微流控芯片外泌体分离分析新方法研究

Exosomes are the nanoscale vesicles in extracellular microenvironment secreted by cells. Recently, their function has been discovered to be related to cell-to-cell communication. It represents a brand new challenge or opportunity for the community of Analytical Chemistry to develop intact, rapid and high performance analysis approach for isolating the exosomes from complex extracellular matrix or body fluids. For this purpose, we proposed in this project a novel microfluidic chip-based method for exosomes separation and analysis. With planted three-dimensional nanostructure based microrods array inside the microchannel and immune-recognition, exosomes were separated from cellular matrix highly efficiently, and were further released for the collection. The collected exosomes were subjected to subsequent analysis with new microfluidic flow cytometry, in which submicro sheath flow was realized for meeting the requirement of exosomes analysis. With this technology, functional proteomics on exosomes membrane was analyzed at single vesicle level. Furthermore, smart and feasible chemical editing method was demonstrated for exosomes tracing and tissue targeting. With this new method, the function of exosomes as signal messenger in cancer (for example, liver cancer, one of the most popular tumor in Chinese populations) angiogenesis was analyzed. This method demonstrated in this project paves a new avenue to early cancer detection and personalized medicine, and will be beneficial for fundamental researches including analytical chemistry in life science, protein science and tumor biology. We hope that it will greatly promote the development of Chinese analytical chemistry or other related disciplines and young talents training.

外泌体是细胞分泌到胞外微环境中的纳米级小囊泡，最近才被证实其主要功能是参与细胞间信号传递。如何从复杂的细胞外基质或体液中无损、快速和高效分离分析外泌体，是分析化学面临的全新挑战和机遇。本项目提出发展基于微流控芯片的外泌体分离分析新方法：在微通道内构建依托微柱阵列的新型三维纳米结构，以免疫识别为基础，捕获和释放外泌体；建立适应于外泌体尺度的微流控芯片流式分析技术，实现亚微米尺度的三维鞘内流，在单囊泡水平分析外泌体膜表面功能蛋白质组；发展新颖的外泌体化学编辑技术，灵活简便地实现外泌体示踪和组织靶向。新方法具有无损、快速和高效分离分析外泌体等优势。采用上述新方法，以中国人群高发癌症之一肝癌为对象，在细胞和活体动物水平分析外泌体作为信号载体在癌症血管新生中的功能。本课题对于生命分析化学、蛋白质科学和癌症等基础研究以及个体化医疗等应用领域具有重要意义，并促进分析化学及其相关交叉学科的发展和人才培养。

外泌体最近被揭示为细胞间信号传递的工具，尤其在肿瘤发育中起关键作用，为肿瘤的诊断与治疗提供了新途径。本研究致力于发展并建立了基于微流控芯片的新方法，实现外泌体的高效分离、富集与分析，该类方法具有对外泌体损伤小、富集效率高和检测灵敏度高等特点；进一步提出并发展了外泌体的化学编辑技术，将靶向配体锚定于外泌体膜表面，实现外泌体的肿瘤靶向和受体介导的内吞作用，采用电转技术将不同的药物装载到外泌体内，实现多模态的肿瘤诊疗，包括化疗、基因治疗、热疗和光动力治疗等手段；基于上述外泌体纯化方法和化学编辑技术，系统发展了基于外泌体的血管新生分析，以及肿瘤靶向与多模态联合治疗等，在对外泌体进行分子水平和细胞水平评价的基础上，在斑马鱼血管模型和小鼠肿瘤模型等体内研究中其功能得到验证。上述研究对于基于外泌体的液体活检和肿瘤靶向治疗提供了新技术支撑，具有重要的科学意义和潜在的临床价值。上述研究结果及相关工作在Adv Mater, Adv Func Mater, Nat Comm, Biomaterials, ACS Nano, Mater Today, Anal Chem等发表项目资助标注的SCI学术论文28篇，其中高水平论文（IF>10）6篇，获得国家发明专利3项，培养博士后1名、博士5名。