assigned to five groups (n = 6 per group): Sham, MIA, Rh-Gel, CCM@PM, and Rh-Gel@CCM@PM. Animals received weekly intra-articular injections of 10 μL PBS or the designated treatment for four consecutive weeks. Doses were standardized across groups: CCM@PM was administered at a final concentration of 2.0 mg/mL, corresponding to 20 μg CCM@PM per joint. For the Rh-Gel@CCM@PM group, CCM@PM was embedded in Rh-Gel at 2 mg/mL gel, and injection of 10 μL delivered an equivalent 20 μg CCM@PM per joint. Behavioral assessments were conducted in week 5, followed by knee joint harvesting for analysis 40.

Behavioral Testing
All behavioral assessments were conducted under blinded conditions between 12:00 and 17:00. In the DMM model, mice underwent three baseline measurements before surgery, followed by weekly testing starting from week 8 post-operation. In the MIA model, behavioral assessments were performed on days 0, 2, 4, 8, 14, 20, and 28 post-injection. Mechanical sensitivity was evaluated using the von Frey test with standard von Frey filaments (Stoelting, USA). Mice were individually placed in transparent Plexiglass chambers on a metal mesh floor and acclimated for 30 minutes. Filaments of increasing force were applied to the plantar surface of the hind paw. Each filament was applied 10 times with a 30-second interval between stimulations. A brisk paw withdrawal was considered a positive response, and the mechanical threshold was defined as the lowest force eliciting at least five positive responses out of ten applications. Open field behavior was assessed using a video tracking system (Smart 3.0, Panlab Harvard Apparatus, Spain). Each mouse was placed in the center of a 45 cm × 45 cm transparent arena and allowed to explore freely for 2 minutes. Locomotor trajectories and total distance traveled were recorded under standard room lighting conditions.

Micro-CT Analysis
After anesthesia, hind limbs were immobilized and scanned using a micro-CT system (Skyscan 1275, Bruker, Germany) at 40 kV tube voltage, 55 μA current, and 10 μm spatial resolution. Three-dimensional reconstruction was performed using Bruker software. Subchondral bone parameters, including bone volume fraction (BV/TV), bone mineral density (BMD), and trabecular thickness (Tb.Th), were quantitatively analyzed.

Histological Staining and Evaluation
Mouse knee joints were fixed in 4% PFA for 24 hours and decalcified in 10% EDTA (pH 7.4) for two weeks. Samples were embedded in paraffin, and serial sections (5 μm) were prepared for hematoxylin and eosin (H&E) staining (G1120, Solarbio, China) and Safranin O/Fast Green (SO/FG) staining (G1371, Solarbio, China). Cartilage morphology was examined under a light microscope (Zeiss) and blindly scored according to the OARSI grading system (0-6). Glycosaminoglycan (GAG) content and articular cartilage thickness were also quantified.

RT-qPCR
Total RNA was extracted from cell or tissue samples using TRIzol reagent (15596018, Invitrogen, USA). One microgram of RNA was reverse transcribed into cDNA using the Hifair II 1st Strand cDNA Synthesis Kit (Yeasen Biotech Co., Ltd., China). Quantitative PCR was performed with the TB Green Fast qPCR Mix Kit (Takara Biomedical Technology Co., Ltd., China) on a QuantStudio 5 real-time PCR system (Thermo Fisher Scientific, USA). The thermal cycling conditions were as follows: initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Relative mRNA expression levels of target genes were calculated using the ΔΔCt method, with β-actin as the internal control. Primer sequences used for qPCR analysis are listed in Supplementary Table 3.

WB Analysis
Samples included CCM@PM and Rh-Gel@CCM@PM nanoparticles, ATDC5 cells, and cartilage tissue. CCM@PM-related samples were lysed in RIPA buffer with protease inhibitors (P0013B, Beyotime, China). ATDC5 cells and cartilage tissues were homogenized and lysed in standard RIPA buffer. All lysates were sonicated on ice and centrifuged at 12,000 × g for 15 minutes at 4°C. Supernatants were collected, and protein concentrations were determined using a BCA Protein Assay Kit (23225, Thermo Fisher Scientific, USA), then standardized to 2 mg/mL. Proteins (30 μg per sample) were separated by 10% SDS-PAGE and transferred to 0.22 μm PVDF membranes (IPVH00010, Millipore, USA). Membranes were blocked at ambient temperature for 1 hour in TBST containing 5% BSA or non-fat milk. After blocking, membranes were incubated overnight at 4°C with the following primary antibodies: CD44 (Rabbit monoclonal, ab189524, Abcam, UK, 1:1000), Integrin β1 (Rabbit monoclonal, ab179471, Abcam, UK, 1:2000), N-cadherin (Rabbit polyclonal, ab98952, Abcam, UK, 1:1000), TFAM (Rabbit monoclonal, ab307302, Abcam, UK, 1:1000), ATPB (Rabbit polyclonal, 17247-1-AP, Proteintech, China, 1:1000), BRD4 (Rabbit monoclonal, ab128874, Abcam, UK, 1:1000), Nav1.7 (Rabbit polyclonal, ab65167, Abcam, UK, 1:1000), IL-6 (Rabbit monoclonal, ab290735, Abcam, UK, 1:1000), SOX9 (Rabbit monoclonal, ab185966, Abcam, UK, 1:1000), COL II (Rabbit polyclonal, ab188570, Abcam, UK, 1:1000), MMP13 (Rabbit polyclonal, ab39012, Abcam, UK, 1:3000), and β-actin (Rabbit monoclonal, ab8226, Abcam, UK, 1:2000). After washing with TBST, membranes were incubated at ambient temperature for 1 hour with HRP-conjugated secondary antibodies, including Goat Anti-Rabbit IgG (A0208, Beyotime, China, 1:5000), Goat Anti-Rabbit IgG H&L (HRP) (ab6721, Abcam, UK, 1:5000), or HRP-conjugated antibodies (111-035-144/115-035-146, Jackson ImmunoResearch, USA, 1:8000). Signal detection was carried out using ECL chemiluminescence reagents (P0018, Beyotime, China; WBKLS0500, Millipore, USA; PE0010, Solarbio, China), and images were captured using imaging systems (Tanon 5200, Tanon, China; ChemiDoc XRS+, Bio-Rad, USA; Science Lab 2003, Fuji, Japan). Densitometric analysis of protein bands was conducted with ImageJ software, and results were averaged from three independent experiments for statistical evaluation.

Immunohistochemical Staining
Immunohistochemical staining was performed on paraffin-embedded mouse knee joint sections to evaluate metabolism-related protein expression. Sections were deparaffinized in xylene and rehydrated through graded ethanol (100%, 95%, 80%). Antigen retrieval was conducted in sodium citrate buffer (pH 6.0, C9999, Sigma-Aldrich, USA) at 95°C for 20 minutes. After cooling, nonspecific binding was blocked with 5% BSA (A2153, Sigma-Aldrich, USA) for 30 minutes. Primary antibodies were applied and incubated overnight at 4°C. The antibodies used included anti-Col2 (Rabbit polyclonal, 1:200, ab34712, Abcam, UK), anti-Mmp13 (Rabbit polyclonal, 1:200, ab39012, Abcam, UK), anti-Aggrecan (Rabbit polyclonal, 1:200, 13880-1-AP, Proteintech, China), and anti-COMP fragment (Mouse monoclonal, 1:200, affinity-purified monoclonal). The following day, sections were washed with PBS and incubated with HRP-conjugated secondary antibody (1:500, A0216, Beyotime, China) at ambient temperature for 1 hour. Color development was performed using a DAB Substrate Kit (ZLI-9019, ZSGB-BIO, China), followed by hematoxylin counterstaining for nuclei. Sections were examined using a light microscope (BX53, Olympus, Japan). Positive staining was semi-quantified using ImageJ (Version 1.51k, Rawak Software, Germany).

