Revealing the Heterogeneity of Extracellular Vesicles: From Population to Single Particle Level.

Extracellular vesicles (EVs) are secreted by cells and enclosed within lipid bilayers. These vesicles contain diverse biomolecular components, including proteins, nucleic acids, lipids, and metabolites. They serve critical roles in intercellular communication and regulate multiple physiological and pathological processes, such as immune modulation, angiogenesis, and tumorigenesis and metastasis. Notably, EVs exhibit marked heterogeneity in both physical characteristics and biomolecular composition. This article will systematically characterize the multidimensional heterogeneity of EVs at the population level through comprehensive analysis of their biogenesis origins, size distribution, surface protein, surface glycan chains, and surface lipid. Conventional population-level analyzes yield averaged molecular profiles that obscure subtype-specific functional correlations, thereby limiting mechanistic insights into EV subpopulation biology. To further understand EV heterogeneity, it is necessary to enhance our understanding about molecular characteristics of EVs from the population to the single particle level. Current single EVs analysis techniques mainly include super-resolution microscopy (SRM), atomic force microscopy (AFM), nanoparticle tracking Analysis (NTA), flow cytometry (FCM), surface enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and proximity barcoding assay (PBA). In this review, we systematically examine population-level EV heterogeneity; evaluate single-particle detection methodologies; and discuss emerging technologies (e.g., click chemistry, Olink proteomics, and molecular imprinting) for resolving single EV heterogeneity.