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-miR-NAs). 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.

Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV‐CATCHER): A customizable purification assay designed for small‐RNA biomarker identification and evaluation of circulating small‐EVs

“…Diagram comparing EV‐CATCHER to 10 commercially available kits and ultracentrifugation for purification of small‐EVs. Three magnetic‐bead purification methods were identified where chemical treatment have been described (1% Formic Acid pH 2.0‐ 4.0, 0.1 M Glycine pH 2.5‐3.0 (Chen et al., 2020; Hisey et al., 2018; Hong et al., 2014), or proprietary buffer (SBI ExoFlow)) for small‐EV release (Barros et al., 2018; Burkova et al., 2019; Gutzeit et al., 2014; Hardin et al., 2018; Huang et al., 2018; Mallegol et al., 2007; Patel et al., 2019; Peterson et al., 2015; Theodoraki et al., 2018; Velandia‐Romero et al., 2020; ). b.…”

“…Whereas the exact neutralizing mechanism of Covid‐19 convalescent circulating small‐EVs requires further evaluation, our observation that small‐EVs isolated from high anti‐spike IgG titer sera possess anti‐SARS‐CoV‐2 neutralizing activity has important implications for enhancement of convalescent plasma therapy or design of novel therapies. It is important to note that recent studies have demonstrated that immuno‐purified small‐EVs can maintain their functional properties (cellular uptake, signalling) with the purifying antibody attached to their surface (Chen et al., 2020; Hardin et al., 2018; Hisey et al., 2018; Hong et al., 2014; Patel et al., 2019; Peterson et al., 2015; Theodoraki et al., 2018; Velandia‐Romero et al., 2020). However, due to the possibility that some small‐EV functions may be compromised by antibody presence, for every new immuno‐purification protocol, it is necessary to initially evaluate their functionality by using small‐EVs purified by ultracentrifugation, as controls.…”

“…The design of an easily customizable low background antibody‐selection assay may not only allow for more sensitive purification of small‐EV subpopulations, but may also provide a versatile tool for the selection of cell‐specific surface biomarkers and in turn fine‐tuned identification of unique disease biomarkers circulating in biofluids (Williams et al., 2013). Finally, and most importantly, although a few low pH elution buffers have been described in the literature (Chen et al., 2020; Hisey et al., 2018; Hong et al., 2014), very few manufacturers provide such buffers for release of the covalently‐bound antibodies and intact release of selected small‐EVs for downstream in vitro analyses (Hardin et al., 2018; Patel et al., 2019; Peterson et al., 2015; Theodoraki et al., 2018; Velandia‐Romero et al., 2020). A recent immunoaffinity‐based exosome isolation assay (exo‐PLA) addressed many of these issues by using an enzymatically degradable DNA linker between the exosome‐binding antibody and the magnetic bead, permitting release of intact exosomes (Löf et al., 2017).…”

Circulating Extracellular Vesicles As Biomarkers and Drug Delivery Vehicles in Cardiovascular Diseases

“…Cheng et al [ 133 ] suggested that expression of miR-126 and miR-21 could be used for early detection of CVD, such as acute myocardial infarction and FH. Another study reported reduced plasma levels of EV-miR-126 in high-risk CVD patients and EV-miR-126 levels were negatively correlated with cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP), suggesting miR-126 as a potential biomarker for CVD [ 103 ].…”

Non-coding RNAs in Cardiac Intercellular Communication

“…However, the relative abundance of cardiac secreted miRNAs within exosomes and/or other vesicles remains debatable. While Chen et al (2020) described higher plasma levels of miR-126 in the exosomal fraction compared to the non-exosomal fraction, another study suggests that plasmatic exosomal miRNAs are only a minority, with 90% of the plasmatic miRNAs being present in a non-membrane-bound form, possibly bound to a ribonucleoprotein complex ( Arroyo et al, 2011 ).…”