cRGD联合TAT穿膜肽介导奥沙利铂纳米粒靶向治疗大鼠肝癌及其机制研究

Oxaliplatin is one of the most commonly used drugs in the interventional therapy on liver cancer. The lack of targeting property of drugs to tumor tissue and sustained-releasing property are the two important factors that affect the therapeutic effect. Our previous studies showed that the oxaliplatin nanoparticles modified by cRGD and TAT peptide had a good targeting ability in vitro tissue of liver cancer. Thus we put forward our hypothesis: The double modified oxaliplatin nanoparticles perfusing by hepatic artery can further improve the drug concentration in liver cancer cells and prolong the time of drug action, which will enhance its anticancer effect. To verify the hypothesis, cell phagocytosis test, antibody blocking test, cytotoxicity test and tissue distribution test will be done to verify the targeting ability of the double modified oxaliplatin nanoparticles on hepatocellular carcinoma tissue and cells in vitro and in vivo. Inhibition rate of tumor, relative density of tumor blood vessels, pathology examination, lymph nodes and lung metastasis effect analysis will be conducted to evaluate the antitumor effect of the nanomedcine by hepatic artery infusion on rats bearing with liver cancer. Moreover, the mechanism of anti-hepatocarcinoma and anti-metastases will be explored in cell and protein levels. In this study, we will use the tumor targeting property of cRGD, the cell targeting property of TAT peptide and the sustained-releasing property of nano-drug delivery system synthetically to improve the anti-tumor effect of oxaliplatin by improving the its preparation，which will provide a new mind for clinical doctor in increasing therapeutic effect on tumor.

奥沙利铂（OXA）是肝癌介入治疗常用药物之一，缺乏肿瘤靶向性和缓释性是影响其疗效的两个重要因素。我们的前期研究提示经cRGD和TAT穿膜肽双重修饰的OXA纳米粒对体外肝癌细胞具有良好的靶向性，其抑瘤作用也显著增强。据此，我们提出假设：经肝动脉灌注双重修饰的OXA纳米粒，可进一步提高肝癌细胞内的药物浓度，延长药物作用时间，从而增强抗癌效果。为验证这一假设，我们将通过细胞吞噬实验、抗体阻断实验、细胞毒性试验和组织分布试验等验证双重修饰的OXA纳米粒对体内外肝癌组织和细胞的靶向结合能力；通过抑瘤率测算、肿瘤血管相对密度计算、病理学检查、淋巴结及肺转移效果分析等评价其经肝动脉给药对原位肝癌大鼠的治疗效果，并从细胞和蛋白水平探讨其作用机制。本研究将协同利用cRGD的肿瘤靶向性、TAT穿膜肽的细胞靶向性和纳米载药系统的缓释性，通过对OXA进行制剂改良提高其抗肝癌效果，为临床提高肿瘤化疗效果提供新思路。

原发性肝细胞癌是我国常见的恶性肿瘤，但目前临床上使用的介入药物缺乏靶向性和缓释性，其作用效果仍有待进一步提高。本研究通过制作OXA@cRGD-TAT纳米粒，通过扫描电镜和红外光谱进行表征，体外释放观察其药物缓释能力；利用纳米粒处理肝癌细胞，通过CCK8检测细胞活力，流式细胞术检测细胞周期和凋亡，划线检测细胞迁移，Western blot检测各组BCL-2、Bax的表达。注射纳米粒处理的Walker-256 细胞，构建大鼠原位移植性肝癌模型，HE检测肿瘤组织和肺组织的病理状态，TUNEL检测肿瘤凋亡情况，Western blot检测肿瘤组织BCL-2、Bax的表达，免疫组化检测肺组织MMP2和MMP9的表达水平。经过优化配比方案，成功制备出OXA@cRGD-TAT-PEG-PLA载药纳米粒，扫描电镜结果下靶向纳米粒在500nm左右，更小一点的在200-300nm，纳米粒稳定，球形规则，且释放过程很平稳，是理想的缓释材料。空白纳米粒处理肝癌细胞对细胞的增殖没有显著影响，说明纳米粒没有细胞毒性。在体外实验中，OXA@cRGD-TAT-PEG-PLA载药纳米粒显著促进肝癌细胞株HepG2和Walker-256的凋亡，并抑制细胞的迁移。在体内实验中，模型对照组肿瘤组织中细胞分布均匀，核大，深染，周围无坏死肿瘤细胞，并可见肿瘤细胞向横纹肌侵袭，血管丰富，凋亡细胞少；且肺组织中可见明显病变，MMP2和MMP9的表达水平最高。各载药组中肿瘤组织可见坏死的肿瘤细胞，凋亡细胞明显增加，且肺泡增厚现象减轻且无出血现象，MMP2和MMP9的表达水平明显下调。总体来说，本研究充分利用纳米载药系统的缓释性、cRGD配体的肿瘤靶向性和TAT穿膜肽的细胞靶向性，成功制备出OXA@cRGD-TAT-PEG-PLA载药纳米粒，多方协同作用来提高OXA的抗肝癌效果，在体内和体外实验中都显示纳米粒能够促进肿瘤细胞的凋亡，减缓肝癌的组织损伤情况。