Hung Michelle E, Lenzini Stephen B, Stranford Devin M, Leonard Joshua N
Interdisciplinary Biological Sciences Program, Northwestern University, Evanston, IL, USA.
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Methods Mol Biol. 2018;1740:109-124. doi: 10.1007/978-1-4939-7652-2_9.
Extracellular vesicles (EVs) are secreted nanoscale particles that transfer biomolecular cargo between cells in multicellular organisms. EVs play a variety of roles in intercellular communication and are being explored as potential vehicles for delivery of therapeutic biomolecules. However, EVs are highly heterogeneous in composition and biogenesis route, and this poses substantial challenges for understanding the role of EVs in biology and for harnessing these mechanisms for therapeutic applications, for which purifying therapeutic EVs from mixed EV populations may be necessary. Currently, technologies for isolating EV subsets are limited by overlapping physical properties among EV subsets. To meet this need, here we report an affinity chromatography-based method for enriching a specific EV subset from a heterogeneous EV starting population. By displaying an affinity tagged protein (tag-protein) on the EV surface, tagged EVs may be specifically isolated using simple affinity chromatography. Moreover, recovered EVs are enriched in the tag-protein relative to the starting population of EVs and relative to EVs purified from cell culture supernatant by standard differential centrifugation. Furthermore, chromatographically enriched EVs confer enhanced delivery of a cargo protein to recipient cells (via enhancing the amount of cargo protein per EV) relative to EVs isolated by centrifugation. Altogether, affinity chromatographic enrichment of EV subsets is a viable and facile strategy for investigating EV biology and for harnessing EVs for therapeutic applications.
细胞外囊泡（EVs）是分泌型纳米级颗粒，在多细胞生物的细胞间传递生物分子货物。EVs在细胞间通讯中发挥多种作用，并正被探索作为治疗性生物分子递送的潜在载体。然而，EVs在组成和生物发生途径上高度异质性，这给理解EVs在生物学中的作用以及利用这些机制进行治疗应用带来了重大挑战，为此可能需要从混合的EV群体中纯化治疗性EVs。目前，分离EV亚群的技术受到EV亚群之间重叠物理性质的限制。为满足这一需求，我们在此报告一种基于亲和色谱的方法，用于从异质的起始EV群体中富集特定的EV亚群。通过在EV表面展示亲和标记蛋白（标签蛋白），可使用简单的亲和色谱法特异性分离标记的EVs。此外，相对于起始的EV群体以及通过标准差速离心从细胞培养上清液中纯化的EVs，回收的EVs中标签蛋白含量更高。此外，与通过离心分离的EVs相比，色谱富集的EVs能增强货物蛋白向受体细胞的递送（通过增加每个EV中的货物蛋白量）。总之，EV亚群的亲和色谱富集是研究EV生物学以及利用EVs进行治疗应用的一种可行且简便的策略。
