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: 10631914
• 负责人: THOMAS R GABORSKI
• 金额: $ 18.5万
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10631914
• 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; 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; 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.

项目摘要 细胞外囊泡（EV）由细胞释放，被认为在细胞增殖中起重要作用。 细胞通讯，包括在疾病中的保护和致病作用。的目的 一项提议是开发一种简单且可扩展的分离技术， 以高纯度和高速度分离细胞外囊泡（EV）亚群。 电动汽车生物物理特征和组成的异质性引入了额外的水平 在研究它们的各种功能时，缺乏将电动汽车破碎成 亚群阻碍了理解EV在细胞间通讯中的功能， 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EV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长时间的 处理.尺寸排阻色谱法（SEC）可以根据大小分离EV亚群，但 导致显著的稀释，并遭受脂蛋白的污染， VLDL，与小型EV大小相同，在血浆中很常见。我们建议解决 通过开发新型纳米袋膜和使用改进的切向 流动过滤（TFF）方法，有效地捕获和释放EV亚群， 对特定的物理特性，同时消除脂蛋白污染物。在目标1中，我们 调整纳米球光刻的使用，以定期将聚苯乙烯纳米球放置在 该方法包括在膜的表面上制备待用作纳米袋的模板的基底。使用 不同的珠大小和蚀刻时间，我们将创建不同的纳米口袋膜 物理属性（囊袋半径、深度、孔径）以捕获EV亚群。在目标2中， 纳米袋膜将被集成到设备中，其中EV捕获和 释放（流体剪切、跨膜压力、释放条件）将被优化。使用 用已知浓度的预纯化EV亚群加标的培养基、血浆和尿液， 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， 增加纳米袋的尺寸。血浆中常见的污染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 如果具有可调尺寸的纳米袋的膜可以捕获和释放小的， 细胞培养基、血浆和尿液中的中型和大型EV，精密度为SEC（在 更高的浓度），同时在<1小时内超过超离心的纯度和产率。