超分子组装非病毒基因载体及其仿生特性的研究

发布时间：2018-06-12 23:57

本文选题：基因治疗 + 超分子组装　；参考：《浙江大学》2007年博士论文

【摘要】： 现代基因技术和人类基因组工程图谱的完成为采用基因分子生物学方法治疗各类疾病，提高人类生命质量提供了广阔的前景。基因治疗的研究已经深入医学研究的各个领域，成为当今最活跃的生物高技术领域之一。采用超分子组装技术制备的非病毒基因传递体系，由于具有合适的纳米尺寸、可控的结构及良好的生物相容性，显示出巨大的发展潜力及应用前景。如何提高非病毒基因载体在体内的稳定传递和高效转染是这类传递体系所需解决的关键的科学问题。病毒是由蛋白质壳层所包被的具有核壳结构的天然超分子组装体，其细胞外稳定存在、细胞内解组装响应并高效转染的特点为非病毒基因传递体系的设计提供了很好的启迪。本论文以构建稳定传递和高效转染的非病毒基因传递体系为研究背景，采用超分子组装技术与基因技术相结合，制备了模拟病毒结构特点的非病毒基因超分子组装体，初步探讨了超分子组装体的结构与基因转染效率的内在联系。聚乙烯亚胺(PEI)具有的“质子海绵效应”有利于其从溶酶体中逃离出来，是目前常用的一类非病毒基因载体。但PEI／DNA组装体在生理盐溶液中不稳定、易聚集，制备PEG化的基因超分子组装体可解决上述问题。本文首先通过含PEG链段的两亲聚合物的自组织共混改性，，采用超分子技术构建了基于疏水作用力的新型PEG化基因超分子组装体。胆固醇—聚乙二醇(CPEG)的加入用量及次序对基因超分子组装体在生理盐溶液中的稳定性产生很大影响。将CPEG与PEI_(25k)的混合溶液加入到等体积的DNA溶液中，制备的PEI_(25k)／CPEG／DNA组装体在生理盐溶液中的稳定性得到很大提高；而在PEI_(25k)／DNA组装体形成之后，再加入CPEG则不能提高其稳定性。这一现象显示新型PEG化基因组装体的形成，与疏水胆固醇参与到疏水核(由PEI_(25k)和DNA静电中和形成)的组装密切相关。研究进一步采用以表达绿色荧光蛋白的质粒pEGFP为模型，将制备的基因超分子组装体在生理盐溶液中放置不同的时间，并转染HEK293T细胞。荧光显微镜及流式细胞仪结果表明：随着放置时间的延长，PEI_(25k)／DNA组装体的转染效率显著降低；而通过CPEG共混改性后的PEI_(25k)／CPEG／DNA基因组装体由于在生理盐溶液中的稳定性得到很大提高，合适的粒子尺寸使其在体外的基因转染效率显著提高。含PEG链段两亲分子的自组织共混为制备具有新型的PEG化基因超分子组装体，提高其在生理盐溶液中的稳定性及基因转染效率，提供了简单有效的新途径。针对基因载体与DNA分子在传递过程中的稳定缔合和在细胞内的解离释放这一对矛盾，本研究依据病毒的蛋白质壳层细胞内外响应传递的特点，开展了生物响应型非病毒基因传递体系的研究。研究利用细胞内高浓度谷胱甘肽对-S-S-键的响应特性，由低分子量PEI_(1800)出发，通过与二甲基3，3’-二硫代-双(丙亚氨酸酯)二氯化氢(DTBP)的交联反应，合成了一系列含-S-S-键的交联聚乙烯亚胺(CLPEI)，通过对交联程度和组装条件的调控，成功制备了谷胱甘肽响应的CLPEI_(50％)／DNA基因超分子组装体。实验结果表明：CLPEI_(50％)具有最佳的缔合DNA分子的能力，并在pH值为7.4到5的范围内，依然具有足够的质子缓冲能力，有利于基因超分子组装体从溶酶体中逃离出来。在pH为6.0、NaCl浓度为20mM的条件下，能与DNA形成直径在150 nm左右的球形粒子。在模拟细胞内谷胱甘肽浓度的条件下，CLPEI_(50％)分子中-S-S-键的断裂导致高分子量聚阳离子转变为低分子量片断，实现组装体的解组装响应。体外细胞培养结果表明：这种仿生交联的基因组装体可显著降低细胞毒性，并有效转染细胞。为进一步增强基因超分子组装体的稳定性和转染效率，研究以病毒组装过程中生物大分子交联的“笼状”结构为启示，开展了“笼状”仿生交联组装体的研究。合成了含巯基的聚乙烯亚胺(HS-PEI)，与DNA组装后，分别通过空气中巯基的氧化交联制备壳层原位交联的基因超分子组装体；通过金纳米粒子(AuNP)与巯基的共价缔合，制备壳层纳米金交联的组装体。荧光光谱测定结果表明：在N／P值为10的条件下，巯基化聚乙烯亚胺(HS-PEI)能有效诱导DNA分子的缔合。在pH为6.0、NaCl浓度为20 mM的HEPEs缓冲溶液中，这种壳层交联的组装体的尺寸在150 nm左右。对于壳层AuNP交联的Au-S-PEI／DNA组装体，由TEM图片我们可清晰观察到壳层金纳米粒子的存在，且随着AuNP加入量的增加，壳层交联的金纳米粒子含量也显著增加。与壳层未交联的基因超分子组装体相比，壳层交联的“笼状”结构可有效提高组装体在生理盐溶液中的稳定性，并显示出很好的谷胱甘肽响应特性。体外细胞培养结果表明：通过选择合适的组装条件，仿生交联的“笼状”非病毒基因传递体系可有效转染。采用超分子组装技术构建仿病毒基因传递体系，使其既具备非病毒载体的低毒性、低免疫原性的特点，又具有病毒载体高效转染的特性，将是未来基因传递体系的发展方向。本论文采用超分子组装手段，通过含PEG链段两亲聚合物的自组织共混改性，制备了新型PEG化基因超分子组装体；利用谷胱甘肽的生物开关效应，设计了含二硫键的聚阳离子，制备了细胞内外响应传递的非病毒基因传递体系；通过制备巯基化聚乙烯亚胺，构建了壳层原位交联和纳米金交联的“笼状”基因超分子组装体，为新型仿病毒基因传递体系的设计提供了切实可行的途径，并在基因治疗领域显示出广阔的应用发展前景。
[Abstract]:The completion of modern gene technology and human genome engineering map provides a broad prospect for the use of gene molecular biology to treat various diseases and improve the quality of human life. The research of gene therapy has already deepened every field of medical research and became one of the most active fields of biotechnology. The non viral gene transfer system, due to the proper nanoscale size, controllable structure and good biocompatibility, shows great potential for development and application prospects. How to improve the stable transmission and efficient transfection of non viral vector in vivo is the key scientific problem to be solved in this