Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

Abstract

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis ; molecular composition ; biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4) ; we identified two exosome subpopulations (large exosome vesicles ; Exo-L ; 90-120 nm; small exosome vesicles ; Exo-S ; 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed 'exomeres' (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia ; microtubule and coagulation proteins as well as specific pathways ; such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways ; and mitotic spindle and IL-2/STAT5 signalling pathways ; respectively. Exo-S ; Exo-L and exomeres each had unique N-glycosylation ; protein ; lipid ; DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns ; suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.