Nanopore Force Spectroscopy and Sorting of Vesicles at Nanoscale

纳米级囊泡的纳米孔力光谱和分选

基本信息

批准号：
10158532
负责人：
MinJun Kim
金额：
$ 17.74万
依托单位：
SOUTHERN METHODIST UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10158532
关键词：
Accreditation Affect Atomic Force Microscopy Attention Biological Biological Markers Biological Process Biology Biomechanics Characteristics Charge Chemicals Cholesterol Clinical Computer Models DNA Data Deoxyribonucleases Development Digestion Discrimination Disease Dose Drops Drug Delivery Systems Drug Design Drug Targeting Electrolytes Encapsulated Endocytosis Engineering Ensure Event Exocytosis Fluorescence Gene Delivery Goals Housing Label Lipids Liposomes Liquid substance Measurement Measures Mechanics Membrane Membrane Fusion Methods Organelles Particle Size Pathologic Pharmaceutical Preparations Physiological Play Polystyrenes Population Procedures Prognosis Property Protocols documentation Psychological reinforcement Reporting Research Research Project Grants Resolution Role Sampling Shapes Signal Transduction Sorting - Cell Movement Spectrum Analysis Structure Surface System Techniques Technology Testing Theoretical model Therapeutic Thinness Time Tissues Travel Vesicle Virus Work base biomaterial compatibility cellular targeting clinically relevant cost density design ds-DNA exosome experimental study feasibility testing gene delivery system improved insight interdisciplinary approach mechanical properties membrane model nanocapsule nanoencapsulated nanoliposome nanoparticle nanopore nanoscale nanosized nanovesicle novel operation particle preservation quality assurance response sensor statistics systemic toxicity theories tool trafficking uptake voltage

项目摘要

PROJECT SUMMARY Soft nanoparticles like exosomes, liposomes and viruses play a vital role in biological and physiological (including therapeutic) functions such as exo- and endocytosis, membrane trafficking, and intercellular signaling. With advancements in targeted drug/gene delivery, cargo carrying vesicles with minimal systemic toxicity, improved uptake and controlled drug release at the cellular/tissue targets have gained substantial attention. A key functional aspect of these soft vesicles is the ability to deform relative to the cargo content, membrane composition and inner/outer media to fuse with cellular organelles and pass through narrow pores. Thus, profiling deformability is critical for understanding their functions and successful drug engineering. The existing classical techniques like atomic force microscopy (AFM) demand laborious and cumbersome procedures, with low throughput. Other available methods to study the cargo encapsulated by the vesicles such as DNase digestion, fluorescence, and UV absorbance suffer from overnight procedures, DNA extraction (and tagging), and DNA standard curve requirements, respectively. In this proposed work, we intend to develop a technique to overcome all these shortcomings to study the deformability of soft nanoparticles by using a nanopore – a nanoscale channel separating two electrolyte chambers through which analyte particles can electrokinetically travel from one chamber to the other in response to an applied bias, one at a time, causing electrical perturbations unique to particle size, shape, and content. Nanopores are robust, sensitive and inexpensive miniature sensors of higher throughput – capable of studying thousands of particles within seconds to minutes. We will first develop a computational model(s) for electro-deformability of the liposomes which depends on conductivity, cargo density, and surface charge (Aim 1a). We propose an automated recapture protocol to study the same particle ~25 times by reversing the voltage bias after each translocation event to re-translocate the same particle to generate a large pool of reliable statistics on deformability of the liposomes. Electro- deformability would be expressed as the relative current drop ratio at forward and reverse biases. The obtained results will be compared with a commercially available, incompressible rigid particle like polystyrene to accredit the results (Aim 1b). It is assumed that the cargo content would affect the deformability of the liposomes by altering the inner solution conductivity and perhaps the shape. We propose to synthesize nanoliposomes housing ss/ds-DNA of known ratios to test this theory (Aim 2a). By studying the electro-deformability of DNA- encapsulated liposomes, in comparison with cargo-free liposomes, we intent to determine the degree of electro- deformability in response to the cargo content (amount and percentage) (Aim 2b). In addition, the automated recapture protocol will enable the discrimination of samples of desired mechanical properties out of a population of vesicles with varied properties. Once parallelized, high throughput data can be obtained to characterize rigidity of any nanoscale soft particle at the single-particle level.

