Enhanced Separation of Extracellular Vesicles Using Capillary Isotachophoresis With Spacer Compounds

Extracellular vesicles (EVs) are pivotal in numerous physiological and pathological processes, such as immune responses, viral pathogenesis, pregnancy, cardiovascular diseases, and cancer progression. Their capacity to influence complex intracellular pathways highlights their therapeutic potential in addressing various conditions, including neurodegenerative diseases and cancer. A novel capillary isotachophoresis (cITP) method was developed for the electrokinetic characterization of pre-isolated EVs. Distinct peaks could be resolved at near-baseline resolution using a novel mixture of spacer ions and laser-induced fluorescence (LIF) detection. The vesicles were effectively separated from the unbound carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) amine-reactive fluorescent stain used to detect them and from residual contaminants. The identity of the peaks shown in the electropherograms was validated via various methods, including incubation with specific antibodies or spiking of putative contaminants, such as proteins and lipoproteins. This report thus provides a detailed proof-of-concept for using cITP-LIF for extracellular vesicle isolation, subtype fractionation, and profiling.