原位降解高分子纳米载体的开发及其靶向基因治疗的研究

Up to now, there are many great challenges to cure terminal cancer tumors in the world. As one of gene therapy methods, RNA interference (RNAi) exhibits bright prospect for terminal cancer treatment. RNA possesses a high molecular weight and multiple negative charges, which prevent passive diffusion across the membrane of most cell types for RNAi, thus necessitating vectors for delivery of siRNA into the cytosol. However, traditional RNA vectors are comprised of non-biodegradable materials which exhibit cytotoxicity due to the accumulation of vectors. At the same time, traditional RNA vectors lack of targeting to cancer cells, which limit their therapeutic effect. Aiming to solve above problems, due to the fact that the cancer cells generate much acidic metabolite (pH ~ 5), new acid-sensitive nanogels will be designed and synthesized in this application, which can be degraded by the acidic metabolite. It is expected that the degradation and low positive charge density of nanogels will decrease the cytotoxicity. RNA will be incorporated into nanogels via electrostatic interaction. The surface of nanogels will be modified using transferrin. The transferrin in nanogel surface interacts with the receptor of transferrin in cancer cell membranes, which accelerates the nanogel uptake process by cancer cells and make the nanogel targeting to cancer cells. HEK293 will be used to investigate the process of RNA delivery and depressing the expression of target genes. The chemical and physical properties of the nanogels will be characterized. The relationship between material structure and uptake effect as well as toxicity will be investigated, which will provide fundamental scientific theory for design of RNA vectors. This application will provide preliminary study for complete terminal cancer cure.

尽管晚期癌症的彻底治愈仍然面临重大挑战，核糖核酸(RNA)干扰治疗为治愈晚期癌症提供一条可行的途径。但RNA是一种聚阴离子，无法通过细胞膜，需要RNA载体将其输送到细胞质内。而传统的RNA载体存在较多问题：不可生物降解，有生物毒性，对癌细胞缺少靶向性。针对以上问题，本项目利用癌细胞代谢产生大量酸性物质(pH~5)这一特点，设计并合成可酸性降解的纳米胶体颗粒作为RNA载体。癌细胞内的酸性物质原位降解纳米载体，同时引发RNA的释放。较低的正电荷密度和纳米载体的降解也降低了纳米载体的生物毒性。然后在纳米载体表面修饰一层转铁蛋白。转铁蛋白与癌细胞膜的转铁蛋白受体相互作用，加速细胞对纳米载体的吞噬，使该体系对癌细胞具有靶向性。选择人体肾脏细胞作为模型细胞，研究该体系的RNA传输过程以及RNA在细胞内抑制基因表达的过程。研究材料的结构对细胞吞噬效率、生物毒性的影响规律。

尽管晚期癌症的彻底治愈仍然面临重大挑战，核糖核酸(RNA)干扰治疗为治愈晚期癌症提.供一条可行的途径。但RNA是一种聚阴离子，无法通过细胞膜，需要RNA载体将其输送到细胞质内。而传统的RNA载体存在较多问题：不可生物降解，有生物毒性，对癌细胞缺少靶向性。针对以上问题，本项目利用癌细胞代谢产生大量酸性物质(pH~5)这一特点，设计并合成可酸性降解的纳米胶体颗粒作为RNA载体。本项研究的核心是基于缩醛结构的季铵性交联剂（BAC）。设计并合成了新型交联剂，随后通过与NIPAm、糖基单体三元共聚，合成功能化的纳米胶体颗粒。随后通过糖基与转铁蛋白之间的生物识别相互作用，对纳米胶体颗粒进行靶向修饰，使其特异性地针对癌细胞。分别使用核磁共振研究了单体的降解过程，并提出降解机理。使用DLS研究了纳米胶体颗粒在不同pH和葡萄糖浓度下的分解过程。负载RNA片段以后，在pH 4-5范围、6个小时内RNA释放效率达到95%。在细胞实验的过程中，癌细胞内的酸性物质原位降解纳米载体，同时引发RNA的释放，6个小时内对绿色荧光蛋白基因的关闭效率达到90%。该较低的正电荷密度和纳米载体的降解也降低了纳米载体的生物毒性。本研究提供了多功能化降解交联剂的设计思路，为以后可降解高分子纳米载体材料的研究奠定一定的理论基础。