脑肿瘤靶向治疗的新策略研究

Noninvasive therapy of brain cancer remains to be a big chalenge due to the blood-brain barrier. Based on our experience, a new therapeutic strategy for targeting experimental brain cancer is proposed, which involves construction of a brain-specifc shRNA expression plasmid (anti-EGFR shRNA) against EGFR under the control of GFAP promoter as well as synthesis and modification of pegylated immuno-lipopolyplexes (PILP). More importantly, the PILP will be made possible to encapsulate both anti-EGFR shRNA and the chemotherapeutic DOX at the same time. A targeting monoclonal antibody will be conjugated to PILP, which will facilitate the transcytosis of the therapeutics across the blood-brain barrier and tumor cell membrane. With all these efforts, we are aiming at an enhanced therapeutic effect for barin cancer, and less side effects. We will first test the new drug system's cytotoxicity and growth inhibitory effect on U87 glioma cells, and then move to in vivo efficacy study in U87 orthotopic brain tumor model. Furthermore, the mechanism of possibly enhanced therapeutic effect and toxicicty will also be investigated. This new PILP system could be a universal system to encapsulate any kinds of shRNA and any positively charged chemotheraputics. Therefore, this project is of grest significance to cancer therapy, espeially brain cancer therapy.

由于血脑屏障的存在，脑肿瘤的非伤害性治疗一直是一个国际性的大难题。本课题创新性地采用GFAP启动子，构建针对EGFR的具有脑特异表达功能的shRNA表达质粒；同时合成和修饰"PEG修饰的免疫脂质复合体"，使它能同时装载shRNA质粒 和化疗药DOX，连接起靶向作用的单抗，使之能通过血脑屏障, 对实验性脑肿瘤进行脑靶向性联合治疗，以期增强治疗效果，降低毒副作用。我们将首先优化制备免疫脂质复合体的条件，然后在体外U87神经胶质瘤细胞上研究比较单一及联合治疗对肿瘤细胞生长的抑制作用，并探讨可能的增强抑制作用机制，然后再同样体内研究脑内接种的U87肿瘤的治疗效果及其毒副作用。本课题研究的免疫脂质复合体可以同时装载各样的shRNA和各样的带正电荷的化疗药物，因而具有广谱性。所以本课题对于探索肿瘤，尤其是脑肿瘤的治疗新策略具有重要理论意义和实际应用价值。

我们已成功建立了基于聚乳酸羟基乙酸共聚物（PLGA）纳米粒的脑肿瘤联合治疗药物转运系统，并且成功引入能够同时穿越血脑屏障和靶向脑胶质瘤细胞的靶向短肽-Angiopep-2。我们首先探究了基于PLGA的纳米粒共载阿霉素（DOX）和（表皮生长因子，EGFR）EGFR siRNA对U87MG脑肿瘤的治疗效果。研究证实该缓释系统能够转运更多的DOX 和siRNA到U87MG细胞内，并且体内静脉注射后能够通过血脑屏障，DOX和EGFR siRNA两者联合能够明显抑制原位接种U87MG细胞的小鼠脑胶质瘤的生长和浸润。然后，我们将该纳米粒系统进行改造，运载化疗药物多西他赛和集成像与光疗于一体的吲哚菁绿，实现近红外荧光示踪下对脑肿瘤化疗和光疗的联合，探索了脑肿瘤诊断治疗一体化的新途径。最后，我们将该系统进一步推广，在装载多西他赛PLGA纳米粒表面还原氯金酸，形成具有X-Ray成像的不连续金壳包被的核壳型载药纳米粒。为了实现纳米粒在体内的长循环特性以及脑肿瘤靶向性，将具有长循环特性的聚乙二醇2000（HS-PEG2000）和功能基团Angiopep-2通过硫金键连接于纳米金表面，制备光热联合化疗与X-Ray成像功能一体化纳米粒。该核壳型纳米粒不但能够实现近红外光触发的远程药物释放，而且将金纳米壳在近红外光照射下的光热转换特性与纳米粒内核所载化疗药物相结合，实现了光热联合化疗治疗脑胶质瘤。通过本项目的实施，到目前为止已有三篇SCI论文发表，一篇发表在药剂学一流期刊Journal of Controlled Release 2015, 220(Pt A), 545-555上，影响因子7.442。另外两篇发表在Journal of Drug Targeting，2015, 23(9), 832-846和Macromolecular Bioscience, 2015, 15(11), 1571-1585上。此外，获得发明专利一项：一种基于聚乳酸-羟基乙酸共聚物的靶向共载药物传递系统纳米粒的制备方法及应用（专利号：ZL201310615647）。通过本项目的实施，培养了四名硕士研究生和一名博士研究生。