Lack of evidence for cargo release of CD63-EVs into recipient cells.

Extracellular vesicles (EVs), including exosomes, are widely believed to mediate intercellular communication by delivering molecular cargo into the cytosol of recipient cells. However, the efficiency and mechanisms of such cargo delivery remain unclear. In this study, we investigated whether EVs bearing the canonical tetraspanin EVs marker CD63 are capable of fusing with recipient cell membranes to release their contents. Using the highly sensitive NanoBiT split luciferase system, we tracked potential fusion events between HiBiT-tagged CD63-EVs and HEK293 cells expressing cytosolic or endosome-localized LgBiT. While HiBiT and LgBiT reconstitution was readily detected when EVs carried the viral fusion protein VSV-G, no luminescence was observed with HiBiT tagged CD63-EVs in absence of VSV-G, despite their uptake or binding by recipient cells. Similarly, EVs carrying HiBiT-HSP70 failed to show evidence of cargo release. These findings demonstrate that unmodified HiBiT-CD63-EVs do not fuse with the plasma or endosomal membranes of recipient cells, suggesting that EV-mediated cargo delivery via membrane fusion is inefficient or absent under standard culture conditions. Our results challenge the prevailing view of EV function in intercellular communication and underscore the need for re-evaluation of EV cargo transfer mechanisms.