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 纳米化的细胞外囊泡和颗粒（EVP）已被确定为细胞的重要手段 与邻近和远处的细胞进行通信。主动调查EVP，以了解其在 癌症，非侵入性疾病诊断和治疗剂。最重要的紧急挑战之一 EVP研究中的克服是了解EVP的异质性。 EVP的大小异质 和分子货物。结果，单个EVP分析已被确定为对破译 单个EVP的异质性及其在各种疾病中的生物学作用。例如， 一个持续的科学问题涉及新发现的细胞外颗粒是否称为外体 和超级人是整体纳米颗粒，富含多个制造商，例如蛋白质，RNA和脂质或 如果它们是不同功能活性纳米颗粒的分布（例如蛋白质，核酸和脂质） 共隔离在一起。广泛使用的分析技术（例如质谱）无法分析 单个EVP，因此这些测定掩盖了EVP的异质性的影响，这使它成为现实 迄今为止无法解决这个问题和其他开放问题。选择单个EV进行分析 无损的方式，必须开发将它们捕获到解决方案中的方法。光学镊子 最近以2018年诺贝尔物理学奖被证明是有效的方法 捕获单细胞和较大的电动汽车。不幸的是，光的衍射极限排除了它们用于捕获的使用 单纳米级电动汽车，最近发现的外域，超级人只有35 nm和25 直径NM。该MIRA研究计划由一系列旨在的项目组成 开发新的光学纳米纳特维式技术，以用于高吞吐量的单纳米化捕获 EVP与增强的拉曼分析相结合，提供有关全球生物分子的独特信息 单个纳米化电动汽车，外体和超级人的组成。随后，我们将调查使用 这些工具可以解决电动汽车研究中正在进行的争议。 首先，我们将基于纳米质结构开发一种新型的光学纳米韦策方法，该方法将启用： （i）在几秒钟内平行捕获数千个单evp； （ii）加强和获取拉曼 单个捕获EVP的信号无损坏，而它们被困在纳米质附近的溶液中 腔； （iii）生物分子成分分析以确定个体的全球生物分子组成 被困的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体和超人是单片还是构成 功能活性纳米颗粒的各种分布。从提议中获得的相关发现 研究计划将大大提高我们了解EVP异质性的能力，解决持续的问题 争议和指导未来的科学问题的性质将在EVP研究领域进行研究。