电荷反转型共载Pt(IV)/siRNA纳米超声-磁共振双模态造影剂用于可视化治疗卵巢癌耐药的研究

Ovarian cancer is the leading cause of death among women with gynecological malignancies. Acquired resistance to chemotherapy is a major limitation for ovarian cancer treatment. New strategies to overcome drug resistance are urgently needed in order to reduce the mortality rate of ovarian cancer. Silencing genes that are involved in drug resistance using RNA interference (RNAi) can reverse cisplatin resistance in ovarian cancer. Successful treatment of ovarian cancer cells with multidrug resistance (MDR) gene-silencing siRNAs and cisplatin requires the development of novel vehicles that can specifically and effectively deliver cisplatin to cell nuclei and siRNAs to cell cytoplasms, respectively. This project has designed a tumor targeted nanoscale phase-change US/MR contrast agents for dual-mode US/MR imaging and the co-delivery of cisplatin and pooled small interfering RNAs (siRNAs) to enhance therapeutic efficacy by silencing multiple drug resistance (MDR) genes and resensitizing resistant ovarian cancer cells to cisplatin treatment. The US/MR contrast agents were loaded with a cisplatin prodrug and MDR gene-silencing siRNAs (Bcl-2, P-glycoprotein [P-gp], and survivin) via encapsulation and surface coordination, respectively. 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. We modified an amino-functionalized nano contrast agents with 2,3-dimethylmaleic anhydride (DMMA), which reacted with the amino group to produce an amide bond and a carboxylic acid group. The resultant amide bond is relatively stable at neutral and alkali pH values, but degrades promptly under slightly acidic conditions to expose positively charged amino groups again. Once accumulated at the tumor site through the enhanced permeation and retention (EPR) effect, the modified nano contrast agents should be activated to be positively charged in response to the pH value. Accordingly, cellular internalization should be enhanced as a result of the strengthened nano contrast agents–cell interaction to promote intracellular cargo release. The US/MR contrast agents exhibited advantages such as better contrast for US imaging, higher backscattering signal as well as and serving as the contrast agent for MR imaging. Enhancement of ultrasound (US)/magnetic resonance (MR) imaging and US triggered drug delivery were evaluated both in vitro and in vivo. The specific treatment target, precise chemosensitization and imaging evaluation of the US/MR contrast agents were studied. The purpose of this study is to establish a new mode of adjuvant therapy in ovarian cancer, which integrates not only effective and safe cell targeted chemotherapy but also a new real time target imaging method.

改善顺铂剂量依赖性毒性、克服顺铂耐药、实现可视性化疗效果评估是卵巢癌治疗中亟待解决的三大问题。申请人已结题的青年基金研究证实毒副作用小的Pt(IV)可替代Pt(II)用于卵巢癌治疗解决顺铂剂量依赖性毒性问题。本项目拟通过设计载体结构电荷反转，提高Pt(IV)/siRNA体内肿瘤靶向性，协同治疗增强顺铂化疗敏感性克服耐药；通过结合药物载体与分子影像技术开发多功能造影剂，实现超声辅助治疗-无创可重复评估疗效。本项目以已有研究为基础，构建电荷反转型共载Pt(IV)/siRNA纳米超声-磁共振双模态造影剂，评价双模态造影剂体内外安全性；探讨超声介导载Pt(IV)/siRNA纳米双模态造影剂对卵巢癌耐药的治疗作用，揭示耐药机制；从动物整体水平上验证Pt(IV)/siRNA的协同治疗效果，评价造影剂增强超声及磁共振显像效果。本课题的完成将证实所构建纳米超声-磁共振造影剂的递送功能、治疗效果和成像质量。

卵巢癌在治疗完成后常常复发，复发性卵巢癌极易产生耐药而难以治疗，最终导致患者死亡。实时准确的监测能对疾病进行再诊断，便于及时调整治疗方案，提升治疗效果。卵巢癌治疗的常规监测手段是定期影像学检查，这具有监测时间的滞后性和监测频率的有限性。因此，研究克服卵巢癌耐药的方法和治疗过程的可视化具有深刻的临床意义。本课题拟构建一种安全有效的共载Pt(Ⅳ)/siRNA纳米超声-磁共振双模态造影剂，用于克服耐药和疾病监测，即诊疗一体化。. 介于前药特性及siRNA的不稳定性，本课题设计共载Pt(IV)和siRNA的纳米粒用于药物、基因递送，同时该纳米粒还包载全氟己烷（PFH）和Fe3O4用于超声和核磁显像。造影剂在体内具有良好的肿瘤靶向性。肿瘤生长曲线、离体肿瘤图片及体内CA125含量三方面结果显示Pt、Pt(IV) NPs-cRGD + US、NPs、NPs-cRGD、NPs-cRGD + US各组体内肿瘤抑制作用逐渐增强，超声联合纳米靶向造影剂能最大程度地克服卵巢癌耐药，其抑瘤率为94%。肿瘤病理切片结果表明超声联合纳米靶向造影剂能最大程度促进肿瘤细胞凋亡、抑制肿瘤细胞增殖、促进促凋亡蛋白的表达、抑制BCl-2及BIRC5的表达。裸鼠体重监测、肝肾功能测定及重要器官HE染色结果证明了超声联合纳米靶向造影剂的安全性。. 本研究成功构建了具有靶向性的共载Pt(Ⅳ)/siRNA纳米超声-磁共振双模态造影剂（NPs-cRGD），它具有安全性高、药物/基因缓释、高效荷载及保护基因、储存及血清稳定性良好的特性。其次，NPs-cRGD具有良好的体内外超声和核磁显像作用。同时，超声联合NPs-cRGD在体外能明显克服卵巢癌耐药，其机制为：增加细胞摄取药物和基因，保证基因成功从溶酶体逃逸，NPs-cRGD内siBIRC5能最大程度抑制耐药细胞内BIRC5的抗凋亡作用，前药Pt(Ⅳ)消耗细胞内GSH并被还原为能与DNA结合的Pt(II)复合物，GSH的减少联合Pt(II)共同破坏细胞内稳态，最终促进促凋亡蛋白的表达和抑制抗凋亡蛋白的表达而克服卵巢癌耐药。最后，动物实验证实超声联合NPs-cRGD能最大程度克服卵巢癌耐药，并且在体内未观察到明显毒副作用。因此，NPs-cRGD实现了可视化克服卵巢癌耐药，它为临床克服耐药的治疗提供新方法，为可视化治疗及多功能造影剂的开发打开了新思路。