Development of size-selective capture and release membranes for purification of extracellular vesicles

项目摘要 细胞外小泡(EV)由细胞释放，被认为在细胞内发挥重要作用。 细胞通讯，包括在疾病中的保护和致病作用。这样做的目的是 建议开发一种简单且可扩展的分离技术，有效地 以高纯度和高速度分离细胞外小泡(EV)亚群。 电动汽车的生物物理特性和组成的异质性引入了额外的水平 研究它们的不同功能时的复杂性。缺乏将电动汽车细分为 亚群阻碍了理解EV在细胞-细胞交流中的功能和 认识到电动汽车在诊断和治疗应用中的潜力。超速离心法 仍然是隔离电动汽车的黄金标准，但串联和密度梯度方法需要 无法多路复用的大型设备需要高技能和长达数小时的 正在处理。大小排除层析(SEC)可以按大小分离EV亚群，但 导致显著稀释，并受到脂蛋白的污染，尤其是 与小型电动汽车大小相同的超低密度脂蛋白，在等离子体中很常见。我们建议解决以下问题 这些限制是通过开发一种新的纳米囊膜和使用改进的切向 流动过滤(TFF)方法，可有效捕获和释放EV亚群 对特定的物理性质，同时消除脂蛋白污染物。在目标1中，我们将 调整纳米球光刻的使用，使聚苯乙烯纳米球规则地放置在 用作膜表面纳米腔模板的底物。vbl.使用 不同的珠粒大小和刻蚀时间，我们会制造出不同的纳米囊膜 物理属性(口袋半径、深度、孔隙大小)以捕获EV亚群。在目标2中， 纳米囊膜将被集成到设备中，在这些设备中，电动汽车捕获和 释放(流体剪切、跨膜压力、释放条件)将得到优化。vbl.使用 培养液、血浆和尿液中添加了已知浓度的预纯化EV亚群， 我们的目标是捕获和释放小型电动汽车以及系列的中型和大型电动汽车 不断增大的纳米腔大小。在等离子体中常见的污染低密度脂蛋白和极低密度脂蛋白，将是 将带负电的葡聚糖硫酸盐珠加到TFF电路中除去。这项工作将 如果具有可调大小的纳米囊的膜能够捕获和释放小的， 来自细胞培养液、血浆和尿液的中型和大型EVS，精度为SEC(at 更高的浓度)，同时在1小时内超过了超速离心法的纯度和产率。

Project Summary Extracellular vesicles (EVs) are released by cells and are thought to play important roles in cell- cell communication, including protective and pathogenic roles in disease. The objective of this proposal is to develop a straightforward and scalable separation technology that effectively fractionates extracellular vesicle (EV) subpopulations with high purity and high speed. Heterogeneity of biophysical characteristics and composition of EVs introduces an extra level of complexity when studying their diverse functions. The lack of ability to fractionate EVs into subpopulations hampers efforts to understand EV function in cell-cell communication and realize the potential of EVs in diagnostic and therapeutic applications. Ultracentrifugation remains the gold standard for isolating EVs, but serial and density gradient approaches require large equipment that cannot be multiplexed, necessitates high skill and many hours of processing. Size exclusion chromatography (SEC) can isolate EV subpopulations by size, but results in significant dilution and suffers from contamination with lipoproteins, particularly VLDLs, which are the same size as small EVs and common in plasma. We propose to address these limitations by developing a novel nanopocket membrane and using a modified tangential flow filtration (TFF) approach that effectively captures and releases EV subpopulations based on specific physical properties, while eliminating lipoprotein contaminants. In Aim 1, we will adapt the use of nanosphere lithography to regularly place polystyrene nanospheres across a substrate to be used as templates for nanopockets on the surface of the membrane. Using different bead sizes and etching times, we will create nanopocket membranes of varying physical attributes (pocket radius, depth, pore size) to capture EV subpopulations. In Aim 2, nanopocket membranes will be integrated into devices where conditions for EV capture and release (fluid shear, transmembrane pressure, release conditions) will be optimized. Using media, plasma and urine spiked with known concentrations of pre-purified EV subpopulations, we will target capture and release of small-EVs as well as medium and large EVs in series with increasing size nanopockets. Contaminating LDLs and VLDLs, common in plasma, will be removed with negatively-charged dextran sulfate beads added to the TFF circuit. This work will be successful if membranes with nanopockets of tunable size can capture and release small, medium and large EVs from cell culture media, plasma and urine with the precision of SEC (at higher concentration), while exceeding the purity and yield of ultracentrifugation in <1 hour.