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

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 ultracentrifuga...

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Bibliographic Details
Published in:PloS one 2020-07, Vol.15 (7), p.e0236508
Main Authors: Arntz, Onno J, Pieters, Bartijn C.H, van Lent, Peter L.E.M, Koenders, Marije I, van der Kraan, Peter M, van de Loo, Fons A.J
Format: Article
Language:English
Subjects:
Albumins
Arthritis
Biological products
Biology and Life Sciences
Biomarkers
Blood & organ donations
Blood plasma
Body fluids
Cell activation
Cell membranes
Cell signaling
Chromatography
Composition
Consent
Diagnosis
Etanercept
Extracellular vesicles
Gene expression
Health aspects
IgG antibody
Immunoassay
Immunoglobulin G
Immunoglobulins
Interleukin 8
Lipids
Medicine and Health Sciences
Membrane vesicles
Methods
Monocytes
Nanoparticles
Nucleotides
Organelles
Phenotypes
Phospholipids
Physiological aspects
Plasma
Plasma proteins
Polyethylene glycol
Polymers
Proteins
Research and Analysis Methods
Rheumatoid arthritis
Rheumatology
Size exclusion chromatography
Tumor necrosis factor-TNF
Tumor necrosis factor-α
Ultracentrifugation
Vesicles
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Description
Summary: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)-[alpha] 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-[alpha] inhibition determined in a TNF-[alpha] 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-[alpha] 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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0236508