Methods and Challenges in Purifying Drug-Loaded Extracellular Vesicles.

Extracellular vesicles (EV) have emerged as promising nanocarriers for drug delivery. However, the efficient loading of therapeutic molecules into EV and the subsequent purification of drug-loaded EV from unloaded drugs remain significant challenges. This review explores the most used methods for EV purification, meaning the separation of drug-loaded EV from unloaded drugs, including ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, dialysis and commercial exosome isolation kits. The principles, advantages and limitations of each method are discussed. Critical parameters such as molecular weight cutoff, membrane composition, and the nature of the loaded molecule are highlighted for their impact on the purification process. The review also addresses the technical aspects, including time, cost and equipment requirements, and emphasizes the need for standardized guidelines to improve reproducibility and comparability across studies. By providing a comprehensive overview of current purification strategies, this review aims to guide researchers in selecting the most appropriate methods for advancing EV-based drug delivery systems.