kind of transmission system. It is a natural supramolecular assembly with nuclear shell structure covered by protein shell, and its extracellular stability exists. The characteristics of intracellular solution assembly response and efficient transfection provide a good inspiration for the design of non viral gene transfer system. This paper is to construct a non viral gene transfer system for stable transmission and efficient transfection. In the context of the combination of supramolecular assembly and gene technology, a non viral gene supramolecular assembly that mimic the structural characteristics of the virus was prepared. The intrinsic relationship between the structure of supramolecular assembly and gene transfection efficiency was preliminarily discussed.
The "proton sponge effect" of polyethyleneimine (PEI) is beneficial to its escape from the lysosome. It is a commonly used non viral gene carrier. However, the PEI / DNA assembly is unstable in the physiological salt solution and is easy to gather. The preparation of PEG gene supramolecular assembly can solve the above problems. First of all, two of the PEG segments are used. A novel PEG gene supramolecular assembly based on hydrophobic interaction was constructed by the self-organized blending modification of the amphiphilic polymer. The dosage and order of the addition of cholesterol and polyethylene glycol (CPEG) had a great effect on the stability of the gene supramolecular assembly in the physiological salt solution. The combination of CPEG and PEI_ (25K) mixed solution was added. In the DNA solution of equal volume, the stability of the prepared PEI_ (25K) / CPEG / DNA assembly in the physiological salt solution is greatly improved, and the addition of CPEG after the formation of PEI_ (25K) / DNA assembly can not improve its stability. This phenomenon shows that the new PEG matrix is formed by the formation of the assembly and hydrophobic cholesterol is involved in hydrophobicity. Nucleation is closely related to the assembly of PEI_ (25K) and DNA electrostatic neutralization. The study further uses plasmid pEGFP expressing green fluorescent protein as a model to place the prepared gene supramolecular assembly in a physiological salt solution for different time and transfect HEK293T cells. The results of fluorescence microscopy and flow cytometry show that with the placement of the fluorescent microscope and flow cytometry The transfection efficiency of the PEI_ (25K) / DNA assembly was significantly reduced, while the PEI_ (25K) / CPEG / DNA genomic assembly modified by CPEG was greatly improved because of the stability in the physiological salt solution. The appropriate particle size made the gene transfection efficiency in the vitro significantly improved. The self group of the two parent molecules containing PEG chain segments was improved. In order to prepare a new PEG gene supramolecular assembly and improve its stability in physiological salt solution and gene transfection efficiency, a new and effective new way is provided.
