Multiparametric Orthogonal Characterization of Extracellular Vesicles by Liquid Chromatography Combined with In-Line Light Scattering and Fluorescence Detection

[Image: see text] Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles with potential as diagnostic markers and carriers for therapeutics. Characterization of EVs poses severe challenges due to their complex structure and composition, requiring the combination of orthogonal analytical techniques. Here, we demonstrate how liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection in one single apparatus can provide multiparametric characterization of EV samples, including concentration of particles, average diameter of the particles, protein amount to particle number ratio, presence of EV surface markers and lipids, EV shape, and sample purity. The method requires a small amount of sample of approximately 10(7) EVs, limited handling of the sample and data analysis time in the order of minutes; it is fully automatable and can be applied to both crude and purified samples.