靶向生物可降解高分子-铂(Ⅳ)/shEZH2纳米胶束治疗耐药性卵巢癌的研究

Ovarian cancer is a common gynecological cancer that seriously threatens women's health and life.Severe side effects, low efficacy of traditional chemotherapy and drug-resistance related problems are the main obstacles to the treatment of ovarian cancer, so targeted delivery, polymer drugs and RNAi technology are becoming new research focuses.Sdudies found that, compared to platinum(Ⅱ), the form of clinical platinum drugs, polymer-platinum(Ⅳ) prodrugs have higher efficacy, lower side effects,etc.,and can more effectively inhibit tumor growth and improve survival.Our previous studies found that EZH2-knockdown through transfection of shEZH2 in drug-resistant ovarian cancer cells can effectively reverse the resistance to platinum drugs.This project intends to design and test a new anti-tumor drug that is prepared by firstly conjugating the Pt(Ⅳ) to a biodegradable polymer molecule, then connecting the Luteinizing Hormone Releasing Hormone (LHRH) as a targeting moiety to the hydrophilic end of the conjugate, and finally co-assembling the congugate with shEZH2 into nanoscale micelles.This new drug is expected to have the advantages of all the polymer drugs,RNAi technology,platinum(Ⅳ) prodrugs and targeted delivery system.Specifically,it can deliver both platinum(Ⅳ) prodrugs and shEZH2 into tumor tissue and cancerous cells and once the micelles are internalized by the cells, The platinum(Ⅳ) prodrugs are rapidly reduced into platinum(Ⅱ),shEZH2 can reverse the resistant to platinum drugs through RNAi mechanism and the biodegradable polymers degrade into water and carbon dioxide.And with tumor-specific accumulation and low toxicity in the circulatory system,its side effects are much lighter and fewer.

卵巢癌是病死率最高的妇科肿瘤，以铂类为基础的化疗是各阶段患者综合治疗的最重要部分。但临床常用二价铂类药物（铂(Ⅱ)）如顺铂、卡铂、奥沙利铂等由于药效低、毒副作用大以及耐药性的产生限制了它们的用量及疗效，是导致治疗失败、生存率低的重要原因。研究发现四价铂前药（铂(Ⅳ)）相比传统铂(Ⅱ)有副作用小、肿瘤组织药物浓度高等优点，能更有效抑制肿瘤生长。申请者前期在体内外实验中发现转染shEZH2降低EZH2的表达后能有效逆转卵巢癌铂类耐药。本课题旨在研究合成一种靶向输送、稳定控释、副作用小并能逆转耐药的全新的纳米抗癌药物：将铂(Ⅳ)键合到可生物降解的高分子上,在其亲水端连接靶向分子促黄体生成素释放激素（LHRH），最后与shEZH2共组装成纳米胶束，利用卵巢癌干细胞、耐药细胞株及动物模型在体内外水平研究该药物治疗耐药性卵巢癌的效果及安全性。并从分子生物学水平阐述药物逆转卵巢癌耐药以及抗肿瘤机制。

铂类抗肿瘤药物应用广泛但其毒副作用及耐药问题成为严重影响患者结局的因素。本项目将常用的顺铂及奥沙利铂制备成对应的四价的衍生物-四价铂（铂(IV)），然后利用制备的生物可降解双亲性高分子运载铂(IV)和其他小分子药物如利尿酸、二氯乙酸及siRNA，联合用药。充分利用纳米药物的被动靶向功能、缓慢释放，四价铂前药的相对惰性以及利尿酸解毒GSH，siRNA下调耐药基因等功能，协同增强药效。我们成功合成了高分子载体MPEG-b-PCL-b-PLL，MPEG-b-P(LA-co-MCC)以及化学还原型四价顺铂及奥沙利铂的前体DACH-Pt。我们先后制成了多种纳米药物：MPEG-b-PCL-b-PLL单独运载四价顺铂的纳米药物MPEG-b-PCL-b-PLL/cisPt(IV)，命名M1，MPEG-b-P(LA-co-MCC)与环己二胺铂及利尿酸键合组装而成的纳米胶束MPEG-b-P(LA-co-MCC/DACHPt)，命名M2，含铂及利尿酸的复合纳米胶束MPEG-b-P(LA-co-MCC/DACHPt/EA)，命名M(EA/Pt)，MPEG-b-P(LA-co-MCC)与四价奥沙利铂键合后再与M(Bcl-2 siRNA)的复合纳米胶束，M(OxaPt(IV)/Bcl-2) 。所制备的纳米胶束呈球形，M1粒径约为150-160 nm，M2粒径约为30-40 nm，M(EA/Pt)粒径120 nm左右，M(OxaPt(IV)/Bcl-2)粒径约为80-90nm，均能利用实体瘤的EPR效应增强肿瘤组织的药物积聚，实现被动靶向功能；都有中性环境释放缓慢、酸性环境（如肿瘤组织）释放加快的特性，有利于延长血液循环中的时间以及在肿瘤组织中的快速释放，提高生物利用度降低副作用。纳米胶束的细胞水平MTT实验均显示比较好的癌细胞杀伤能力：纳米胶束M1效果是顺铂的7倍；M2的效果是奥沙利铂的2-3倍，M(EA/Pt)里利尿酸和铂的协同作用强于小分子联合用药，M(OxaPt(IV)/Bcl-2)能有效下调Bcl-2的表达，其协同作用也优于siRNA转染+小分子或高分子铂的联合应用组。此外纳米胶束在细胞内铂的积聚以及DNA-Pt加合物形成均多于对应的小分子药物。M(EA/Pt)动物皮下移植瘤模型结果显示其有高效、低毒的优势。本课题的研究结果为恶性肿瘤的药物治疗提供了新的可能。