New strategies for the isolation and characterization of extracellular vesicles in human plasma

Abstract

Extracellular vesicles (EVs), especially exosomes, have attracted considerable attention in recent years as promising biomarkers for biomedical and clinical applications. These nanosized vesicles take part in intercellular communication and carry a wide variety of biomolecules, including proteins, lipids and nucleic acids, which reflect the physiological or pathological state of the cells from which they originate. Because of these characteristics, exosomes are increasingly explored for disease diagnosis, prognosis and therapeutic monitoring, especially in cancer, neurodegenerative disorders and inflammatory diseases. However, the isolation and characterization of EV from human biological fluids remain major analytical challenges. Biological matrices such as plasma hold high concentrations of proteins, lipoproteins and other nanoscale particles that can interfere with EV purification and analysis. In addition, the intrinsic heterogeneity of EV in size, composition and origin complicates the development of standardized and reproducible methodologies. Consequently, the selection of a proper isolation strategy is a critical factor that directly influences the quality, purity and analytical performance of the obtained samples. In the present study, different precipitation- and chromatography-based isolation methods were evaluated and compared to decide their suitability for EV extraction from plasma samples. The isolated fractions were characterized by Dynamic Light Scattering (DLS) and capillary electrophoresis with UV/Vis diode array detection (CE-UV/Vis-DAD). These analytical techniques were employed to assess the size distribution, colloidal behaviour and electrophoretic profiles of the obtained vesicles, as well as to evaluate the capability of the methodologies to isolate exosome-enriched fractions. The results obtained proved that the choice of isolation method has a direct impact on the physicochemical properties of the extracted vesicles, including particle size distribution, signal intensity and electrophoretic behaviour. Differences seen between the analysed samples suggest that each isolation approach may preferentially enrich distinct vesicle populations or co-isolate different plasma components. Furthermore, the study highlights the relevance of complementary analytical techniques for a more reliable characterization of EV and for the detection of possible contaminants or aggregation phenomena associated with the isolation process. Overall, this work emphasizes the importance of carefully selecting the isolation strategy according to the intended analytical or clinical application. The findings contribute to ongoing efforts to optimize and standardize EV characterization methodologies, supporting the development of more robust protocols for exosome-based biomarkers research