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
基本信息
- 批准号:10432803
- 负责人:
- 金额:$ 22.19万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAffinity ChromatographyBackCell CommunicationCell Culture TechniquesCellsChargeChromatographyComplexCulture MediaDevelopmentDevicesDextran SulfateDiagnosticDiseaseEquipmentField Flow FractionationFiltrationFractionationGeometryGoalsGoldHeterogeneityHourHumanIon ExchangeLipoprotein (a)LipoproteinsLiquid substanceLow-Density LipoproteinsMLLT2 geneMembraneMesenchymal Stem CellsMolecular Sieve ChromatographyNanospherePathogenicityPatientsPerformancePlasmaPlayPolystyrenesProcessProteinsRadialResearchRoleSeriesSpeedStructureSurfaceTechniquesTechnologyTestingTherapeuticTimeTractionUltracentrifugationUrineValidationVery low density lipoproteinWestern BlottingWorkbasebiophysical propertiesdensityextracellular vesiclesfeasibility testinghypercholesterolemiaimprovedlithographynovelparylenephysical propertypressureskillssuccessvesicular release
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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THOMAS R GABORSKI其他文献
THOMAS R GABORSKI的其他文献
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{{ truncateString('THOMAS R GABORSKI', 18)}}的其他基金
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 - 财政年份:2022
- 资助金额:
$ 22.19万 - 项目类别:
Using nanopocket membranes to capture bacterial outer membrane vesicles from biofluids
使用纳米袋膜从生物液中捕获细菌外膜囊泡
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10288527 - 财政年份:2021
- 资助金额:
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Using nanopocket membranes to capture bacterial outer membrane vesicles from biofluids
使用纳米袋膜从生物液中捕获细菌外膜囊泡
- 批准号:
10425443 - 财政年份:2021
- 资助金额:
$ 22.19万 - 项目类别:
Plasma clearance of water soluble and albumin bound toxins using graphene oxide nanoengineered laminates
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9387567 - 财政年份:2017
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Transparent Ultrathin Nanomembranes for Barrier Cell Models and Novel Co-Culture Systems
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用于细胞微阵列和体外测定的纳米多孔膜
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8200228 - 财政年份:2011
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Microfabricated porous TEM grids for improved phase contrast and CryoEM imaging
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7802530 - 财政年份:2010
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