Virus-Free Method to Control and Enhance Extracellular Vesicle Cargo Loading and Delivery

[Image: see text] Extracellular vesicles (EVs)—including exosomes and microvesicles—are involved in cell–cell communication. EVs encapsulate different types of molecules such as proteins or nucleotides and are long-lasting contenders for the establishment of personalized drug delivery systems. Recent studies suggest that the intrinsic capacities for uptake and cargo delivery of basic EVs might be too limited to serve as a potent delivery system. Here, we develop two synergistic methods to, respectively, control EV cargo loading and enhance EV cargo delivery through fusion without requirement for any viral fusogenic protein. Briefly, cargo loading is enabled through a reversible drug-inducible system that triggers the interaction between a cargo of interest and CD63, a well-established transmembrane EV marker. Enhanced cargo delivery is promoted by overexpressing Syncytin-1, an endogenous retrovirus envelop protein with fusogenic properties encoded by the human genome. We validate our bioengineered EVs in a qualitative and quantitative manner. Finally, we utilize this method to develop highly potent killer EVs, which contain a lethal toxin responsible for protein translation arrest and acceptor cell death. These advanced methods and future downstream applications may open promising doors in the manufacture of virus-free and EV-based delivery systems.