A rapid capillary-channeled polymer (C-CP) fiber spin-down tip approach for the isolation of plant-derived extracellular vesicles (PDEVs) from 20 common fruit and vegetable sources
A rapid capillary-channeled polymer (C-CP) fiber spin-down tip approach for the isolation of plant-derived extracellular vesicles (PDEVs) from 20 common fruit and vegetable sources
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DOI：
10.1016/j.talanta.2022.123779
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发表时间：
2022-08-19
期刊：
影响因子：
6.1
通讯作者：
Marcus, R. Kenneth
中科院分区：
文献类型：
--
作者：
Jackson, Kaylan K.;Mata, Carolina;Marcus, R. Kenneth
在新兴的植物纳米技术领域中，现在已知30-200 nm的植物来源的细胞外囊泡（PDEV）含有活性生物分子，其以与哺乳动物细胞中的外泌体非常相似的方式介导细胞与细胞的通信过程。跨细胞膜递送货物的能力表明，植物系统可用于治疗载体的大规模生产，以将外源分子转运到人类细胞中。由于缺乏有效的方法来分离和表征这些纳米囊泡，PDEV的基本生化特征仍然知之甚少。本文描述了一种快速PDEV分离方法，该方法使用在毛细管通道聚合物（C-CP）纤维旋下尖端上进行的基于疏水相互作用色谱（HIC）的提取。C-CP固相萃取法使用标准台式离心机进行，能够分离和浓缩PDEV（100 μ L样品中> 1 x 10（10）个颗粒）。从20种常见的水果和蔬菜中获得了平均直径为189 nm的PDEV。使用透射电子显微镜（TEM）、多角度光散射（MALS）、吸光度定量、蛋白质纯度测定和针对PEN 1 PDEV表面标志物蛋白的酶联免疫吸附测定（ELISA）评估回收的PDEV的大小、完整性和纯度。HIC C-CP尖端分离方法允许在合理的时间尺度（< 15 min）和低成本（
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 x 10(10) particles from 100 mu L 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 x 10(11) EVs) on reasonable time scales (< 15 min) and low cost (