Biophysical Characterization and Drug Delivery Potential of Exosomes from Human Wharton's Jelly-Derived Mesenchymal Stem Cells
Biophysical Characterization and Drug Delivery Potential of Exosomes from Human Wharton's Jelly-Derived Mesenchymal Stem Cells
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DOI：
10.1021/acsomega.9b01180
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发表时间：
2019-08-01
期刊：
影响因子：
4.1
通讯作者：
Choudhury, Sangeeta
中科院分区：
文献类型：
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作者：
Chopra, Neha;Arya, Braham Dutt;Choudhury, Sangeeta
作为生物“纳米载体”的细胞来源的外泌体（30-200 nm）由于其在体内生物系统中内化的能力（即，细胞）。虽然它们可以从包括干细胞在内的各种来源收获，但需要适当的分离和表征方案来获得“纯的”外泌体群体。对于潜在的临床应用，了解外泌体的功能能力及其纯度，即不含微泡、凋亡小体和蛋白质聚集体，是先决条件。为了从人沃顿商学院来源的间充质干细胞（hWJ-MSC）中获得高纯度和高产量的尺寸范围为30-200 nm的外泌体，我们进行并比较了三种分离方法：超离心（UC）、蔗糖垫（SC）和市售试剂（CR）。使用纳米颗粒跟踪分析（NTA）、场发射扫描电子显微镜（FESEM）和原子力显微镜（AFM）表征分离的外泌体。此外，为了理解hWJ-MSC衍生的外泌体（hWJ-ME）靶向胰腺肿瘤细胞的治疗潜力，已经使用共聚焦显微镜和流式细胞术在MiaPaCa-2细胞系上评估了内化功效。NTA结果显示，与UC（40.5%; p = 0.050）和CR（38%; p = 0.050）相比，蔗糖垫是用于高纯度（86.8%）的外泌体分离的最佳方法。通过FESEM和AFM的光学分析显示，SC分离的外泌体呈现球形形态，而UC和CR分离的外泌体表现出不均匀的形态。此外，来自共聚焦图像和流式细胞术的数据显示，hWJ-ME被MiaPaCa-2内化，证明了外泌体作为“潜在纳米载体”的可行性。因此，我们的研究表明，NTA（产量），AFM（尺寸），和FESEM（形态和地形）的组合可以提供灵敏的生物物理特性的hWJ-ME。在未来，富集的外泌体可以用作运载工具，运输靶向特异性药物或基因沉默构建体的肿瘤。
Cell-derived exosomes (30-200 nm) as biological "nanocarriers" have attracted a great deal of interest for therapeutic applications due to their ability to internalize in in vivo biological systems (i.e., cells). Although they can be harvested from various sources including stem cells, yet an appropriate isolation and characterization protocol to obtain "pure" exosomal population is needed. For potential clinical applications, understanding the functional ability of exosomes and their purity, that is, free from microvesicles, apoptotic bodies, and protein aggregates, is a pre-requisite. To achieve high purity and yield of exosomes from human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) in the size range of 30-200 nm, we have performed and compared three isolation procedures: ultracentrifugation (UC), sucrose cushion (SC), and commercially available reagent (CR). The isolated exosomes were characterized using nanoparticle tracking analysis (NTA), field emission scanning electron microscopy (FESEM), and atomic force microscopy (AFM). Furthermore, to understand the therapeutic potential of the hWJ-MSC-derived exosomes (hWJ-ME) to target pancreatic tumor cells, the internalization efficacy has been evaluated on the MiaPaCa-2 cell lines using confocal microscopy and flow cytometry. The NTA results showed sucrose cushion to be an optimal method for exosome isolation with high purity (86.8%), as compared to UC (40.5%; p = 0.050) and CR (38%; p = 0.050). Optical analysis by FESEM and AFM revealed that SC-isolated exosomes presented a spherical morphology, whereas UC- and CR-isolated exosomes exhibited an uneven morphology. Furthermore, the data from confocal images and flow cytometry showed that hWJ-ME were internalized by MiaPaCa-2, demonstrating the feasibility of exosomes as a "potential nanocarrier". Thus, our study suggests that a combination of NTA (yield), AFM (dimensions), and FESEM (morphology and topography) could provide sensitive biophysical characterization of hWJ-ME. In the future, enriched exosomes could be used as a delivery vehicle to transport target-specific drugs or gene-silencing constructs to tumors.