可形成无规线团和胶束结构的聚阳离子基因递送性能研究

发布时间：2018-05-21 17:02

本文选题：无规线团 + 胶束结构　；参考：《天津大学》2015年硕士论文

【摘要】：基因治疗有望成为治疗癌症等疾病的有效方法,基因治疗获得临床应用的关键是高效递送基因药物的载体材料。作为非病毒基因载体的一种,聚阳离子能够有效的避免酶解并促进细胞摄取。虽然许多聚阳离子在很多方面表现出较低的免疫原性、制备简单以及成本低等优点,但是较低的基因转染效率仍然是基因治疗的瓶颈。为提高基因递送效率,采取了许多种化学修饰的方法。本研究我们利用PVP具有协同递送基因药物的性能及PDMAEMA的递送基因药物的优良性能制备具有不同PVP链长的双亲水性聚合物PVP-g-PDMAEMA。我们研究了亲水链长度对基因递送的影响,优化亲水端长度。在此基础上制备两亲性聚合物——PVP-g-PDMAEMA-b-PMMA。探究了以PVP-g-PDMAEMA形成的无规线团结构和PVP-g-PDMAEMA-b-PMMA形成的胶束结构对基因递送的影响。PVP-g-PDMAEMA-b-PMMA具有较低的CMC值,约为6.3×10-3 mg/mL。PVP-g-PDMAEMA-b-PMMA/pDNA形成的纳米粒粒径与电位均比PVP-g-PDMAEMA/pDNA明显小。与PVP-g-PDMAEMA相比,PVP-g-PDMAEMA-b-PMMA显示出较好的基因转染效率,在293T细胞上以较低的N/P比约为3:1时明显的超过PEI的转染效率。因此,结果表明PVP-g-PDMAEMA-b-PMMA是一种高效的基因载体。聚阳离子虽然体外具有较高的转染效率,但体内转染效率较低。为了解决此问题,我们发展了基于BSA的电荷反转和二硫键可逆形成断裂的方法。采用巯基乙胺修饰的牛血清白蛋白(SH-BSA)与PVP-g-PDMAEMA-b-PMMA/pDNA通过静电引力结合,形成三元复合物,且SH-BSA表面的巯基氧化交联成二硫键,提高其稳定性。SH-BSA的等电点从4.7提高到5.3。在生理条件下,SH-BSA带有负电荷;在pH为5.0时,SH-BSA带有正电荷。SH-BSA随着pH的变化发生电荷反转作用,与PVP-g-PDMAEMA-b-PMMA/pDNA由静电引力变成静电斥力,但由于交联二硫键的存在,SH-BSA无法从PVP-g-PDMAEMA-b-PMMA/pDNA表面分离。当加入谷胱甘肽后,二硫键断裂,SH-BSA从PVP-g-PDMAEMA-b-PMMA/pDNA分离。所以,具有适当稳定性的SH-BSA有利于基因材料的转移和释放。
[Abstract]:Gene therapy is expected to be an effective method for the treatment of cancer and other diseases. The key to the clinical application of gene therapy is the efficient delivery of carrier materials for gene drugs. As a non-viral gene vector, polycations can effectively avoid enzymatic hydrolysis and promote cell uptake. Although many polycations have the advantages of low immunogenicity, simple preparation and low cost in many aspects, the low efficiency of gene transfection is still the bottleneck of gene therapy. In order to improve the efficiency of gene delivery, many methods of chemical modification have been adopted. In this study, we prepared PVP-g-PDMAEMA-a double hydrophilic polymer with different PVP chain lengths by using PVP as a synergistic delivery gene drug and PDMAEMA as a delivery gene drug. We studied the effect of hydrophilic chain length on gene delivery and optimized the hydrophilic end length. On this basis, amphiphilic polymer PVP-g-PDMAEMA-b-PMMA was prepared. The effect of random coil structure formed by PVP-g-PDMAEMA and micelle structure formed by PVP-g-PDMAEMA-b-PMMA on gene delivery. PVP-g-PDMAEMA-b-PMMA has a lower CMC value. The particle size and potential of the nanoparticles formed by about 6.3 脳 10-3 mg/mL.PVP-g-PDMAEMA-b-PMMA/pDNA are obviously smaller than that of PVP-g-PDMAEMA/pDNA. Compared with PVP-g-PDMAEMA, PVP-g-PDMAEMA-b-PMMA showed better gene transfection efficiency, and the transfection efficiency of PEI on 293T cells was significantly higher than that of PEI when the ratio of N / P was about 3:1. Therefore, the results show that PVP-g-PDMAEMA-b-PMMA is an efficient gene vector. Although polycation has high transfection efficiency in vitro, in vivo transfection efficiency is low. In order to solve this problem, we developed a method of charge inversion and reversible fracture of disulfide bond based on BSA. Bovine serum albumin (SH-BSA) modified by mercaptoethylamine was combined with PVP-g-PDMAEMA-b-PMMA/pDNA by electrostatic force to form a ternary complex, and the sulfhydryl group on the surface of SH-BSA was crosslinked to form disulfide bond. The isoelectric point of SH-BSA was increased from 4.7 to 5.3. Under physiological conditions, SH-BSA has negative charge, and when pH is 5.0, SH-BSA has positive charge .SH-BSA changes with pH, and changes from electrostatic gravity to electrostatic repulsion with PVP-g-PDMAEMA-b-PMMA/pDNA, but SH-BSA can not be separated from PVP-g-PDMAEMA-b-PMMA/pDNA surface because of the existence of cross-linked disulfide bond. When glutathione was added, the disulfide bond was separated from PVP-g-PDMAEMA-b-PMMA/pDNA. Therefore, SH-BSA with proper stability is conducive to the transfer and release of genetic materials.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450;O631

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