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

开发用于纯化细胞外囊泡的尺寸选择性捕获和释放膜

• 批准号: 10432803
• 负责人: THOMAS R GABORSKI
• 金额: $ 22.19万
• 依托单位: ROCHESTER INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10432803
• 关键词: Address; Affinity Chromatography; Back; Cell Communication; Cell Culture Techniques; Cells; Charge; Chromatography; Complex; Culture Media; Development; Devices; Dextran Sulfate; Diagnostic; Disease; Equipment; Field Flow Fractionation; Filtration; Fractionation; Geometry; Goals; Gold; Heterogeneity; Hour; Human; Ion Exchange; Lipoprotein (a); Lipoproteins; Liquid substance; Low-Density Lipoproteins; MLLT2 gene; Membrane; Mesenchymal Stem Cells; Molecular Sieve Chromatography; Nanosphere; Pathogenicity; Patients; Performance; Plasma; Play; Polystyrenes; Process; Proteins; Radial; Research; Role; Series; Speed; Structure; Surface; Techniques; Technology; Testing; Therapeutic; Time; Traction; Ultracentrifugation; Urine; Validation; Very low density lipoprotein; Western Blotting; Work; base; biophysical properties; density; extracellular vesicles; feasibility testing; hypercholesterolemia; improved; lithography; novel; parylene; physical property; pressure; skills; success; vesicular release

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.

项目概要 细胞外囊泡（EV）由细胞释放，被认为在细胞- 细胞通讯，包括疾病中的保护作用和致病作用。此举的目的 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囊泡 (EV) 亚群。 EV 的生物物理特征和组成的异质性引入了额外的水平 研究其不同功能时的复杂性。缺乏将电动汽车细分为 亚群阻碍了理解细胞间通讯中 EV 功能的努力 实现电动汽车在诊断和治疗应用中的潜力。超速离心 仍然是隔离电动汽车的黄金标准，但串行和密度梯度方法需要 无法多路复用的大型设备，需要高技能和大量时间 加工。尺寸排阻色谱 (SEC) 可以按尺寸分离 EV 亚群，但是 导致显着稀释并受到脂蛋白污染，特别是 VLDL，与小型 EV 大小相同，常见于血浆中。我们建议解决 通过开发新型纳米袋膜并使用改进的切向膜来克服这些限制 流动过滤 (TFF) 方法可有效捕获和释放基于 EV 亚群的 特定的物理特性，同时消除脂蛋白污染物。在目标 1 中，我们将 采用纳米球光刻技术将聚苯乙烯纳米球定期放置在 基底用作膜表面纳米袋的模板。使用 不同的珠子尺寸和蚀刻时间，我们将创建不同的纳米袋膜 物理属性（口袋半径、深度、孔径）来捕获 EV 亚群。在目标 2 中， 纳米袋膜将被集成到具有 EV 捕获和条件的设备中 释放（流体剪切、跨膜压力、释放条件）将得到优化。使用 培养基、血浆和尿液中掺有已知浓度的预纯化 EV 亚群， 我们的目标是捕获和释放小型电动汽车以及中型和大型电动汽车 增加纳米口袋的尺寸。血浆中常见的污染性 LDL 和 VLDL 将被 用添加到 TFF 电路中的带负电荷的硫酸葡聚糖珠去除。这项工作将 如果具有可调尺寸纳米袋的膜能够捕获和释放小分子， 具有 SEC 精度（at 更高浓度），同时在 <1 小时内超过超速离心的纯度和产量。