Immunofluorescence Staining
Cells were seeded on glass coverslips in 24-well plates, while tissue sections underwent standard deparaffinization and rehydration. Cells were fixed with 4% PFA for 15 minutes and permeabilized with 0.1% Triton X-100 (T8787, Sigma-Aldrich, USA) for 10 minutes following PBS washing. Tissue sections were incubated with 0.1% trypsin at 37°C for 30 minutes, then treated sequentially with 0.25 U/mL chondroitinase ABC (C3667, Sigma-Aldrich, USA) and 1 U/mL hyaluronidase (H3560, Sigma-Aldrich, USA) for 60 minutes. After blocking with 1% BSA for 30 minutes, samples were incubated overnight at 4°C with the following primary antibodies: rabbit anti-N-Cadherin (Rabbit monoclonal, 1:50, ab321897, Abcam, UK), rabbit anti-E-Cadherin (Rabbit monoclonal, 1:50, ab314063, Abcam, UK), rabbit anti-Integrin αV (Rabbit monoclonal, 1:50, ab150361, Abcam, UK), rabbit anti-Integrin β1 (Rabbit monoclonal, 1:50, ab183666, Abcam, UK), rabbit anti-Integrin α3 (Rabbit monoclonal, 1:50, ab131055, Abcam, UK), rabbit anti-CD44 (Rabbit monoclonal, 1:100, ab243894, Abcam, UK), rabbit anti-IL-6 (Rabbit monoclonal, 1:50, ab290735, Abcam, UK), rabbit anti-SOX9 (Rabbit monoclonal, 1:50, ab185966, Abcam, UK), rabbit anti-COL II (Rabbit polyclonal, 1:50, ab34712, Abcam, UK), rabbit anti-MMP13 (Rabbit polyclonal, 1:100, ab39012, Abcam, UK), rabbit anti-ATPB (Rabbit polyclonal, 1:300, 17247-1-AP, Proteintech, China), rabbit anti-TFAM (Rabbit monoclonal, 1:100, ab307302, Abcam, UK), rabbit anti-Nav1.7 (Rabbit polyclonal, 1:50, ASC-027, Alomone Labs, USA), rabbit anti-BRD4 (Rabbit monoclonal, 1:200, ab128874, Abcam, UK), and rabbit anti-ACAN (Rabbit polyclonal, 1:200, 13880-1-AP, Proteintech, China). The following day, samples were washed with PBS and incubated for 1 hour at ambient temperature with either Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) or Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (ab150115). Nuclei were counterstained with DAPI. Imaging was conducted using a laser scanning confocal microscope.

Statistical Analysis and Sample Size Determination
No statistical methods were used to predetermine sample size. Sample sizes were determined based on previous experience with similar experimental models and standard practices in the field of osteoarthritis research 43, 44. Specifically, for in vivo studies, a sample size of n = 6 mice per group was chosen to account for biological variability in surgical outcomes and disease progression, providing sufficient power to detect significant differences in histological scores and behavioral parameters.

All data were obtained from at least three independent experiments and are presented as Mean ± SD. Comparisons between two groups were performed using an independent-samples t-test, while comparisons among three or more groups were conducted using one-way analysis of variance (ANOVA). All statistical analyses were carried out using GraphPad Prism 9.5.0 (GraphPad Software, Inc.) and R 4.2.1 (R Foundation for Statistical Computing). A two-tailed p-value < 0.05 was considered statistically significant, whereas p > 0.05 was regarded as not significant.

Blinding and Randomization
For in vivo studies, animals were randomly assigned to treatment groups. Investigators performing the surgeries, intra-articular injections, and behavioral assessments were blinded to group allocation.

For histological and micro-CT analyses, samples were assigned random identification numbers to ensure blinded evaluation during data acquisition and quantification.

For in vitro molecular biology experiments (e.g., RT-qPCR, Western blot), blinding was not performed during sample processing due to the requirement for specific labeling of treatment conditions; however, data analysis was conducted using automated software (e.g., ImageJ, QuantStudio) to minimize operator bias.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors present an innovative rhein-based hydrogel (Rh-Gel) incorporating chondrocyte cell membrane–coated, peptide-modified mesoporous silica nanoparticles (CCM@PM) to deliver the BRD4 PROTAC dBET1 intra-articularly for osteoarthritis (OA) treatment. By integrating scRNA-seq, bulk RNA-seq, WGCNA, and functional assays, they identify a previously unrecognized BRD4/Nav1.7 signaling axis, in which BRD4 enhances histone acetylation at the Scn9a (gene encoding Nav1.7) promoter, thereby driving mitochondrial dysfunction, catabolic activation, and inflammatory responses in chondrocytes. This discovery is notable not only for advancing OA biology but also for linking epigenetic regulation with sodium channel–mediated pain mechanisms. The biomaterial-based delivery platform achieves efficient BRD4 degradation, restores anabolic activity, suppresses inflammation, and alleviates disease severity in both DMM and MIA models. This is an ambitious and technically comprehensive study that spans multi-omics analysis, molecular and cellular dissection, biomaterials engineering, and in vivo validation. Its novelty lies in connecting BRD4-driven transcriptional regulation with Nav1.7 biology—an axis of broad interest to researchers in arthritis, pain, and sodium channel physiology—while also demonstrating a clinically relevant drug delivery strategy with high translational potential. While the biological mechanism (BRD4→Nav1.7) and human relevance require further validation, and several textual overstatements should be tempered, with appropriate revisions this exciting paper would merit publication in Nature Communications.\nSpecific Comments\n• Much of the mechanistic work was done in ATDC5 chondrogenic progenitors. Key findings should be replicated in human chondrocytes from arthroplasty samples, including testing dBET1 hydrogel effects in primary chondrocytes or cartilage explants.\n• BRD4 is expected to regulate multiple downstream targets, with Nav1.7 being one among them. Additional assays should clarify whether BRD4’s regulatory effects in chondrocytes and OA depend specifically on Nav1.7. For example, test whether BRD4 loses efficacy in Nav1.7-deficient chondrocytes and/or mice.\n• Only TNF-α was tested. IL-1β is equally central to OA pathophysiology. Please examine BRD4 activity and dBET1 rescue in IL-1β–stimulated chondrocytes.\n• Cytotoxicity window: Cytotoxicity was assessed only at 24 h. Extend testing to ≥72 h and preferably 7 days in both chondrocytes and synoviocytes.\n• Tone down statements of “first demonstration” of Nav1.7 expression in chondrocytes. Nav1.7’s expression and regulatory role in chondrocyte biology were recently reported (Fu et al., Nature 2024).\n• Title/claim accuracy. The manuscript repeatedly implies “Nav1.7-mediated histone acetylation,” including in the title. However, your own data and abstract state that BRD4 enhances histone acetylation at the Scn9a (Nav1.7) promoter, i.e., acetylation is BRD4-dependent, not Nav1.7-dependent. Please revise the title and text to reflect BRD4-dependent histone acetylation leading to increased Nav1.7 transcription.\n• Clarify replicates and statistics. Provide n values, exact p-values, scatter plots with CIs.\n• English editing required for grammar and typos (e.g., 'chondorcytes').", "reviewer_2": "This study designed and validated a rhein-based hydrogel nanoplatform (Rh-Gel@CCM@PM) for delivering BRD4 PROTAC. By targeting and degrading BRD4 to inhibit Nav1.7 expression, the platform significantly alleviates the pathological progression of osteoarthritis (OA).\nYet, the Rh-Gel, MSN, cell membrane coating, and the targeting peptide are already established in other works, and the study presents them as a parallel assembly. The work does not yet show that each module is indispensable, that the combination is synergistic, or that it clearly outperforms works. Please address the points below and clarify the design’s distinctiveness and necessity.Until these key issues are fully addressed, the manuscript does not meet the requirements for publication.\n1. The depth of the mechanism needs to be strengthened。\nThe mechanism of how BRD4 specifically recognizes and binds to the Scn9a promoter region is not clear. How NAV1.7 specifically affects mitochondrial function and metabolic state through molecular pathways remains to be elucidated.\n2. Appropriateness of scRNA-seq design and batch correction.\nThe sample of each group uses n = 1, which does not allow separation of biological variation from technical or batch effects. The application of Harmony integration under this design may introduce a risk of over-correction. Please justify this choice or provide an analysis that avoids cross-sample batch correction, and clarify how this decision affects clustering and differential analyses.\n3. In vivo dosing form and injectability.\nPlease state the actual formulation delivered into the joint. Indicate whether the administration used an injectable sol with in situ gelation of Rh-Gel@CCM@PM or a pre-formed gel. Provide formulation-appropriate handling data, including shear-thinning or thixotropy, storage and loss moduli where applicable, the relationship between viscosity and shear rate, compatible needle gauge, stability in synovial-mimicking media at 37 °C, and feasibility of repeat dosing.