Selective peptide–guided transcytosis enhances extracellular vesicle–mediated siRNA delivery across the blood–brain barrier

Extracellular vesicles (EVs) have clinically emerged as promising biocompatible vesicles for delivering therapeutic siRNAs to the central nervous system. Among targeting strategies, the rabies virus glycoprotein (RVG) peptide is the most commonly used modification on the EV surface to enable efficient systemic delivery of EVs. Although RVG is widely believed to facilitate blood-brain barrier (BBB) through receptor interactions, the underlying mechanism remains indirect and equivocal. Similarly, cell-penetrating peptide (CPP) modifications have been used to enhance BBB transport of various vehicles, such as CPP.16, which improves the brain delivery efficiency of adeno-associated virus 9 capsids. However, whether CPP.16 retains its delivery efficacy when applied to EVs remains unclear, raising concerns about carrier-specific limitations. In this study, we investigate the mechanisms underlying the transcytosis and delivery efficiency of RVG- and CPP.16-modified small EVs (sEVs) loaded with siRNAs. Using an in vitro BBB model, we found that these modifications do not alter the internalization of siRNAs by endothelial cells. Instead, these modifications appear to divert sEVs and siRNAs into transcytotic pathways, enabling their release into abluminal cells and subsequent target gene silencing. Moreover, RVG-sEVs primarily interact with the receptor and are internalized via clathrin-mediated endocytosis, leading to more efficient BBB penetration compared with CPP.16-sEVs. Consistently, in vivo studies demonstrate that RVG-sEVs deliver siRNAs more efficiently to both neurons and astrocytes compared with unmodified or CPP.16-sEVs. Our findings support the clinical potential of BBB-targeting peptides and provide critical insights for the rational selection of guiding peptides in central nervous system drug delivery.