Department of Chemical and Biomolecular Engineering, Lehigh University.
Department of Chemical and Biomolecular Engineering, Lehigh University;
J Vis Exp. 2021 Mar 31(169). doi: 10.3791/62429.
The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of "large" (350 nm) OMVs and "small" (<150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.
革兰氏阴性菌的细胞壁由内层（细胞质）和外层膜（OM）组成，两者之间隔着一层薄薄的肽聚糖层。在整个生长过程中，外层膜可以起泡形成球形的外膜囊泡（OMVs）。这些 OMVs 参与许多细胞功能，包括向宿主细胞输送货物和与细菌细胞进行通讯。最近，人们开始探索 OMVs 的治疗潜力，包括将其用作疫苗和药物输送载体。尽管 OMVs 源自 OM，但人们早就认识到 OMVs 的脂质和蛋白质货物与 OM 有很大的不同，通常差异很大。最近，有证据表明细菌可以释放多种类型的 OMVs，并且有证据表明大小会影响它们被宿主细胞摄取的机制。然而，该领域的研究受到难以有效分离异质大小 OMVs 的限制。密度梯度离心（DGC）传统上用于此目的；然而，该技术耗时且难以扩展。另一方面，体积排阻色谱（SEC）不太繁琐，适合 OMVs 治疗用途的必要未来扩展。在这里，我们描述了一种 SEC 方法，该方法可重复分离异质大小的囊泡，以 Aggregatibacter actinomycetemcomitans 产生的 OMVs 为例，其直径范围从小于 150nm 到大于 350nm。我们通过动态光散射（DLS）证明了“大”（350nm）OMVs 和“小”（<150nm）OMVs 的分离。我们建议基于 SEC 的技术优于基于 DGC 的技术，因为前者易于使用、可重现（包括用户之间）并且有可能扩展。
