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

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 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.

细胞外囊泡（Extracellular vesicles, EVs）因其在多种生物学过程及医学应用中具备潜在作用，而受到学界日益广泛的关注。然而，细胞外囊泡的分离在技术层面颇具挑战，主要原因在于其尺寸微小且异质性强，且分离过程中常伴随污染物共析出。为此，我们开发了一种基于磁珠的两步分离法（two-step magnetic bead-based, 2MBB），可从微量样本中富集部分细胞外囊泡及其微小核糖核酸（microRNAs, miRNAs）。该方法依托包被有识别细胞外囊泡表面标志物（如CD63）的捕获分子的磁珠开展操作，捕获得到的细胞外囊泡可从磁珠上洗脱，或直接裂解以用于后续分析。本研究中，我们使用第二组包被有互补寡核苷酸的磁珠，分离与细胞外囊泡结合的微小核糖核酸（EV-associated microRNAs, EV-miRNAs）。通过掺入外源性cel-miR-238分子的逆转录聚合酶链式反应（reverse transcription-polymerase chain reaction, RT-PCR），对2MBB法的操作效率进行了评估。实验结果表明，该方法在细胞外囊泡富集（74 ± 7%，n=4）与微小核糖核酸提取（91 ± 4%，n=4）方面均具有较高效率。透射电子显微镜（Transmission electron micrographs, TEM）与纳米颗粒追踪分析（nanoparticle tracking analysis, NTA）结果显示，经2MBB法富集的捕获细胞外囊泡可被释放，且其纯度高于差速超速离心法（differential ultracentrifugation, DUC）（p n = 3）。作为一项预实验，我们对8份临床血浆样本中的EV相关微小核糖核酸126-3p（EV-miR126-3p）与总循环游离微小核糖核酸126-3p（circulating cell-free miR126-3p, cf-miR126-3p）进行了检测，并与蛋白标志物水平开展对比分析。与cf-miR126-3p相比，EV-miR126-3p与心肌肌钙蛋白I（cardiac troponin I, cTnI）及B型钠尿肽N末端前体（N-terminal propeptide of B-type natriuretic peptide, NT-proBNP）之间的相关性显著升高。此外，健康志愿者（n=18）与高危心血管疾病（cardiovascular disease, CVD）患者（n=10）的血浆样本中EV-miR126-3p水平存在显著差异（p=0.006），提示EV-miR126或可作为心血管疾病的潜在生物标志物。2MBB法操作简便、通用性强，可为细胞外囊泡及其结合型核酸分子的富集提供高效途径。