CAS OpenIR  > 中科院上海应用物理研究所2004-2010年
基因转染载体M-PEIs纳米凝胶的合成
Alternative TitleThe preparation of M-PEIs nanogels as gene transfection vectors
徐冬梅
Subtype博士
Thesis Advisor盛康龙
2007-05-23
Degree Grantor中国科学院上海应用物理研究所
Place of Conferral上海应用物理研究所
KeywordFenton反应 聚乙烯亚胺纳米凝胶 基因转染 光化学
Abstract基因治疗是一种新兴发展起来的治疗手段,它为遗传性、恶性及传染性等疾病的治愈提供了可能,但制备高效安全的基因转染载体仍是基因治疗的一个瓶颈。由于非病毒类载体毒副作用小,具有纳米结构的阳离子型聚合物的合成和使用成为近年来该研究领域的研究热点之一。 聚乙烯亚胺(PEI)是一种研究广泛的阳离子聚合物,它和其他载体的不同点在于其分子骨架上每三个原子就有一个可质子化的N原子,因此,PEI具有较高的pH缓冲能力,易被细胞内吞和从内涵体中逃逸,可以将载带的DNA从载体中释放到细胞质中。许多研究小组研究了直线型和枝杈型PEI的制备方法,并且使用PEG化或与其他功能分子共聚等手段对PEI分子进行修饰,以提高PEI的生物相容性、亲水性、生物降解性或靶向性。 制备PEI通常使用单体氮丙啶的阳离子聚合方法,但制备特定分子量PEI的合成、纯化步骤复杂,体系中单体及修饰剂残留不利于生物和医学应用。Fenton反应产生高反应活性羟基自由基,常用于引发有机物抽氢和加成反应,也用于废水中有机物的氧化降解处理。在紫外光诱导下的光助Fenton反应其氧化能力明显提高,羟基的生成速率和量子产额因Fe2+的存在而显著提高。 本论文主要研究内容为:在室温下使用光助Fenton反应制备M-PEIs纳米凝胶作为基因转染载体。使用PEI预聚物为合成试剂,使用低压汞灯(辐射253.7 nm的紫外光)为辐照源,反应体系持续通N2以排除O2对合成反应的干扰。通过改变pH值、反应时间和体系中各种试剂浓度,控制 M-PEIs纳米凝胶粒径及其zeta电位。所得样品经0.45 μm水相针式滤器过滤纯化后,再用透析袋截留分子量大于10000的样品。研究表明纯化后的样品具有更好的稳定性。本论文详细讨论了三种合成体系获得的结果,发现体系B不仅粒径可控性更强,且样品的粒径分布更均匀。 为了探索M-PEIs纳米凝胶粒径对细胞基因转染效率的影响规律,选取38, 75, 87, 121, 132 和167 nm六种粒径样品,以肝癌细胞Bel-7402、肺癌细胞A549、胃癌细胞BGC-823和卵巢癌细胞Hela四种人类高发肿瘤细胞为细胞模型,进行细胞基因转染研究。MTT实验中没有发现M-PEIs/DNA复合物具有明显的细胞毒性。以pLEGFP-C1为转染基因,使所有研究样品载带质粒DNA质量相同,选择最优M-PEIs:DNA比以使DNA-载体复合物带有少量的正电荷,结果发现75 和87 nm的M-PEIs纳米凝胶样品具有最高的基因转染效率。 本论文使用各种谱学对合成的样品进行了系列表征,对光助Fenton反应合成M——PEIs纳米凝胶机理进行了探索,对M-PEIs纳米凝胶粒径及表面电位与细胞基因转染效率的相关性进行了讨论,也对γ射线辐照法制备M-PEIs纳米凝胶方法作了尝试,为阳离子纳米凝胶载体合成及其基因转染研究提供了有益启示。
Other AbstractGene therapy is a new and exciting therapeutic concept that offers a promise of cure for an array of inherited, malignant and infectious disorders. However, gene delivery vectors are still the main problem to be improved in gene therapy. There are considerable interests in using nano-structured cationic polymers as non-viral vectors, which have less serious side effects compared to viral vectors. As a well-known cationic polymer, PEI is different from the others in that every third atom of its backbone is a protonable nitrogen atom which makes it have high buffering capacity to osmotic swelling and rupture of endosomes resulting in the release of the vector into the cytoplasm. Some scientist groups made their efforts to synthesize linear or branched PEI, and modified PEI with hydrophilic groups by PEGylation or copolymerize PEI with other functional reagents to make them more biocompatible, amphiphilic, biodegradable or targetable. PEI could be obtained by cationic polymerization of aziridine. However, the method required complex steps to prepare PEI with designed molecular weight. And it was difficult to remove residual monomers and additives from sample which were toxicity used in biology and medicine. Highly reactive hydroxy radicals could be generated from fenton system to react with the organic compounds (H abstraction, addition, and so on). It was also used used for degradation of organic pollutants in waste water. Oxidation ability of the Fenton mixtures can be greatly enhanced via UV irradiation. The production rate of hydroxyl radicals and quantum yield can be enhanced by the rebirth of ferrous ions. In this paper, photo-Fenton reaction (hoto-induced polymerization) was utilized to synthesize M-PEIs nanogels as gene delivery vector at room temperature. PEI pre-polymer was used as the initiate reagent and low-pressure Hg lamps which emit 253.7 nm UV light were used to illuminate the systems. During the reaction, the solution was protected from O2 by bubbling N2 continuously. By changing reaction conditions, such as the pH value, reaction time and concentrations of solutes, sizes and zeta potentials of M-PEIs nanogels could be controlled. After polymerization, the products were purified with membrane filters of 0.45 μm and treated further by dialysis bags with size exclusion below 10 kDa in order to make them more stable for storage. The photo-Fenton reaction system (System B) of preparing M-PEIs nanohydrogel was also compared to other systems. The results showed that the sizes of M-PEIs in system B could be controlled easily and most samples had narrow size distributions. In order to study the size effect of M-PEIs nanogels on gene transfection efficiency, six M-PEIs samples with size being 38, 75, 87, 121, 132 and 167 nm were chosen as transfection vectors. The same quantities of plasmid DNA (pLEGFP-C1) were loaded into human liver cancer cell (Bel-7402), human lung cancer cell ( A549), human gastric cancer cell (BGC-823) and human cervix cancer cell (Hela). It has been found that all of the DNA complexes had no obvious cytotoxicity in MTT assays and theie surface charges showed positive at the optimum weight ratio of M-PEIs and DNA. The samples of 75 and 87 nm had the highest transfection efficiency over 30% with no dependent on cell lines. Kinds of methods were used to characterize the samples, the synthesized mechanism was discussed and the affect of size and zeta potentials on gene transfection efficiency were also studied. γ-ray irradiation has also been explored to synthesize M-PEIs. The results may be useful for deep study on preparation of cationic nanogels and its application in gene delivery.
Language中文
Document Type学位论文
Identifierhttp://ir.sinap.ac.cn/handle/331007/7194
Collection中科院上海应用物理研究所2004-2010年
Recommended Citation
GB/T 7714
徐冬梅. 基因转染载体M-PEIs纳米凝胶的合成[D]. 上海应用物理研究所. 中国科学院上海应用物理研究所,2007.
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