基于环糊精构建高性能基因载体材料

发布时间：2018-06-29 17:18

本文选题：基因治疗 + 阳离子聚合物基因载体　；参考：《北京化工大学》2016年博士论文

【摘要】：基因治疗指将核酸分子作为药物递送进入患者细胞,对疾病的基因表达进行调控(目标基因的表达或沉默)以治疗疾病。作为全新治疗途径,基因治疗在重大疾病的治疗中发挥着举足轻重的作用,但其研究现状是受限于缺乏安全且高效率的基因载体。与高转染效率、但安全性差的病毒类载体相比,阳离子聚合物为主要代表的非病毒类载体具有一些显著优点,例如安全性高(低免疫原性),可以大规模生产和聚合物设计的灵活多样性。因此,开发新型阳离子聚合物基因载体材料一直吸引着人们强烈的研究兴趣。近年来,原子转移自由基聚合(ATRP)和超分子组装被广泛应用于生物医用高分子材料的制备。生物相容性多糖(包括壳聚糖,葡聚糖,环糊精等)的功能化为新型基因载体材料的设计提供了多样化的途径。由于具有密集的分子结构且保持适度的分子柔韧性这一优点,星状阳离子聚合物在非病毒基因载体研究中引起了广泛的研究兴趣。第二章中,利用ATRP制备了由不带电的p-环糊精核、二硫键连接低分子量阳离子侧链组成的可剪切星状聚合物载体(CD-SS-PGEA)。首先,在p-环糊精上引入二硫键连接的ATRP引发位点;随后,利用ATRP制备了聚甲基丙烯酸缩水甘油酯(PGMA)侧链,并利用开环反应制备了PGMA的乙醇胺开环产物——具有丰富不带电羟基和可质子化仲胺基团的阳离子聚合物(PGEA)。在细胞内还原性酶作用下,二硫键连接的PGEA臂可以从环糊精核上断裂、解离下来。这种可剪切的星状聚合物载体在不同细胞系中表现出良好的质粒DNA络合能力,低毒性和高转染效率。基于非共价键相互作用组装形成的超分子聚合物,广泛应用于基因载体的临床前研究和临床试验,优点在于：其合适的结合强度非常有利于功能分子递送,并且简便的自组装过程有利于载体的简便制备和结构、功能优化。β-环糊精主体分子和金刚烷客体分子之间的主客体组装是一种经典的超分子组装体系,过程非常简便,且已经被充分证实和应用。第三章中,利用主客体组装手段,组装环糊精为核、PGEA为侧链的星状聚合物(CD-PGEA)和金刚烷分子修饰的线性PGEA合成了阳离子聚合物为骨架的超分子梳状结构载体(1-PGEA-Ad/CD-PGEA),是一种以非共价键连接的星状聚合物为侧链的独特梳状结构——与组装前星状CD-PGEA和线性1-PGEA相比,1-PGEA-Ad/CD-PGEA显示出更好的DNA络合能力。同时,超分子梳状聚合物载体具有和星状、线性载体类似的低毒性,显著提高的转染效率。设计不同的骨架和聚合物侧链、并引入可细胞内剪切的二硫键,可以进一步证明超分子梳状聚合物的新颖结构在基因递送中的优势,并探索转染效率提高的内在机理。第四章中,组装环糊精为核、二硫键连接聚甲基丙烯酸N,N-二甲氨基乙酯(PDMAEMA)侧链的可剪切星状阳离子聚合物(CD-SS-pDM)和二硫键连接的金刚烷修饰的葡聚糖(Dex-SS-Ad),合成了可剪切的超分子梳状聚合物载体(Dex-SS-Ad/CD-SS-pDM);同时,制备无二硫键的对照组：将非剪切的星状聚合物与金刚烷直接修饰的葡聚糖组装,制备非剪切的超分子梳状载体Dex-Ad/CD-pDM。与组装前载体相比,两种葡聚糖骨架的超分子梳状载体显示出类似的低细胞毒性,但转染效率显著提高,原因在于超分子梳状载体具有显著提高的细胞内吞效率。由于引入刺激响应性的二硫键,可剪切的超分子梳状载体转染效率明显高于非剪切的对照组,并且体外实验证明前者介导的自杀基因治疗系统具有优异的抗肿瘤性能。在阳离子聚合物中引入聚甲基丙烯酸聚乙二醇(乙醚)酯(PPEGEEMA)可以提高转染性能。然而,在阳离子聚合物中更合适地引入PPEGEEMA的方法需要进一步设计,PPEGEEMA提高转染效率的内在机理也需要进一步研究。第五章中,组装不同分子量可剪切的环糊精为核星状阳离子聚合物(CD-SS-pDM)和金刚烷为端基线性PPEGEEMA,合成了一系列可剪切的超分子嵌段聚合物载体(CD-SS-pDM/Ad-pPEG),是一种在阳离子聚合物中更合适地引入PPEGEEMA的方法。与星状载体相比,引入PPEGEEMA未降低CD-SS-pDM/Ad-pPEG载体的DNA络合能力和二硫键的细胞内可剪切性,同时降低了毒性、显著地提高了转染效率。进一步的无血清条件下细胞转染、细胞内吞以及动物体内基因治疗实验证明PPEGEEMA的关键作用在于抑制蛋白非特异性吸附、提高血清中稳定性,进一步提高内吞效率。综上所述,本论文利用ATRP和超分子组装两大合成方法,选择环糊精作为基质设计了多种类型的新颖结构的聚合物。本论文通过ATRP和超分子组装两大合成方法,构建了一系列基于环糊精的新颖结构阳离子聚合物载体,体外细胞和体内动物实验证明了载体材料的高性能。这一系列高性能阳离子聚合物基因载体,对新型基因载体材料的构建和环糊精为基质聚合物的更广泛应用均具有重要意义。
[Abstract]:Gene therapy refers to the delivery of nucleic acid molecules as drugs into the patient's cell, regulating the gene expression of the disease (the expression or silence of the target gene) to treat the disease. As a new therapeutic approach, gene therapy plays an important role in the treatment of major diseases, but its research status is limited to the lack of safety and efficiency. Compared with high transfection efficiency and poor safety viral vectors, cationic polymers have some significant advantages, such as high safety (low immunogenicity), and flexible diversity in mass production and polymer design. Therefore, a new type of cationic polymer gene is developed. In recent years, atomic transfer radical polymerization (ATRP) and supramolecular assembly have been widely used in the preparation of biomedical polymer materials. The