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

项目摘要 纳米尺寸的细胞外囊泡和颗粒（EVP）已被鉴定为细胞粘附的重要手段。 与邻近和遥远的细胞进行通信。执行副总裁积极调查，以了解他们的作用， 癌症、非侵入性疾病诊断和治疗。最重要的紧迫挑战之一是， EVP研究的一个难点是理解EVP的异质性。EVP在规模上是异质的 和分子货物含量。因此，单一EVP分析已被确定为破译 个体EVP的异质性，并了解它们在不同疾病中的生物学作用。作为一个例子， 一个正在进行的科学问题是，新发现的细胞外颗粒， 超微粒子是富含多种标记物（如蛋白质、RNA和脂质）的整体纳米颗粒， 如果它们是不同功能活性纳米颗粒（例如蛋白质、核酸和脂质）的分布， 一起隔离。广泛使用的分析技术如质谱法不能分析 个体EVP，因此这些测定掩盖了EVP异质性的影响，这使得它 无法解决这个问题和其他悬而未决的问题。要选择单个EV进行分析， 非破坏性的方式，必须开发将它们捕获在溶液中的方法。光镊 最近获得2018年诺贝尔物理学奖的方法已被证明是有效的方法， 捕获单个细胞和较大的EV。不幸的是，光的衍射极限排除了它们用于捕获 单个纳米电动汽车，以及最近发现的exomeres，以及只有35 nm和25 nm的supermeres， nm的直径。这个MIRA研究计划由一系列项目组成， 开发新的光学纳米镊子技术，用于单个纳米尺寸的高通量并行捕获 EVP结合增强型拉曼分析，提供全球生物分子的独特信息 单个纳米EV、外泌体和超泌体的组成。随后，我们将调查使用 这些工具来解决EV研究中持续存在的争议。 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镊子方法，该方法将使： (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 当单个捕获的EVP被捕获在纳米等离子体激元附近的溶液中时， 生物分子成分分析，以确定个体的整体生物分子组成 被困的EVP其次，我们将利用已开发的技术来解决EVP中持续存在的问题 研究包括新发现的exomeres和supermeres是否是单片或包括一个 功能活性纳米颗粒的多样性分布。从拟议的调查中获得的有关结果 一项研究计划将大大提高我们理解EVP异质性的能力， 争议和指导未来的科学问题的性质，以调查在EVP研究领域。

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.