Quantifying the Shape and Stiffness of Single Extracellular Vesicles in Aqueous Solution via Membrane Diffusivity...

Quantifying the shape and stiffness of extracellular vesicles (EVs) is essential for understanding their biophysical properties and roles in intercellular communication. However, achieving single-particle resolution under physiological conditions remains a significant challenge. Here, we introduce an approach that integrates single-molecule diffusivity mapping (SMdM) with diffusion models for spherical and discoidal shapes to quantify the geometric and mechanical properties of individual liposomes and EVs in aqueous solution. Our findings identify charged lipids and cholesterol as critical factors that enhance liposome stiffness, driving their shapes closer to spheres. Applying this method to EVs reveals that those derived from tumor cells exhibit lower stiffness compared to EVs from normal cells, consistent with the biomechanical characteristics of their parent cells. This rapid, high-throughput strategy for characterizing the shape and stiffness of single EVs in aqueous solution offers promising applications in cancer biomarker discovery and the development of EV-based therapeutics.