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

Abstract

Extracellular vesicles (EVs)�including exosomes and micro-vesicles�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 wellestablished 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.

Engineered Exosome for Drug Delivery: Recent Development and Clinical Applications

“…After the donor cells are loaded with target molecules, the MVBs can deliver active molecules to extracellular vesicles via ILVs, as shown in Figure 2 . 19 , 20 Notably, the co-incubation method is one of the most widely used drug loading strategies, and they put the drugs and donor cells together, which induces new biological characteristics in exosomes by altering culture conditions and the incubation environment of donor cells. Critically, this method involves selecting suitable donor cells, such as fibroblasts and hematopoietic stem cells, and then incubating them with drugs suspended in the culture medium.…”

Extracellular Vesicles: A New Star for Gene Drug Delivery

“…For instance, Es-Haghi et al 134 developed a fusion protein of EVs membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2) to achieve the RNA loading, and the results indicated that EVs contained high levels of miRNA and these engineered EVs could also transfer RNA cargo to recipient cells more efficiently. Bui et al 135 achieved endogenous cargo loading by interacting with CD63 and improved the delivery efficiency of EVs by fusion without requiring any viral fusogenic protein. The results indicated that the delivery efficiency of the cargo was significantly improved by the overexpressing of Syncytin-1.…”

Bioengineering extracellular vesicle cargo for optimal therapeutic efficiency

“…A VGV-G-free method of rapamycin-FKBP-FRB EV cargo loading has been developed through the use of Syncytin-1, an endogenous human fusogen protein. 117 …”