Understanding the Heterogeneity of Nanoscale Extracellular Vesicles, Exomeres, and Supermeres using Next Generation Optical Nanotweezers

使用下一代光学纳米镊子了解纳米级细胞外囊泡、外泌体和 Supermeres 的异质性

• 批准号: 10714221
• 负责人: Justus Chukwunonso Ndukaife
• 金额: $ 36.74万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714221
• 关键词: Address; Biological; Biological Assay; Cells; Collection; Communication; Detection; Diameter; Disease; Disease Marker; Distant; Future; Heterogeneity; Individual; Light; Lipids; Malignant Neoplasms; Masks; Mass Spectrum Analysis; Methods; Molecular; Nature; Nobel Prize; Nucleic Acids; Optics; Organism; Pathogenesis; Physics; Proteins; RNA; Raman Spectrum Analysis; Research; Role; Signal Transduction; Structure; Techniques; Technology; Therapeutic; design; disease diagnosis; extracellular; extracellular vesicles; high throughput technology; improved; individual variation; laser tweezer; nanoparticle; nanoplasmonic; nanoscale; nanosized; next generation; novel; optic tweezer; parallelization; particle; programs; tool

Project Summary Nanosized extracellular vesicles and particles (EVPs) have been identified as an important means for cells to communicate with neighboring and distant cells. EVPs are actively investigated to understand their roles in cancer, non-invasive disease diagnosis, and therapeutics. One of the most significant urgent challenges to overcome in EVP research is understanding the heterogeneity of EVPs. EVPs are heterogeneous in their size and molecular cargo contents. As a result, single EVP analysis has been identified as crucial to deciphering the heterogeneity of individual EVPs and understanding their biological roles in diverse diseases. As an example, an ongoing scientific question concerns whether the newly discovered extracellular particles called exomeres and supermeres are monolithic nanoparticles enriched with multiple makers such as proteins, RNA and lipids or if they are a distribution of different functionally-active nanoparticles (such as proteins, nucleic acid and lipids) co-isolated together. The widely used analysis techniques such as mass spectrometry are incapable of analyzing individual EVPs and hence these assays mask the impact of the heterogeneity of EVPs, which has made it impossible to address this question and other open questions to date. To select individual EVs for analysis in a non-destructive manner, it is imperative to develop methods for trapping them in solution. Optical tweezers recently recognized with a 2018 Nobel Prize in Physics have been demonstrated as effective approaches for trapping single cells and larger EVs. Unfortunately, the diffraction limit of light precludes their use for the trapping of single nanosized EVs, and the recently discovered exomeres, and supermeres that are only 35 nm and 25 nm in diameter, respectively. This MIRA research program is comprised of a collection of projects designed to develop new optical nanotweezer technologies for high throughput parallelized trapping of single nanosized EVPs combined with enhanced Raman analysis to provide unique information on the global biomolecular composition of individual nanosized EVs, exomeres and supermeres. Subsequently, we will investigate the use of these tools to address ongoing controversies in EV research. First, we will develop a novel optical nanotweezer approach based on nanoplasmonic structures that will enable: (i) parallelized trapping of thousands of single EVPs within seconds; (ii) enhancement and acquisition of Raman signals from single trapped EVPs nondestructively while they are trapped in solution near nanoplasmonic cavities; and (iii) biomolecular component analysis to determine the global biomolecular composition of individual trapped EVPs. Secondly, we will utilize the developed technologies to address ongoing questions in EVP research including whether the newly discovered exomeres and supermeres are monolithic or comprise a diverse distribution of functionally active nanoparticles. The pertinent findings to be obtained from the proposed research program will greatly improve our ability to understand the heterogeneity of EVPs, address ongoing controversies and guide the nature of future scientific questions to be investigated in the EVP research field.

项目摘要 纳米级的胞外囊泡和颗粒(EVP)已被认为是细胞 与邻近和远距离的小区进行通信。积极调查EVP以了解它们在 癌症、非侵入性疾病诊断和治疗。面临的最重大的紧迫挑战之一 EVP研究的难点在于了解EVP的异质性。EVP的大小是不同的 和分子货物含量。因此，单一的EVP分析被认为是破译 个体EVP的异质性及其在不同疾病中的生物学作用。举个例子， 一个正在进行的科学问题是关于新发现的称为外显子的细胞外颗粒 而超级单体是富含多种分子的整体式纳米颗粒，如蛋白质、RNA和脂类或 如果它们是不同功能活性纳米颗粒(如蛋白质、核酸和脂类)的分布 共同隔离在一起。广泛使用的分析技术，如质谱仪，无法进行分析。 单个的EVP，因此这些检测掩盖了EVP的异质性的影响，这使得 到目前为止，不可能解决这个问题和其他未决问题。要选择单个电动汽车在 为了以非破坏性的方式将它们捕获，必须开发将它们捕获在溶液中的方法。光镊子 最近获得2018年诺贝尔物理学奖的一些方法被证明是有效的 捕获单细胞和更大的电动汽车。不幸的是，光的衍射极限排除了它们用于捕获的可能性 单个纳米尺寸的电动汽车，以及最近发现的仅为35 nm和25 nm的外显子和超级聚体 直径分别为NM。米拉研究计划由一系列项目组成，旨在 开发高通量并行捕获单个纳米级的光学纳米捕获器新技术 EVPS与增强拉曼分析相结合，提供关于全球生物分子的独特信息 单个纳米电动汽车、外显子和超级单体的组成。随后，我们将调查其使用情况 这些工具用于解决电动汽车研究中正在进行的争议。 首先，我们将开发一种基于纳米等离子体结构的新型光学纳米消除器方法，该方法将能够： (I)在几秒钟内并行捕获数千个单个EVP；(Ii)增强和获取拉曼光谱 单个捕获的EVP在纳米等离子体附近的溶液中被捕获时的信号是无损的 以及(Iii)生物分子成分分析，以确定个体的全球生物分子组成 被困EVP。其次，我们将利用开发的技术来解决EVP中正在进行的问题 研究包括新发现的外显子和超分子是否为整体或包含一个 功能活性纳米颗粒的多样化分布。拟从建议中获得的相关结果 研究计划将极大地提高我们了解EVP异质性的能力，解决正在进行的 在EVP研究领域中，对将要研究的未来科学问题的性质进行争论和指导。