Clinical feasibility of a method for enrichment of exosome-like extracellular vesicles from human plasma for miRNA biomarker research.

초록

Exosomes are extracellular vesicles (EVs) that are secreted from most cells through sorting pathways and characterized with a diameter of approximately 30 ? 150 nm and a density of 1.13 ? 1.19 g/mL. Exosome-like extracellular vesicles (ELVs) containing various molecular compositions such as proteins, lipids and nucleic acids have a potential of biomarker development, which reflect their origin and regulate functions of adjacent or remote cells via intercellular communication. However, it is difficult to enrich ELVs with consistently high yield and purity from biologic fluids. In particular, plasma is a complex matrix that contains a variety of proteins, lipids and circulating nucleic acids. Therefore, to profile exosomal miRNAs in body fluid for disease diagnosis, it is critical to isolate ELVs with high yield and purity. Polymer-based precipitation (PP) is the most commonly used method to purify exosomes from body fluids. However, there are several issues such as low purity with co-precipitated contaminants such as lipoproteins when isolates ELVs using PP kits, which hampers the development of valid ELV-associated miRNAs biomarkers. In the present study, we tested the modified ELV isolation protocols of 3 commercially available PP kits using proteinase K (PK) treatment to deplete protein contaminants and non-vesicular miRNAs. Our modified method, as compared to the original protocols without PK treatment, showed the excellent yield and purity of ELVs that were characterized by immunoblot of exosomal and non-exosomal proteins, electron microscopy and size distribution analysis. When we selected a kit with the highest purity and yield, we successfully quantified ELV-associated miRNAs (miR-30c-5p, miR-126 and miR-192) with acceptable variability in 0.6 mL of human plasma. The quantity of enriched ELV miRNAs was lower (45 ? 65%) than that of miRNAs in total plasma. Collectively, our ELV enrichment method using a commercially available kit with PK treatment appears suit