Elucidation of extracellular vesicles behavior during capillary isoelectric focusing

In this study, we investigated the behavior of extracellular vesicles (EVs), during capillary isoelectric focusing (cIEF). For that, we used different approaches, imaging cIEF with a whole-column imaging detection (WCID) and conventional cIEF as well as different detection methods (LIF after EV labelling, native fluorescence and UV). Our study reveals that EVs exhibit significant aggregation during their migration toward, and upon reaching, their isoelectric point (pI). By optimizing key parameters such as voltage and the addition of solubilizers, we successfully reduced this issue, particularly with bovine milk EVs. Our findings also showed distinct pI regions observed for EVs isolated from different sources: bovine milk EVs shows acidic pI characteristics (4.0–4.1), while pig and human plasma EVs exhibit more basic pI zones (4.7–4.9 and 5.8–6.7, respectively). The study was extended to cIEF coupled to laser induced fluorescence detection (LIF) using intra-vesicular CFDA-labeled EVs, to better understand their susceptibilities. Prolonged mobilization time due to long capillary lengths adversely affected EV’s integrity in conventional cIEF. Our study reveals the necessity to specific cIEF optimization for each EV source due to variations in charge distribution and aggregation behavior across different pI regions. The use of a short capillary length (<10 cm), low electric field and solubilizers such as Tween-20 is recommended to preserve EVs integrity during cIEF-EV studies.