Phospholipid‐Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting

Abstract Extracellular vesicles (EVs) are recognized as potential candidates for next‐generation drug delivery systems. However, the inherent cancer‐targeting efficiency is unsatisfactory, necessitating surface modification to attach cell‐binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide‐containing primary alcohol, the authors successfully anchored ligands onto milk‐derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano‐flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long‐term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost‐effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.