Unravelling Ligand Conjugation Performance in Extracellular Vesicles: A Quantitative Assessment of Lipid, Protein,...

Surface functionalization is an effective approach for enhancing the cancer-targeting efficiency of extracellular vesicles (EVs). However, the lack of direct comparisons between functionalization strategies has hindered the rational design of EV delivery systems. To address this gap, we developed a nano-flow cytometry-based methodology to quantitatively evaluate ligand conjugation and its relationship to targeting efficiency across three representative post‑production EV engineering strategies: lipid modification, protein modification, and membrane insertion. All three strategies achieved high conjugation efficiencies (>90%) with ligand densities ranging from several to tens of ligands per 100 nm² under optimized conditions. Beyond ligand density, functionalization strategies resulted in varying degrees of ligand clustering and reduced accessibility of endogenous cell-binding proteins on EVs, such as MFGE8, leading to differences in targeting performance. For milk-derived EVs, lipid modification achieved the highest ligand density, the most uniform conjugation, and minimal disruption to surface protein accessibility, yielding superior cancer-targeting efficiency. These findings highlight the importance of precise quantification of ligand conjugation performance and provide a robust methodology for optimizing surface functionalization to advance EV-based drug delivery.