Augmenting Intracellular Cargo Delivery of Extracellular Vesicles in Hypoxic Tissues through Inhibiting Hypoxia-Induced Endocytic Recycling

As mesenchymal stem-cell-derived small extracellular vesicles (MSC-sEVs) have been widely applied in treatment of degenerative diseases, it is essential to improve their cargo delivery efficiency in specific microenvironments of lesions. However, the interaction between the microenvironment of recipient cells and MSC-sEVs remains poorly understood. Herein, we find that the cargo delivery efficiency of MSC-sEVs was significantly reduced under hypoxia in inflammaging nucleus pulposus cells due to activated endocytic recycling of MSC-sEVs. Hypoxia-inducible factor-1 (HIF-1)-induced upregulated RCP (also known as RAB11FIP1) is shown to promote the Rab11a-dependent recycling of internalized MSC-sEVs under hypoxia via enhancing the interaction between Rab11a and MSC-sEV. Based on this finding, si-RCP is loaded into MSC-sEVs using electroporation to overcome the hypoxic microenvironment of intervertebral disks. The engineered MSC-sEVs significantly inhibit the endocytic recycling process and exhibit higher delivery efficiency under hypoxia. In a rat model of intervertebral disk degeneration (IDD), the si-RCP-loaded MSC-sEVs successfully treat IDD with improved regenerative capacity compared with natural MSC-sEV. Collectively, the findings illustrate the intracellular traffic mechanism of MSC-sEVs under hypoxia and demonstrate that the therapeutic capacity of MSC-sEVs can be improved via inhibiting endocytic recycling. This modifying strategy may further facilitate the application of extracellular vesicles in hypoxic tissues.