An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization.

Onno J Arntz,Bartijn C H Pieters,Fons A J Van De Loo,Peter M Van Der Kraan,Peter L E M Van Lent,Marije I Koenders,Aftab A Ansari

doi:10.1371/journal.pone.0236508

Onno J Arntz, Bartijn C H Pieters + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0236508

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Abstract

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.

Highlights

Extracellular vesicles (EVs) are a heterogeneous group of membrane-covered nanoparticles of diverse sizes and shapes produced by almost all cells
From four etanercept-treated rheumatoid arthritis (RA) patients (p1-2-3-4) plasma EVs (pEVs) were isolated by three conventional isolation methods (UC, Polyethylene Glycol precipitation (PEG) or size exclusion chromatography (SEC)) in parallel
In the isolated pEV samples the presence of patients owns immunoglobulin G (IgG) levels were detected by ELISA and these levels were markedly lower in SEC isolated pEVs (Fig 1D)

Summary

Introduction

Extracellular vesicles (EVs) are a heterogeneous group of membrane-covered nanoparticles of diverse sizes and shapes produced by almost all cells. There are three different types of EVs: exosomes (30-100nm), microvesicles (100-1000nm) and apoptotic bodies (1–10μm) which are classified based on their origin. Apoptotic bodies originate from apoptotic cells, microvesicles from the plasma membrane and exosomes from multivesicular bodies [1,2,3]. EVs can be found in all body fluids such as breast milk, saliva, urine and blood [4] and their lipid bilayer membrane protect their content from proteases and nucleases present in body fluids. EV content consist out of different proteins, lipids, RNA and DNA molecules [5] and their role as important cell-to-cell communicators is widely accepted [6]. For instance transfer of proteins and/or RNA by EVs can affect gene expression in acceptor cells including genes that are involved in auto-immune diseases [7] and inflammatory processes [8]

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