Development of Small Interfering RNA-Loaded Extracellular Vesicles via a High-Pressure Homogenization Approach

Extracellular vesicles (EVs) have garnered significant interest as potential bioderived drug delivery systems for RNA therapeutics owing to their distinctive characteristics, which include their inherent capability to transport various biological molecules throughout the body. However, several issues still pose challenges, such as the limited loading of exogenous RNAs and poor scalability of conventional techniques. High-pressure homogenization (HPH) is a useful alternative for the scalable preparation of EVs encapsulating small-molecule therapeutic agents via a one-step pharmaceutical process. However, whether HPH can be applied to macromolecular drugs, such as small interfering RNA (siRNA), remains unclear. Herein, we report the applicability of using HPH to prepare siRNA-loaded EVs and their subsequent cytoplasmic siRNA delivery. Simultaneous HPH of bovine milk-derived EVs (mEVs) with distearoylphosphatidylethanolamine-polyethylene glycol-polyethyleneimine and cholesterol-conjugated siRNA allowed siRNA loading onto the mEVs. Modification of the mEVs with the tumor-targeting ligand, cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD), showed high affinity for a human glioma cell line, resulting in remarkable intracellular siRNA delivery. Moreover, siRNA delivered with the cRGD-modified mEVs showed a higher knockdown efficiency than that of the unmodified mEVs. The present findings suggest, for the first time, that HPH holds potential for the development of siRNA-loaded EV therapeutics that could be useful for cytoplasmic siRNA delivery.