基于多糖接枝聚天冬氨酸构建新型可降解基因载体

发布时间：2018-01-12 04:19

本文关键词：基于多糖接枝聚天冬氨酸构建新型可降解基因载体　出处：《北京化工大学》2015年硕士论文　论文类型：学位论文

【摘要】：基因治疗(Gene therap y)在治疗先天基因缺陷以及获得性疾病方面展现出巨大的潜力。基因治疗依赖于安全高效的基因载体,基因载体通常分为病毒性载体和非病毒性载体两大类。两者相比,非病毒性基因载体无传染性,载体容量大,结构容易控制,适合大规模生产,毒性低、免疫反应低,但是现有的基因载体转染效率还不够理想。因此,发展安全高效的非病毒性载体仍面临着挑战。天然多糖可再生,生物相容性良好,基于多糖构建基因载体是设计安全高效载体的一种有效手段。聚天冬氨酸(Poly (aspartic acid))毒性低,生物相容性好,易生物降解,是制备基因载体的理想材料。本文研究内容如下：1、我们首先以环糊精(Cyclodextrin, CD)、葡聚糖(Dextran)和壳聚糖(Chitosan, CS)为代表探究了一种多糖接枝聚天冬氨酸的普适性方法。以乙二胺(Ethylene diamine, ED)修饰的CD和Dextran以及本身含有-NH2的CS为引发剂,引发单体天冬氨酸-p-苄酯-N-羰基内酸酐(BLA-NCA)的开环聚合反应得到多糖接枝聚天冬氨酸苄酯聚合物(Polysaccharide-PBLA),再通过ED与Polysaccharide-PBLA上苄基的胺解反应构建了一系列基因载体(Polysaccharide-PAsp-ED)。通过降解,细胞毒性,海肾荧光素酶等实验证明Polysaccharide-PAsp-ED具有良好的降解性,较低的毒性,尤其是基于壳聚糖的基因载体具有较优异的转染效率。2、为进一步提高基因载体的转染效率,构建具有靶向性的基因载体,我们又进行了下面的课题。在CS上引入了CD,形成CS-CD多糖骨架,以通过ED修饰的CS-CD为引发剂,引发单体BLA-NCA的开环聚合反应,得到CS-CD-PBLA,通过与ED的胺解反应,得到阳离子聚合物CS-CD-PAsp-ED (CCPE)。通过接有叶酸(Folic acid, FA)的金刚烷胺(Adamantanamine, Ad)与CD空腔之间的自组装赋予阳离子聚合物靶向性(CCPE-FA)。并且将CCPE和C CPE-FA聚合物通过layer-by-layer方式包裹DNA形成靶向性三元复合物。实验结果表明,在具有叶酸靶向的Hela、C6细胞系中靶向性三元复合物具有较高的转染效率以及显著靶向性。
[Abstract]:Gene therapy (Gene therap Y) shows great potential in the treatment of congenital gene defects and acquired diseases. Gene therapy relies on safe and efficient gene vectors. Gene vectors are usually divided into viral vectors and non-viral vectors. Compared with the two vectors, non-viral gene vectors are non-infectious, the vector capacity is large, the structure is easy to control, suitable for mass production, low toxicity. The immune response is low, but the transfection efficiency of the existing gene vectors is not ideal. Therefore, the development of safe and efficient non-viral vectors is still facing challenges. Natural polysaccharides can be regenerated and have good biocompatibility. The construction of gene vector based on polysaccharide is an effective way to design a safe and efficient vector. Poly aspartic acido) has low toxicity and good biocompatibility. It is an ideal material for the preparation of gene vectors. In this paper, we use cyclodextrin Cyclodextrin (CDCD) as follows. Dextranan and chitosan. A universal method for grafting polyaspartic acid with Polysaccharide was studied. Ethylenediamine ethylene diamine was used. D) modified CD and Dextran and CS containing -NH2 as initiators. Poly (benzyl aspartate) was grafted by the ring-opening polymerization of the monomer aspartic acid -p-benzyl ester -N-carbonyl intraanhydride (BLA-NCA). Polysaccharide-PBLAs. A series of gene vectors were constructed by the amination reaction of Ed with benzyl on Polysaccharide-PBLA. By degradation. Cytotoxicity, sea kidney luciferase and other experiments proved that Polysaccharide-PAsp-ED has good degradability and low toxicity. Especially, chitosan based gene vector has excellent transfection efficiency. In order to further improve the transfection efficiency of gene vector, construction of targeted gene vector. We also carried out the following tasks: the introduction of CDS on CS to form a CS-CD polysaccharide skeleton with CS-CD modified by Ed as initiator. The ring-opening polymerization of monomer BLA-NCA was initiated to obtain CS-CD-PBLA.Through the amination reaction with Ed, CS-CD-PBLAwas obtained. The cationic polymer CS-CD-PAsp-ED was prepared by grafting folate Folic acid. The amantanamine of amantanamine. Self-assembly between Adand and CD cavity endows CCPE-FAA with cationic polymer targeting property. The CCPE and C CPE-FA polymers were encapsulated with DNA by layer-by-layer to form targeted ternary complexes. The targeting ternary complexes have high transfection efficiency and significant targeting in Hela- C6 cell lines with folic acid targeting.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450;R318.08

【共引文献】