\n4. Necessity of Rh-Gel and rationale for MSN as the core carrier.\nRh-Gel based on rhein self-assembly has been reported in prior studies (10.1038/s41467-019-09601-3; 10.1016/j.colsurfb.2020.111260; 10.1002/adhm.202400841). The manuscript should clarify non-redundant functions that Rh-Gel contributes in this system, such as staged release, joint retention, or protection of the payload under shear, and support these claims with matched ablation comparisons of PM or CCM@PM with and without Rh-Gel. In addition, please explain the choice of MSN over liposomes or polymeric carriers, and present specific advantages in loading stability, performance, safety, manufacturing control, and translational potential.\n5. Effective concentration and dose of CCM@PM.\nReport the final CCM@PM concentration and the administered amount per joint or per animal for each in vivo experiment. Provide loading or encapsulation efficiency to convert to an effective drug dose. In addition, it is necessary to specify the particle content when it is embedded in Rh-Gel, in order to facilitate dose normalization and result comparison across experiments.\n6. Inconsistency between “grafting” and physical loading.\nFTIR is interpreted as successful grafting of BRD4-PROTAC onto the nanoparticle surface, while the described preparation uses negative-pressure drying and rehydration to load PROTAC, which indicates physical loading. Reconcile these descriptions. If covalent attachment is intended, provide direct chemical evidence and resistance to washing under physiological conditions. If non-covalent loading is intended, revise the terminology and provide loading capacity and leaching data under physiological conditions.\n7. Release and activity of the PROTAC\nCurrent release studies mainly track CCM@PM or composite signals. Quantitative release profiles of dBET1 or FITC-labeled BRD4-PROTAC are not shown, nor is intracellular availability demonstrated, including endosomal escape and retention in the cytosol. Please provide release kinetics from PM or CCM@PM and from Rh-Gel@CCM@PM, cellular localization, and post-release activity readouts that confirm BRD4 degradation relative to an exposure-matched free dBET1 control.\n8. Whether and how PROTAC is released from CCM@PM\nClarify the microenvironmental triggers that drive PROTAC dissociation from CCM@PM, such as hydrophobic partitioning, pH, redox conditions, or enzymatic processes. Provide mechanistic evidence and release kinetics, and link these data to the time course of functional BRD4 degradation.\n9. Source and applicability of neonatal chondrocyte membranes\nCCM is derived from P1 primary chondrocyte. Please address potential differences between neonatal and adult or diseased cartilage with respect to receptor and adhesion profiles. Provide evidence that supports applicability in diseased adult cartilage, or comparative data using adult or OA-derived membranes.\n10. In vivo distribution and clearance\nOnly local joint retention imaging is presented at several time points. Whole-body biodistribution, organ tropism, clearance routes, and systemic safety have not been established.", "reviewer_3": "This study is the first to reveal the pathological mechanism of osteoarthritis (OA) in which BRD4 regulates Nav1.7 expression through histone acetylation. It proposes a novel regulatory axis of \"BRD4-histone acetylation-Nav1.7-metabolic imbalance,\" broadening the research perspective on epigenetic regulation in OA. While the study demonstrates certain innovativeness, the following critical shortcomings and issues need to be carefully addressed before considering acceptance:\nAs an epigenetic regulator, BRD4 may participate in the progression of OA by regulating multiple downstream target genes. In addition to Nav1.7, are there other key target genes (such as inflammation-related genes IL-6 and MMP13) that collaboratively mediate the pro-OA effects of BRD4? GSEA analysis of whole transcriptome data after BRD4 knockout should be supplemented to clarify the weight of Nav1.7 in the downstream pathways of BRD4.\nNav1.7 is traditionally known as a pain-related channel in neurons, and its functional validation in chondrocytes requires more rigorous investigation. For example, are the electrophysiological characteristics of Nav1.7 in chondrocytes consistent with those in neurons? Beyond mitochondrial function and metabolic imbalance, does Nav1.7 participate in OA by regulating chondrocyte mechanosensation or calcium signaling?\nThe targeting specificity of the materials used by the authors is insufficient. For chronic diseases, targeting specificity is a critical issue due to the need for long-term use. Therefore, the authors should conduct in-depth research on targeting specificity and long-term toxic side effects.\nThere are many small molecule inhibitors of BRD4. Why must a BRD4 degrader be used? Have small molecule inhibitors been compared for the treatment of OA?"}, "author_response": "Response to Reviewers’ Comments\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors present an innovative rhein-based hydrogel (Rh-Gel) incorporating chondrocyte cell\nmembrane–coated, peptide-modified mesoporous silica nanoparticles (CCM@PM) to deliver the\nBRD4 PROTAC dBET1 intra-articularly for osteoarthritis (OA) treatment. By integrating scRNA-seq,\nbulk RNA-seq, WGCNA, and functional assays, they identify a previously unrecognized BRD4/Nav1.7\nsignaling axis, in which BRD4 enhances histone acetylation at the Scn9a (gene encoding Nav1.7)\npromoter, thereby driving mitochondrial dysfunction, catabolic activation, and inflammatory responses\nin chondrocytes. This discovery is notable not only for advancing OA biology but also for linking\nepigenetic regulation with sodium channel–mediated pain mechanisms. The biomaterial-based delivery\nplatform achieves efficient BRD4 degradation, restores anabolic activity, suppresses inflammation, and\nalleviates disease severity in both DMM and MIA models.\nThis is an ambitious and technically comprehensive study that spans multi-omics analysis, molecular\nand cellular dissection, biomaterials engineering, and in vivo validation. Its novelty lies in connecting\nBRD4-driven transcriptional regulation with Nav1.7 biology—an axis of broad interest to researchers\nin arthritis, pain, and sodium channel physiology—while also demonstrating a clinically relevant drug\ndelivery strategy with high translational potential. While the biological mechanism (BRD4→Nav1.7)\nand human relevance require further validation, and several textual overstatements should be tempered,\nwith appropriate revisions this exciting paper would merit publication in Nature Communications.\nSpecific Comments\n• Much of the mechanistic work was done in ATDC5 chondrogenic progenitors. Key findings should\nbe replicated in human chondrocytes from arthroplasty samples, including testing dBET1 hydrogel\neffects in primary chondrocytes or cartilage explants.\nResponse: We thank the reviewer for this valuable suggestion. In response, we have performed\nadditional validation experiments in human primary articular chondrocytes (HACs), which are now\nincluded as newly added Figures S19 and S20. The overall trends of these results are largely consistent\nwith those observed in ATDC5 cells, further supporting the robustness and translational relevance of\nour findings.\n• BRD4 is expected to regulate multiple downstream targets, with Nav1.7 being one among them.\nspecifically on Nav1.7. For example, test whether BRD4 loses efficacy in Nav1.7-deficient\nchondrocytes and/or mice.\nResponse: We thank the reviewer for this insightful comment. In the current manuscript, to clarify the\nfunctional role of the BRD4–Nav1.7 axis in TNF-α–treated ATDC5 cells, we performed rescue\nexperiments by activating Nav1.7 in the context of BRD4 knockdown. As shown in the revised Figures\nS6 and 7, treatment with the Nav1.7 activator OD1 following BRD4 silencing markedly exacerbated\nTNF-α–induced mitochondrial dysfunction, suppressed anabolic activity, and promoted catabolic\nresponses in ATDC5 cells. Notably, the overall phenotypic trends observed in the sh-BRD4 + OD1\ngroup were largely comparable to those in the sh-NC group, indicating that Nav1.7 activation\nsubstantially rescued the phenotypic effects induced by BRD4 knockdown. These findings suggest that\nNav1.7 functions as a major downstream effector of BRD4 in regulating oxidative stress, anabolic\nmetabolism, and catabolic processes in ATDC5 chondrocytes.\n• Only TNF-α was tested. IL-1β is equally central to OA pathophysiology. Please examine BRD4\nactivity and dBET1 rescue in IL-1β–stimulated chondrocytes.