双功能肽tLyP-1-NLS修饰的DNA纳米胶束的构建及其抗肿瘤作用研究

肿瘤基因治疗的瓶颈是尚未找到理想的转基因载体，其最主要障碍是体内转染效率明显偏低，而根本原因在于DNA复合物的体内靶向性和细胞核递送能力有待提高。基于前期研究，首先，采用两亲性壳聚糖OTMCS连接低分子量聚乙烯亚胺(PEI)，得到转染效率高且细胞毒性小的OTMCS-PEI衍生物；然后，利用跨膜蛋白受体NRP在人多数肿瘤细胞高表达的特点，选择在体内可特异亲和并高效区分该受体的tLyP-1肽，与核定位信号肽NLS连接，合成具有靶向于NRP和提高核递送能力的含tLyP-1、NLS的双功能肽tLyP-1-NLS（即K12）；最后，利用交联技术将K12与OTMCS-PEI偶联，从而构建新型非病毒基因载体系统OTMCS-PEI-K12，并考察该系统细胞核转运机制。本课题旨在降低PEI细胞毒性的基础上，通过提高其在体内的靶向性和核递送能力，从而提高体内转染效率，为基因治疗最终应用于临床探索一条有效途径。

The key to gene therapy on cancer clinically is finding an ideal gene-delivery vector，and the supreme barrier is the lower transfection efficiency in vivo. As fundamental reason, tumor targeting in vivo and nuclear uptake of DNA complexes should be improved. Based on previous study，at first a kind of polyethylenimine (PEI) derivates OTMCS-PEI with high transfection efficiency and low cytotoxicity will be synthesized by cross-linking low molecular weight PEI with the amphiphilic chitosan OTMCS. The NRP receptors are highly expressed on human tumor cells and the peptide tLyP-1 has specific affinity and effective discrimination to NRP. Then we will use NRP-targeting peptide tLyP-1, in conjunction with the nuclear localization signal NLS to yield a bifunctional peptide tLyP-1-NLS named K12 which can improve cell selection and increase nuclear uptake, and at last adopt K12 to modify OTMCS-PEI so as to prepare a new polymeric gene vector OTMCS-PEI-K12. Mechanisms of nuclear transport of this vector will be also investigated. The purpose of the present study is to increase transfection efficiency in vivo on the premise of ensuring low cytotoxicity by improving its tumor targeting in vivo and nuclear uptake, and then enhance the therapeutic effect of gene therapy on cancer. This study will find an available way to gene therapy on cancer clinically.