双氟链修饰的高分子作为基因和蛋白质载体的研究

发布时间：2018-06-23 00:10

本文选题：DNA + siRNA　；参考：《华东师范大学》2016年硕士论文

【摘要】：基因和蛋白质治疗是一种新兴的疾病治疗手段,它相比于传统的治疗方法,具有靶向性强,副作用小,治疗效果明显等优势,因此成为当今生物医学领域的研究热点。基因和蛋白质自身很难进入细胞,在实际应用过程中需要载体来递送。阳离子高分子是当前研究最多的一类基因和蛋白质载体。然而,这些高分子载体普遍存在转染效率不高,毒副作用大等问题,因而需要对其进行各种功能化修饰来改善这些载体的性能。常用的高分子载体改性方法包括脂肪链修饰,糖基化修饰.,氨基酸修饰,小肽和蛋白质修饰,纳米材料修饰等。但这些修饰方法无法从根本上解决阳离子高分子转染过程中带来的细胞毒性等问题。因此,开发高效、低毒的基因和蛋白质载体一直是该研究领域亟待解决的关键问题。氟化修饰是一种改善阳离子高分子基因转染效率的新策略。这种方法修饰的高分子材料可以在极低的氮磷比条件下实现高效基因转染,低氮磷比也会降低复合物表面的电荷密度,从而缓解阳离子高分子对细胞的毒副作用。但是,氟化修饰的高分子材料要获得高转染效率往往需要在高分子表面接枝高比例的含氟脂肪链,这样会带来一系列的问题。比如,高表面接枝率会造成高分子表面产生严重的空间位阻,影响高分子材料与核酸分子结合,也限制了在高分子材料上进行其它的功能化修饰；另外,接枝大量的含氟脂肪链也可能会产生严重的细胞毒性。本论文针对这些问题,拟采用在高分子表面修饰含双氟链的化合物来使得高分子可以在低接枝率条件下实现高转染效率。即在不同的高分子材料表面修饰2-氯-4,6-双[3-(全氟己基)丙氧基]-1,3,5-三嗪(CBT),探索其作为DNA, siRNA和蛋白质载体的可行性。论文的主要研究结果介绍如下：在不同代数(Generation, G)的聚酰胺-胺树形高分子(PAMAM)和分子量为1800 Da的支化聚乙烯亚胺(bPEI1.8K)上修饰不同比例的CBT分子,并通过茚三酮和氟元素分析等方法表征这些材料表面CBT的平均接枝数量。所得材料分别定义为GI-CBT1.9, G2-CBT1.5,G5-CBTi.3和bPEI1.8K-CBTi.3 (CBT后的数字下标为CBT的平均接枝数)。研究结果表明所有CBT修饰的高分子材料均可以紧密结合DNA。仅仅修饰1-2条CBT后,所有的高分子材料转染绿色荧光蛋白和荧光素酶的效率均大幅提高。其中,GI-CBT1.9的转染效率提高最为显著,在HEK293细胞中的转染效率达到了70%以上,与商业化转染试剂Lipofectamine2000近乎相当,显著优于SuperFect和PolyFect。这些CBT修饰的高分子材料除了高转染效率外,在转染条件下几乎未对细胞产生细胞毒性。除了具有较高的DNA转染能力,CBT修饰的高分子材料还能够有效递送siRNA。比如,G1-CBT1.9, G2-CBT1.5, G5-CBT1.3和bPEI1.8K-CBTi.3在稳定表达荧光素酶的HeLa和MDA-MB-231细胞中均能有效干扰荧光素酶基因的表达水平,而且在RNA干扰过程中未检测到脱靶现象。其中,GI-CBT1.9的RNA干扰效率与Lipofectamine 2000相当,而且基因沉默过程中细胞毒性更低。GI-CBT1.9不仅在体外具有较高的RNA干扰水平,在小鼠体内也展示了一定的基因沉默能力,显著优于未进行CBT修饰的G1树形高分子材料。这一结果表明GI-CBT1.9具有作为潜在的基因治疗载体的可能。虽然氟化修饰的高分子材料作为DNA和siRNA的递送载体已有相关报道,但是其作为蛋白质载体的可行性还未进行探索。研究结果表明GI-CBT1.9, G2-CBT1.5和bPEI1.8K-CBTi.3均可将修饰绿色荧光素的牛血清蛋白(BSA-FITC)递送进HeLa细胞。三种载体中,GI-CBT1.9对BSA-FITC递送效率最高,这与前面DNA和siRNA的转染结果一致,表明GI-CBT1.9递送这几种生物分子采用了类似的机理。除了BSA-FITC之外,GI-CBT1.9还可以将β-半乳糖苷酶(β-Galactosidase,β-Gal)递送进入细胞质中。综上所述,本论文提供了一种新的氟化修饰方法用于改善阳离子高分子对基因和蛋白质的递送性能。相对于传统的含氟脂肪链修饰,双氟链修饰法可以在极低的氟链接枝率(1-2条CBT分子)条件下实现高效、低毒的基因和蛋白质转染。这种方法简单易行,具有较高的商业化应用前景。该研究进一步完善了氟化修饰高分子的基因递送系统,并且拓展了氟化高分子在蛋白质递送中的应用。
[Abstract]:Gene and protein therapy is a new means of treatment for disease. Compared with the traditional therapy, it has the advantages of strong targeting, small side effect and obvious therapeutic effect. Therefore, it has become a hot topic in the field of biomedicine. Gene and protein are difficult to enter cells. In practical application, the carrier is needed to deliver. Ionic polymers are the most widely studied genes and protein carriers. However, these polymer carriers generally have problems such as low transfection efficiency and toxic and side effects. Therefore, various functional modifications are needed to improve the performance of these carriers. The commonly used polymer modified methods include fatty chain modification and glycosylation. Modification, amino acid modification, small peptide and protein modification, nanomaterial modification, etc. but these modification methods can not fundamentally solve the cytotoxicity of cationic polymers in the process of transfection. Therefore, the development of high efficient, low toxic genes and protein carriers has been the key problem to be solved urgently in this field. Fluorination modification. It is a new strategy to improve the efficiency of cationic polymer gene transfection. This modified polymer can achieve efficient gene transfection at very low nitrogen and phosphorus ratio. The low N and P ratio will also reduce the charge density on the surface of the compound, thus relieving the side effects of the cationic polymer on the cells. However, the high grade of fluorinated modification is high. In order to obtain high transfection efficiency, a high ratio of fluorine fat chain is often needed to be grafted on the surface of the polymer, which will lead to a series of problems. For example, the high surface grafting rate will cause serious space hindrance on the surface of the polymer, the binding of polymer to nucleic acid