In view of the contradiction between the stable association of gene carriers and DNA molecules during the transfer process and the release of intracellular release, this study carried out a study on the biological response non viral gene transfer system based on the characteristics of the response transmission of the protein shell inside and outside of the virus, and studied the use of high concentration of intracellular glutathione to the -S-S- key. A series of crosslinked polyethylenimines (CLPEI) containing -S-S- bonds were synthesized by crosslinking reaction with two methyl 3,3 '- two thiosulfate (malonic acid) two hydrogen chloride (DTBP) by the response characteristic of low molecular weight PEI_ (1800). The CLPEI_ (50%) / DNA base response of glutathione response was successfully prepared by the regulation of the crosslinking degree and the assembly conditions. The results of the supramolecular assembly show that CLPEI_ (50%) has the best ability to associate DNA molecules, and still has enough proton buffering ability in the range of 7.4 to 5 of pH, which is beneficial to the escape of the gene supramolecular assembly from the lysosome. Under the condition of pH and 20mM, the diameter of DNA can be 15. 0 nm spherical particles. Under the condition of simulated cell Uchiya Ka concentration, the fracture of the -S-S- bond in the CLPEI_ (50%) molecule leads to the transformation of high molecular weight polycation to a low molecular weight fragment and the assembly response to the assembly. The results of cell culture in vitro show that this crosslinked genome assembly can significantly reduce cytotoxicity. Sex, and transfection of cells effectively.
In order to further enhance the stability and transfection efficiency of gene supramolecular assembly, the study of "cage like" bionic crosslinking assembly was carried out by the "cage like" structure of biological macromolecule crosslinking in the process of virus assembly. The polyethylenimide (HS-PEI) containing sulfhydryl group was synthesized. After assembling with DNA, the sulfhydryl oxygen in the air was carried out respectively. The in-situ crosslinked gene supramolecular assembly of shell was prepared by chemical crosslinking; the shell nanoscale crosslinked assembly was prepared by covalent Association of gold nanoparticles (AuNP) with the sulfhydryl group. The results of fluorescence spectrometry showed that under the condition of N / P 10, mercapto polyethyleneimine (HS-PEI) could effectively induce the association of DNA molecules. In pH, 6, NaCl In the HEPEs buffer solution with a concentration of 20 mM, the size of the shell crosslinked assembly is about 150 nm. For the AuNP crosslinked Au-S-PEI / DNA assembly of the shell layer, the existence of the shell gold nanoparticles can be clearly observed from the TEM picture. With the increase of AuNP addition, the content of the gold nanoparticles crosslinked by the shell is also significantly increased. Compared with the non crosslinked gene supramolecular assembly, the shell crosslinked "cage" structure can effectively improve the stability of the assembly in the physiological salt solution, and show a good response characteristic of glutathione. In vitro cell culture results show that the "cage like" non viral gene transfer through the selection of suitable assembly conditions and bionic crosslinking The system can be transfected effectively.
Using supramolecular assembly technology to construct a virus gene transfer system, which not only has the characteristics of low toxicity, low immunogenicity, and high efficiency of transfection of virus carriers, which will be the development direction of the gene delivery system in the future. This paper uses the supramolecular assembly method, through the two amphiphilic polymer containing PEG segment.
A novel PEG gene supramolecular assembly was prepared by self tissue blending, and a polycation containing two sulfur bonds was designed by using the biological switching effect of glutathione, and the non viral gene transfer system was prepared in vitro and in vitro. By preparing mercapto polyethyleneimine, the shell in situ crosslinking and nanoscale crosslinking were constructed. The "cage" gene supramolecular assembly provides a practical way for the design of a new type of virus like gene transfer system, and has shown a broad prospect of application in the field of gene therapy.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R346

【引证文献】