Mitochondrial and bioenergetic processes are enriched in large-but are reciprocally depleted in small extracellular vesicles

Extracellular vesicles (EVs) are a promising class of drug delivery carriers for a broad range of therapeutic applications. We have previously demonstrated that brain endothelial cell (BEC)-derived large EVs (>200 nm in particle diameter) increased mitochondrial function in recipient BECs and improved therapeutic outcomes in a mouse model of ischemic stroke. In the current study, we demonstrate using size-resolved quantitative proteomics and gene-set enrichment analysis that mitochondrial and bioenergetic processes are enriched in large- but are reciprocally depleted in small EVs (exosomes, <200 nm in particle diameter). These results are striking and reveal the selective packaging of mitochondrial cargo in large but not small EVs. These strong mechanistic data anchor the rationale for the continued development of large EV mitochondria as a potential approach for the treatment of multiple diseases associated with mitochondrial dysfunction.