\nResponse: We thank the reviewer for this constructive suggestion. In the present study, we not only\nemployed TNF-α–stimulated ATDC5 cells as an in vitro inflammatory chondrocyte model, but also\nexpanded our validation to human primary articular chondrocytes (HACs). The corresponding results\nhave been newly added as Figures S19 and S20, providing robust in vitro evidence for the functional\nregulation of the BRD4–Nav1.7 axis and the therapeutic efficacy of Rh-Gel@CCM@PM. In addition,\nour conclusions were further validated in vivo using a destabilization of the medial meniscus (DMM)–\ninduced osteoarthritis mouse model (Figures 7 and 8). We acknowledge that IL-1β is another widely\nused inflammatory stimulus in osteoarthritis research. Notably, previous studies have reported that\nTNF-α and IL-1β exhibit comparable expression levels and exert similar regulatory roles in\nosteoarthritis pathogenesis (PMID: 40818538; PMID: 37188001; PMID: 33650224). Based on this\nestablished evidence, TNF-α was selected as the representative inflammatory factor in the current work.\nWhile we anticipate that IL-1β stimulation would produce similar effects, we did not include additional\nIL-1β–based experiments in the present manuscript in order to maintain focus and avoid excessive\nexpansion of an already comprehensive dataset. Nevertheless, we fully agree that incorporating\nadditional induction models would further strengthen the conclusions, and we plan to include such\nstudies in future work.\n2\n• Cytotoxicity window: Cytotoxicity was assessed only at 24 h. Extend testing to ≥72 h and preferably\n7 days in both chondrocytes and synoviocytes.\nResponse: We thank the reviewer for this valuable suggestion. The relevant experiments have now\nbeen added to the revised manuscript: “ In addition, long-term toxicity was evaluated using 1 μM\ndBET1. The results showed that treatment with 1 μM dBET1 exerted no long-term cytotoxic effects on\nATDC5 cells or MSCs (Figure S8G–H)”.\n• Tone down statements of “first demonstration” of Nav1.7 expression in chondrocytes. Nav1.7’s\nexpression and regulatory role in chondrocyte biology were recently reported (Fu et al., Nature 2024).\nResponse: We thank the reviewer for this important feedback. We have revised the description\nregarding Nav1.7 expression in chondrocytes by removing the wording “for the first time,” thereby\nensuring accuracy and appropriately acknowledging prior studies.\n• Title/claim accuracy. The manuscript repeatedly implies “Nav1.7-mediated histone acetylation,”\nincluding in the title. However, your own data and abstract state that BRD4 enhances histone\nacetylation at the Scn9a (Nav1.7) promoter, i.e., acetylation is BRD4-dependent, not Nav1.7-\ndependent. Please revise the title and text to reflect BRD4-dependent histone acetylation leading to\nincreased Nav1.7 transcription.\nResponse: We thank the reviewer for this important clarification regarding title and claim accuracy.\nWe agree that histone acetylation in our study is BRD4-dependent and occurs at the Scn9a (Nav1.7)\npromoter, rather than being mediated by Nav1.7 itself. We have therefore revised the title into: “A\ncartilage-targeting herbal hydrogel delivers PROTACs to degrade BRD4, suppress BRD4-dependent\nhistone acetylation and Nav1.7 transcription, and alleviate osteoarthritis”.\n• Clarify replicates and statistics. Provide n values, exact p-values, scatter plots with CIs.\nResponse: Thank you for the suggestion. We have added the sample size (n) for independent replicates\nand clarified the significance annotations in all figure legends. In addition, single data points are now\nshown (scatter + Mean ± SD) to better illustrate the data distribution and statistical outcomes. The\ncorresponding revisions have been updated in the revised manuscript.\n• English editing required for grammar and typos (e.g., 'chondorcytes').\nas 'chondorcytes,' which has been corrected to 'chondrocytes.' Thank you for highlighting this issue.\nThis study designed and validated a rhein-based hydrogel nanoplatform (Rh-Gel@CCM@PM) for\ndelivering BRD4 PROTAC. By targeting and degrading BRD4 to inhibit Nav1.7 expression, the\nplatform significantly alleviates the pathological progression of osteoarthritis (OA).\nYet, the Rh-Gel, MSN, cell membrane coating, and the targeting peptide are already established in\nother works, and the study presents them as a parallel assembly. The work does not yet show that each\nmodule is indispensable, that the combination is synergistic, or that it clearly outperforms works.\nPlease address the points below and clarify the design’s distinctiveness and necessity.Until these key\nissues are fully addressed, the manuscript does not meet the requirements for publication.\n1. The depth of the mechanism needs to be strengthened。\nThe mechanism of how BRD4 specifically recognizes and binds to the Scn9a promoter region is not\nclear. How NAV1.7 specifically affects mitochondrial function and metabolic state through molecular\npathways remains to be elucidated.\nResponse: We thank the reviewer for this valuable comment. In response, we have added\nclarifications and expanded the discussion in the revised manuscript: “BRD4 contains two\nbromodomains (BD1 and BD2) that recognize acetylated lysine residues and mediate chromatin\nassociation, as well as a histone acetyltransferase (HAT) domain capable of catalyzing histone lysine\nacetylation 37. In the present study, TNF-α stimulation markedly increased histone H3ac at the Scn9a\npromoter, whereas BRD4 knockdown significantly reduced H3ac enrichment at this locus (Figure 3E).\nIn conjunction with previous reports, these findings suggest that BRD4-mediated regulation of Scn9a\npromoter activity may be closely associated with changes in local H3 acetylation. Moreover, BRD4 has\nbeen shown to bind chromatin by recognizing acetylated histone lysine residues and to directly recruit\ndistinct transcription factors, thereby facilitating transcriptional activation of target promoters 38-40.\nConsistent with this mechanism, we observed that TNF-α markedly enhanced Scn9a promoter activity,\nwhile BRD4 silencing led to a pronounced suppression of promoter-driven transcription (Figure 3F).\nCollectively, these data support a model in which BRD4 activates Scn9a transcription through histone\nacetylation–dependent chromatin engagement and transcription factor recruitment. Identification of the\n5\nspecific transcription factors involved in this BRD4-dependent regulatory process will be a key focus\nof future studies.\nNav1.7 is a voltage-gated sodium channel that has traditionally been regarded as a key mediator of\nelectrical signal transmission and pain perception in neurons 41, 42. In recent years, a limited number of\nstudies have reported Nav1.7 expression in non-neuronal tissues, although its functional relevance in\nthese contexts has remained largely unclear 14. Notably, emerging evidence has demonstrated the\npresence of Nav1.7 in chondrocytes and implicated its involvement in joint degeneration, highlighting\nthe potential importance of ion channels in regulating pathological metabolism within joint tissues 14.\nPrevious studies have suggested that voltage-gated sodium channels may influence cellular metabolic\nhomeostasis by modulating membrane excitability, ionic influx, and mitochondrial membrane potential\ndynamics 33. Nevertheless, the precise signaling nodes through which Nav1.7 operates in chondrocytes\nremain to be elucidated. In the present study, we establish that elevated Nav1.7 expression in\nchondrocytes is functionally significant and closely associated with mitochondrial dysfunction and\nmetabolic imbalance. These findings extend the role of Nav1.7 beyond that of a canonical “pain target”\nto that of a potential “metabolic regulator” and implicate Nav1.7 as a previously underappreciated\ncontributor to cartilage degeneration. More broadly, this work provides new insight into the\ninvolvement of ion channels in non-neuronal pathological processes. However, the specific molecular\npathways by which Nav1.7 upregulation impacts mitochondrial function and cellular energy\nmetabolism warrant further investigation.”.\n2. Appropriateness of scRNA-seq design and batch correction.\nThe sample of each group uses n = 1, which does not allow separation of biological variation from\ntechnical or batch effects. The application of Harmony integration under this design may introduce a\nrisk of over-correction. Please justify this choice or provide an analysis that avoids cross-sample batch\ncorrection, and clarify how this decision affects clustering and differential analyses.\nseq) design and batch correction strategy. In this study, scRNA-seq (n = 1 per group) was primarily\nintended to identify major cell types and cellular states within cartilage tissue and to exploratorily\ncharacterize degeneration-associated chondrocyte populations, rather than to perform formal cross-\ngroup statistical comparisons. To ensure comparability of the two samples in a shared low-dimensional\nspace, we applied the Harmony algorithm solely for technical batch correction based on sample identity\n(orig.ident). Importantly, this correction was restricted to visualization and consistent cell annotation,\nand was not used for statistical inference. All differential expression and enrichment analyses were\nconducted using bulk RNA-seq datasets with adequate biological replication (n = 6 per group), and the\nkey findings were further supported by multiple layers of experimental validation. Moreover,\nconservative clustering parameters were applied (top 30 principal components and a resolution of 0.4),\nand no evidence of overcorrection or loss of biologically meaningful signals was observed. Therefore,\nthe use of Harmony does not affect the main conclusions of this study. The scRNA-seq data serve an\nauxiliary role for cell-type localization and visualization, whereas the central mechanistic insights and\nstatistically supported conclusions are derived from well-replicated population-level analyses.