Targeted delivery of engineered extracellular vesicles to simultaneously promote vascularization and muscle regeneration in ischemic limbs.

Critical limb ischemia (CLI) leads to a high rate of limb amputation. Regenerating vasculature and skeletal muscles can save the affected limbs. Therapy using stem cell-derived extracellular vesicles (EVs) has emerged as a promising approach. However, the therapeutic efficacy is limited because EVs were not engineered to simultaneously possess the optimal composition of proangiogenic and promyogenic factors necessary to effectively support the survival, migration, and morphogenesis of endothelial and skeletal muscle cells under ischemic conditions. We discovered that the proangiogenic and promyogenic factors, including miR-126, miR-21, miR-296, miR-182, PDGF-BB, VEGF, bFGF, and HGF, can be concurrently upregulated in EVs derived from human iPSC-derived mesenchymal stem cells (iMSCs) by enhancing either N-cadherin-mediated or RGD-mediated interactions between the cells and matrix. Notably, enhancing N-cadherin interaction was more effective in upregulating these factors. The EVs from enhanced N-cadherin interaction markedly improved survival, migration, and morphogenesis of endothelial cells and myoblasts under the CLI-like conditions. To ensure targeted delivery to ischemic limbs, these EVs were cloaked with platelet membranes modified with an ischemia-homing peptide. Following intravenous delivery in a murine model of ischemic hindlimb, the EVs fully restored blood perfusion within 28 days, and significantly promoted skeletal muscle regeneration. These results underscore the potential of EVs with simultaneously upregulated proangiogenic and promyogenic factors in effectively treating CLI.