Investigation of Lipid-PEG Anchorage of Proteins onto Mesenchymal Stem Cell Derived Extracellular Vesicles for Intracellular Delivery: A Quantitative Assessment

Extracellular Vesicles (EVs) are promising natural therapeutic vectors for delivery of cargos. However, loading of proteins into/onto EVs remains a major challenge limiting their interest as protein delivery system. Major hurdles are low loading efficiency and lack of absolute metrics regarding protein loading and cellular uptake. Here, interest of Lipid-PEG anchorage to trigger cargo protein association to murine mesenchymal stem cells derived EVs for intracellular delivery was investigated. HRP was used as a functional model protein to focus this study on absolute quantitative metrics. Impact of different structural parameters such as lipid anchor nature (cholesterol and DSPE), PEG linker molecular weight (MW) and physical parameters (temperature, cargo concentration) were studied. In contrast to native HRP, Lipid-PEG-HRP was significantly and stably associated to EVs and intracellularly delivered. Cholesterol anchor allowed an association of approximately 1000 HRP per EVs which was 3.5-folds higher than DSPE, while highest MW PEG linker impacted cell internalization by steric hindrance. Control of the HRP per EV ratio yielded to an efficient cellular uptake in three carcinoma cell lines (PANC-1, A549 and SKBR3 cells). By thoroughly characterizing Lipid-PEG-protein anchorage, this study evidences the compatibility of this approach for EV protein delivery in addition to provide useful metrics.