\n3. In vivo dosing form and injectability.\nPlease state the actual formulation delivered into the joint. Indicate whether the administration used an\ninjectable sol with in situ gelation of Rh-Gel@CCM@PM or a pre-formed gel. Provide formulation-\nappropriate handling data, including shear-thinning or thixotropy, storage and loss moduli where\napplicable, the relationship between viscosity and shear rate, compatible needle gauge, stability in\nsynovial-mimicking media at 37 °C, and feasibility of repeat dosing.\nResponse: We sincerely thank the reviewer for the critical comments regarding the in vivo dosing\nformulation and injectability. In the revised manuscript, we have explicitly clarified that the\nformulation administered in the animal experiments was Rh-Gel@CCM@PM in an injectable sol state.\nUpon intra-articular injection, this sol undergoes in situ gelation under physiological temperature and\nionic conditions, thereby forming a localized drug reservoir within the joint cavity. In addition, in\nresponse to the reviewer’s suggestion, we have systematically added rheological and handling data\nclosely related to injectability and practical operability to support the feasibility of this administration\nstrategy: “To systematically evaluate the mechanical responsiveness and injectability of the Rh-\nmeasurements, the hydrogel exhibited a typical viscoelastic response in the low-strain region; with\nincreasing strain, the storage modulus gradually decreased and eventually reached a plateau, indicating\nstructural yielding (Figure S15A). Cyclic shear tests demonstrated that under alternating low- and high-\nshear conditions, both the storage modulus and loss modulus displayed reproducible and periodic\nvariations, reflecting the reversible mechanical behavior of the hydrogel (Figure S15B). Frequency\nsweep analysis showed that across the frequency range of 0.1-100 Hz, the storage modulus consistently\nexceeded the loss modulus, indicating stable frequency-dependent viscoelastic properties (Figure\nS15C). Time sweep measurements further revealed that under constant strain, both moduli remained\nrelatively stable over time, suggesting good structural integrity (Figure S15D). In addition, viscosity-\nshear rate analysis indicated pronounced shear-thinning behavior, with viscosity continuously\ndecreasing as the shear rate increased (Figure S15E), supporting the suitability of the hydrogel for\ninjectable applications.\nWith respect to injectability, the hydrogel could be smoothly extruded through syringes equipped\nwith needles of different gauges. Continuous filamentous or droplet-like gel structures were readily\nformed using 23G, 25G, and 27G needles (Figure S15F). Photographs of the injection process further\nillustrated the flow behavior and shape retention of the hydrogel under applied mechanical force\n(Figure S15G). To evaluate stability under simulated joint cavity conditions, the hydrogel was\nincubated in synovial fluid–mimicking solution and monitored over time. Macroscopic observation\nrevealed progressive swelling and volumetric changes, accompanied by a gradual color transition of the\nsurrounding medium from transparent to yellow (Figure S15H).\nTo assess long-term storage stability and suitability for repeated administration, samples stored\nunder a nitrogen atmosphere at 4 °C for 0, 1, 2, 4, and 8 weeks were systematically evaluated for\nappearance, injectability, and rheological properties. Throughout the 0–8 week storage period, the\n8\nhydrogel maintained a uniform brown appearance without visible macroscopic alterations (Figure\nS16A). Samples from all storage time points could be smoothly extruded through a 27G needle,\nexhibiting consistent filament formation and droplet behavior (Figure S16B). Shear rheological\nanalysis further demonstrated that all samples retained stable shear-thinning characteristics, with\nviscosity decreasing as shear rate increased and no noticeable shifts in the viscosity profiles across\ndifferent storage durations (Figure S16C–G).”.\nIn addition, to evaluate potential systemic responses associated with long-term and repeated\nadministration, we have supplemented comprehensive biocompatibility and tolerability assessments\n(Figure S18). Following intra-articular injection once weekly for 8 consecutive weeks, hematological\nand serum biochemical analyses were performed. White blood cell counts, red blood cell parameters, as\nwell as liver and kidney function indicators all remained within normal ranges (Figure S18A–B).\nFurthermore, histological examination (H&E staining) of major organs (heart, liver, spleen, lung, and\nkidney) and joint tissues revealed no apparent tissue damage or inflammatory infiltration (Figure\nS18C). These results indicate that Rh-Gel@CCM@PM exhibits favorable in vivo tolerability and\nbiosafety under conditions of long-term administration and potential repeated dosing.\nCollectively, in response to the reviewer’s suggestions, we have added and clearly presented\ndata regarding the dosing form, rheological properties, injectability, needle gauge compatibility,\nstability in synovial fluid–mimicking conditions, feasibility of long-term storage and repeated\nadministration, as well as in vivo safety evaluation. These additions substantially strengthen the\nreproducibility, practical feasibility, and translational credibility of Rh-Gel@CCM@PM as an\ninjectable in vivo delivery system. We sincerely appreciate the reviewer’s insightful comments, which\nhave significantly helped improve the rigor and completeness of this study.\n4. Necessity of Rh-Gel and rationale for MSN as the core carrier.\nRh-Gel based on rhein self-assembly has been reported in prior studies (10.1038/s41467-019-09601-3;\n10.1016/j.colsurfb.2020.111260; 10.1002/adhm.202400841). The manuscript should clarify non-\nredundant functions that Rh-Gel contributes in this system, such as staged release, joint retention, or\nprotection of the payload under shear, and support these claims with matched ablation comparisons of\nPM or CCM@PM with and without Rh-Gel. In addition, please explain the choice of MSN over\nliposomes or polymeric carriers, and present specific advantages in loading stability, performance,\n9\nsafety, manufacturing control, and translational potential.\nResponse: We thank the reviewer for this valuable comment. In the revised manuscript, we have\nadded a dedicated paragraph in the Discussion section to explicitly clarify the non-redundant and\nindispensable roles of Rh-Gel in the current delivery system: “In addition, although self-assembled\nrhein-based hydrogels (Rh-Gel) have been reported in previous studies 34-36, Rh-Gel in the present\nsystem does not merely serve as a generic hydrogel matrix but instead fulfills indispensable and non-\nredundant functions. First, Rh-Gel exhibits characteristic shear-thinning behavior and in situ gelation\ncapability, enabling smooth injectability while rapidly transitioning into a gel state after intra-articular\nadministration. This property markedly prolongs residence on the cartilage surface and establishes a\nstaged release microenvironment, thereby ensuring stable and sustained BRD4 PROTAC activity\nwithin the joint. Second, compared with PM or CCM@PM alone, incorporation of Rh-Gel significantly\nreduces mechanical washout during joint motion, providing effective “shear protection” for the\nencapsulated payload. This contribution is reflected in the graded therapeutic efficacy observed among\nthe PM, CCM@PM, and Rh-Gel@CCM@PM groups.”.\nIn addition, we have supplemented a clear rationale for selecting MSNs as the core carrier:\n“Meanwhile, MSNs were selected as the core carrier primarily due to their tunable and well-defined\npore architecture, which is particularly suitable for efficient loading of macromolecular PROTACs.\nTheir favorable surface chemistry also facilitates stable cell membrane coating, thereby conferring\ncartilage-targeting capability. Moreover, the controlled release behavior of MSNs is well aligned with\nthe in situ sustained-release mechanism of Rh-Gel, allowing synergistic coupling between the two\ncomponents. In contrast, liposomal systems are prone to membrane destabilization in synovial fluid,\nwhereas polymer-based carriers often suffer from batch-to-batch variability and inconsistent\ndegradation profiles, which may compromise payload stability, injection reproducibility, and\ntranslational controllability. Collectively, the integration of Rh-Gel and MSNs achieves a more\n10\nfavorable balance among stability, sustained release, local retention, and safety, providing clear\nadvantages for further preclinical development.”