Application of Intelligent Targeting System for Cancer Gene Therapy

1)包载硼的隐形脂质体在体内硼中子俘获治疗模型中抑制肿瘤细胞生长的应用：硼中子俘获治疗（BNCT）中的肿瘤细胞破坏是由于^ B与热中子之间的核反应<10>。有效的BNCT需要<10>在肿瘤细胞中积累大量的B原子。我们制备了一种聚乙二醇（PEG）结合脂质体（DPPC/胆固醇/DSPC-PEG 2000）包载的β-<10>B化合物作为给药系统。我们用热中子照射的方法评价了静脉注射β-<10>B-PEG-脂质体对人胰腺癌裸鼠移植瘤的细胞毒作用。在注射^<10>B-裸脂质体或^<10>B-PEG-脂质体的小鼠的热中子照射后，相对于对照，AsPC-1肿瘤生长被抑制。在<10>热中子照射下，注射β-B-PEG-liposome对肿瘤的抑制作用最强<10>。 ...更多信息 脂质体可增加肿瘤细胞对~（125）<10>B原子的滞留，从而抑制肿瘤细胞的生长。2）脂质体基因转移的体外肿瘤治疗：非病毒载体如阳离子脂质体和阳离子聚合物具有无免疫原性、无潜在重组、无互补性等优点。在肿瘤基因治疗中应用非病毒载体时，需要提高转染效率。我们制备了新型的质粒DNA-阳离子脂质复合物，称为四元复合物（“Qplex”），并评估了DNA递送效率。Qplex由阳离子脂质体、pDNA、鱼精蛋白和转铁蛋白组成。脂质体M-（α-三甲基氨乙酰基）-双十二烷基-D-天门冬氨酸（TMAG）/二月桂酰磷脂酰胆碱（DLPC）/二油酰磷脂酰乙醇胺（DOPE）（1：2：2）。Xgal染色显示，Qplex将lac Z基因的转染效率从传统阳离子lipoplex的4%提高到67%。Tob（Transducer of Erb B-2）是一种新发现的肿瘤抑制因子，可与包括Erb B-2在内的酪氨酸激酶受体相互作用并产生干扰。将tob基因导入NIH 3 T3细胞中导致细胞生长受到抑制。Tob质粒被捕获到“Qplex”中。AsPC-1胰腺癌细胞的肿瘤生长抑制表现为50%的胸苷摄取消退。荧光素酶质粒/壳聚糖复合物的转染及转染机制分析：本研究用荧光素酶质粒/壳聚糖复合物转染肿瘤细胞A549、B16和Hela。该复合物显示出比质粒/脂质体复合物更高的转染活性。虽然聚半乳糖胺是与壳聚糖相同的氨基多糖，但质粒/聚半乳糖胺复合物未显示任何基因表达。并对壳聚糖与多聚半乳糖胺复合物的相互作用机理进行了分析。我们合成了FITC标记的荧光素酶质粒和德克萨斯红标记的壳聚糖，以评估质粒/壳聚糖复合物的转染效率、细胞摄取和亚细胞分布。采用凝胶迁移实验研究了复合物在Dnase I和阴离子表面活性剂存在下的稳定性。少

1) Application of boron entrapped stealth liposome to tumour cell growth inhibition in in vivo boron neutron capture therapy model : The tumor cell destruction in boron neutron-capture therapy (BNCT) is due to the nuclear reaction between ^<10>B and thermal neutrons. It is necessary for effective BNCT to accumulate of ^<10>B atoms in the tumor cells. We prepare a polyethylene-glycol (PEG) binding liposome (DPPC/cholesterol/DSPC-PEG2000) entrapped ^<10>B compound for the delivery system. We evaluated the cytotoxic effects of intrveneously injected ^<10>B-PEG-liposome on human pancreatic carcinoma xenografts in nude mice with thermal neutron irradiation. After thermal neutron irradiation of mice injected with ^<10>B- bare liposome or ^<10>B-PEG-liposome, AsPC-1 tumour growth was suppressed relative to controls. Injection of ^<10>B-PEG-liposome caused the greatest tumour suppression with thermal neutron irradiation in vivo These results suggest that intraveneous injection of ^<10>B-PEG-li … More posome can increase the retention of ^<10>B atoms by tumor cells, causing tumor growth suppression in vivo upon thermal neutron irradiation.2) Liposomal gene delivery using novel lipoplexes called "Qplex" for cancer therapy in vitro : Non-viral vectors, such as cationic liposomes and cationic polymers have developed in the merits without immunogenicity, potential recombination, nor complementation. It is necessary to increase the transfectional efficiency when we use non-viral vector on cancer gene therapy. We have prepared new typed plasmid DNA-cationic lipoplexes, called quatenary complex ("Qplex"), and evaluated DNA delivery efficiency. Qplex is composed with cationic liposome, pDNA, protamine and transferrin. The lipid of liposome M-(α-trimethylammonioacetyl)-didodecyl-D-glutamatechloride (TMAG)/dilauroyl-phospatidylcholine (DLPC)/dioleoylphospatidylethanolamine (DOPE) (1 : 2 : 2). The transfectional efficiency of lac Z gene is increased to 67% with Qplex from 4% with conventional cationic lipoplexes in Xgal staining. The transfection efficiency is superior in the existence of serum.Tob (Transducer of Erb B-2) is a newly identified tumor suppressor that may interact and interfere with tyrosine kinase receptors including Erb B-2. Introduction of tob gene into NIH3T3 cells results in suppression of growth of the cells. Tob plasmid was entrapped into the "Qplex". The tumor growth suppression of AsPC-1 pancreatic cancer cells was shown in 50% of thymidine uptake regression. These results, suggest that the Qplex has an candidate for non viral vector of gene therapy for treatment of cancer.3) Transfection with Luciferase Plasmid/Chitosan Complex and Analyses of Transfection Mechanism : In this study, we transfected tumor cells (A549, B16 and Hela) with the plasmid/chitosan complex. The complex showed higher transfection activity than the plasmid/lipofectin complex did. Although the polygalactosamine is the same amino polysaccharide as the chitosan, the plasmid/polygalactosamine complex did not show any gene expression. So we analysed the travsfection mechanism between chitosan and polygalactosamine complex. We synthesized FITC-labeled luciferase plasmid and Texas Redlabeled chitosan to evaluate the transfection efficiency, cell uptake and sub-cellular distribution of the plasmid/chitosan complex. Gel shift assay was used to evaluate the stability of the complexes in the presence of Dnase I and anionic surfactant.. Less