Single Particle Characterization and Isolation of Extracellular Vesicles

细胞外囊泡（EVs）是亚微米大小的生物颗粒，由所有生命形式分泌，如细胞、细菌和酵母。电动汽车从释放它们的细胞中携带作为货物的分子（例如，蛋白质、脂质、核酸）。这些物质反过来又能在吸收它们的细胞中发挥生物效应，如诱导生长、增殖、迁移，甚至分泌可溶性生物活性因子。电动汽车在压力、损伤和疾病的情况下被释放，但也可以作为生物实体之间完成特定任务的一般通信手段被释放。单个电动汽车表面的蛋白质和标记引导它们走向预定的靶细胞，然后将它们吞没。在复杂的环境中，如细菌生物膜和生物流体，系统中所有细胞的ev一起释放到细胞外环境中。由于电动汽车具有几乎相同的生物物理特性，目前的纯化方法将电动汽车作为一个整体分离。因此，研究EV多样性或从系统中特定细胞中分离EV几乎是不可能的。因此，人们对EV多样性、亚群以及它们如何影响复杂和异质系统中受体细胞的行为知之甚少。******我的研究项目旨在了解电动汽车的功能以及它们如何在复杂环境中改变细胞。为了实现这一目标，我建议：****** I)建立一个名为NanoFlow的新技术分析平台，使单颗粒表征和分离成为可能。*** i)分析表面蛋白和脂质，并表征在各种复杂环境和生物流体中分泌的ev的多样性。*** iii)通过NanoFlow分选分离特异性EV群体，分析其蛋白、脂质和核酸含量，并测定其对受体细胞的生物学效应。******通过详细的EV表征提供的见解将有助于更好地理解EV如何用于细胞之间的信息交流和协调生物效应。此外，我的研究项目将开发的技术的专业性将为学员提供一套独特的可翻译和市场技能，这无疑将对他们未来在学术研究和工业领域的职业生涯有用。

Extracellular vesicles (EVs) are submicron-sized biological particles secreted by all forms of life such as cells, bacteria and yeast. EVs carry as cargo molecules from the cells from which they were released (e.g., proteins, lipids, nucleic acids). These can in turn exert biological effects in cells that uptake them such as inducing growth, proliferation, migration and even the secretion of soluble bioactive factors. EVs are released in instances of stress, damage and disease, but can also be released as a means of general communication between biological entities to accomplish a specific task. Proteins and markers at the surface of individual EVs guide them towards their intended target cells that will then engulf them. In complex environments such as bacterial biofilms and biological fluids, EVs from all the cells in the system are released together into the extracellular environment. Because EVs share nearly identical biophysical properties, current purification methods isolate EVs as a bulk. It is therefore nearly impossible to study EV diversity or isolate EVs emanating from specific cells in the system. As such, very little is known about EV diversity, subpopulations and how they impact the behavior of recipient cells in complex and heterogeneous systems. ******My research program aims to understand the function of EVs and how they alter cells in complex environments. To achieve this goal, I propose to:****** i) Establish a new technological analysis platform called NanoFlow that enables single particle characterization and isolation. *** i) Profile surface proteins and lipids, and characterize the diversity of EVs secreted in various complex environments and biological fluids. *** iii) Isolate specific EV populations by NanoFlow sorting, analyze their protein, lipid and nucleic acid content, and determine their biological effects on recipient cells.******The insight provided by detailed EV characterizations will help to better understand how EVs are used to communicate information between cells and coordinate biological effects. Furthermore, the specialized nature of the technology that will be developed by my research program will provide trainees with a unique set of translatable and marketable skills that will no doubt be useful for their future careers in academic research and industry.