functionalization of biocompatible polysaccharides, including chitosan, dextran, cyclodextrin, and so on, has provided a variety of design for the design of new gene carrier materials. Due to the advantages of dense molecular structure and moderate molecular flexibility, stellate cationic polymers have attracted extensive interest in the study of non viral gene carriers. In the second chapter, a shear star consisting of an uncharged p- cyclodextrin nucleus and a two sulfur bond connected to a low molecular weight cation side chain was prepared by ATRP. CD-SS-PGEA. First, the ATRP initiation site was introduced on p- cyclodextrin with two sulfur bonds. Then, the side chain of polyglycidyl methacrylate (PGMA) was prepared by ATRP, and the open ring product of the ethanolamine was prepared by the ring opening reaction - a cation with rich non charged hydroxyl and protonated secondary amine groups. Polymer (PGEA). Under the action of intracellular reductive enzymes, the two sulfur bond linked PGEA arm can be broken from the cyclodextrin nucleus and dissociated. This shear star polymer carrier shows good plasmid DNA complexation, low toxicity and high transfection efficiency in different cell lines. Subpolymers, widely used in preclinical and clinical trials of gene carriers, have the advantage that the appropriate binding strength is very beneficial to functional molecular delivery, and the simple self-assembly process is beneficial to the simple preparation and structure of the carrier and the function optimization. The host and guest group between the beta cyclodextrin host molecules and the adamantane guest molecules. The assembly is a classic supramolecular assembly system. The process is very simple and has been fully proved and applied. In the third chapter, the assembly of host and guest assembly means the assembly of cyclodextrin as the core, the PGEA as the side chain star polymer (CD-PGEA) and the linear PGEA modified by the amantadine molecule into the supramolecular comb structure of the cationic polymer as the skeleton. Body (1-PGEA-Ad/CD-PGEA) is a unique comb like structure of a star shaped polymer connected by a non covalent bond. Compared with the pre assembled stellate CD-PGEA and linear 1-PGEA, 1-PGEA-Ad/CD-PGEA shows a better DNA complexing ability. At the same time, the supramolecular comb polymer carrier has a similar low toxicity to the star shaped and linear vector. High transfection efficiency. The design of different skeleton and polymer side chains and the introduction of two sulfur bonds with intracellular shear can further prove the advantages of the novel structure of the supramolecular comb polymer in gene delivery and explore the intrinsic mechanism of the enhancement of transfection efficiency. In the fourth chapter, the group is packed with cyclodextrin as the core, and the two sulfur bond connects polymethacrylic acid N. N- two methylamino ethyl (PDMAEMA) side chain, the shear star shaped