靶向肿瘤微环境的功能蛋白、microRNA和阿霉素联合投递纳米微囊设计及肿瘤抑制研究

Tumor microenvironment plays fundamental roles in tumor growth and metastasis. The tumor infiltrate composed of immune cells, angiogenic vascular cells, lymphatic endothelial cells, cancer-associated fibroblastic cells and the hypoxia and acidity environment contributes actively to cancer progression. Thus in the clinical setting the targeting of the tumor microenvironment to encapsulate or destroy cancer cells in their local environment has become mandatory. However, the varieties of stromal cells, the complexity of the molecular components of the tumor stroma, and the hypoxia and abnormal vasculature which present huge challenges for the delivery of therapeutants targeting the tumor microenvironment by nanoparticles need synergetic multi-target therapy. MicroRNAs (miRs) have recently emerged as important players involved in regulating multiple aspects of cancer biology and the tumor microenvironment. And von Hippel-Lindau (VHL) protein has been proved to be an efficient suppressor for tumor hypoxia by degrading of hypoxia-inducible factor (HIF), which is a transcription factor that plays a central role in the regulation of tumor hypoxia. In present proposal, we design a microenvironment-responsive nanocapsule for co-delivery of VHL, miR and DOX based on single-protein nanocapsules technology. The nanocapsules were prepared through in-site radical polymerization. Phosphatidylcholine (PC) groups were conjugated to the surface of nanocapsules via enzyme-cleavable peptide or acid-degradable spacer, facilitating the long-circulation in vivo and passive-targeting and penetration of nanocapsules in tumor. After the detachment of PC shell triggered by microenvironment, the expose of target ligand or positive-charges promote the internalization of nanocapsules by intratumoral cells, releasing VHL, miR and DOX to synergistically regulate hypoxia, normalize of tumor vasculature and extracellular matrix, and inhibit tumor. The influence of nanocapsule structure on their intratumoral penetration and drug distribution, and the mechanism of synergetic therapy will be investigated systematically.

肿瘤微环境在肿瘤发生发展中发挥着重要作用，靶向肿瘤微环境需要多靶点协同治疗和可克服肿瘤屏障的药物载体。本课题以单蛋白纳米微囊技术为基础，制备包载VHL-GC/DOX-反义miR结合体的纳米微囊，通过微囊表面修饰磷脂酰胆碱壳层促进纳米微囊体内长循环、肿瘤被动靶向和肿瘤内渗透，进一步利用肿瘤微环境高表达酶或弱酸环境使间隔臂断链并脱去表面壳层，形成表面带靶向因子或呈正电性纳米微囊，利于肿瘤细胞吞噬，并释放VHL调控肿瘤缺氧，释放DOX抑制肿瘤细胞增殖，释放miR抑制VEGF和LOX表达促进血管和细胞外基质正常化，同时协同VHL调控缺氧并增敏DOX化疗，解决肿瘤信号通路的冗余性问题。课题将系统研究纳米载体结构设计与其克服肿瘤屏障在肿瘤内渗透、药物释放、肿瘤微环境调控与肿瘤抑制的关系，同时深入探讨肿瘤微环境调控对纳米载体靶向肿瘤微环境的影响，为开发新型药物载体和靶向肿瘤微环境治疗提供理论依据。

构建药物联合投递载体对联合治疗意义重大。本项目通过原位自由基聚合的方法成功合成了反义miRNA的纳米微囊。研究结果表明，这种新型miRNA负载方法，具有高转染效率，低生物毒性，经高分子壳层的降解miRNA可以高效释放，具有高抑瘤活性。随后，我们以纳米微囊为基础构建了蛋白、miRNA和化疗药的联合投递载体，为多种药物联合调解微环境提供基础。..纳米药物载体在肿瘤组织中渗透也是目前肿瘤治疗的关键。本项目利用原位自由基聚合的方式合成粒径相同的电性不同的纳米微囊，并在其表面修饰聚乙二醇（PEG）以保证它们具有相近的血液循环时间。结果显示，纳米粒子的渗透与肿瘤的大小及纳米粒子的电性有关。.单抗以其特异性强和安全性高的特点成为临床治疗肿瘤不可或缺的药物。本项目构建了一种通过酶响应多肽组装的磷酰胆碱壳层材料，用于包裹负载单克隆抗体。结果表明，抗体表面磷酰胆碱含量为58.3%时，该壳层可以有效地抑制细胞对其的内吞，而在酶过度表达的情况下，能于细胞外释放单克隆抗体，高效抑制肿瘤。.药物对肿瘤的高效靶向可以降低药物的毒副作用，提高抑制肿瘤的效果。本项目构建了一种基于外泌体的肿瘤靶向体系，利用其负载化疗药物阿霉素。该体系可以有效实现外泌体的分离与提纯，并在外加磁场的作用下，负载药物的外泌体可以高效地靶向肿瘤组织，增强了肿瘤的治疗效果。.药物联合治疗是治疗癌症的重要手段。本项目采用miR-21抑制剂和DOX联合投递，结果表明miRNA21抑制剂/DOX联合投递同时下调了肿瘤细胞和肿瘤干细胞的miRNA21表达量。随后，我们验证三种药物（蛋白类药物，基因类药物和化疗药）联合治疗肿瘤的效果。结果显示，DOX，反义miRNA 21和VHL联合用药可以有效降低细胞内缺氧相关的HIF-1α蛋白含量和β-catenin的含量，并且降低成血管相关的VEGF，表明联合用药可以对肿瘤细胞缺氧和成血管进行调控，并增加细胞凋亡相关的caspase-3和BcL-2的含量，进而有效抑制肿瘤细胞增殖，具有综合调控微环境的能力。