Daniel Levy, David Wang, Haseeb Afzali, Mai Anh Do, Jiayi Zhang, Renceh Flojo, Joy Ku, Kyle Asano, David Diebold, Aijun Wang, Biao Lu
Small extracellular vesicles (sEVs) are emerging as versatile, biologically derived nanocarriers for precision drug delivery, yet strategies to enhance their targeting efficiency remain limited. Inspired by viral tropism, this study investigates whether viral envelope glycoproteins (GPs) can co-opt native biogenesis pathways to functionalize sEV membranes. Diverse viral GPs from both DNA and RNA viruses-including VSVG, HSV-gpB, SARS-CoV-1 spike, and RD114A-are shown to efficiently incorporate into sEV membranes in human cells, independent of viral assembly. Live-cell confocal imaging and co-localization with canonical sEV markers (CD63, XPACK) reveals that these GPs hijacked endosomal trafficking routes to access the sEV biogenesis machinery. Strikingly, truncation of the ectodomain does not impede sEV sorting, indicating the transmembrane and cytoplasmic domains as primary determinants of incorporation. Functionally, sEV bearing VSVG exhibit over a 2-fold increase in uptake by recipient cells compared to unmodified sEVs. These findings uncover a conserved mechanism by which viral GPs exploit host sEV pathways and establish a modular strategy for sEV surface engineering. This work paves the way for the rational design of targeted, virus-inspired sEV therapeutics for cancer, neurological disease, and gene delivery applications.