多重靶向酶响应型载Au-Pt(IV)纳米载体的构建及对卵巢癌的治疗研究

Ovarian carcinoma is the fifth leading cause of death from cancer in women and still ranks the most lethal of the gynecological malignancies. Ovarian cancer is a highly metastatic disease that is rarely detected. The long-term survival rate of ovarian cancer patients treated by conventional modalities, such as surgical resection, chemotherapy, and radiation remains far from satisfactory. Conventional drug delivery is rarely successful because only a limited dosage of the chemotherapeutic drugs target ovarian cancer sites, even when administered at a high dose. Most chemotherapeutic drugs act on normal cells, inhibiting their growth or even killing, which makes the patient extremely weak and can even result in death. Cisplatin (CDDP), a platinum containing compound, is a key drug widely used to treat ovarian cancer. Pt(IV) complexes provide an attractive alternative to Pt(II) compounds because their inertness results in fewer side effects. Pt(II)-based anticancer drugs are associated with higher reactivity and thus lower biological stability. Pt(IV) complexes are reduced in the intracellular milieu to yield the cytotoxic Pt(II) species through reductive elimination of axial ligands. Thus, Pt(IV) complexes provide an attractive alternative to the existing portfolio of Pt(II) drugs. For increasing drug efficacy, targeted delivery to cancer cells is desirable. RGD motifs are present in many extracellular matrix components such as fibronectin and vitronectin and bind to integrins on the cell surface. There is an increasing interest in nanocarriers such as inorganic and polymeric nanoparticles, nanotubes, nanophosphors, liposomes, and quantum dots coated with RGD analogues as targeting and imaging agents. In order to circumvent pathways that deactivate a platinum-based drug by premature metabolism and protect non-cancerous cells from its effects, more inert Pt(IV) complexes can be used as redox-activatable prodrugs for Pt(II). Polymeric NPs can be used as a preferred nano-scale drug delivery vehicle especially for their excellent endocytosis, passive tumor-targeting, high encapsulation efficiency, and high stability, allowing for extended time in the circulatory system. GSH-mediated release represents an alternative non-enzymatic strategy for the selective intracellular activation of prodrugs. In this work, we developed a strategy that significantly improves the encapsulation efficiency and drug loading of Au-Pt(IV) in biodegradable nanoparticles of RGD-PEG1000-PLGA/mPEG5000-matrixmetalloproteinase (MMP)-substrate peptide-PLGA. We further study on the in vitro cytotoxicity and the in vivo efficacy of the construct in an ovarian carcinoma xenograft model.

卵巢癌是女性生殖系统三大恶性肿瘤之一，常规化疗药物顺铂Pt(II)毒副作用大。本项目的研究是在前期工作的基础上，拟将纳米缓释技术、药物治疗技术相结合优势互补，构建多重靶向、纳米金辅助治疗、谷胱甘肽介导释药的酶响应型RGD-PEG1000-PLGA/mPEG5000-肽-PLGA载Au-Pt(IV)的纳米递送系统（Au-Pt(IV)@NPs-RGD）。以卵巢癌细胞为模型细胞，裸鼠卵巢癌模型为动物模型，从建立靶向递送系统、实现纳米粒分级释放及纳米金辅助前药Pt(IV)治疗三方面入手，系统性研究和评价所构建载药系统对卵巢癌的治疗效果。达到改善并提高Pt在体内的靶向分布、克服硫醇介导的药物抗性的目的。本项目的完成将证实构建的RGD-PEG1000-PLGA/mPEG5000-肽-PLGA载Au-Pt(IV)纳米递送系统的递送功能和治疗效果，为该载体的临床化应用提供重要的基础。

卵巢癌是女性生殖系统三大恶性肿瘤之一，常规化疗药物顺铂 Pt(II)毒副作用大。本项目设计了一种肿瘤微环境敏感性药物递送系统将药物靶向递送到肿瘤部位，发挥抗肿瘤作用。.（1）肿瘤微环境敏感型药物递送系统的构建及表征：本课题将Au-Pt(IV)包载到 RGD-PEG1K-PLGA/PEG5K-多肽-PLGA中，制备了Au-Pt(IV)@NPs-cRGD药物递送系统。载药纳米粒粒径约为180nm，呈电中性。形态呈均匀分散球形；稳定性良好；Au-Pt(IV)@NPs-cRGD中Pt释放对酸不敏感，呈GSH浓度依赖性。与游离药物快速释放相比，载药纳米粒有一定的缓释能力。.（2）肿瘤微环境敏感型药物递送系统体外抗肿瘤作用：PEG5K断链研究结果表明，PEG5K未断链会影响到纳米粒进入细胞；同时也证实，细胞分泌的MMP-2能将纳米粒表面接枝的PEG5K 断链。入细胞途径研究显示，纳米粒进入细胞的途径主要为网格蛋白和小窝蛋白介导的内吞途径。纳米粒被细胞内吞后，细胞器与纳米粒共定位结果显示，纳米粒能躲避或从溶酶体中逃逸，将药物释放到细胞外。载药纳米粒能有效抑制SKOV-3细胞生长，增强药物的抗肿瘤作用。.（3）肿瘤微环境敏感型药物递送系统抗肿瘤作用与细胞周期及凋亡的相关性：对 Au-Pt(IV)@NPs-cRGD抑制SKOV-3细胞生长的作用机制进行分析。在非同步化模型中，检测药物作用后细胞周期的变化。结果显示，Au-Pt(IV)@NPs-cRGD将细胞周期阻滞在S期；对照组Pt(II)以及纳米粒无明显的周期阻滞作用。在同步化模型中，检测药物作用后细胞周期的变化。结果显示，Au-Pt(IV)@NPs-cRGD将各期同步化细胞阻滞在S期。载药纳米粒更容易诱导细胞凋亡。.（4）肿瘤微环境敏感型药物递送系统体内抗肿瘤效果评价：以SKOV-3裸鼠皮下瘤为模型，活体成像观察包载荧光染料的纳米粒在荷瘤鼠体内随时间的分布。结果显示，随着循环时间延长，载药纳米粒多聚集在肿瘤部位。表明载药纳米粒有显著的长循环效果和肿瘤靶向性。考察载药纳米粒对SKOV-3细胞裸鼠皮下移植瘤治疗效果。结果显示，Au-Pt(IV)@NPs-cRGD能明显抑制肿瘤生长。组织病理和细胞凋亡分析显示，该载药纳米粒能更有效地诱导肿瘤细胞的凋亡。对其安全性进行研究，结果显示该载药纳米粒无明显的脏器毒性，荷瘤鼠耐受性良好。