叶酸分子靶向GSH/pH双重敏感星型聚合物共载多烯紫杉醇与TFPI-2质粒治疗鼻咽癌的研究

Co-delivery of chemotherapy drugs and gene has become research hotspots for the tumor treatment nowadays. In order to enhance its anti-tumor effect and reduce the toxicity furtherly, in view of higher expression of folate receptor(FR)、higher GSH concentration and acid environment in tumor tissue, folic acid molecular targeted star type polymer modified by histidine(His) and poly(L-lysine) dendron(PLLD) with GSH/pH dual-sensitivity(FA-CD-NH-NH=CH-SS-PLLD-His) will be constructed to co-delivery Docetaxel(DOC) and TFPI-2 expressing plasmid, and FA-CD-NH-NH=CH-SS-PLLD-His/DOC/TFPI-2 nanocomposites will be used for targeted dual-sensitive therapy for nasopharyngeal carcinoma(NPC) in this project. Furthermore, not only the physicochemical characteristics, gene transfection efficiency, NPC-targeting capacity of the nanocomposite and its distribution in vivo tissue of NPC-bearing nude mice, but also the expression of mRNA and protein of TFPI-2 in FR positive HNE-1 cell before and after gene transfection、its toxicity in vivo and inhibiting effect on NPC in vitro and in vivo will be studied by HPLC, in vivo imaging, RT-PCR, Western-blot, MTT assay, flow cytometry, Matrigel invasion test, HE staining, immunohistochemistry and TUNEL. The purpose of this study is to explore the feasibility and validity of the molecular targeted dual-sensitive system co-delivering drug and gene to treat NPC, and provide an experimental basis for clinical application.

共载药物及基因治疗已成为肿瘤治疗研究热点。为进一步增强共载体系抗肿瘤效应及减轻毒性，针对肿瘤高表达叶酸受体、高浓度谷胱甘肽和酸性环境特点，本项目构建叶酸分子靶向组氨酸及树枝状聚赖氨酸修饰的具有谷胱甘肽/pH双重敏感的星型聚合物(FA-CD-NH-NH=CH-SS-PLLD-His)，其共载多烯紫杉醇及TFPI-2质粒的纳米复合物对鼻咽癌进行靶向双重敏感综合治疗，通过HPLC、活体成像、RT-PCR及Western-blot法、MTT法、流式细胞术、Matrigel侵袭实验、HE染色、免疫组化、TUNEL法等，研究该复合物理化特征、基因转染效率、对鼻咽癌的靶向性、在荷鼻咽癌裸鼠体内分布、转染前后叶酸受体阳性的HNE-1细胞TFPI-2 mRNA和蛋白表达、对体内器官组织的毒性以及对NPC的体内外抑制效应。探索分子靶向双重敏感共载药物及基因治疗NPC的可行性和有效性，为临床应用提供实验依据。

共载药物及基因治疗已成为肿瘤治疗研究热点。为进一步增强共载体系抗肿瘤效应及减轻毒性，针对肿瘤高表达叶酸受体（FR）、高浓度谷胱甘肽和酸性环境特点，本项目制备了FA靶向GSH/pH双敏感纳米载体（FA-DS-Polymer），该载体具良好的血液相容性和低细胞毒性，对DOC的载药量是19.8 μg/mg，与基因有良好的结合能力，具良好的GSH/pH双重敏感释药性能。该载体对FR阳性的HNE-1细胞基因转染效率明显高于FR阴性的CNE-2细胞。在无血清条件下，载体对HNE-1细胞转染效率约42%；在有血清条件下，转染效率约26%，明显高于PEI（约6%）。其共载体系较单一载DOC或TFPI2的HNE-1细胞生长抑制率和凋亡率明显增强及细胞侵袭能力明显下降，且较无靶向的DS-Polymer/DOC/TFPI2的细胞凋亡率也明显增加，细胞侵袭能力明显下降。FA-DS-Polymer/DOC较DOC体内肝毒性明显降低。经荷HNE-1及CNE-2瘤块同一裸鼠尾静脉注射FA-DS-Polymer/DOC/TFPI2，HPLC方法分析肝肺心脾肾中DOC浓度随血液循环时间延长而衰减，脑组织中未检测出。瘤块中DOC浓度随血液循环时间延长而增加，HNE-1瘤块DOC浓度明显较CNE-2瘤块高。经尾静脉注射复合物转染24 h，活体成像显示HNE-1瘤块荧光面积及积分光密度明显较CNE-2瘤块强，冰冻切片可见肝肺心脾肾中有GFP表达，脑组织无GFP表达，HNE-1瘤中表达明显较CNE-2瘤明显。体内HNE-1瘤块抑制实验显示，FA-DS-Polymer/DOC/TFPI2抑瘤效果较DS-Polymer/DOC/TFPI2明显，瘤块PCNA表达也明显降低。研究结果表明FA-DS-Polymer/DOC/TFPI2具良好的体内外肿瘤靶向双敏感抑制效应。研究成果在ACS Appl. Bio Mater刊物发表。本项目构建了一类新颖的生物相容性好，载药量及基因转染效率较高的靶向GSH/pH双敏感载体，其共载系列显示出良好的肿瘤治疗效应，在肿瘤治疗方面具重要的临床应用前景。此外，在此基础上，申请人在该国家自然基金资助下积极进行了一些新的探索性工作，如：对NPC的发病机制进行研究；制备了其他系列针对NPC的靶向纳米药物，这些研究成果分别在J MATER CHEM B、Drug Delivery等刊物发表。