Scalable Preparation of Extracellular Vesicles Encapsulating Anticancer Drugs by High-Pressure Homogenization for Targeted Cancer Therapy

Extracellular vesicles (EVs) hold great potential as drug delivery platforms owing to their unique properties involved in intercellular communication. However, several limitations, including limited drug encapsulation efficiency and low scalability of conventional methods, pose challenges to the clinical translation of EVs as delivery vehicles. Given the success achieved in the manufacture of lipid-based nanoparticles to date, high-pressure homogenization (HPH) has the potential to be an alternative approach for the scale-up production of drug-encapsulated EV therapeutics. Herein, the usefulness of HPH was investigated for the scalable preparation of EVs encapsulating the anticancer drug paclitaxel (PTX) and their application in targeted cancer therapy. Encapsulation of PTX into bovine milk-derived EVs (mEVs) was efficiently achieved by simultaneously mixing distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), PTX, and mEVs prior to HPH. Process conditions optimized in a laboratory-scale instrument enabled the scale-up preparation of PEG-modified PTX-encapsulated mEVs (PTX-mEVs) at a 100 mL scale using a larger industrial-scale instrument. In addition, enhanced delivery of encapsulated PTX to cancer cells was achieved by modifying PTX-mEVs with cyclo(Arg-Gly-Asp-d-Phe-Lys) (cRGD) grafted DSPE-PEG using HPH. Furthermore, the intravenous administration of cRGD-modified PTX-mEVs significantly suppressed tumor growth in subcutaneous Colon-26 tumor-bearing mice and exhibited more potent antitumor efficacy than PEG-modified PTX-mEVs. These results suggest, for the first time, that the application of HPH offers great potential for the scalable preparation of drug-encapsulated EV therapeutics via a one-step pharmaceutical process and that the prepared EV therapeutics should be useful for cancer therapy.