聚合物修饰的叶酸靶向磁性纳米传递系统的制备及其对鼻咽癌的抑制和成像作用

发布时间：2018-01-13 23:11

本文关键词：聚合物修饰的叶酸靶向磁性纳米传递系统的制备及其对鼻咽癌的抑制和成像作用　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：鼻咽癌(Nasopharyngeal carcinoma,NPC)为我国南方地区常见的转移性恶性肿瘤。放疗是鼻咽癌各个分期的标准疗法,但并发症较多。鼻咽癌引入化疗可提高总生存率,但化疗药也存在靶向性低、副作用大的问题。基因治疗是治疗肿瘤的另一种有效策略,但基因的如何传递至靶细胞也是一个重要挑战。利用纳米技术将药物与基因载于纳米粒子内部,在体内靶向进入肿瘤细胞后再将基因与药物释放可以有效解决上述问题。本课题组前期研究表明,叶酸受体在鼻咽癌细胞内高表达,并在此基因上合成了叶酸靶向载顺铂磁性纳米粒子,可靶向杀伤鼻咽癌细胞。但该磁性纳米粒子存在摄取效率低的问题。本研究在前期研究的基础上制备了两种叶酸靶向磁性纳米载体,以期解决上述问题。制备阳离子聚合物mPEG-PLL-FA,并通过静电相互作用包裹带负电的载顺铂的磁性纳米粒子ASA-CDDP-MNP,并吸附含有抑癌基因TFPI2质粒,合成一种叶酸靶向载顺铂和TFPI2基因的多功能靶向磁性纳米共传递系统mPEG-PLL-FA-TFPI2-ASA-MNPs-CDDP。利用核磁氢谱、动态光衍射、低温透射电镜、磁强计检测磁性纳米共传递系统的结构及理化特性;血细胞凝集实验、MTT法、琼脂糖凝胶电泳分别检测其生物相容性及对DNA的结合和保护能力;普鲁氏蓝染色、荧光显微镜、Western blot分别检测磁性纳米共传递系统的靶向性及基因转染效率;MTT、划痕实验、流式细胞术、平板克隆形成实验检测磁性纳米共传递系统对鼻咽癌细胞HNE-1的靶向抑制作用;MRIT2加权成像检测磁性纳米共传递系统在细胞内的成像效果。结果显示,磁性纳米共传递系统具有良好的生物相容性、优良的靶向性、对鼻咽癌细胞HNE-1有较强的抑制作用和成像效果。但该体系存在转染效率低的问题。为了克服这一问题,我们用叶酸靶向修饰的季铵阳离子化羟乙基纤维素包裹磁性纳米粒子并吸附质粒DNA,形成复合物catHEC-FA@SPIO/pDNA。通过核磁共振氢谱、红外光谱、紫外光谱、热失重、X射线衍射、磁化曲线、透射电镜等手段对产物进行表征;MTT法、琼脂糖凝胶电泳分别检测其生物相容性及对DNA的结合和保护能力;普鲁氏蓝染色、荧光显微镜、Western blot分别检测磁性纳米粒靶向性及基因转染效率;MTT、流式细胞术、检测磁性纳米粒对鼻咽癌细胞HNE-1的靶向抑制作用;MRI T2加权成像检测磁性纳米共传递系统在细胞内的成像效果。结果显示,catHEC-FA@SPIO/pDNA具有良好的生物相容性、优良的靶向性、较高的基因转染效率,对鼻咽癌细胞HNE-1有较强的抑制作用和成像效果。叶酸靶向修饰的季铵阳离子化羟乙基纤维素包裹磁性纳米粒子复合物catHEC-FA@SPIO可实现基因传递与磁共振成像双功能化。
[Abstract]:Nasopharyngeal carcinoma (Nasopharyngeal carcinoma NPC) is a common malignant tumor of the southern region of China. Radiotherapy is the standard treatment for nasopharyngeal carcinoma in various stages, but more complications. The introduction of nasopharyngeal carcinoma chemotherapy can improve the total survival rate, but also has targeted chemotherapy, and severe side effects. Gene therapy is another effective the strategy for the treatment of tumors, but how to transfer the gene to target cells is an important challenge. Using nanotechnology to drug and gene contained in the nanoparticles inside, the in vivo targeting into tumor cells after gene and drug release can effectively solve the above problems. Ourprevious studies showed that high expression of folate receptor in nasopharyngeal carcinoma in the cell, and this gene was synthesized with cisplatin loaded magnetic nanoparticles to target folic acid, can be targeted killing of nasopharyngeal carcinoma cells. But the magnetic nanoparticles are the problem of low efficiency of uptake This study. On the basis of the previous research has prepared two kinds of folate targeting vectors of magnetic nanoparticles, in order to solve the above problems. The preparation of cationic polymer mPEG-PLL-FA, and through the electrostatic interaction of cisplatin loaded magnetic nanoparticles coated with negative ASA-CDDP-MNP, and adsorption of tumor suppressor gene TFPI2 containing plasmid, multifunctional targeted drug cisplatin the gene expression of TFPI2 and synthesis of a folate targeted delivery system consists of mPEG-PLL-FA-TFPI2-ASA-MNPs-CDDP. magnetic nanoparticles by 1H NMR, dynamic light diffraction, cryo TEM, magnetometer detection magnetic nano delivery system total structure and physicochemical properties; blood cell agglutination test, MTT assay, agarose gel electrophoresis were used to detect the compatibility and the combination of DNA and the protection ability of the biological; Prussian blue staining, fluorescence microscope, Western blot was used to detect the total magnetic nano delivery system and the efficiency of gene transfection to target ; MTT, scratch test, flow cytometry, colony formation assay total magnetic nano delivery system on nasopharyngeal carcinoma HNE-1 cell targeting inhibition; MRIT2 weighted imaging detection system imaging effect of magnetic nano co transmission in cells. The results showed that the total magnetic nano delivery system has good biocompatibility and targeting excellent, inhibit and imaging strong effect on nasopharyngeal carcinoma cells HNE-1. But there is the problem of low transfection efficiency of the system. In order to overcome this problem, we use the folate modified quaternary ammonium cationic hydroxyethyl cellulose coated magnetic nanoparticles and adsorption of plasmid DNA, catHEC-FA@SPIO/pDNA. complexes formed by hydrogen nuclear magnetic resonance spectrum, infrared spectroscopy, UV spectroscopy, thermogravimetric analysis, X ray diffraction, magnetization curve, the structure of product was characterized by transmission electron microscope; MTT assay, agarose gel electrophoresis respectively. To test its biocompatibility and the combination of DNA and protection capacity; the Prussian blue staining, fluorescence microscope, Western blot was used to detect the magnetic nanoparticles targeting and gene transfection efficiency; MTT, flow cytometry, detection of magnetic nanoparticles on nasopharyngeal carcinoma HNE-1 cell targeting inhibition; MRI T2 weighted imaging detection of magnetic nanoparticle the effect of system transfer imaging in cells. The results show that catHEC-FA@SPIO/pDNA has good biocompatibility, excellent targeting, higher gene transfection efficiency, inhibition and stronger effect on the imaging of nasopharyngeal carcinoma cells HNE-1. Folate modified quaternary ammonium cationic hydroxyethyl cellulose coated magnetic nanoparticle composites can achieve catHEC-FA@SPIO gene transfer and dual functional magnetic resonance imaging.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943;R96

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