Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study

Abstract

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

Fig 1. The schematic diagram of the experimental procedure.

EVs in human plasma samples were first isolated using anti-CD63 antibody-coated magnetic beads. Captured EVs were then eluted for whole particle assays, such as transmission electron microscopy and nanoparticle tracking analysis. In addition, captured EVs were lysed for studying their molecular contents, including proteins and RNAs. EV-miRNAs were extracted using oligonucleotide-conjugated magnetic beads and subjected to subsequent analysis.

Fig 2. Nanoparticle analysis (NTA) of supernatants and eluates.

NTA was utilized to determine the number (A) and size profile (B) of nanoparticles in the starting platelet-poor plasma (PPP), supernatant after EV enrichment, eluate after formic acid-mediated EV release from magnetic beads. Data are expressed as mean ± SEM (n = 3, ** p < 0.01, * p < 0.05, Student’s t-test).

Fig 3. Nanoparticle and protein analysis of MB eluates and DUC concentrates.

Transmission electron micrographs of negatively stained EVs isolated and eluted from magnetic beads (MB) or pelleted by differential ultracentrifugation (DUC) methods from PPP. Scale bars represent 100 nm. The double-layer character of EV membranes could be discerned at increased magnification (insets) (A). Enumeration of nanoparticles in MB eluate and DUC concentrate by NTA (B). Protein levels were quantified using MicroBCA assays. The ratio of nanoparticle concentration to μg protein was plotted as a relative measure of purity (C). Data are expressed as mean ± SEM from three measurements (** p < 0.01, Student’s t-test).

Fig 4. miRNA levels in plasma samples.

Levels of miRNAs hsa-miR-21-5p (n = 4) (A) and hsa-miR-126-3p (n = 5) (B) extracted from platelet-poor plasma, EVs captured on magnetic beads (MB), and supernatant after MB concentration were measured relative to the spike-in exogenous cel-miR-238-3p by RT-qPCR. Data are expressed as mean ± SEM (** p < 0.01, * p < 0.05, Student’s t test).

Fig 5. Levels of EV-miR-126-3p and cf-miR-126-3p were quantified and compared with the levels of conventional CVD biomarkers.

Concentrations of cTnI and NT-proBNP were determined by enzyme-linked immunosorbent assays (ELISA) performed in NCKUH. EV-derived miR-126-3p showed negative correlations with cTnI (n = 6) (A) and NT-proBNP (n = 8) (B), while cell-free miR-126-3p showed weaker correlations with both protein biomarkers (C and D).

Fig 6. Levels of EV-miR-126-3p in plasma samples from healthy volunteers and high-risk CVD patients.

A box-and-whisker chart shows measured EV-miR-126-3p concentrations in plasma samples from healthy volunteers (n = 18) and CVD patients with elevated levels of cTnI (> 0.5 ng/mL) or NT-proBNP (> 0.125 ng/mL) (n = 10) (A). The sensitivity and specificity were 100% and 94%, respectively, when the threshold EV-miR-126-3p concentration was 50 fM. The area under the receiver operating characteristic (ROC) curve was close to 1 and much greater than that of a randomly selected case shown as a straight line at a 45° angle (B).