Loading of ”cocktail siRNAs” into extracellular vesicles via TAT-DRBD peptide for the treatment of castration-resistant prostate cancer

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules between cells. As natural vehicles, EVs may exhibit higher delivery efficiency, lower immunogenicity, and better compatibility than existing RNA carriers. A major limitation of their therapeutic use is the shortage of efficient, robust, and scalable methods to load siRNA of interest. Here, we report a novel strategy using polycationic membrane-penetrating peptide TAT to encapsulate siRNAs into EVs. Three TAT peptides were co-expressed with DRBD as 3TD fusion protein. The sequence-independent binding of DRBD facilitates multiplex genes targeting of mixed siRNAs. Functional assays for siRNA-mediated gene silencing of CRPC were performed after engineered EVs treatment. EVs were isolated using differential centrifugation from WPMY-1 cell culture medium. The increase of merged yellow fluorescence in the engineered EVs showed by TIRFM and the decrease in zeta potential absolute values certified the co-localization of siRNA with EVs, which indicated that siRNA had been successfully delivered into WPMY-1 EVs. qRT-PCR analysis revealed that the mRNA level of FLOH1, NKX3, and DHRS7 was dramatically decreased when cells were treated with engineered EVs loaded with siRNAs mixtures relative to the level of untreated cells. Western and flow cytometry results indicate that delivery of siRNA mixtures by engineered EVs can effectively downregulate AR expression and induce LNCaP-AI cell apoptosis. The uptake efficiency of the EVs and the significantly downregulated expression of three genes suggested the potential of TAT as efficient siRNA carriers by keeping the function of the cargoes.