基于刺激相应性聚合物的基因运输载体的研究

发布时间：2018-02-02 18:58

本文关键词： 响应性聚合物基因运输纳米载体 MRI造影增强　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：刺激响应性聚合物能够对外界的环境刺激产生响应,这一独特的理化性质使得其在药物递送、基因运输、组织工程等领域具有重要的应用前景;本论文主要研究了环境响应性聚合物在基因运输方面的潜在应用。在第一章中,我们简要概述了响应性聚合物及其在基因输运领域应用近来来的发展;第二章主要研究了基于β-环糊精的星形聚合物用于基因输运和核磁共振造影(MRI)增强;第三章我们构筑了还原环境敏感的聚合物基因载体;在第四章中,我们构筑了具有温敏性异质壳层结构的聚离子复合物用于基因输运,具体来说,本论文的包含以下三个方面的研究工作:1.在基因诊疗中,如何实现基因诊疗一体化一直是一个挑战。因此我们针对诊疗一体化的问题,通过在β-CD上进行不对称改性合成出一星形高分子聚合物,(DOTA-Gd)7-CD-(PDMAEMA)14。该星型聚合物标记了能够用于MR造影成像的DOTA-Gd基元和能够络合DNA的阳离子聚合物PDMAEMA链段。在N/P = 2时,该阳离子星型聚合物即可与pDNA有效的络合;在N/P比为8时,该星型聚合物能够达到最佳转染效果,其转染率与PEI标准接近。另一方面,MRI测试结果发现星型聚合物的驰豫率相对于商业化的小分子T1纵向弛豫率达到了约10.9 s-1mM-1,MRI造影增强功能具有明显的提高。2.基因诊疗中,基因运输载体释放治疗型基因的效率一直是一个重大的挑战。因此我们通过RAFT聚合和ATRP技术合成了一种新型的氧化还原响应的阳离子型刷状嵌段共聚物 P(OEGMA-co-FAMA)-b-P(BSSMA-g-PDMAEMA)。在该嵌段聚合物刷中PDMAEMA侧链基元通过可降解的二硫键连接,该刷状嵌段共聚物可作为可生物降解的癌细胞靶向非病毒基因传送载体。实验发现该嵌段聚合物刷在N/P ≥ 4的条件下能够有效的络合pDNA;在还原环境下,由于二硫键在还原性环境下发生断裂,嵌段聚合物刷能够生成分子量较小的PDMAEMA均聚物,从而触发络合的pDNA释放使DNA的释放效率大大增强。此外,我们发现引入叶酸靶向基元能够显著增强胞内的转染效率,而MTT测试证明了刷状嵌段共聚物的具有较低细胞毒性。因此,利用氧化还原反应响应性刷状嵌段共聚物可用作具有高转染效率和低细胞毒性的靶向基因传送纳米载体。3.基因载体的稳定性一直是限制基因载体性能的最重要因素之一。通过原子转移自由基聚合(ATRP)和NCA开环聚合合成使用两种两嵌段共聚物PME02 MA-b-PAsp(DET)和 POEGMA-b-PAsp(DET)。通过将嵌段共聚物 POEGMA-b-PAsp(DET)和PMEO2MA-b-PAsp(DET)以等摩尔比混合,并以不同的N/P值络合DNA,所产生的具有异质表面的复合胶束对DNA有着优良的保护作用。络合过程在20 ℃下进行,其中POEGMA和PMEO2MA链段均具有水溶性。然而,当将温度升至37 ℃时,PMEO2MA嵌段(LCST约为26 ℃)由亲水性转变为疏水性,从而塌陷在复合胶束的表面上,进一步压缩pDNA,使得在生物环境中形成更紧凑的结构,并且由于形成了异质壳层纳米结构,可以进一步提高该复合胶束在盐和蛋白质介质中的稳定性。
[Abstract]:Stimuli responsive polymer to environmental stimuli response, this unique physicochemical properties of the gene in the drug delivery, transportation, has important application prospect in tissue engineering field; this paper mainly studies the environment responsive polymer in the potential applications of gene transport. In the first chapter, we give a brief overview the development of polymer and its response in gene transport applications in recent years; the second chapter mainly studies based on cyclodextrin star polymer for gene delivery and magnetic resonance imaging (MRI) enhancement; the third chapter we have constructed a polymer gene vector sensitive reductive environment; in the fourth chapter, we built a temperature sensitive heterogeneous shell structure of polyion complex for gene transport, specifically, this thesis includes the following three aspects of research: 1. in gene diagnosis and treatment, how to To achieve the integration of gene therapy has been a challenge. So we are aiming at the problem of theranostics, the asymmetry in the beta -CD modified synthesis of star polymers, (DOTA-Gd) 7-CD- (PDMAEMA) 14. of the star polymer can be used as MR contrast markers like DOTA-Gd primitives and able to complex DNA cations PDMAEMA polymer chain segment. At N/P = 2, the cationic star polymer and pDNA can be effective in the complex; the N/P ratio is 8, the star polymer can achieve the best effect of transfection, the transfection efficiency of PEI and close to the standard. On the other hand, the MRI test results showed that the star polymer relaxation rate is small molecular relaxation relative to the commercial T1 longitudinal relaxation rates reached about 10.9 s-1mM-1, MRI contrast enhancement function shows a significant increase in.2. gene diagnosis, gene carriers release efficiency of therapeutic genes has been a major Challenges. We therefore synthesized RAFT polymerization and ATRP P cationic copolymer brush a new redox responsive block (OEGMA-co-FAMA) -b-P (BSSMA-g-PDMAEMA). In the block polymer brush in PDMAEMA side chain element by connecting two disulfide bond degradation, the brush copolymers can be as a cancer cell target biodegradable transfer to non viral gene vectors. The experimental results showed that the pDNA complex can effectively block polymer brush in N/P more than 4 conditions; under reducing conditions, due to the two disulfide bond rupture in a reducing environment, diblock polymer brushes can generate a smaller molecular weight PDMAEMA the complexation of pDNA homopolymer, thereby triggering the release to release efficiency of DNA is greatly enhanced. In addition, we found that the introduction of folate targeted element can significantly enhance the intracellular transfection efficiency, and MTT test proved the block copolymer brush With low cell toxicity. Therefore, the reduction reaction response of brush copolymers could be used as high transfection efficiency and low cytotoxicity targeting gene transfer vector.3. gene stability of nano carrier has been one of the most important factors limiting gene carrier performance by oxidation. Via atom transfer radical polymerization (ATRP) and NCA ring opening polymerization using two two block copolymer PME02 MA-b-PAsp (DET) and POEGMA-b-PAsp (DET). The block copolymer POEGMA-b-PAsp (DET) and PMEO2MA-b-PAsp (DET) with equal mole mixture with different N/P values, and complex DNA micelles with heterogeneous surface produced with excellent protective effect the complex process of DNA. Under the temperature of 20 DEG C, wherein POEGMA and PMEO2MA segments are water soluble. However, when the temperature rose to 37 degrees, PMEO2MA block (LCST is about 26 degrees Celsius) changed from hydrophilic Further compressing pDNA for hydrophobicity and collapsing on the surface of compound micelles, resulting in a more compact structure in the biological environment, and further improving the stability of the composite micelles in salt and protein medium due to the formation of heterogeneous shell nanostructures.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R73-3;O631

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