【作者】 张磊；

【导师】 张敏；

【作者基本信息】 安徽大学 ， 生态学， 2016， 硕士

【摘要】 基因治疗作为当今生物治疗的重要组成部分,已经成为医学领域里一个新的研究热点。与传统治疗方法相比,基因治疗从根源上修正了引起疾病的异常基因,并且能选择性地治疗多种严重威胁人类健康的疾病。然而安全、高效、靶向表达的基因载体是基因治疗成功的关键。在基因治疗的基础研究和临床应用中,经常使用的传递载体包括病毒载体(Viral vector)和非病毒载体(Non-viral vector)两类,其中,阳离子聚合物载体由于具有设计灵活、易于大规模生、低毒性、目的基因携带量大、基因尺寸不受限制且费用较低等优点,近年来受到研究者极大的关注。而在众多合成材料中,聚乙烯亚胺(Polyethylenimine, PEI)由于具备较高的电荷密度以及良好的质子缓冲能力已成为阳离子聚合物基因载体中的研究热点之一。其中,枝化PEI (25kDa)被称为高分子基因载体领域的“黄金标准”。尽管高分子量PEI转染效率较高,但因为其细胞毒性较大、不可生物降解等问题制约了PEI在临床治疗方面的应用。为了获得细胞毒性较低且转染性能优良的PEI载体,研究者们尝试了许多方法,近些年来,通过可降解键交联低分子量PEI获得性能优越的基因载体得到科学工作者们的持续关注。本论文通过一种简单、经济、高效的合成工艺制备了一种具有酸敏感性的丙烯酸类原酸酯单体(OEAc),之后通过控制投料比(1：1,1.75：1,3.5：1)与低分子量PEI (600 Da)发生迈克尔加成反应,合成了一系列主链含原酸酯的新型酸敏感型基因治疗载体(POEI 1、POEI 2、POEI 3)。并且采用多种实验方法对聚合物进行了表征并研究了它们的理化性质,核磁结果分析证明OEAc以及聚合物结构正确；凝胶渗透色谱(GPC)测定了聚合物数均分子量及分子量分布；通过测定聚合物伯胺含量证明了迈克尔加成反应的有效性,并且POEI 1的伯胺含量最高；酸碱滴定结果表明,POEI 1-3与25 kDa PEI相比具有更强的质子缓冲能力,且缓冲能力随着投料比的增加而增大,其中POEI 3的缓冲能力最强。原酸酯键具有高度酸敏感性,在酸性条件下能快速水解,因此聚合物主链酸响应性由1HNMR测定,结果显示POEI 1-3均具有较强的酸敏感性,三种聚合物主链原酸酯在pH=5.0环境下4小时内即完全降解,核磁分析其降解机制属于环外降解机制。在正常生理条件下,POEIs与质粒DNA通过静电作用相互结合,并有效压缩DNA形成复合物纳米粒子。通过琼脂糖凝胶电泳实验以及肝素置换实验评价聚合物DNA压缩及保护能力的强弱,结果表明,POEI 1-3均能有效压缩质粒DNA,并且DNA压缩能力与聚合反应投料比例有关,反应比例越大压缩能力越差,同时采用DNase Ⅰ降解实验进一步验证了POEI 1和POEI 2能够保护DNA免受DNase Ⅰ的酶解。动态光散射(DLS)结果显示POEI 1和POEI 2与DNA在质量比为2-32条件下能有效包裹质粒DNA形成粒径为200～300 nm、表面带有正电荷的纳米复合物颗粒,适合介导质粒进入细胞；同时由DLS分析验证了POEI/DNA复合物同样表现出良好的酸敏感性,在pH=5.0条件下,三种复合物粒径均发生不同程度的增大,表明复合物稳定性减弱,进而有利于DNA释放。此外,以25kDa PEI为对照物,通过一系列测试探讨了POEI作为基因载体的潜力和性能。本工作采用体外细胞毒性试验(MTT法)评价了POEI 1-3的生物相容性,与PEI (25 KD a)相比,三种聚合物(POEIs)与293T、SH-SY5Y、及HeLa细胞共培养均表现出较低的细胞毒性,其中POEI 3在高浓度下(100μg/mL)与三种细胞共培养后的细胞成活率也均超过80%。本文还通过流式细胞术定量测定法和荧光显微镜直接观察法进行体外细胞基因转染效率检测,结果显示,POEI 1 和 POEI2对于293T细胞系具有一定的转染性能,POEI 1与质粒DNA质量比为4时在293T细胞系中染效率最高达到7.63%。此外,在SH-SY5Y细胞系中,POEI 1和POEI 2的基因复合物在质量比为8时具有最佳转染效率,分别是25 kDa PEI的2.42倍和1.15倍,转染性能均优于25 kDa PEI, 而 POEI 3 在这两种细胞系中几乎没有转染性能。综上所述,这种基于低分子量PEI的pH敏感型基因载体(POEI 1口POEI2)在提高基因转染效率的同时降低了其细胞毒性、提高了生物相容性,再加上其优良的DNA结合能力,因此在基因治疗领域具有良好的应用前景。更多还原

