Simultaneous targeting of TGF-β1/PD-L1 via a hydrogel-nanoparticle system to remodel the ECM and immune microenvironment for limiting adhesion formation

Adhesion seriously affects the recovery of tendon gliding function. Our group previously found that inhibition of TGF-β1, which is closely related to adhesion formation, effectively attenuated adhesions but did not eliminate them, suggesting that there may be other mechanisms involved in adhesion formation. In this study, we considered that uncontrolled and excessively proliferating fibroblasts undergo immune escape, which aggravates the deposition of extracellular matrix during the adhesion formation. We found that the expression of the immune checkpoint PD-L1 was significantly elevated after injury and may be involved in adhesion formation. Therefore, we intended to silence both TGF-β1 and PD-L1 to improve the immune advantage in the microenvironment after flexor tendon injury to further reduce adhesion. We constructed the nanoparticle/TGF-β1 or/and PD-L1 siRNA complexes and verified their high biocompatibility and high transfection efficiency. We found that CD8⁺ T cells had a greater killing effect on the excessively proliferating cells that were transfected with nanoparticle/TGF-β1 or/and PD-L1 siRNAs. The hydrogel-nanoparticle/TGF-β1 or/and PD-L1 siRNA system could effectively improve the gliding function of the tendons without weakening the mechanical properties in injured rat FDL tendon and chicken FDP tendon models. In addition, the potential of CD8⁺ T cells to encircle the adhesion cells on the tendon surface was observed, which resulted in increased levels of cell apoptosis. Thus, our study confirmed that combined knockdown of TGF-β1 and PD-L1 could activate immunodominance after flexor tendon repair and provided a potential treatment to limit adhesion formation and improve gliding function. STATEMENT OF SIGNIFICANCE: Adhesion seriously affects the recovery of tendon gliding function. TGF-β1 is related to adhesion formation as it regulates the production of extracellular matrix. We found that excessively proliferated fibroblasts might undergo immune escape, which aggravated the deposition of extracellular matrix. Therefore, we constructed a hydrogel-nanoparticle/TGF-β1 and PD-L1 siRNAs system for silencing TGF-β1 and PD-L1 to improve the immune advantage in the microenvironment after tendon injury. This system could improve the gliding function of tendons without weakening the mechanical property and increase the killing effect of CD8⁺ T cells. Combined knockdown of TGF-β1 and PD-L1 could activate immunodominance after tendon repair and provide a potential treatment to limit adhesion formation.