cationic polymer (CD-SS-pDM) and the two sulfur bond linked adamantane modified glucan (Dex-SS-Ad), synthesized a shear supramolecular comb polymer carrier (Dex-SS-Ad/CD-SS-pDM); at the same time, a control group without two sulfur bonds was prepared: the non shear star polymer and the adamantane Directly modified glucan assembly, the preparation of the non shear supramolecular comb carrier Dex-Ad/CD-pDM. compared with the pre assembled carrier, the two glucan skeleton supramolecular comb carriers showed similar low cytotoxicity, but the transfection efficiency was significantly improved because the supramolecular comb carrier had a significant increase in the endocytosis efficiency. The transfection efficiency of the two sulfur bond with irritation responsiveness, the transfection efficiency of the shear supramolecular comb carrier is significantly higher than that of the non shear control group, and in vitro experiments have proved that the former has excellent anti-tumor performance. The introduction of polymethacrylic acid polyethylene glycol (ether) ester (PPEGEEMA) in cationic polymers can be improved. Transfection performance. However, the more appropriate introduction of PPEGEEMA in cationic polymers needs further design. The internal mechanism of PPEGEEMA to improve transfection efficiency also needs further study. In the fifth chapter, the assembly of cyclodextrins with different molecular weights is nuclear star cationic polymerization (CD-SS-pDM) and adamantane as the end group linear PPEGEEMA, A series of shear supramolecular block polymer carriers (CD-SS-pDM/Ad-pPEG) is synthesized, which is a more suitable method for introducing PPEGEEMA in cationic polymers. Compared with stellate carriers, the introduction of PPEGEEMA does not reduce the DNA complexation capacity of the CD-SS-pDM/Ad-pPEG carrier and the intracellular cleavage of the two sulfur bond, and reduces the toxicity. The transfection efficiency is improved. Further serum-free cell transfection, endocytosis and animal gene therapy experiments show that the key role of PPEGEEMA is to inhibit non specific adsorption of protein, improve the stability of serum, and further improve endocytosis efficiency. In this paper, two combinations of ATRP and supramolecular assembly are used in this paper. A series of novel structural polymers were designed by cyclodextrin as matrix. In this paper, a series of novel structural cationic polymer carriers based on cyclodextrin were constructed by two synthetic methods of ATRP and supramolecular assembly. In vitro and in vivo animal experiments proved the high performance of the carrier materials. The performance of cationic polymer gene carriers is of great significance for the construction of new gene carrier materials and the wider application of cyclodextrins as matrix polymers.
【学位授予单位】：北京化工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R450;O636.12

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