Quantitative Capillary Electrophoresis for Analysis of Extracellular Vesicles (EVqCE)

Yuchu Dou,Lixuan Ren,Emil Zaripov,Prabir Kulabhusan,Maxim Berezovski

doi:10.3390/separations8080110

Abstract

Extracellular Vesicles (EVs) gained significant interest within the last decade as a new source of biomarkers for the early detection of diseases and a promising tool for therapeutic applications. In this work, we present Extracellular Vesicles Quantitative Capillary Electrophoresis (EVqCE) to measure an average mass of RNA in EVs, determine EV concentrations and the degree of EV degradation after sample handling. We used EVqCE to analyze EVs isolated from conditioned media of three cancer cell lines. EVqCE employs capillary zone electrophoresis with laser-induced fluorescent detection to separate intact EVs from free nucleic acids. After lysis of EVs with a detergent, the encapsulated nucleic acids are released. Therefore, the initial concentration of intact EVs is calculated based on a nucleic acid peak gain. EVqCE works in a dynamic range of EV concentrations from 108 to 1010 particles/mL. The quantification process can be completed in less than one hour and requires minimum optimization. Furthermore, the average mass of RNA was found to be in the range of 200–400 ag per particle, noting that more aggressive cancer cells have less RNA in EVs (200 ag per particle) than non-aggressive cancer cells (350 ag per particle). EVqCE works well for the degradation analysis of EVs. Sonication for 10 min at 40 kHz caused 85% degradation of EVs, 10 freeze-thaw cycles (from −80 °C to 22 °C) produced 40%, 14-day storage at 4 °C made 32%, and vortexing for 5 min caused 5% degradation. Presently, EVqCE cannot separate and distinguish individual EV populations (exosomes, microvesicles, apoptotic bodies) from each other. Still, it is tolerant to the presence of non-EV particles, protein-lipid complexes, and protein aggregates.

Highlights

Extracellular Vesicles (EVs) are biological particles released from various cells and found in large numbers in the body fluid, including blood, saliva, amniotic fluid, and urine.Most EVs consist of different molecules like proteins and nucleic acids, delimited by a phospholipid bilayer
We extended our previously developed method, viral quantitative capillary electrophoresis (Viral qCE), [31,32] for separation of intact EVs from impurities, for measuring RNA amount per EV, concentrations of EVs, and their degradation levels after isolations from cancer cells
In the flow cytometry analysis, anti-CD63 APC antibody and anti-CD9 PE antibody were used to mark the positive subpopulation of EVs

Summary

Introduction

Extracellular Vesicles (EVs) are biological particles released from various cells and found in large numbers in the body fluid, including blood, saliva, amniotic fluid, and urine.Most EVs consist of different molecules like proteins and nucleic acids (mRNA, miRNA, DNA), delimited by a phospholipid bilayer. Extracellular Vesicles (EVs) are biological particles released from various cells and found in large numbers in the body fluid, including blood, saliva, amniotic fluid, and urine. EVs have a diameter range from 50–1000 nm, which can be distinguished as three broad classes, including exosomes (50–150 nm, originating from the endosomal pathway) and microvesicles (100–1000 nm, released from the plasma membrane) and apoptotic bodies (>1000 nm, budded during cell death) [1,2,3,4]. It is difficult to isolate different sub-categories of EVs since the collected samples contain heterogeneous EV populations. Accurate quantitative analysis of EVs and their contents is considered an essential and challenging topic [6,7,8,9,10,11]. EV quantification has proven technically challenging due to their small size and heterogeneity in size and composition

Methods

Findings

Discussion

Conclusion