多功能化聚合物超分子自组装基因载体系统的研究

Gene therapy is a promising approach to treat human genetic diseases and cancer diseases. Its major technical difficulty at present is the availability of gene delivery systems. Cationic polymer is one kind of important non-viral gene vectors, which should be degradable and multifunctional with low toxicity. Our strategy is to design the supramolecular self-assembly of many degradable functionalized polymers via host-guest interaction for complexing plasmid DNA or siRNA into nanoparticles. These supramolecular nanoparticles will release the payloads genes due to partial disassembly or fast degradation after their cellular internalization of special cells in a targeted manner, to increase gene expression in special cells and enhance the therapeutic effect. . Linear beta-cyclodextrin polymers （PCD) and mono-adamantane-terminated functional polymers (ADPs), such as mono-adamantane-terminated disulfide-containing cationic polymers with low molecular weight, have been synthesized successfully. Supramolecular self-assembly inclusion complexes were formed based on host-guest interaction of main chain PCDs with ADPs. The inclusion complexes were able to bind and condense plasmid DNA into nanoparticles. The resulted nanoparticles, showing good stability and reduction-sensitivity, were demonstrated by the in vitro experiment to have high transfection efficiency and low cytotoxicity. In this work, a series of PCDs and various types of degradable ADPs, such as mono-adamantane-terminated disulfide-containing cationic polymers with low molecular weight and mono-adamantane-terminated targeted functional peptides, will be synthesized as the basic units of the self-assembling systems for gene vectors. Based on the applications in gene vectors, programming assembly, disassembly and the regulation rules of the inclusion complexes formed from host-guest interaction of PCDs with ADPs will be investigated in details. Through optimization of the simple combination of these basic units of the self-assembling systems in aqueous solution, supramolecular self-assembly of multifunctional polymers as gene vectors with high transfection efficiency and low cytotoxicity will be obtained.

基因治疗对于根治遗传性疾病和癌症等有很好的前景，但目前主要技术难点在于基因的传递系统。聚阳离子是一种重要的基因载体，它必须多功能化并且低毒可降解。我们的策略是设计多种功能化的高分子通过主客体自组装，可有效复合基因核酸（DNA或siRNA），该基因复合体系通过靶向作用进入目标细胞后，部分解组装或者快速降解释放基因，减少毒性，提高基因在特定细胞中的表达和治疗效果。申请者已初步合成了倍他环糊精聚合物和含金刚烷端基和双硫键连接的低分子量聚阳离子等功能化聚合物，它们有明显主客体作用，可与DNA复合形成超分子自组装包合物纳米粒子，该复合基因粒子有好的稳定性和还原敏感性。本项目拟制备环糊精改性聚合物系列和含金刚烷端基和双硫键的功能化聚合物和靶向多肽系列的组装模块，并结合基因载体的功能应用研究其程序化组装和解组装规律，在水溶液中通过多功能化组装模块的简单组合优化获得高转染效率和低毒性的基因载体材料。

基因治疗对于根治遗传性疾病和癌症等有很好的前景，但目前主要技术难点在于基因的传递系统。聚阳离子是一种重要的基因载体，它必须多功能化并且低毒可降解。如何设计多功能结构的简单引入和简单组合，研究和发展新的高分子基因载体系统实现多功能化（高的转染效率，低的毒性，靶向性等）的目的，具有重要的学术创新意义和实际应用价值。本项目通过分子设计合成了三类β-环糊精改性聚合物作为主体模块；合成了二类含金刚烷端基的低分子量聚阳离子，含有双硫键和多个端基偶氮苯的阳离子支化聚合物及含有罗丹明荧光基团的偶氮苯支化聚阳离子，和三类系列含金刚烷端基和功能靶向基团的聚乙二醇PEG作为客体聚合物。通过金刚烷或者偶氮苯与环糊精之间的主客体相互作用，利用简单的物理混合进行PEG化和靶向多功能化，在水溶液中自组装一系列多功能化基因载体系统。研究了其主客体超分子自组装的过程、解组装、稳定性及其调控规律。探索了这些多功能化聚合物超分子自组装体系复合核酸（DNA和siRNA）纳米颗粒的稳定性和在细胞内还原条件下的释放特性，及其对多种细胞系的转染效率和毒性。通过多种功能化的高分子模块单元的简单组合优化，得出了在支化阳离子聚合物中引入双硫键，增加其支化度，引入环糊精聚合物和使用紫外光辐照均能提高该光/还原双重敏感主客体超分子自组装体系基因载体的细胞内吞和转染效率的结果，及延长复合物与细胞的孵育时间可提高PEG化的基因传递系统的转染效率。以斑马鱼为模型，初步探索了这些基因纳米载体粒子的体内毒性明显低于对照物25-kDa 聚乙烯亚胺PEI。相对于PEI，本项目制得了多种具有高转染效率和低毒性的聚合物超分子自组装基因载体材料，为进一步研发临床可应用的高分子基因载体材料奠定了实验和理论基础。已发表标注SCI论文22篇，其中影响因子大于3的SCI论文19篇，已获3项中国专利授权，3篇文章已在线发表。

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