以PEI衍生物为载体构建肿瘤基因递送体系：从小分子到大分子的修饰
发布时间：2018-08-13 14:30
【摘要】：聚乙烯亚胺（PEI）作为最典型的阳离子聚合物之一，目前已经广泛用作基因转染的载体，其中尤以分子量为25KDa的超支化PEI（b-PEI），以其优异的转染性能被称为基因递送的“黄金标准”。但由于b-PEI25K较高的阳性电荷密度，导致其细胞毒性大，血液相容性差等缺点，从而严重制约了其进一步的应用。天然化合物如氨基酸、蛋白质及糖胺聚糖，往往具有良好的生物相容性、较低的免疫源性及细胞毒性，部分天然化合物（如硫酸软骨素）还能够特异性结合肿瘤细胞表面的抗原分子（CD-44），，是PEI修饰改性的良好配体选择并能够实现靶向性递送。本论文结合天然化合物的多种优势，设计并合成了三种以天然化合物改性的b-PEI25K衍生物，并将三种衍生物分别与DNAzyme、pDNA、microRNA等不同基因药物组装，重点评价了衍生物的理化性质、衍生物与基因药物的组装及相互作用、载体的生物相容性、细胞毒性及载体在体外水平的转染性能，进而考察了基因药物在体外转染后对细胞的影响及药效。本文的研究重点分为以下几个方面: （1）采用EDC/NHS催化的方法，以N-乙酰-亮氨酸为原料，与b-PEI25K的伯氨基进行偶联，合成了不同比例的疏水性修饰的PEI衍生物，基因载体N-Ac-L-Leu-PEI。对合成载体的结构及理化性质进行了表征，检测了衍生物载体的细胞毒性和生物相容性，及其与pDNA、DNAzyme的结合能力，评价了衍生物载体与DNAzyme的复合物的胞内行为及对细胞增殖、迁移及浸润的影响。相关实验结果均表明衍生物具备较低的细胞毒性及良好的转染效率和生物相容性，转染DNAzyme进入细胞后有效的抑制了肿瘤细胞的增殖和迁移。 （2）通过化学键合的手段，以具有红色荧光的京尼平作为交联剂，将低毒性的嗜热组蛋白与高转染效率的PEI25K进行键合，合成了低细胞毒性，高转染效率的具有协同效应的蛋白质-高分子杂合基因传输载体。对组蛋白－PEI杂合基因载体进行了结构与性质的表征，验证了蛋白质与高分子的有效键合，并检测了新型载体与pDNA的组装能力、细胞毒性及转染效率，实验结果表明该新型杂合基因载体具备良好的DNA包覆能力，基因转染效率高以及较低的细胞毒性。 （3）以天然糖胺聚糖硫酸软骨素为配体通过Michael加成反应与b-PEI25K实现偶联，利用硫酸软骨素能够特异性识别肿瘤基质中高表达的CD44受体的特点，构建了低毒、高效、具有肿瘤靶向能力的新型基因递送体系，并实现抑癌microRNA药物miR-34a对肿瘤细胞的靶向递送。对该载体的理化性质、载药能力、细胞毒性及靶向能力进行了评估，并对转染基因药物进入细胞后的胞内定位、相关蛋白表达量及细胞迁移能力进行了测定。实验结果表明该新型靶向型基因载体通过CD44介导的细胞内吞能够有效的将miR-34a递送到肿瘤细胞内，通过诱导肿瘤细胞的凋亡及抑制肿瘤细胞的迁移达到双重抗瘤的效果。 综上所述，本论文中合成的基于PEI与天然化合物的多种基因载体体系，在具有良好的生物相容性、较低细胞毒性的同时能够保持高的转染效率，并能有效介导所搭载的基因药物在胞内发挥生物学功能，因此可望成为高效安全的新型基因载体。
[Abstract]:Polyethylenimide (PEI), as one of the most typical cationic polymers, has been widely used as a vector for gene transfection, especially hyperbranched PEI (b-PEI) with molecular weight of 25 KDa, which is called the "golden standard" for gene delivery because of its excellent transfection performance. Natural compounds, such as amino acids, proteins and glycosaminoglycans, often have good biocompatibility, low immunogenicity and cytotoxicity, and some natural compounds (such as chondroitin sulfate) can specifically bind to antigens on the surface of tumor cells. CD-44 is a good ligand selection for PEI modification and can achieve targeted delivery. In this paper, three kinds of b-PEI 25K derivatives modified by natural compounds were designed and synthesized, and three derivatives were assembled with DNA zyme, pDNA, microRNA and other gene drugs, with emphasis on the evaluation of derivatives. The physical and chemical properties, the assembly and interaction of derivatives and gene drugs, the biocompatibility of the carriers, cytotoxicity and the transfection performance of the carriers in vitro were investigated. The effects of gene drugs on cells after transfection in vitro were investigated.
(1) Hydrophobically modified PEI derivatives with different proportions were synthesized by coupling N-acetyl-leucine with primary amino group of b-PEI 25K using EDC/NHS as catalyst. The structure and physicochemical properties of the synthesized carriers were characterized, and the cytotoxicity and biocompatibility of the carriers were tested. The binding ability of the derivatives with pDNA and DNA zyme was evaluated. The intracellular behavior of the derivatives with DNA zyme and their effects on cell proliferation, migration and infiltration were evaluated. Cell proliferation and migration.