.\n5. Effective concentration and dose of CCM@PM.\nReport the final CCM@PM concentration and the administered amount per joint or per animal for each\nin vivo experiment. Provide loading or encapsulation efficiency to convert to an effective drug dose.\nIn addition, it is necessary to specify the particle content when it is embedded in Rh-Gel, in order to\nfacilitate dose normalization and result comparison across experiments.\nResponse: We sincerely thank the reviewer for the critical comments regarding the effective\nconcentration, in vivo dosing, and dose normalization of CCM@PM. In response to these suggestions,\nwe have systematically clarified and supplemented all dosing parameters related to CCM@PM in the\nrevised manuscript. Specifically, we now explicitly report the final injection concentration, per-joint\ninjection dose, the effective drug dose calculated based on encapsulation efficiency and drug loading,\nas well as the actual content of nanoparticles embedded within the hydrogel matrix. These additions\nensure that dosing information is traceable, quantifiable, and comparable across experiments. In this\nstudy, the encapsulation efficiency of CCM@PM was 82.43 ± 2.16%, and the drug loading capacity\nwas 7.52%. For both the DMM- and MIA-induced osteoarthritis models, intra-articular administration\nwas performed using Rh-Gel@CCM@PM in an injectable sol state. The final injection concentration\nwas standardized at 2.0 mg/mL, with an injection volume of 10 μL per joint, corresponding to a single-\ndose amount of 20 μg CCM@PM per joint. To facilitate dose normalization across different\nexperimental settings, we further specified the nanoparticle content within the hydrogel system. The\nCCM@PM content in Rh-Gel was 2 mg/mL gel, which has now been explicitly indicated in the main\ntext to enable normalized comparisons across different models and experimental batches. All relevant\nsections of the manuscript have been updated accordingly, including the dosing parameters for both\nDMM and MIA models, as well as the encapsulation efficiency and drug loading values. With these\nrevisions, the dosing strategy, effective concentration conversion, and administration details are now\nmore rigorous, reproducible, and standardized, fully addressing the reviewer’s concerns regarding dose\naccuracy and comparability.\n6. Inconsistency between “grafting” and physical loading.\nFTIR is interpreted as successful grafting of BRD4-PROTAC onto the nanoparticle surface, while the\n11\ndescribed preparation uses negative-pressure drying and rehydration to load PROTAC, which indicates\nphysical loading. Reconcile these descriptions. If covalent attachment is intended, provide direct\nchemical evidence and resistance to washing under physiological conditions. If non-covalent loading is\nintended, revise the terminology and provide loading capacity and leaching data under physiological\nconditions.\nResponse: We thank the reviewer for the careful and rigorous evaluation of the BRD4-PROTAC\nloading strategy. In this study, dBET1 was not covalently grafted onto the carrier; instead, it was\nphysically encapsulated within the mesoporous structure through hydrophobic interactions, pore\nadsorption, and other noncovalent interactions. In the original manuscript, imprecise wording\ninadvertently linked the characterization of the cartilage-targeting peptide with the PROTAC loading\nstrategy, which may have led to the misunderstanding that dBET1 was covalently conjugated to the\ncarrier. To directly address this concern, we have added new structural and release validation data in\nthe revised manuscript and clearly delineated the supporting evidence as follows: (1) FTIR analysis\nwas used exclusively to confirm the successful modification of the cartilage-targeting peptide on the\nsurface of mesoporous silica nanoparticles (Figure S10B) and is not indicative of the dBET1 loading\nmechanism. (2) Newly added nitrogen adsorption-desorption isotherms (BET) and density functional\ntheory (DFT) pore size distribution analyses demonstrate that PepM possesses a well-defined\nmesoporous architecture with high specific surface area and pore sizes suitable for small-molecule\ndiffusion (Figure S11A-B), structurally supporting physical encapsulation rather than chemical bonding.\n(3) Newly added elution experiments (Figure S12) show that both PM and CCM@PM exhibit\nreversible release profiles under physiological conditions (PBS with 10% FBS at 37 °C), lacking the\nwash-resistance characteristic of covalent grafting, thereby directly confirming that dBET1 is\nnoncovalently encapsulated within the mesoporous framework. Based on these additional data, all\ndescriptions related to “grafting” has been systematically revised throughout the manuscript.\n7. Release and activity of the PROTAC\nCurrent release studies mainly track CCM@PM or composite signals. Quantitative release profiles of\ndBET1 or FITC-labeled BRD4-PROTAC are not shown, nor is intracellular availability demonstrated,\nincluding endosomal escape and retention in the cytosol. Please provide release kinetics from PM or\nCCM@PM and from Rh-Gel@CCM@PM, cellular localization, and post-release activity readouts that\nconfirm BRD4 degradation relative to an exposure-matched free dBET1 control.\nResponse: We thank the reviewer for this important comment. We fully agree that it is essential to\n12\nprovide experimental evidence for the release kinetics, intracellular availability, and functional activity\nof the PROTAC. Accordingly, in the revised manuscript, we have added comprehensive experimental\ndata addressing these aspects. The detailed results are presented in the newly added Figure S14.\n8. Whether and how PROTAC is released from CCM@PM\nClarify the microenvironmental triggers that drive PROTAC dissociation from CCM@PM, such as\nhydrophobic partitioning, pH, redox conditions, or enzymatic processes. Provide mechanistic evidence\nand release kinetics, and link these data to the time course of functional BRD4 degradation.\nResponse: We thank the reviewer for the careful evaluation of the PROTAC release mechanism from\nCCM@PM. In the present system, dBET1 (BRD4-PROTAC) is physically encapsulated within\nPM/CCM@PM through noncovalent interactions, including hydrophobic interactions and pore\nadsorption, without the involvement of enzyme-sensitive or redox-cleavable linkers. Accordingly,\nPROTAC dissociation is primarily governed by hydrophobic partitioning gradients and by changes in\ncarrier stability following endocytosis in mildly acidic intracellular environments. To comprehensively\naddress this comment, we have systematically added experimental data in the revised manuscript to\nclarify the release mechanism across four interconnected levels: structural basis, in vitro release\nbehavior, intracellular trafficking, and functional BRD4 degradation. First, to confirm successful\nsurface modification of the cartilage-targeting peptide, Fourier transform infrared spectroscopy (FTIR)\nwas performed to characterize the chemical structure of PepM. The appearance of characteristic C=O,\nC–N, and C–H peaks confirmed successful peptide conjugation to the mesoporous silica nanoparticle\nsurface (Figure S10B), which is independent of the PROTAC loading mechanism. We further\ncharacterized the specific surface area and pore architecture of PepM using nitrogen adsorption–\ndesorption isotherms (BET) and density functional theory (DFT) pore size distribution analysis. These\nresults demonstrate a well-defined mesoporous structure with pore sizes suitable for dBET1 loading\nand diffusion (Figure S11A–B), providing structural support for a noncovalent physical encapsulation\nand diffusion-based release mechanism. In addition, we introduced elution experiments conducted\nunder physiological conditions (PBS with 10% FBS at 37 °C). Quantitative analysis of dBET1 in the\nsupernatant revealed slow and reversible release profiles for both PM and CCM@PM, lacking the\nwash-resistant behavior characteristic of covalent systems (Figure S12). These findings experimentally\nconfirm that dBET1 is noncovalently retained within the mesoporous framework and can gradually\ndissociate under physiological conditions. Second, to directly compare PROTAC release behaviors\nacross delivery systems, we evaluated the in vitro release kinetics of dBET1 from PM and CCM@PM.