Abstract
Small extracellular vesicles (EVs), including exosomes, in body fluids have important applications in the noninvasive liquid biopsy-based diagnosis of cancer. Current EV-based diagnostic techniques still face practical challenges, such as inefficient EV isolation. Here, we report an efficient, resource-free pre-enrichment approach using (–)-epigallocatechin-3-gallate (EGCG), a polyphenolic biomolecule, to isolate and detect exosomal microRNAs (miRNAs) in human blood plasma samples. Our system comprises three steps: (1) EGCG-mediated EV aggregation, (2) filter-based EV isolation, and (3) molecular beacon-based detection of target miRNA in EVs. Using blood samples from cancer patients with gastric cancer or hepatocellular carcinoma, we constructed an EGCG-assisted miRNA diagnostic system. For both cancers, the levels of target miRNAs (miR-21, -27a, and -375) in EVs were strongly correlated with those in the publicly available GEO database. Our approach, an easy-to-use method for efficient EV isolation and the detection of miRNA in clinical samples, is applicable for molecular diagnostics in precision medicine.
Highlights
Extracellular vesicles (EVs) are spherical bilayered phospholipid vesicles released from cells that contain various biocomponents, such as RNA, DNA, proteins, and lipids[1,2,3,4]
The other polyphenolic biomolecules catechin (1 mg/mL in 50% ethyl alcohol), gallic acid (GA, 1 mg/mL in 50% ethyl alcohol), tannic acid (TA, 1 mg/mL in distilled water), and naringenin (1 mg/mL in 50% ethyl alcohol) formed precipitates containing EVs in conditioned media (CM) at a higher rate than Dulbecco’s phosphate-buffered saline (DPBS), which was used as a negative control (Fig. S2)
We observed differences in the quantity of centrifuge-precipitated EVs (Fig. 2a) and confirmed that the visibly significant increase in precipitation corresponded to increased levels of the exosome surface markers CD9 and CD63 (Fig. 2b) upon analysis. These results indicate that EGCG is more reactive with the biomolecules in CM than the polymers used in commercialization kits are, resulting in increased precipitation
Summary
Extracellular vesicles (EVs) are spherical bilayered phospholipid vesicles released from cells that contain various biocomponents, such as RNA, DNA, proteins, and lipids[1,2,3,4]. Several studies have suggested that tumor-derived exosomes are important cancer markers that can be serially detected with minimal invasion[5,6]. They have been significantly improved by micro- and nanoengineering strategies[7,8,9,10,11,12,13,14,15,16,17], exosome-based diagnostics still struggle to meet the demands of clinical applications involving point-of-care testing (PoCT)[18], which requires a resource-free. A recent study demonstrated that green tea-derived polyphenolic molecules interact with isotropic bicelle lipid membranes; likewise, the gallol ring
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