Clemson University, Department of Chemistry, Clemson, SC, 29634, USA.
California State Polytechnic University, Pomona, CA, USA.
Talanta. 2023 Jan 15;252:123779. doi: 10.1016/j.talanta.2022.123779. Epub 2022 Aug 13.
In the emerging field of phyto-nanotechnology, 30-200 nm plant-derived extracellular vesicles (PDEVs) are now known to contain active biomolecules that mediate cell-to-cell communication processes in a manner very similar to exosomes in mammalian cells. The ability to deliver cargo across cellular membranes suggests that botanical systems could be used in the mass production of therapeutic vectors to transport exogenous molecules into human cells. The fundamental biochemical characteristics of PDEVs remain poorly understood due to the lack of efficient methods to isolate and characterize these nanovesicles. Described here is a rapid PDEV isolation method using a hydrophobic interaction chromatography (HIC)-based extraction performed on a capillary-channeled polymer (C-CP) fiber spin-down tip. The C-CP solid-phase extraction method is performed using a standard table-top centrifuge, enabling the isolation and concentration of PDEVs (>1 × 10 particles from 100 μL of sample). PDEVs of 189 nm average diameter were obtained from 20 common fruit and vegetable stocks. The size, integrity, and purity of the recovered PDEVs were assessed using transmission electron microscopy (TEM), multi-angle light scattering (MALS), absorbance quantification, a protein purity assay, and an enzyme-linked immunosorbent assay (ELISA) to the PEN1 PDEV surface marker protein. The HIC C-CP tip isolation method allows for concentrated PDEV recoveries (up to 2 × 10 EVs) on reasonable time scales (<15 min) and low cost (<$1), with the purity and integrity fit for fundamental research and downstream applications.
在植物纳米技术这一新兴领域中,人们现在知道 30-200nm 的植物衍生细胞外囊泡(PDEV)中含有活性生物分子,这些分子以非常类似于哺乳动物细胞外体的方式介导细胞间通讯过程。穿过细胞膜传递货物的能力表明,植物系统可用于大量生产治疗载体,将外源性分子输送到人类细胞中。由于缺乏有效分离和表征这些纳米囊泡的方法,PDEV 的基本生化特性仍未得到充分了解。本文描述了一种使用基于疏水相互作用层析(HIC)的提取方法从毛细管通道聚合物(C-CP)纤维离心管尖端快速分离 PDEV 的方法。C-CP 固相萃取方法使用标准台式离心机进行,能够从 100μL 样品中分离和浓缩 PDEV(>1×10 个颗粒)。从 20 种常见的水果和蔬菜库存中获得了平均直径为 189nm 的 PDEV。使用透射电子显微镜(TEM)、多角度光散射(MALS)、吸光度定量、蛋白质纯度测定和酶联免疫吸附测定(ELISA)对回收的 PDEV 的尺寸、完整性和纯度进行评估,以检测 PEN1 PDEV 表面标记蛋白。HIC C-CP 尖端分离方法可在合理的时间范围内(<15 分钟)和低成本(<$1)下实现浓缩的 PDEV 回收(高达 2×10 EV),其纯度和完整性适合基础研究和下游应用。
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