cRGD engineered MSC-derived small extracellular vesicles as targeted delivery vehicles of miR-373 for liver fibrosis therapy

Liver fibrosis represents a major pathological consequence of chronic metabolic and inflammatory liver diseases, including Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), there is no currently available anti-fibrotic drugs. Small extracellular vesicles (sEV) are a novel cell-free therapy that contain abundant cell-derived miRNAs, mRNA, and proteins, which exert various biological effects. However, the precise roles and mechanisms of sEV derived from mesenchymal stem cells (MSC-sEV) in liver fibrosis remain inadequately understood. In this study, we aimed to investigate the effects of miR-373 contained in MSC-sEV on hepatic stellate cell (HSC) activation and collagen deposition in fibrotic liver tissues. We employed a series of in vitro and in vivo experiments, including the isolation of MSC-sEV through ultracentrifugation and the establishment of a liver fibrosis model in C57BL/6 mice using carbon tetrachloride (CCl 4 ). The efficacy of MSC-sEV and engineered MSC-sEV with cyclic arginine-glycine-aspartate (cRGD) and miR-373 was evaluated through Western blot, immunofluorescence analysis, and histological assessments. Our study demonstrates that miR-373 from MSC-sEV can suppress the expression of TGFβR2 in HSCs, resulting in the downregulation of p -Smad2/3 and HSC activation markers. Ultimately, this leads to the inhibition of collagen deposition in fibrotic liver tissues. Compared to MSC-sEV, MSC-sEV engineered with cRGD and miR-373 (cRGD/miR-373-MSC-sEV) significantly inhibited TGFβR2/ p -Smad2/3 signaling and HSC activation. Importantly, the injection of cRGD/miR-373-MSC-sEV did not cause any adverse effects on the structural integrity of the heart, spleen, lung, and kidney tissues in mice. Our research suggests that the transfer of miR-373, facilitated by MSC-sEV, can effectively inhibit HSC activation by targeting TGFβR2 and suppressing the Smad2/3 signaling pathway. Therefore, cRGD/miR-373-MSC-sEV holds promise as a promising therapeutic candidate requiring further efficacy and safety studies for the treatment of liver fibrosis.