Abstract
The aim of the study was to explore the polyethylene glycol–dextran two-phase polymer system formed in human plasma to isolate the exosome-enriched fraction of plasma extracellular nanovesicles (ENVs). Systematic analysis was performed to determine the optimal combination of the polymer mixture parameters (molecular mass and concentration) that resulted in phase separation. The separated phases were analyzed by nanoparticle tracking analysis and Raman spectroscopy. The isolated vesicles were characterized by atomic force microscopy and dot blotting. In conclusion, the protein and microRNA contents of the isolated ENVs were assayed by flow cytometry and by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), respectively. The presented results revealed the applicability of a new method for plasma ENV isolation and further analysis with a diagnostic purpose.
Highlights
Circulating plasma has a complex composition including electrolytes, cyclic compounds, nucleic acids, proteins, lipoproteins of various densities, glycosides, and other compounds
The complex composition of plasma interfered with the polymer solution and affected the conditions required for phase separation
Bright lower phases (LP) dissolved in phosphate buffered saline (PBS) were formed in plasma two-polymer system (PTPS) with polyethylene glycol (PEG) (20 kDa) and DEX (450–650 kDa)
Summary
Circulating plasma has a complex composition including electrolytes, cyclic compounds, nucleic acids, proteins, lipoproteins of various densities, glycosides, and other compounds. Extracellular nanovesicles (ENVs) are minor components of plasma. Their quantitative and qualitative patterns are still poorly understood, but the emerging biological significance of ENVs is attracting growing scientific interest. When ENVs were isolated by the standard ultracentrifugation procedure, the concentration of plasma ENVs differed by 2–3 orders of magnitude [4,5]. Such obvious discrepancies in the quantification of plasma ENVs may reflect the imperfection of both the isolation methods and the calculation technologies
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