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

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.