【Abstract】 Gene therapy, as an important component of current biotherapy, has drawn a lot of attention in the field of medicine research. Compared with traditional therapeutic methods, gene therapy has shown great potentials for the treatment of both inherited and acquired diseases by delivering therapeutic genes to diseased cells. However, developing an efficient and safe delivery system that delivers the therapeutic genes to a specific target tissue or organ is vital for the success of gene therapy. The present vectors used for gene therapy in basic studies and clinical trials are usually divided into two categories:viral vectors and non-viral vectors. Especially, non-viral vectors based on cationic polymers are receiving a tremendous amount of attention as gene delivery vehicles on account of their advantages, such as ease of manipulation and large scale manufacture, low immunogenicity, low cost and high flexibility regarding the size of the transgene delivered. Among the various synthetic polycations exploited, polyethylenimine (PEI) has become one of the most promising and widely studied gene vectors due in large part to high positive charge density and efficient escape from the endocytic pathway through the "proton-sponge" mechanism. As a matter of fact, branched PEI (25 kDa) is applied as golden standards for non-viral gene transfection. However, high molecular weight PEIs (HMW PEIs) are often associated with pronounced toxicity and poor biocompatibility in vitro and in vitro, which have restricted its clinical applications. To circumvent such dilemma, modifications of PEI to achieve high transfection efficiency as well as low toxicity have been investigated in recent years. One promising strategy has been developed by cross-linking low molecular weight PEIs (LMW PEIs) with stimuli-responsive linkages.In this paper, we have designed and developed a new type of acid-labile ortho ester linkage (OEAc) containing carbon-carbon double bonds through a simple and effective synthetic method. Then, a series of pH-sensitive LMW PEI-based gene vectors (POEI 1, POEI 2 and POEI 3) were synthesized via Michael addition between vinyl groups of ortho ester linkage (OEAc) and primary amines in LMW PEI (M.W. 600) by feed molar ratio of 1:1,1.75:1 and 3.5:1. The 1H and 13C NMR spectra confirmed OEAc was structurally correct and pure. The structure of obtained polycations (POEI 1-3) was also identified by’H NMR analysis, and the molecular weight of polymers was determined by gel permeation chromatography (GPC). To estimate the primary amine content of POEIs, the ninhydrin colorimetric method was carried out, and results revealed that POEI 1 had the highest primary amine content. Besides, the buffering capacities of polymers were assessed by acid-base titration, and all three polymers have slightly higher buffering capacity than 25 kDa PEI, and the buffering capacity was found to rise as the reaction molar ratio increased. Ortho ester structures are highly hydrolytically labile and acid-sensitive. Thus, we used 1H NMR to explore acid-triggered degradation and kinetics of ortho ester groups in main-chains of POEIs, confirming that POEI 1, POEI 2 and POEI 3 were highly sensitive to mildly acid pH (e.g. at endosomal pH) and follow an exocyclic cleavage mechanism.The polycations could efficiently bind and condense DNA into nanosized particles based on the electrostatic interaction with the negatively charged phosphate group of DNA under physiological condition. In this research, agarose gel retardation and heparin replacement assay were adopted to assess DNA condensation ability of POEIs. All POEIs showed effective DNA condensation ability from the weight ratio (POEI/DNA, w/w) of 0.5. And DNA condensation ability of POEI 1-3 was in the following order:POEI 1> POEI 2> POEI 3, suggesting that amide groups might benefit the interaction. The DNA protection by POEIs against DNase I was further studied, and the results indicated that POEI 1 and POEI 2 could protect DNA against enzymatic degradation by nucleases. Subsequently, Dynamic light scattering (DLS) assay was carried out to study the particle sizes and zeta potentials of all formed POEIs/DNA polyplexes, and results revealed that POEI 1 and POEI 2 could efficiently condense plasmid DNA into spherical nanoparticles with appropriate sizes around 200-300 nm and zeta-potentials about+15 mV, facilitating the interaction between the polyplexes and cell membrane and leading to more efficient cellular uptake. At the same time, the stability of POEIs/DNA polyplexes at mildly acid condition was investigated through analyzing particle size changes by DLS assay. The ortho ester group in main-chains of POEI hydrolyzed at pH 5.0, resulting in dissociation of POEIs/DNA polyplexes, which is expected to enhance endosomal escape of polyplexes and modulate release of DNA.In addition, using unmodified 25 kDa PEI as a contrast, the properties of newly synthesized polycations (POEI 1-3) as potential gene vectors were discussed, including cytotoxicity and in vitro transfection efficiency. Cell viability of POEI 1 to 3 was evaluated on 293 T, SH-SY5Y, and HeLa cells by using MTT assay at various concentrations, which cover the range used in gene transfection assays. MTT results demonstrated that cell viabilities of studied polymers (POEI 1-3) were distinctly higher than that of 25 kDa PEI at all concentrations after 24 h incubation, suggesting that these polycations have better biocompatibility. Especially, POEI 3 exhibited remarkably low cytotoxicity in all three cell lines, and above 80% cell viability was observed even at a high dose of 100 μg/mL. Finally, to inspect the transfection performance of the newly prepared polymers, the transfection efficiency of the POEIs/DNA polyplexes was quantitatively measured by flow cytometry, meanwhile, the transfection efficiency and the transgene expression level were directly visualized by observation of pEGFP-positive cells using the inverted fluorescence microscope. In 293T cells, POEI 1-3 showed lower transfection efficiencies in the absence of serum in comparison with 25 kDa PEI at its optimal N/P ratio of 10 (w/w= 1.4). And for these new cationic polymers, POEI 1 gave the highest transfection efficiency at w/w ratio of 4, reaching up to 7.63%. The transfections mediated by POEIs in SH-SY5Y cells were subsequently processed. We found that POEI 1 and POEI 2 showed much better EGFP expression in comparison with 25 kDa PEI, and they gave 2.42 and 1.15 times higher tansfection efficiency at w/w ratio of 8 than PEI, respectively. Meantime, POEI 3 exhibited poor transfection ability in all tested cell lines. This research thus provides an adoptable plan to address the toxicity-efficiency contradiction of non-viral gene delivery, and the results demonstrate that such acid-labile LMW PEI-based polymers (POEI 1 and POEI 2) with efficient DNA condensation ability might be promising candidates for improved gene therapy.更多还原