肿瘤微环境响应的细胞内、细胞核双重定位共转运系统的研究

Many researchers have been focusing on gene/drug combinational treatment for tumor.But how to achieve efficient gene/drug co-delivery to tumor cells, intracellular drug releasing and gene nucleus localization simultaneously is still unsolved problem. In this project, two novel materials would be synthesized and a nanoparticle complex be prepared to solve the problem. Matrix metalloproteinases-2 (MMP-2) substrate peptide would be used to link PEG and PLGA copolymers which contain disulfide bond to obtain PEG-MPPp-PSSP material. The other material, W5R4K-PEG-pHis, would be produced from cyclic cell-penetrating peptides W5R4K modified pHis. The nanoparticle complex loading Curcumin and pGenesil-Survivin plasmid would be prepared using these two materials. This nanoparticle complex delivery system may have the capabilities of avoiding being cleared by the reticuloendothelial-system, detaching PEG coat responsively in tumor milieu, penetrating the cell efficiently with the mediate of W5R4K, and releasing Curcumin quickly when trigged by the reduction environment in the cell. Moreover, with the breaking of disulfide bond, the nanoparticle size would be decrease due to the holding effect of gene loaded, which would benefit the subsequent nucleus gene delivery. It is an original research on intracellular and nucleus dual localization of nanoparticle using W5R4K as a ligand. And for the first time, we discover and utilize the properties of nanoparticle size decrease resulted from the breaking of disulfide bond in mixed materials, which would be a new efficient way to transport gene. Therefore, this project is novel, and will be a beneficial trail for gene/drug co-delivery system.

基因/药物联用是肿瘤治疗的新兴手段，如何实现其高效共转运至肿瘤细胞，并同时实现胞内释药、递送基因至细胞核，仍需解决。本项目拟以MMP-2底物多肽连接PEG与含二硫键PLGA共聚物制得新材料PEG-MPPp-PSSP、以W5R4K穿膜环肽修饰多聚组氨酸制得新材料W5R4K-PEG-pHis，两种材料共混制得多元纳米粒包载姜黄素和pGenesil-Survivin质粒。该纳米粒预期能逃逸RES吞噬；在肿瘤微环境中由MMP-2响应脱PEG外衣；由W5R4K介导高效入胞；在胞质内由二硫键还原响应定位快速释放姜黄素；并因二硫键断裂，纳米粒在所载基因牵附作用下变小，再由W5R4K介导穿过细胞核膜，实现基因细胞核定位。本项目首次采用W5R4K环肽作配体实现细胞内和细胞核双重定位，首次发现并利用复合材料中二硫键断裂后纳米粒变小的性质进行更高效的基因递送，具有原创性；是对基因/药物共转运新型系统的有益尝试。

基因/药物联用是肿瘤治疗的新兴手段，高效共转运联用的基因/药物至肿瘤细胞，对其协同发挥抗癌作用至关重要。本课题针对基因与化疗药物的不同特性，设计制备了两种聚合物：PEG5K-MMP-PLGA和W5R4K-PEG2K-pHis，并以其构建多功能的非病毒基因/药物载体。该载体的PEG5K水化层提供了“隐形”功能，延长了纳米粒子在血液中的循环时间；到达肿瘤部位后，肿瘤微环境中高表达的MMP-2脱掉PEG5K外衣，增加了纳米粒子的摄取；暴露的W5R4K环肽可促进纳米内吞、逃逸溶酶体、到达细胞核。本文研究表明，pHis嵌段可以响应pH的微小变化，其在酸性环境下质子化后具有“质子海绵”效应，可以使得纳米粒载体快速逃逸溶酶体。使用二硫键对纳米载体骨架材料PLGA进行交联后，结果也显示在高浓度GSH下纳米粒子易解体，快速释放出药物或基因。在此研究基础上，本课题筛选出该安全高效的载体作为抗凋亡蛋白SurvivinshRNA/姜黄素药物的共传递系统，研究表明该系统在体内外均具有较好的抗肿瘤效果，且SurvivinshRNA/姜黄素协同治疗肿瘤作用显著。本研究为肿瘤的协同治疗提供了一种新的思路和方法，具有借鉴意义。