基于响应肿瘤微环境逐级释药的适体共载大分子药物纳米微囊的研究

With Tumorigenesis and development, the microenvironment and protein expression have greatly different to normal tissues. In this project, we have integrated innovative strategies into a drug delivery system(DDS) to bridge some obstacles in the treatment of breast cancers. The targeting component is an aptamer PNDA which against periostin in microenvironment. Literature reports that periostin is overexpressed in breast cancers and associate with tumor metastasis. The macromolecules drug component of this DDS is Gelonin which have high antitumor activity and not able to penetrate the cell by itself. The designed microenvironment responsive nanocapsules for Gelonin bases on single protein nanocapsules technology. The nanocapsules were prepared through noncovalent-bond in-situ polymerization. F3 peptides were conjugated to the surface of nanocapsules facilitating the tumor target and penetration. PNDA were conjugated to F3-nanocapsules via PEG and enzyme-cleavable peptide facilitating the long circulation in vivo and nanocapsules release. After DDS triggered by microenvironment enzyme, the exposure of F3 peptide promote the internalization of nanocapsules, then drugs were released by low pH in cells. The DDS which co-delivery of PNDA and Gelonin could inhibit tumor growth by and metastasis. The project investigated the relationship of nanocapsules structure, drug stability and tumor microenvironment to overcome some obstacles in the treatment of cancers before.

肿瘤发生发展过程中会使其组织内特异性蛋白和微环境与正常组织有较大差异，其特征可作为靶向药物传递系统的构建依据。本项目以乳腺癌为研究对象构建新型智能靶向药物传递系统。引入的靶向肿瘤微环境的核酸适体PNDA，可以解决给药系统靶向效率低的问题,且该适体具有抗肿瘤转移的功能。选择的高效低毒大分子药物Gelonin可以解决小分子效价低的问题，并且自身没有入胞能力。以纳米微囊技术为基础，通过静电力结合的方式修饰大分子药物构建的单蛋白纳米微囊，可使药物具有良好的体内和胞内稳定性、pH选择释放性。微囊表面修饰的乳腺癌细胞穿膜肽F3，可以改进微囊药物的细胞渗透性。通过组装合理的药物传递系统，可使共载药物PNDA与Gelonin聚集在肿瘤部位后，分别响应肿瘤微环境高表达酶和弱酸环境，进而层次化释药，达到抗肿瘤转移和抑肿瘤增殖的疗效。该项目将系统研究纳米载药体系设计与肿瘤微环境调控及大分子药物稳定性之间的关系。

乳腺癌是一种发病率高的恶性肿瘤，被认为是对人类健康的主要威胁，我国是世界上乳腺癌发病率最高的国家，乳腺癌的治疗方法主要有手术、化疗、放射治疗和免疫治疗，乳腺癌的治疗方法主要有化疗药物紫杉醇、阿霉素和顺铂，可有效地杀死分化的乳腺癌细胞来发挥治疗作用，但它们往往具有体内毒性，接受这些药物的患者在治疗后很容易复发。大分子药物疗效已显示出巨大的前景，可克服小分子抗癌药物的局限性。但是肿瘤细胞选择性的大分子胞内传递仍然是其成功临床应用的主要障碍。因此为了实现组织内更深入的渗透，需要对药物和药物载体进行调控，然而调节这些特性的原则仍然有待摸索。在此项目中，我们研究了纳米微粒在肿瘤中的递送性能，通过适体进行靶向修饰。由肿瘤归巢F3肽和白树毒素组成的融合蛋白（F3-Gel）在体外和体内具有抗癌活性。F3-Gel纳米微粒是基于非共价键原位聚合制备的蛋白纳米微粒技术。我们成功地制备了负载F3-Gel蛋白的PNC纳米微粒，通过选择最佳的聚阳离子和蛋白质比例来优化纳米微粒性能，通过动态光粒度分析仪，观察了PNC纳米颗粒表结构的透射电镜（TEM），并对其粒径和zeta电位进行了表征。结果表明，我们成功制备了均匀分布的纳米颗粒，并在基质金属蛋白酶（MMP）介质作用下体外释放肿瘤组织。适体通过PEG与纳米微粒结合，促进体内长循环、靶向和释放。最后，BALB/c小鼠的4T1乳腺癌动物模型进行体内抗癌研究。结果表明，APT-PEG-PNC组能显著抑制肿瘤体积。APT-PEG-PNC组对乳腺癌细胞有较强的抑制作用，使用适体PNDA和药物F3-Gel可提高乳腺癌的协同杀伤作用，并可提高乳腺癌的治疗效果。本研究根据肿瘤的特异性，选择具有适当表面电荷的药物载体作为肿瘤治疗的潜在策略，为临床治疗打下理论基础。