PNNL

Abstract

Small extracellular vesicles (sEVs), also referred as exosomes, are cell-released vesicles ranging from 30-150nm in size. They have been a focus of increasing attention because of their potentials in disease diagnosis and therapeutics. The diversity of sEVs derives from their biological composition and cargo content. Currently, the isolations of sEV subpopulations are primarily based on bio-physical and affinity-based approaches. As a standardized definition for sEV subpopulations is yet to be fully established, it is important to further understand the correlation between the biomolecular composition of sEVs and their physical properties. In this study, we employed a platform combining single-vesicle based surface enhanced Raman spectroscopy (SERS) and machine learning to examine individual sEVs isolated based on their size. The biomolecular composition of each vesicle examined was reflected by its corresponding SERS spectral features (biomolecular “fingerprints”) with their roots in the composition of their collective Raman-active bonds. Origins of the SERS spectral features were validated through a comparative analysis between SERS and mass spectrometry (MS). SERS fingerprinting of individual vesicles was effective to overcome the challenge posed by EV population averaging, opening the possibility of analyzing the variations in biomolecular composition between the vesicles of similar and/or different sizes. Using this approach, we uncovered that each of the sizebased subpopulation of sEVs contained particles with predominantly similar SERS spectral features. Over 84% of the vesicles residing within in a particular group that were clearly distinguishable from that of the other EV sub-populations despite some features of spectral variations within each sub-population. Our results suggest that there exists a correlation between sEV size-based subpopulations and their biomolecular composition from the perspective of individual vesicles. Our findings therefore highlight the possibility that the biogenesis and respective biological functionalities of the various sEV subpopulations, as distinguished by the vesicle-size discrepancies, may be inherently different.