叶酸受体靶向的高分子-赛特铂/二氯乙酸纳米胶束克服卵巢癌铂类耐药的应用及机制

Ovarian cancer is the most lethal gynecological cancer that seriously threatens women's health and life. The current standard therapy is debulking surgery plus platinum based chemotherapy. However, severe side effects, low efficacy of traditional chemotherapy and drug-resistance related problems are the main obstacles to the treatment of ovarian cancer and thus caused most deaths. So far the most prominent and best understood mechanisms caused drug resistance are: reduced drug uptake, increased drug inactivation, increased DNA adduct repair, reprogram of cell energy metabolism, etc. So new drugs are urgently needed. Satraplatin is one of the most promising platinum analogues emerging recently. It is a fourth-generation platinum analogue with activity against cisplatin-resistant tumors and with a relatively milder toxicity profile. It has shown similar antitumor activity to that of cisplatin and carboplatin in in vitro and in vivo studies, as well as shown activity in some platinum-resistant in vitro tumor models. And the lipophilicity and stability of satraplatin making it easier to get into drug-resistant cells, the very slow or negligible reaction of satraplatin with GSH reduced the deactivation, the asymmetrical stable ligands of satraplatin alter its DNA-adduct profile, leading to less likelihood of being recognized by DNA-mismatch repair or high-mobility group proteins. Dichloroacetate(DCA) is an orphan drug used as an investigational treatment for various acquired and congenital disorders of mitochondrial intermediary metabolism. Recently researches found that it can reverse the aerobic glycolysis (Warburg effect) of most solid tumors. Our previous study found that DCA can sensitize ovarian cancer cells to cisplatin. Therefore, this project intends to design and test a new anti-tumor nanodrug that is prepared by firstly conjugating the satraplatin and DCA to a biodegradable polymer molecule respectively, and co-assembling the conjugates into nanoscale micelles. This new drug is expected to have the advantages of all the polymer drugs, satraplatin and DCA. Specifically, it can deliver both satraplatin and DCA into tumor tissues and cancerous cells which overexpressing folate receptor, and once the micelles were internalized by the cells, the satraplatin and DCA would be released from the polymer carriers and function as what they were expected, and the biodegradable polymers degrade into water and carbon dioxide. We will study the antitumor efficacy and mechanisms of this new nanomicelles in platinum-resistant ovarian cancer cells and xenograft models. This project may provide a new strategy to the treatment of the drug-resistant ovarian cancer.

铂类耐药是导致卵巢癌复发、治疗失败的重要原因，我们前期研究发现：四价铂药赛特铂抑瘤率强，副作用小，作用持久，能够规避DNA损伤修复、GSH解毒所致耐药，具有克服耐药的潜能；二氯乙酸（DCA）能够促进线粒体凋亡通路的活化，能够有效逆转细胞凋亡障碍所致耐药；卵巢癌细胞表面高表达叶酸受体，接枝叶酸的高分子纳米载体可实现主动靶向特性，能进一步减少毒副作用，同时能够改善细胞内药物蓄积不足所致疗效不佳。根据目前卵巢癌多对传统铂药产生多药耐药，以致复发快、病死率高的现状，我们提出综合赛特铂、DCA及靶向高分子载体的优势，合成针对多种耐药机制的靶向高分子四价铂药复合物，充分发挥各自优势及之间协同作用，于细胞水平及顺铂耐药的荷瘤小鼠模型上检测其抗癌效果、化疗反应性、安全性，并研究其克服耐药的分子机制，为卵巢癌临床化疗提供新方向、新策略。

卵巢癌铂类耐药是导致治疗失败、患者病死率高的重要原因，本项目运用制备的高分子材料运载赛特铂和二氯乙酸DCA，联合用药。充分利用赛特铂与GSH反应速率很低、降低细胞内解毒作用，DCA逆转细胞凋亡障碍所致耐药以及纳米药物的被动靶向功能、缓慢释放，协同增强药效。我们成功合成了高分子载体MPEG-b-P(LA-co-MCC)及TPGS-PCL共聚体，制成了多种高分子纳米胶束药物：得到键合药 MPEG-b-P(LA-co-MCC-Pt)，简称P(Pt)，铂药含量约为10%，组装成纳米胶束SatPt-NPs。载体与DCA的键合物MPEG-b-P(LA-co-MCC/DCA)，简称P(DCA)，核磁图谱证实合成成功，DCA含量约为9%。 所制备的纳米胶束呈球形，与赛特铂键合后纳米胶束直径约为40nm，与DCA键合后纳米胶束直径约为59nm。纳米胶束在ph5.0和ph7.4条件下，72 h时铂的累积释放率分别为96%和46%，显示了中性环境缓慢释放、酸性环境快速释放、肿瘤组织积聚的特性，实现被动靶向功能及延长药物清除时间。比较亲本株及耐药EOC细胞中药物摄取及细胞毒性实验。如在浓度为10µM作用72小时候,顺铂、赛特铂和纳米胶束SatPt-NPs处理组的细胞存活率分别为47%，36%和13%。对于亲本株A2780、耐药株A2780DDP顺铂的IC50值1.62µM和8.8µM， 赛特铂的 IC50值分别为1.70和4.50µM，而SatPt-NPs的IC50值分别为0.19和0.08µM。而计算出A2780DDP细胞对三个药物的耐药倍数分别为5.43，2.65和0.44倍。显示纳米胶束SatPt-NPs克服耐药的潜力。实验结果也显示赛特铂及纳米胶束SatPt-NPs抑制细胞克隆形成能力显著优于顺铂，相同条件处理下细胞摄取纳米胶束SatPt-NPs细胞内铂含量明显高于相同浓度小分子顺铂给药结果，且耐药株内铂含量更接近亲本株。也提示纳米胶束进入细胞内有另外的机制。而卵巢癌动物模型结果显示SatPt-NPs抑制肿瘤生长药效优于对照组。而药物在肿瘤、肝脏、脾脏、肾脏中蓄积量最多。提示主要是经肝脏脾脏肾脏代谢，同时发现肿瘤中单位蓄积量与肝脏相当，甚至超过肾脏。肿瘤并非代谢器官，集中蓄积的原因可能与肿瘤的EPR被动靶向以及细胞内吞等有关。本课题为卵巢癌的药物治疗提供了新的可能。

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