外泌体是一种由细胞分泌至胞外的直径为30-150 nm的纳米囊泡。肿瘤来源的外泌体中含有肿瘤特异性蛋白、mRNAs、microRNAs和DNA等，因此有望作为肿瘤早期诊断的生物标志物。然而，传统的外泌体分离检测方法耗时费力，操作繁琐而且需要庞大的仪器，使外泌体的临床应用受到极大限制。本文提出了一些新颖的技术来解决上述问题，主要包括以下几部分： （1）开发了一种基于介电电泳（DEP）的外泌体分离方法，该方法可以在30分钟内从血浆中分离得到外泌体，分离的外泌体的回收率和纯度均优于超速离心（UC）。本研究工作的创新点在于首次将外泌体分离和外泌体裂解集成在一张芯片上，裂解产物可直接用于RT-PCR检测。实验发现外泌体miR-21和miR-192在肺癌患者血浆中高表达，未来有可能作为肺癌诊断的生物标志物。 （2）开发了一种基于阴离子交换（AE）的外泌体高效分离方法，该方法可以在30分钟内从血浆、尿液样品和细胞培养液中分离得到外泌体。AE法分离的外泌体粒径分布均一，回收率高（＞90%），纯度高于UC组。本研究工作的创新点在于首次利用AE磁珠分离外泌体，并首次将AE法与微流控技术相结合，利用外泌体分离芯片实现自动化地分离血浆外泌体，为后续的生物研究提供了很大便利。 （3）开发了一种基于磁纳米颗粒的外泌体可视化检测方法，该方法可以在30分钟内实现外泌体的可视化检测。本研究工作的创新点在于首次利用磁纳米颗粒实现前列腺特异性抗原（PSA）-外泌体的可视化检测，与现有的可视化检测方法对比，最低检测限降低了145倍。该方法未来有可能用于基于外泌体的前列腺癌早期诊断。 （4）开发了一个外泌体载药平台用于肺腺癌的靶向治疗。本研究工作的创新点在于首次探索了利用iRGD修饰的牛奶外泌体靶向杀伤肺腺癌细胞的可能性。实验发现载有紫杉醇（PAC）的外泌体对肺腺癌细胞的杀伤作用强于直接给药组，而且对正常细胞的毒副作用小于直接给药组。该外泌体载药平台在肺腺癌的靶向治疗方面具有很大潜力。 综上所述，本研究开发了一系列外泌体分离和检测新技术，并开发了一个外泌体载药平台，有利于实现基于外泌体的肿瘤早期诊断和靶向治疗。
Exosomes are nano-sized extracellular vesicles with sizes of 30-150 nm. Tumor-derived exosomes contain tumor specific proteins, mRNAs, microRNAs and DNA, which provide promising biomarkers for the early diagnosis of cancer. However, the clinical applications of exosomes have been limited by the fact that traditional exosome isolation methods are tedious, non-standardized, and require bulky instrumentation. This paper proposes several innovative approaches to solve the above problems: (1) A dielectrophoresis (DEP)-based isolation method was developed to isolate exosomes from diluted plasma samples. With this method, we could isolate exosomes with higher recovery efficiency and purity compared with ultacentrifugation (UC) within 30 minutes. To facilitate subsequent analysis, the DEP chip integrated the isolation and in situ lysis of exosomes together, and the lysate could be directly used for RT-PCR. The levels of exosomal miR-21 and miR-192 in lung cancer plasma samples were found to be higher compared to those from healthy plasma samples. Which are potential biomarkers for early diagnosis of lung cancer. (2) In order to isolate exosomes directly from plasma, an anion-exchange (AE)-based isolation method was proprosed for the first time. The AE-based isolation method is able to isolate exosomes directly from plasma samples, urine samples and cell-culture medium within 30 minutes. Exosomes isolated with AE magnetic beads had higher recovery efficiency (>90%) and less protein impurities than those isolated by UC. The AE method could be combined with microfluidic technology for automatic exosome isolation. Which provides great convenience for subsequent biological research. (3) Apart from exosome isolation, appropriate quantitative detection methods for exosomes are also necessary for the better study of exosomes. Thus, a visual detection method for prostate-specific antigen (PSA) exosomes was firstly proprosed by using Fe3O4 nanoparticles. Using this method, exosomes in the plasma samples from prostate cancer patients were detected in a visual, label-free, and quantitative manner within 30 minutes, demonstrating the potential of this detection method in the early diagnosis of prostate cancer. (4) A drug delivery platform using exosomes was developed for targeted tumor therapy. Milk exosomes were modified with tumor-specific peptides, and then loaded with the paclitaxel (PAC) via electroporation. After that, the modified exosomes loaded with PAC was used for tumor therapy. It was found that the modified exosomes loaded with PAC could kill lung adenocarcinoma cells well and had no obvious toxicity to normal cells. Therefore, the drug delivery platform has the great potential for targeted therapy of lung adenocarcinoma. Overall, the studies mentioned above provide promising solutions for the early diagnosis and targeted therapy of cancer.