molecules, and other restrictions on the polymer materials. In addition, the grafting of large amounts of fluorine fat chains may also produce severe cytotoxicity. In this paper, we propose to use compounds containing double fluorine chains on the surface of polymer to make high efficiency of high transfection efficiency under low grafting rate. That is, the surface modification of 2- chlorine -4 on the surface of different polymer materials 6- double [3- (perfluoro hexyl) promethoxy]-1,3,5- three azine (CBT) as a DNA, siRNA and protein carrier is explored. The main results of this paper are as follows: modified polyamide amine tree (PAMAM) and branched polyethylenimide (bPEI1.8K) of different Algebras (Generation, G) and molecular weight of 1800 Da (bPEI1.8K) The average grafting number of CBT on the surface of these materials was characterized by the method of BT and fluorine analysis. The obtained materials were defined as GI-CBT1.9, G2-CBT1.5, G5-CBTi.3 and bPEI1.8K-CBTi.3 (the number of digital subscript after CBT was the average grafting number of CBT). The results showed that all the CBT modified polymer materials could be tightly combined. The efficiency of all polymer materials transfected to green fluorescent protein and luciferase was greatly improved after only 1-2 CBT modified by DNA.. The transfection efficiency of GI-CBT1.9 was the most significant. The transfection efficiency in HEK293 cells was more than 70%, which was almost equal to that of commercial transfection reagent Lipofectamine2000, which was significantly better than SuperFect and Po. LyFect. in addition to high transfection efficiency, these CBT modified polymer materials have no cytotoxicity to the cells under the transfection condition. In addition to high DNA transfection capacity, CBT modified polymer materials can also deliver siRNA., such as G1-CBT1.9, G2-CBT1.5, G5-CBT1.3 and bPEI1.8K-CBTi.3, in the stable expression of luciferase He. Both La and MDA-MB-231 cells could effectively interfere with the expression level of luciferase gene, and no miss target was detected during RNA interference. The RNA interference efficiency of GI-CBT1.9 was equal to that of Lipofectamine 2000, and the lower cytotoxicity of.GI-CBT1.9 in the process of gene silencing was not only with a high level of RNA interference in vitro. The mice also showed a certain gene silencing ability, which was significantly better than the G1 tree without CBT modification. This result indicates that GI-CBT1.9 has the potential as a potential vector for gene therapy. Although fluorinated polymer materials have been reported as delivery carriers of DNA and siRNA, it is used as a protein carrier. The results show that GI-CBT1.9, G2-CBT1.5 and bPEI1.8K-CBTi.3 can deliver the bovine serum protein (BSA-FITC) modified with green fluorescein (BSA-FITC) into HeLa cells. Among the three carriers, GI-CBT1.9 has the highest delivery efficiency to BSA-FITC, which is in accordance with the transfection results of the previous DNA and siRNA, indicating that GI-CBT1.9 delivery is a few kinds of birth. In addition to BSA-FITC, GI-CBT1.9 can also deliver beta galactosidase (beta -Galactosidase, beta -Gal) into the cytoplasm. To sum up, this paper provides a new fluorinated modification method to improve the delivery performance of cationic polymers to genes and proteins. Adipose chain modification and double fluorine chain modification can be used to achieve high efficiency, low toxic gene and protein transfection at a very low fluorine linked branching rate (1-2 CBT molecule). This method is simple and has a high commercial application prospect. This study further perfected the gene delivery system of fluorinated high fraction and expanded fluorinated polymers. Application in protein delivery.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q789

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