(2) Protein-macromolecule hybrid gene delivery vectors with low cytotoxicity and high transfection efficiency were synthesized by chemical bonding and crosslinking with genipin with red fluorescence as crosslinking agent. Histone-PEI hybrid gene delivery vectors were introduced. The structure and properties of the hybrid gene vector were characterized to verify the effective binding between protein and polymer. The assembly ability, cytotoxicity and transfection efficiency of the new vector with pDNA were tested. The results showed that the hybrid gene vector had good DNA coating ability, high gene transfection efficiency and low cytotoxicity.
(3) Using natural glycosaminoglycan chondroitin sulfate as ligand to couple with b-PEI25K by Michael addition reaction, a novel gene delivery system with low toxicity, high efficiency and tumor targeting ability was constructed by using chondroitin sulfate as a specific recognition of CD44 receptor in tumor matrix, and the anti-cancer microRNA drug microRNA microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs micro The physical and chemical properties, drug loading capacity, cytotoxicity and targeting ability of the vector were evaluated. The localization, expression of related proteins and cell migration ability of the transfected gene drug after entering the cell were determined. The experimental results showed that the novel targeting gene vector was fine mediated by CD44. Intracellular endocytosis can effectively deliver microRNA-34a to tumor cells and achieve dual antitumor effects by inducing apoptosis and inhibiting migration of tumor cells.
In summary, the PEI-based gene delivery systems synthesized in this paper have good biocompatibility, low cytotoxicity and high transfection efficiency, and can effectively mediate the biological functions of the gene drugs carried in the cells. Therefore, PEI-based gene delivery systems are expected to be a new type of genes with high efficiency and safety. Carrier.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R91;R914.5
本文编号：2181266
[Abstract]:Polyethylenimide (PEI), as one of the most typical cationic polymers, has been widely used as a vector for gene transfection, especially hyperbranched PEI (b-PEI) with molecular weight of 25 KDa, which is called the "golden standard" for gene delivery because of its excellent transfection performance. Natural compounds, such as amino acids, proteins and glycosaminoglycans, often have good biocompatibility, low immunogenicity and cytotoxicity, and some natural compounds (such as chondroitin sulfate) can specifically bind to antigens on the surface of tumor cells. CD-44 is a good ligand selection for PEI modification and can achieve targeted delivery. In this paper, three kinds of b-PEI 25K derivatives modified by natural compounds were designed and synthesized, and three derivatives were assembled with DNA zyme, pDNA, microRNA and other gene drugs, with emphasis on the evaluation of derivatives. The physical and chemical properties, the assembly and interaction of derivatives and gene drugs, the biocompatibility of the carriers, cytotoxicity and the transfection performance of the carriers in vitro were investigated. The effects of gene drugs on cells after transfection in vitro were investigated.
(1) Hydrophobically modified PEI derivatives with different proportions were synthesized by coupling N-acetyl-leucine with primary amino group of b-PEI 25K using EDC/NHS as catalyst. The structure and physicochemical properties of the synthesized carriers were characterized, and the cytotoxicity and biocompatibility of the carriers were tested. The binding ability of the derivatives with pDNA and DNA zyme was evaluated. The intracellular behavior of the derivatives with DNA zyme and their effects on cell proliferation, migration and infiltration were evaluated. Cell proliferation and migration.
(2) Protein-macromolecule hybrid gene delivery vectors with low cytotoxicity and high transfection efficiency were synthesized by chemical bonding and crosslinking with genipin with red fluorescence as crosslinking agent. Histone-PEI hybrid gene delivery vectors were introduced. The structure and properties of the hybrid gene vector were characterized to verify the effective binding between protein and polymer. The assembly ability, cytotoxicity and transfection efficiency of the new vector with pDNA were tested. The results showed that the hybrid gene vector had good DNA coating ability, high gene transfection efficiency and low cytotoxicity.
(3) Using natural glycosaminoglycan chondroitin sulfate as ligand to couple with b-PEI25K by Michael addition reaction, a novel gene delivery system with low toxicity, high efficiency and tumor targeting ability was constructed by using chondroitin sulfate as a specific recognition of CD44 receptor in tumor matrix, and the anti-cancer microRNA drug microRNA microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs micro The physical and chemical properties, drug loading capacity, cytotoxicity and targeting ability of the vector were evaluated. The localization, expression of related proteins and cell migration ability of the transfected gene drug after entering the cell were determined. The experimental results showed that the novel targeting gene vector was fine mediated by CD44. Intracellular endocytosis can effectively deliver microRNA-34a to tumor cells and achieve dual antitumor effects by inducing apoptosis and inhibiting migration of tumor cells.
In summary, the PEI-based gene delivery systems synthesized in this paper have good biocompatibility, low cytotoxicity and high transfection efficiency, and can effectively mediate the biological functions of the gene drugs carried in the cells. Therefore, PEI-based gene delivery systems are expected to be a new type of genes with high efficiency and safety. Carrier.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R91;R914.5
【参考文献】
相关期刊论文 前3条
1 张赛群;彭剑青;;PAMAM作为抗肿瘤药物载体研究进展[J];数理医药学杂志;2012年03期
2 王启钊;吕颖慧;费凌娜;许瑞安;;肿瘤基因治疗的研究进展与思考[J];中国肿瘤临床;2010年15期
3 韩冬;蒋经柱;肖亮;周汉新;;肿瘤多药耐药细胞株诱导方法研究进展[J];医学综述;2014年22期
本文编号：2181266
本文链接：https://www.wllwen.com/yixuelunwen/yiyaoxuelunwen/2181266.html
最近更新
教材专著
