Evaluation of size-exclusion and multimodal chromatography for enrichment of extracellular vesicles from blood plasma

Abstract

Extracellular vesicles (EVs) are bilayer lipid membrane-enclosed vesicles shed by cells into physiological fluids. EVs serve as important mediators of cell-cell communication, which can transfer proteins, messenger RNA, microRNAs, metabolites and other biological molecules. There is a significant interest in EVs as potential novel tools for liquid biopsy-based disease diagnostics and therapy delivery. In this work, we developed and evaluated new techniques for EV enrichment from human blood plasma using size-exclusion chromatography (SEC) and multimodal chromatography. In brief: 1. we evaluated different column bed volumes for the SEC-based EV enrichment, and the column packed with 4 mL of the SEC stationary phase Sepharose CL-2B showed the most efficient EV separation; 2. The maximum protein binding capacity of multimodal chromatography resins CaptoCore 400 and CaptoCore 700 in both suspension mode and the column format were determined. 3. We combined the size-exclusion chromatography and multimode chromatography approaches to further increase the purity of EV enrichment from blood plasma and decrease the levels of free plasma proteins in EV isolates. The developed biphasic 'sandwich' columns packed with two layers of SEC and multimode CaptoCore stationary phases placed on top of each other and sequential depletion of plasma proteins by the CaptoCore beads were evaluated for EV enrichment from blood plasma. Higher purity of EV isolation by the 'sandwich' column approach was confirmed by EV count/size distribution and free protein concentration measurements. However, the "sandwich" columns packed with a layer of CaptoCore 700 demonstrated a better plasma protein binding capacity but lower recovery rate of vesicles, while "sandwich" columns packed with a layer of CaptoCore 700 showed a higher EV recovery rate, but a little lower plasma protein binding capacity. The EV isolates prepared using the developed enrichment techniques were evaluated by means of transmission electron microscopy (TEM), nano-flow cytometry, proteomics profiling using nanoscale liquid chromatography coupled to tandem mass spectrometry, and glycomic profiling of released N-glycans using capillary electrophoresis coupled to tandem mass spectrometry. The microscopy- and molecular profiling-based techniques confirmed that the developed techniques could effectively enrich EVs from complex physiological fluids at high purity.