基于磁热疗法的cRGD介导的超顺磁长循环纳米脂质体用于维吾尔药去氢骆驼蓬碱抗癌及其对癌细胞p53基因的研究

In order to target cancer cells and decrease side effects of harmine, a uygur anti-cancer drugs, A kind of cRGD modified magnetoliposomes which encapsulates ultrasmall superparamagetic nanoparticles(USPIOs) and harmine into its aqueous core will be studied. This kind of magnetoliposomes will entrap not only USPIOs but also harmine which is a kind of common Uygur anti-cancer drugs, and cRGD peptide will be conjugated with the lipid at the out of the space of liposomes, because cRGD will be highly expressed in cancer cells. This kind of magnetoliposomes owns several functions at the same time. One is that it can target to cancer cells with the aid of external magnetic fields and can serve as MRI contrast agents. Another function is that it can actively target to cancer cells because cRGD at the surface of this magnetoliposme can bind with αγβ3 existing the cancer cells.And also this kind of magnetoliposomes can increase the content of USPIOs at the tumor sites and prolong the mean residual time of USPIOs.So it can overcome some disadvantages of USPIOs when it is used in clinic. When this magnetoliposomes reach to cancer cells, it will burst out becuase the magnetical effects creat heat and lead to membrane of liposomes breaks, and then harmine will release and kill cancer cells,which is named as hyperthermia. If that, harmine will focus on cancer cells because of this targeting delivery system and subsequently kill cancer cells after this kind of magnetoliposomes enter the cancer cells, which will decrease the side effect of harmine on the nervous system. So this drug delivery system is a multifuntional preparation. In this study, we will focus on the characteristics of magnetoliposomes in vitro and in vivo through detecting DLS, entrap efficiency, cell intake, cell apoptosis,MTT etc. And we also want to be know if harmine can kill brain tumor cells since harmine has strong side effects on brain when it is used. Futhermore, we are also want to concentrate on the anti-cancer mechanism of harmine and its magnetoliposomes through cell and molecular biology experiments such as Western-blot and RT-PCR,especially their effects on p53 gene because there are few researchers who are studying its anti-cancer mechanism.

为使维吾尔药抗癌成分去氢骆驼蓬碱更好地靶向癌细胞，减少其毒副作用，本研究拟首次构建一种cRGD修饰的去氢骆驼蓬碱超顺磁长循环多功能脂质体。该脂质体将超顺磁纳米粒包裹入长循环脂质体，同时包裹维吾尔药抗癌成分去氢骆驼蓬碱，并在脂质体表面偶联RDG肽。利用磁纳米粒的磁靶向作用，在外部磁场下，将脂质体靶向到肿瘤细胞，又利用cRGD与肿瘤表面αγβ3受体的结合，使系统主动靶向到肿瘤细胞，并利用磁热效应，在肿瘤部位使脂质体膜破裂，药物释放，从而杀灭癌细胞。该磁纳米脂质体，可使磁纳米粒在肿瘤部位富集，克服磁纳米粒在肿瘤部位浓度低的问题，同时利用脂质体表面修饰达到磁纳米粒的长循环作用，还提高药物靶向性，降低其毒副作用。本研究对脂质体的可行性从体外和体内进行考察。另外针对去氢骆驼蓬碱对神经系统的毒性，利用脑胶质细胞瘤为模型细胞，考察去氢骆驼蓬碱及脂质体对脑癌的作用。并考察其对肿瘤细胞p53基因的作用机制。

为增强去氢骆驼蓬碱的体内抗癌效果，降低其毒性。本课题采用cRGD和转铁蛋白两种配体分别作为靶头，构建了cRGD修饰的去氢骆驼蓬碱长循环磁纳米脂质体和转铁蛋白修饰的去氢骆驼蓬碱长循环磁纳米脂质体两种磁纳米脂质体，并对两种磁纳米脂质体的体外理化性质进行了表征。在此基础上，采用HepG2肝癌和U87脑癌细胞两种细胞模型，通过MTT细胞毒性实验、流式细胞凋亡实验、Hoechst染色实验探讨了构建的两种磁纳米脂质体对肿瘤细胞的抑制作用，通过激光共聚焦显微镜研究制剂对肿瘤细胞的靶向摄取。进一步地，采用RT-PCR和Western blot方法探讨了制剂对p21、p53、Caspase-3、bax、bcl-2等抑癌基因的作用，从机理上探讨制剂对肿瘤的作用，最后采用荷瘤小鼠模型，探讨了去氢骆驼蓬碱长循环磁纳米脂质体的抑瘤效果和体内分布。为探索不同的剂型的作用，课题组在完成上述工作的基础上，又制备了去氢骆驼蓬碱磁纳米脂质颗粒和去氢骆驼蓬碱透明质酸聚合物胶束两种剂型。初步探讨了体外的理化性质和肿瘤细胞抑制作用。经过研究，两种配体修饰的去氢骆驼蓬碱长循环磁纳米脂质体已形成，平均粒径均为200nm左右，脂质体荷负电，包封率较高，磁纳米粒包封率按Fe2+含量计算为131.1±1.8 μg.mL-1。脂质体稳定性良好，一个月内未见明显沉淀，释放缓慢持久，磁响应性良好，具备强的T2 成像效果。细胞毒性结果显示，制得的两种磁纳米脂质体对HepG2细胞和U87细胞具良好的细胞毒性，细胞毒作用较原料药相比缓慢持久，促细胞凋亡作用明显，对肿瘤细胞具有好的靶向作用。配体修饰的磁纳米脂质体使得p21、Caspase-3和p53相关凋亡基因明显上调，对Bcl-2作用不明显。体内抗肿瘤结果显示，磁纳米脂质体有很好的T2成像效果，肿瘤抑制作用与原料药相比，有显著性差异，磁纳米脂质体和原料药均在肝、脾部位浓度较高，磁纳米脂质体与原料药相比，降低炎症的效果更显著。以上结果证实了该剂型的可行性。