叶酸-穿膜肽修饰的磁性纳米载体介导p53靶向治疗肝癌的实验研究

In this study, we are trying to synthesize a kind of nonferrous metal doped ferrite magnetic nanoparticles with chemical process, which were modified with hydrophilic molecules. The physical, chemical and biological properties of these magnetic nanoparticles were investigated using modern analytical techniques and biological technology. When the magnetic nanoparticles were conjugated by Folate and coupled by TAT-EGFP fusion proteins, we name them as Folate-TAT coupled magnetic nanoparticles (FTMN).The FTMN will acquire both active target function and transmembrane activity, which could be observed by fluorescence microscope. The negatively wt-p53 were complexed with the positively charged FTMN via electrostatic interactions to prepare wt-p53 loaded FTMN complexes (wt-p53- FTMN). The potency of combining wt-p53 gene and resisting DNase I digestion of FTMN was analyzed by agarose gel electrophoresis and the effect of gene transefection on cells proliferation and apoptosis rates were measured in vitro. After the model of transplanted hepatocellular carcinoma(HCC) has been established in nude mice, magnetic field was applied to investigate the target distribution of wt-p53-FTMN in transplanted tumor in nude mice. When subjected to an alternating magnetic field, magnetic nanoparticles would produce heat. Hyperthermia and gene therapy are combined to investigate synergistic therapeutic effects on HCC. This is the first time to build a kind of double target magnetic nanoparticles which were connected with both folic acid and transmembrane peptides. We are mean to provide a high efficiency and new carrier for gene therapy of HCC.

本研究拟用化学方法制备有色金属掺杂的铁氧体纳米磁粒，采用亲水性小分子对其进行表面修饰，运用分析技术与生物学技术对其进行分析，确定其理化性质与生物性能。通过嫁接技术把叶酸连接到纳米磁粒上，赋予其主动靶向功能；运用偶联技术把融合有报告基因的穿膜肽TAT连接到纳米磁粒上，赋予其跨膜性能。荧光显微镜观察叶酸/穿膜肽纳米磁粒(FTMN)的靶向性能与穿膜活性。凝胶电泳分析FTMN结合wt-p53基因及抵抗DNase I消化的能力，体外细胞实验考察FTMN转染wt-p53基因后对细胞增殖、凋亡的影响。建立肝癌裸鼠模型后，在外部磁场引导下，进行体内靶向定位实验，通过检测各组织中的纳米磁粒含量，考察基因载体的靶向能力。在交变磁场下，磁致生热，使热疗与基因治疗结合起来，探讨其协同治疗的疗效。本研究首次将叶酸和穿膜肽同时连接到纳米磁粒上，拟构建具跨膜功能的双重靶向纳米磁粒，为肝癌的基因治疗提供高效、新型的载体。

本研究采用化学方法制备出铁氧体纳米磁粒，把叶酸和穿膜肽TAT对铁氧体纳米磁粒进行了表面修饰，制备出一种新型纳米磁粒—FTMN。红外分析证实叶酸和穿膜肽已被成功地修饰在磁性纳米粒子表面。扫描电镜观察显示纳米粒呈均匀分散的球形颗粒，平均粒径为250nm。FTMN颗粒表现出顺磁性。DNA结合试验和保护试验证实FTMN纳米粒能有效结合p53基因并能抵抗血清酶的消化作用。荧光显微镜观察、Western印迹法分析显示FTMN纳米粒能将p53基因高效地转染入HepG2细胞中并表达相应蛋白。体内抑瘤实验显示FTMN能介导p53基因进入瘤体，并有效抑制肝癌裸鼠移植瘤的生长，且在FTMN处理组的裸鼠移植瘤内证实了p53蛋白的表达。因此，制得的FTMN纳米粒可作为p53基因的有效载体，有望成为肝癌基因治疗的新的辅助手段。