\nwhereas CCM@PM displayed significantly reduced release during the first 12 h (Figure S14A). These\ndata indicate that the CCM coating introduces an additional diffusion barrier, consistent with a release\nmechanism driven by hydrophobic partitioning and structural stabilization rather than chemical bond\ncleavage. Third, to visualize intracellular trafficking and dissociation of PROTAC from\nendosomal/lysosomal compartments into the cytosol, we employed FITC-labeled BRD4-PROTAC\ntogether with immunofluorescence labeling of Rab5 (early endosomes) and LAMP1 (lysosomes),\nfollowed by time-resolved confocal imaging and Pearson’s correlation analysis. At early time points,\nBRD4-PROTAC fluorescence strongly colocalized with Rab5-positive vesicles, indicating uptake\nthrough classical endocytic pathways into early endosomes. With prolonged incubation, the\nfluorescence pattern gradually transitioned from punctate vesicular localization to a more diffuse\ncytosolic distribution, accompanied by reduced colocalization with LAMP1 (Figure S14B–E). These\nobservations indicate partial lysosomal escape and progressive cytosolic accumulation of PROTAC,\nconsistent with a microenvironment-triggered dissociation mechanism, whereby mild acidic conditions\nweaken hydrophobic core stability and facilitate drug release. Finally, to directly link release and\nintracellular transport to functional BRD4 degradation, we added time-course Western blot analyses\ncomparing free dBET1, PM, and CCM@PM treatments. Free dBET1 induced a marked reduction in\nBRD4 protein levels starting at 6 h, with near-complete depletion by 24–48 h. In contrast, PM- and\nCCM@PM-treated cells exhibited time-dependent BRD4 downregulation with slower and more\ngradual degradation kinetics (Figure S14F–G). Importantly, the temporal profiles of BRD4 degradation\nclosely matched the corresponding release kinetics of each carrier, demonstrating that PROTAC\nreleased from the nanocarrier remains functionally active and that release dynamics are temporally\ncoupled to downstream protein degradation. Collectively, through the addition of FTIR\ncharacterization, BET–DFT pore structure analysis, elution assays (Figure S10B, 11, and 12), as well\nas release kinetics, intracellular distribution, and BRD4 degradation time-course analyses (Figure\nS14A–G), we systematically elucidate the noncovalent, microenvironment-responsive release\nmechanism of PROTAC from CCM@PM and Rh-Gel@CCM@PM. These integrated data establish a\nclear temporal and mechanistic link between structural features, release behavior, intracellular\navailability, and functional BRD4 degradation, thereby fully addressing the reviewer’s concern.\n9. Source and applicability of neonatal chondrocyte membranes\nCCM is derived from P1 primary chondrocyte. Please address potential differences between neonatal\n14\nand adult or diseased cartilage with respect to receptor and adhesion profiles. Provide evidence that\nsupports applicability in diseased adult cartilage, or comparative data using adult or OA-derived\nmembranes.\nResponse: We thank the reviewer for this constructive comment. In the revised manuscript, we have\nadded a detailed rationale in the Methods section (“Preparation of cartilage cell membrane (CCM)”)\nto clarify the selection of P1 primary chondrocytes as the membrane source. Specifically, P1 primary\nchondrocytes were chosen to ensure membrane integrity and batch-to-batch consistency, thereby\nproviding a stable adhesion and receptor phenotype that minimizes preparation-related variability. We\nfurther clarify that the isolated CCM retains key membrane proteins and adhesion-related molecules\nafter preparation, including receptors, integrins, and glycosaminoglycan-binding components, which\nare essential for constructing the subsequent membrane-camouflaged delivery system. Regarding\napplicability, based on the results of the present study, Rh-Gel@CCM@PM fabricated using\nmembranes derived from P1 primary murine chondrocytes exhibited good biocompatibility in adult\nmice (Supplementary Figure 17) and effectively alleviated cartilage degeneration and pain-related\nbehaviors in both DMM- and MIA-induced osteoarthritis models (Figures S7 and S8). These findings\nsupport the feasibility of using P1 primary chondrocyte-derived membranes for applications in adult\nmice. Nevertheless, we have also added a discussion of potential limitations in the Discussion section:\n“With regard to membrane source-dependent variability, neonatal and adult or osteoarthritic cartilage\nmay differ in receptor density, integrin/adhesion profiles, and glycosylation patterns, which could\ninfluence interactions between membrane-camouflaged nanoparticles and cartilage matrix or cells.\nIn this study, P1-derived CCM was employed to ensure membrane structural integrity and preparation\nconsistency. Notably, the observed intra-articular retention and therapeutic efficacy in disease models,\ntogether with molecular and phenotypic validation in human primary chondrocytes, support the\ntranslational relevance of this strategy in adult and OA contexts. Future work will systematically\ncompare membrane characteristics derived from adult or OA cartilage sources and evaluate their\nimpact on cellular uptake, tissue retention, and therapeutic outcomes, thereby strengthening the\ntranslational foundation of this delivery platform”.\n10. In vivo distribution and clearance\nOnly local joint retention imaging is presented at several time points. Whole-body biodistribution,\norgan tropism, clearance routes, and systemic safety have not been established.\nResponse: We thank the reviewer for this insightful suggestion. The primary objective of this study\n15\nwas to evaluate the intra-articular retention behavior of Rh-Gel@CCM@PM within the joint\nmicroenvironment and its therapeutic effects targeting the BRD4/Nav1.7 axis. Accordingly, our in vivo\nexperiments initially focused on local retention and therapeutic efficacy within the joint cavity. As the\njoint space represents a relatively enclosed compartment with limited fluid exchange, its drug delivery\nbehavior differs substantially from that of systemic circulation, and local retention directly reflects\neffective intra-articular exposure of the delivery system. In response to the reviewer’s comment, we\nhave revised Figure S17C by replacing the localized joint images with whole-body in vivo fluorescence\nimages. These data clearly show no appreciable signal accumulation in other major organs. In addition,\nwe performed ex vivo fluorescence imaging of harvested tissues to further assess biodistribution. The\nresults demonstrate that none of the nanoparticle formulations exhibited detectable fluorescence\nenrichment in other organs (Figure S17E). Notably, among all tested formulations, Rh-\nGel@CCM@PM exhibited the longest intra-articular retention time, the highest fluorescence intensity,\nand the slowest signal decay rate. Quantitative fitting analysis further revealed a significantly\nprolonged intra-articular half-life for Rh-Gel@CCM@PM (Figure S17D). Furthermore, in\nSupplementary Figure 18, we evaluated the in vivo biocompatibility of Rh-Gel@CCM@PM, showing\nno evident adverse effects on major organs, thereby supporting its favorable biosafety profile.\nReviewer #3 (Remarks to the Author):\nThis study is the first to reveal the pathological mechanism of osteoarthritis (OA) in which BRD4\nregulates Nav1.7 expression through histone acetylation. It proposes a novel regulatory axis of \"BRD4-\nhistone acetylation-Nav1.7-metabolic imbalance,\" broadening the research perspective on epigenetic\nregulation in OA. While the study demonstrates certain innovativeness, the following critical\nshortcomings and issues need to be carefully addressed before considering acceptance:\nAs an epigenetic regulator, BRD4 may participate in the progression of OA by regulating multiple\ndownstream target genes. In addition to Nav1.7, are there other key target genes (such as inflammation-\nrelated genes IL-6 and MMP13) that collaboratively mediate the pro-OA effects of BRD4? GSEA\nanalysis of whole transcriptome data after BRD4 knockout should be supplemented to clarify the\nweight of Nav1.7 in the downstream pathways of BRD4.\nResponse: We thank the reviewer for this valuable suggestion. We fully agree that, as an epigenetic\nregulator, BRD4 is likely to exert its pro-osteoarthritis (OA) effects through multiple downstream\ntargets. As described in the manuscript, our multidimensional experimental data indicate that BRD4 not\n16\nonly regulates Nav1.7 transcription but also positively modulates the expression of inflammatory\nmediators (such as IL-6 and IL-1β) and matrix-degrading enzymes, including MMP13. In the TNF-α–\ninduced chondrocyte model, BRD4 knockdown significantly reduced the expression of IL-6 and IL-1β\nwhile concomitantly upregulating cartilage anabolic markers SOX9 and type II collagen (COL II).\nIn contrast, pharmacological activation of Nav1.7 partially reversed the anti-inflammatory and pro-\nanabolic effects induced by BRD4 silencing, suggesting that BRD4 contributes to OA progression\nthrough coordinated regulation of the Nav1.7 pathway and inflammatory signaling networks.\nConsistently, immunohistochemical analyses in vivo further demonstrated that either BRD4\nknockdown or PROTAC-mediated BRD4 degradation led to decreased expression of MMP13 and IL-\n6, providing additional support for this conclusion. In response to the reviewer’s recommendation, we\nhave also added a genome-wide transcriptomic analysis following BRD4 knockout, including gene set\nenrichment analysis (GSEA) (new Figure S4I in the revised manuscript). The results show that BRD4\ndepletion is associated with coordinated suppression of pathways related to ion transmembrane\ntransport, cellular energy metabolism, and extracellular matrix remodeling. These transcriptomic\nchanges are in good agreement with our in vitro and in vivo observations, including reduced Nav1.7\nexpression and decreased IL-6 and MMP13 levels. Collectively, these findings further support the\nnotion that BRD4 acts as an upstream regulatory hub in OA pathogenesis, exerting multi-layered\ncontrol over metabolic, inflammatory, and matrix-remodeling processes.