Lipid Chain Length-dependent Anchoring of Artificial Lipidated Proteins Enables Cell-Selective Uptake of Extracellular Vesicles

Abstract Extracellular vesicles (EVs) are promising drug delivery carriers; however, systematic and minimally invasive strategies for EV surface protein display remain limited. Here, we report a modular approach based on microbial transglutaminase-mediated site-specific lipidation to prepare molecularly uniform lipid–protein conjugates with precisely defined alkyl chain lengths. Quantitative analysis revealed lipid chain length-dependent EV membrane anchoring. Extension to a human epidermal growth factor receptor (HER2)-targeting affibody enabled selective EV uptake modulation, enhancing internalization in HER2-positive cells while suppressing nonspecific uptake in HER2-negative cells. This strategy provides a tunable platform for rational EV surface engineering.