An anti-FAP-scFv-functionalized exosome-carrying hydrogel delivers SKI mRNA to fibrotic nucleus pulposus cells to alleviate intervertebral disc degeneration by regulating FOXO3.

Rationale: Nucleus pulposus (NP) fibrosis is a contributing factor to intervertebral disc degeneration (IVDD), which lacks an effective treatment. This study focuses on elucidating the role and mechanisms of the TGF-β signaling repressor SKI in NP fibrosis and exploring its therapeutic potential. Methods: Single-cell RNA sequencing (scRNA-seq) was used to investigate fibrotic nucleus pulposus cell (NPC) subsets and assess TGF-β signaling activation. Two recombinant plasmids encoding single-chain variable fragments (scFvs) targeting the fibrotic NPC marker FAP and SKI mRNA were co-transfected into HEK-293T cells to yield functionalized exosomes (EX(ski+scFv)). The addition of EX(ski+scFv) to a gelatin/oxidized sodium alginate hydrogel produced a pH-responsive exosome/hydrogel system named Gel@EX(ski+scFv). The therapeutic effects and underlying mechanism of Gel@EX(ski+scFv) were evaluated by RNA sequencing, molecular docking and coimmunoprecipitation. Results: A fibrotic NPC subset was characterized by elevated FAP and diminished SKI expression, along with activation of the TGF-β signaling pathway. SKI overexpression reduced fibrosis in TGF-β-treated NPCs. EX(ski+scFv) successfully delivered SKI mRNA to FAP-expressing fibrotic NPCs. Gel@EX(ski+scFv) possessed good mechanical properties, degradability, injectability, and biocompatibility. Gel@EX(ski+scFv) effectively alleviated NP fibrosis and IVDD in rats. RNA sequencing, molecular docking and coimmunoprecipitation revealed that SKI could interact with FOXO3 to suppress the TGF-β signaling pathway. Conclusion: This study elucidates the mechanism by which SKI-mediated TGF-β signaling inhibition alleviates NP fibrosis. The development of the Gel@EX(ski+scFv) system for the targeted delivery of SKI mRNA offers a promising therapeutic strategy to alleviate NP fibrosis and IVDD in the future.