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

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.

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。

肿瘤基因治疗的瓶颈是尚未找到理想转基因载体，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。本课题采用两亲性壳聚糖OTMCS连接小分子聚乙烯亚胺(PEI)，得到OTMCS-PEI衍生物；然后选择在体内可特异、高效亲和肿瘤细胞表面NRP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双功能肽K12，与OTMCS-PEI偶联后，即得新型非病毒基因载体系统OTMCS-PEI-K12。旨在降低PEI毒性基础上，通过提高其在体内的靶向性和核递送能力，提高体内转染效率，为基因治疗最终应用于临床探索有效途径。. 首先采用固相法合成K12，通过三光气+琥珀酰亚胺法活化OTMCS并交联PEI 2KDa，进而选择SMCC法将K12与OTMCS-PEI偶联形成终产物，核磁等表明目的产物修饰度高且纯度好。OTMCS-PEI-K12在 37℃约 60h缓慢水解成无毒小分子，MTT实验显示细胞毒性极低；其DNA复合物呈椭球形，粒径100-600nm，电位适中，与DNA质量比约为1时可完全结合，并可抵抗高达200μg/mL肝素钠、50%FBS及50 U DNase I/DNA 解离，表现出极强体液稳定性；对U87细胞及B16细胞的转染效率远高于PEI 2 KDa，也高于PEI 25 KDa。作为新型基因载体材料，OTMCS-PEI-K12设计思路达到了先进水平，主要性质展示了其应用于体内转染的巨大潜力，在细胞移植模型中，活体成像表明其在体内有较强靶向能力，体内转染结果则展示了良好诱导肿瘤细胞凋亡、抑制肿瘤生长的效果。在细胞核转运机制研究中，K12的修饰大大增强了细胞核递送能力，此过程主要是由importinα和importinβ二者协作完成，这对于加深阳离子基因载体认识、设计安全高效的载体并应用于临床，具有一定理论意义。