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

Extracellular vesicles (EVs) are membrane-enclosed biological nanoparticles withpotential as diagnostic markers and carriers for therapeutics. Characterizationof EVs poses severe challenges due to their complex structure andcomposition, requiring the combination of orthogonal analytical techniques.Here, we demonstrate how liquid chromatography combined with multi-anglelight scattering (MALS) and fluorescence detection in one single apparatuscan provide multiparametric characterization of EV samples, includingconcentration of particles, average diameter of the particles, proteinamount to particle number ratio, presence of EV surface markers andlipids, EV shape, and sample purity. The method requires a small amountof sample of approximately 10(7) EVs, limited handling ofthe sample and data analysis time in the order of minutes; it is fullyautomatable and can be applied to both crude and purified samples.

Automated summary

This study demonstrates that liquid chromatography combined with multi-angle light scattering (MALS) and fluorescence detection can provide multiparametric characterization of extracellular vesicles (EVs) in one single apparatus. The method allows for analysis of EV concentration, average diameter, protein amount to particle number ratio, presence of surface markers and lipids, EV shape, and sample purity. It is fully automatable and can be applied to both crude and purified samples with limited sample handling and data analysis time.