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 small interfering RNAs (siRNA) to the central nervous system (CNS). 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 peptides (CPPs) modifications have been used to enhance BBB transport of various vehicles, such as CPP.16, which improves the brain delivery efficiency of AAV9 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 receptor and are internalized via clathrin-mediated endocytosis, leading to more efficient BBB penetration compared to CPP.16-sEVs. Consistently, in vivo studies demonstrate that RVG-sEVs deliver siRNAs more efficiently to both neurons and astrocytes compared to unmodified or CPP.16-sEVs. Our findings support clinical potential of BBB-targeting peptides and provide critical insights for the rational selection of guiding peptides in CNS drug delivery.