Designing liposomal EV mimetics by tuning mechanical properties

Extracellular vesicles (EVs) exhibit unique biological performance in intercellular communication and drug delivery, yet their clinical translation is hindered by limited scalability, compositional heterogeneity, and regulatory complexity. Here, we report the rational design at the nanoscale of fully synthetic liposomal EV mimetics (Lipo-EVs) engineered to replicate the key biomechanical properties of small EVs while retaining the manufacturability and compositional control of synthetic systems. By systematically tuning membrane lipid composition, with a particular focus on cholesterol content, we identify a formulation that closely reproduces the nanoscale membrane packing, structure-property relationships, and nanomechanical behavior of HEK293-derived EVs. Advanced mechano-fluorescence assays and atomic force microscopy reveal that Lipo-EVs interact with model membranes in a manner closely resembling native EVs, especially under cholesterol-rich conditions, highlighting key principles of nanoscale membrane organization and interface mechanics. The optimized Lipo-EVs further display high colloidal stability, robust nanoscale integrity, compatibility with lyophilization and recovery, and efficient, homogeneous cellular uptake without detectable cytotoxicity.