\nNav1.7 is traditionally known as a pain-related channel in neurons, and its functional validation in\nchondrocytes requires more rigorous investigation. For example, are the electrophysiological\ncharacteristics of Nav1.7 in chondrocytes consistent with those in neurons? Beyond mitochondrial\nfunction and metabolic imbalance, does Nav1.7 participate in OA by regulating chondrocyte\nmechanosensation or calcium signaling?\nResponse: We thank the reviewer for this professional and thoughtful suggestion. The primary\nobjective of the present study was to elucidate the epigenetic regulatory cascade linking BRD4,\nhistone acetylation, and Scn9a (Nav1.7) transcription, and to evaluate its impact on chondrocyte metabolic\nhomeostasis and tissue-level phenotypes. Accordingly, our experimental design prioritized molecular\nand functional readouts, including BRD4/Nav1.7 expression, cartilage anabolic and catabolic markers\n(COL II and MMP13), mitochondrial and metabolic indices, and in vivo therapeutic outcomes.\nElectrophysiological characterization (e.g., membrane currents or action potentials), as well as\nmechanobiological or calcium signaling-focused assays, were therefore beyond the intended scope of\n17\nthe current work. Notably, our existing data demonstrate that TNF-α–induced sodium currents in\nATDC5 chondrocytes are predominantly mediated by Nav1.7 (Figures 2F–K), which is consistent with\npreviously published reports (PMID: 39559752; PMID: 32128393; PMID: 38172636). Regarding the\npossibility that Nav1.7 contributes to osteoarthritis (OA) progression through mechanosensation or\ncalcium signaling, recent work by Chuan-ju Liu et al. (PMID: 38172636) has shown that Nav1.7\nblockade modulates intracellular Ca²⁺ signaling and the chondrocyte secretome, thereby influencing\nchondrocyte biology and OA progression. These findings support the notion that Nav1.7 may\nparticipate in OA pathogenesis via mechanical or calcium-dependent pathways. While direct\ninvestigation of these signaling modalities was not included in the present study, we agree that they\nrepresent important and compelling directions for future research. Building upon the current findings,\nwe plan to further explore the mechanistic links between Nav1.7 activity, calcium signaling,\nmechanotransduction, and cartilage degeneration in subsequent studies.\nThe targeting specificity of the materials used by the authors is insufficient. For chronic diseases,\ntargeting specificity is a critical issue due to the need for long-term use. Therefore, the authors should\nconduct in-depth research on targeting specificity and long-term toxic side effects.\nResponse: We thank the reviewer for this important and thoughtful suggestion. The primary objective\nof this study was to validate the pathogenic cascade linking BRD4, histone acetylation, and Scn9a\n(Nav1.7) transcription, and to evaluate the feasibility of local intra-articular intervention targeting this\naxis. Accordingly, at the materials design level, we prioritized a strategy combining intra-articular local\nadministration, in situ gelation–mediated retention, and cartilage cell membrane (CCM) camouflage to\nachieve functional “on-site enrichment” within the joint cavity. Using multimodal evidence, we\ndemonstrated effective local exposure and favorable tolerability of the delivery system. Specifically,\nSupplementary Figure 17 shows that CCM@PM exhibits strong adhesion to cartilage tissue, enhanced\npenetration, and prolonged intra-articular retention, with minimal off-target accumulation, supporting\nits cartilage-specific distribution. In Supplementary Figure 18, we further demonstrate that Rh-\nGel@CCM@PM does not induce detectable adverse effects in major organs following repeated intra-\narticular administration. In addition, we included long-term in vitro cytotoxicity assessments, which\nshowed that treatment with 1 μM dBET1 did not elicit long-term cytotoxic effects in ATDC5\nchondrocytes or murine synovial mesenchymal stem cells (MSCs) (Supplementary Figure 8G–H).\nCollectively, these results provide initial evidence that Rh-Gel@CCM@PM possesses favorable tissue\ntargeting and a low risk of systemic toxicity. At the same time, we fully acknowledge that the present\n18\nfindings do not immediately support clinical translation. We agree that, particularly in the context of\nchronic diseases, long-term targeting specificity and cumulative toxicity are critical considerations.\nDue to the scope of the current study, we did not perform detailed quantitative analyses of receptor- or\nadhesion-mediated specificity, nor comprehensive systemic pharmacokinetic and toxicological\nevaluations. We have supplemented the future direction in our revised Discussion section:\n“Nevertheless, several limitations remain. Long-term toxicity and immunogenicity have not yet been\nsystematically evaluated. Future studies will focus on quantifying targeting specificity through\nreceptor-blocking or competitive binding assays, as well as comparisons between homologous and\nheterologous membrane sources. Comprehensive evaluations of systemic biodistribution,\npharmacokinetics, and organ toxicity, including long-term and repeated dosing studies, will also be\nrequired. In addition, comparative validation using adult- or OA-derived membrane sources will be\nperformed to further strengthen evidence for targeting precision and long-term safety”.\nThere are many small molecule inhibitors of BRD4. Why must a BRD4 degrader be used? Have small\nmolecule inhibitors been compared for the treatment of OA?\nResponse: We thank the reviewer for this important suggestion. The choice of a BRD4 degrader rather\nthan a small-molecule inhibitor in this study was driven by both mechanistic considerations and the\nintended application context. Currently, the clinical development of pan-BRD4 inhibitors has been\nlimited by adverse effects, with several BET inhibitors (e.g., MK-8628) reported to induce dose-\nlimiting toxicities such as thrombocytopenia, diarrhea, nausea, and vomiting (PMID: 39918129; PMID:\n26367796). In contrast, BD1- or BD2-selective BRD4 inhibitors have been developed to reduce\ntoxicity by more precise modulation of bromodomain activity, thereby minimizing off-target effects\nand pathway bypass activation, and have shown improved safety profiles compared with pan-BRD4\ninhibitors (doi:10.1016/j.rechem.2025.102670). However, BRD4 contains not only two bromodomains\n(BD1 and BD2), which mediate recognition of acetylated lysine residues and chromatin association,\nbut also a histone acetyltransferase (HAT) domain capable of catalyzing histone lysine acetylation\n(PMID: 30466442). In addition, BRD4 plays non–bromodomain-dependent scaffold roles in\ntranscriptional complex assembly and phase separation. Therefore, selectively blocking bromodomain\ninteractions alone may be insufficient to fully abrogate BRD4 function. In contrast, BRD4 degraders\neliminate the BRD4 protein itself via the ubiquitin–proteasome pathway, enabling more comprehensive\nsuppression of the upstream regulatory cascade linking BRD4, histone acetylation, and Scn9a (Nav1.7)\ntranscription, while reducing the risk of functional rebound due to residual BRD4 activity. In the\n19\ncontext of osteoarthritis, a chronic and localized disease, our strategy combines intra-articular injection\nwith an in situ gelation system to achieve sustained local exposure. Under these conditions, an event-\ndriven degradation strategy is particularly advantageous compared with occupancy-driven inhibition, as\nit allows prolonged pathway suppression at lower dosing frequency. Although we did not directly\ncompare BRD4 degraders with small-molecule BRD4 inhibitors in the present study, we agree that\nsuch comparisons would be valuable for further optimization of therapeutic strategies. Accordingly, we\nplan to conduct comparative efficacy and safety evaluations between dBET1 and representative BRD4\ninhibitors under equivalent local exposure conditions in future work to further support translational\ndevelopment.\n20"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed this reviewer’s comments by either performing new experiments or providing clarification.", "reviewer_2": "The authors have addressed all of my questions thoroughly and clarified my concerns.", "reviewer_3": "I recommend acceptance. The authors have addressed the main concerns by adding human cell data and rescue experiments, as well as clarifying the material properties (such as physical loading and injectability). These additions improve the study. I suggest checking the statistics in the figure legends and updating the abstract."}, "author_response": ""}]

Decision letter