外泌体单体的精准检测和多模态表征技术

Exosomes are small membrane bound organelles that are released by cells into the extracellular space as an integral part of cell-cell communication. They carry functional RNA and DNA, as well as associated proteins, and are heavily implicated in diseases such as cancer and cancer metastasis. They have high potential for use as a minimally-invasive liquid biopsy as well as potential to be used as a targeted drug carrier for personalized therapy. However, because of exosomes’ diverse roles in normal biology, they are highly heterogeneous in both structure and function. In order to fully understand the relationship between the origins and functions of exosomes, we need the comprehensive knowledge of individuals to perform statistical analysis. Current analysis methods are restricted to looking at exosomes as a bulk population rather than as a collection of heterogeneous individual vesicles. The available single particle analysis techniques offer either the morphological or chemical information, but not both. Thus， they are limited in profiling the exosomes. In this application we will develop a multifunctional instrument that can study individual exosomes, characterizing them both morphologically and chemically. The individual exosome is trapped by laser tweezers, which also generate both Raman and elastic scattering. Raman scattering measures the chemical information while size information is probed through elastic scattering, using the same instrument and laser beam. We will use this to study individual exosomes released from cells under various conditions such as cell starvation or Rab27 knockdown. This will help unravel critical questions related to the function and biogenesis of subpopulations of exosomes from different cell lines. Furthermore, we will explore the possibility of using our instrument to characterize exosomes from patient samples, to develop sorting criteria, as the first step in developing a robust liquid biopsy biomarker.

外泌体是细胞释放到胞外，进行长距离通讯的重要介质。它携带RNA、DNA及蛋白质, 是一把双刃剑，同时参与免疫和癌扩散。其起源、功能、大小和化学成分具有多样性。为了能从根本上理解外泌体的作用和机制，首先需要获得其个体的详细信息，才能统计分析各子群的起源与功能之间的关系。目前的标准分析方法需要对批量进行分析，是大量外泌体的平均结果。仅有的单粒子表征方法不能同时收集物理和化学信息，模态单一。我们将针对这一问题，开发一个多功能系统，它可以捕捉外泌体个体，并同时测量其形态和化学信息。利用该多模态表征系统，我们将研究来自不同细胞系，如饥饿培养或Rab27敲低处理的细胞系，所分泌的单个外泌体，以期阐明其功能和起源相关的关键问题。此外，我们也将与安徽医科大学合作，研究来自癌症病人体液的外泌体，在个体信息的基础上区分外泌体，为分类技术的发展提供思路和参数，促进外泌体的临床应用。

胞外囊泡是由细胞释放的用于短程和长程细胞间通讯的重要的功能性生物纳米颗粒。它们在癌细胞生物学中有特别重要价值，并且与免疫调节以及准备便于癌细胞向远处转移的生态位有关。而且它们就会被癌细胞大量释放，并且可以在患者血液和其他体液中循环发现他们的踪迹。但是当前表征胞外囊泡的方法通常仅限于批量分析的方法，例如质谱和其它通过检测聚合或者“平均”囊泡的详尽信息的“组学”技术。在本项目中，我们通过将拉曼光谱和高速感知激光势阱中的纳米粒子相结合，开发了一种独特的激光捕获系统，“拉曼纳米粒子跟踪分析，R-NTA”。单个胞外囊泡的拉曼光谱提供化学信息，同时运动感知系统提供尺寸、折射率和聚合状态（即单体与聚合颗粒）之类的物理参数。通过同时将化学和形态学信息相结合，可以确定像是层状数（颗粒内脂质层的层数）之类的具备高度特异性的参数。此外，当R-NTA技术被应用于单个胞外囊泡的研究时，尺寸归一化的拉曼光谱提供了一种精确表征来自正常细胞和癌性头颈细胞的囊泡的手段。通过敲除离子通道蛋白TRPP2（在头颈癌细胞中过度表达）来调节这些细胞系的表达，来展示在敲除TRPP2之后，癌系细胞释放的囊泡和正常细胞释放的囊泡更加相似。而且，利用R-NTA系统提供的独特的化学成分特征，使得我们能够鉴别出几种独特的囊泡亚型。因此，R-NTA平台被证明是对外泌体和其他生物纳米颗粒进行完整的形态-化学表征的理想的方法。