Análise física e bioquímica de vesículas extracelulares isoladas com depleção de lipoproteínas plasmáticas

Abstract

Extracellular vesicles (EVs) are small membranous vesicles shed by all cell types. EVs are able to transport several biomolecules from parental cell and transfer then to target cells in an objective way. Therefore, EVs participate in many biological processes mediating cells communication, immune response and homeostasis. Previous attempts of isolating EVs from plasma have shown contamination with metabolic and inflammatory responses. Therefore, our aim was to standardize protocols for EVs isolation avoiding lipoprotein contamination. In the first work addressed in this dissertation, we present a protocol standardized by our group for the concomitant isolation of EVs and low or very low-density lipoproteins (LDL or VLDL) from plasma through ultracentrifugation of a density- based gradient (G-UC). In the following work, we tested different methods for isolating EVs without lipoprotein contamination following lipoprotein depletion by G-UC. Firstly, plasma was applied to G-UC for lipoprotein depletion and then subjected to serial centrifugation (SC) or applied to a size exclusion column (SEC) for isolation of EVs. The analysis of EVs population was made through nanoparticle tracking analysis (NTA) and flow cytometry. The absence of lipoproteins in the isolated EVs was confirmed through quantification of cholesterol and detection of apolipoprotein B100 (apoB-100) through western blot analysis. We employed bottom-up proteomics aiming the large-scale analysis of proteins in EVs isolated through the different approaches. We showed that SEC separates EVs from high-density lipoproteins (HDL) but not from LDLs or VLDLs, which remained contaminants. G-UC was efficient in separating lipoproteins from the EVs-enriched plasma fraction, allowing subsequent isolation of EVs depleted from lipoprotein contamination. Combined analysis from EVs proteomics, cholesterol quantification and apo B-100 detection confirmed the elimination of LDL and VLDL contamination by G-UC before EVs isolation. Proteomic analysis identified similar protein numbers and similar biological pathways in isolated EVs regardless of lipoprotein depletion, which was consistent with similar cellular source identified by flow cytometry. Taken together our results demonstrate that the combination of G-UC followed by SEC can provide lipoprotein-free EVs without bias of cellular origin and function, allowing the obtention of high pure EVs with potential implications for functional assays and lipidomic analysis.