项目概要外泌体、脂质体和病毒等软纳米粒子在生物和生理学中发挥着至关重要的作用（包括治疗）功能，例如外吞作用和内吞作用、膜运输和细胞间信号传导。随着靶向药物/基因递送的进步，载货囊泡具有最小的全身毒性，改善细胞/组织靶点的吸收和控制药物释放已引起广泛关注。这些软囊泡的关键功能方面是相对于货物内容物、膜的变形能力成分和内部/外部介质与细胞器融合并穿过狭窄的孔，因此，分析变形能力对于理解其功能和成功的药物工程至关重要。原子力显微镜 (AFM) 等经典技术需要费力且繁琐的程序，其他可用的方法来研究囊泡封装的货物，例如 DNase。消化、荧光和紫外吸光度会受到过夜程序、DNA 提取（和标记）、和 DNA 标准曲线要求，在这项拟议的工作中，我们打算开发一种技术。克服所有这些缺点，通过使用纳米孔来研究软纳米粒子的变形能力 - 分隔两个电解质室的纳米级通道，分析物颗粒可以通过该通道进行电动运动响应于所施加的偏压，从一个室移动到另一个室，一次一个，从而产生电纳米孔独特的扰动是稳健、敏感且廉价的。更高通量的微型化——能够在几秒到几分钟内研究数千个粒子。我们将首先开发脂质体电变形性的计算模型，该模型取决于我们提出了一种自动重新捕获协议来研究。通过在每次易位事件后反转电压偏置来重新易位相同的粒子〜25次相同的颗粒产生大量关于脂质体变形性的可靠统计数据。变形能力可表示为正向和反向偏压下的相对电流降比。结果将与市售的不可压缩刚性颗粒（如聚苯乙烯）进行比较，以验证结果（目标 1b）假设货物含量会影响脂质体的可变形性。我们建议改变内部溶液的电导率以及形状。容纳已知比例的单链/双链 DNA 来测试这一理论（目标 2a）。封装的脂质体，与无货物脂质体相比，我们打算确定电-的程度响应货物含量（数量和百分比）的变形性（目标 2b）。重新捕获协议将能够从群体中区分出具有所需机械性能的样本一旦并行化，就可以获得高通量数据来表征。任何纳米级软颗粒在单颗粒水平上的刚性。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fine-tuning-based Transfer Learning for Characterization of Adeno-Associated Virus.

DOI：
10.1007/s11265-022-01758-3
发表时间：
2022-12
期刊：
JOURNAL OF SIGNAL PROCESSING SYSTEMS FOR SIGNAL IMAGE AND VIDEO TECHNOLOGY
影响因子：
1.8
作者：
Khan, Aminul Islam;Kim, Min Jun;Dutta, Prashanta
通讯作者：
Dutta, Prashanta

Assessment of 1/f noise associated with nanopores fabricated through chemically tuned controlled dielectric breakdown.

DOI：
10.1002/elps.202000285
发表时间：
2021-04
期刊：
Electrophoresis
影响因子：
2.9
作者：
Saharia J;Bandara YMNDY;Karawdeniya BI;Alexandrakis G;Kim MJ
通讯作者：
Kim MJ

Adeno-associated virus characterization for cargo discrimination through nanopore responsiveness.

DOI：
10.1039/d0nr05605g
发表时间：
2020-12-08
期刊：
Nanoscale
影响因子：
6.7
作者：
Karawdeniya BI ;Bandara YMNDY ;Khan AI ;Chen WT ;Vu HA ;Morshed A ;Suh J ;Dutta P ;Kim MJ
通讯作者：
Kim MJ