Department of Chemistry, Biosystems Research Complex, Clemson University, Clemson, SC, 29634, USA.
Department of Bioengineering, Life Sciences Facility, Clemson University, Clemson, SC, 29634, USA.
Anal Chim Acta. 2019 Nov 15;1082:186-193. doi: 10.1016/j.aca.2019.07.035. Epub 2019 Jul 22.
Exosomes are one class of extracellular vesicles (30-150 nm diameter) that are secreted by cells. These small vesicles hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. On a cellular level, exosomes are attributed to playing a key role in intercellular communication, and may eventually be exploited for targeted drug delivery. In order for exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Plasma from peripheral blood is an excellent source of exosomes, as it is easily collected and the process does not normally cause undue discomfort to the patient. Unfortunately, blood plasma content is complex, containing abundant amounts of soluble proteins and aggregates, making exosomes extremely difficult to isolate in high purity from plasma. Most current exosome isolation methods have practical challenges including being too time-consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from a human plasma sample. Initial results demonstrate the ability to isolate exosomes with comparable yields and size distributions and on a much faster time scale when compared to traditional isolation methods, while also alleviating concomitant proteins and other impurities. As a demonstration of the potential quantitative utility of the approach, a linear response (particles injected on-column vs peak area) using a commercial exosome standard was established using a standard UV absorbance detector. Based on the calibration function, the concentration of the original human plasma sample was determined and subsequently confirmed by NTA measurement. The potential for scalable separations covering sub-milliliter spin-down solid phase extraction tips to the preparative scale is anticipated.
外泌体是一类细胞外囊泡（直径 30-150nm），由细胞分泌。这些小囊泡在疾病诊断中有很大的应用前景，因为它们显示出与其起源细胞相同的蛋白质生物标志物。在细胞水平上，外泌体被认为在细胞间通讯中发挥关键作用，并可能最终被用于靶向药物递送。为了使外泌体在疾病诊断中变得有用，并成为新兴的药物递送平台，它们必须在不影响其结构的情况下高效、有效地被分离。外周血血浆是外泌体的极好来源，因为它易于采集，而且该过程通常不会给患者带来不适。不幸的是，血浆内容物复杂，含有大量可溶性蛋白质和聚集体，使得外泌体极难从血浆中以高纯度分离。目前大多数外泌体分离方法都存在实际挑战，包括耗时过长、劳动强度大、对囊泡有破坏性，或者成本过高，不适合临床使用。为此，本研究探讨了在疏水相互作用色谱（HIC）方案中使用聚对苯二甲酸乙二醇酯（PET）毛细管通道聚合物（C-CP）纤维从人血浆样品中分离外泌体的方法。初步结果表明，与传统的分离方法相比，该方法能够更快地分离出外泌体，且具有可比的产率和粒径分布，同时还能减轻伴随的蛋白质和其他杂质。作为该方法潜在定量应用的证明，使用商业外泌体标准品建立了一个线性响应（注入柱上的粒子与峰面积之比），使用标准紫外吸收检测器。基于校准函数，确定了原始人血浆样品的浓度，随后通过 NTA 测量进行了确认。预计该方法具有从亚毫升级离心固相萃取小柱到制备级的可扩展分离潜力。
