Application of Intelligent Targeting System for Cancer Gene Therapy

智能靶向系统在癌症基因治疗中的应用

• 批准号: 10470254
• 负责人: YANAGIE Hironobu
• 金额: $ 8.06万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470254/
• 关键词: Drug Delivery System; Non-viral vector; Gene Therapy; Tumor suppressor gene; Stealth liposome; Cationic liposome; Qplex; Chitosan; 非ウイルスベクター; ドラッグデリバリーシステム; 遺伝子治療; 癌抑制遺伝子; カチオニックリボソーム; ステルスリボソーム; キトサン; スチルスリポソーム; カチオニックリポソーム; プラスミドDNA

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

1)硼包载隐身脂质体在体内硼中子俘获治疗模型中对肿瘤细胞生长抑制的应用：硼中子俘获治疗（BNCT）中肿瘤细胞的破坏是由于^<10>B与热中子之间的核反应。有效的BNCT需要在肿瘤细胞中积累^<10 b>个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-PEG2000）包裹的^<10>B化合物用于递送系统。研究了热中子辐照下，静脉注射^<10> b - peg脂质体对人胰腺癌异种移植裸鼠的细胞毒性作用。在热中子照射小鼠注射^<10>B-裸脂质体或^<10>B- peg -脂质体后，与对照组相比，AsPC-1肿瘤的生长受到抑制。体内注射^<10>B- peg -脂质体对热中子辐照下肿瘤的抑制作用最大，提示静脉注射^<10>B- peg -li .更多的脂质体可增加肿瘤细胞对^<10>B原子的保留，从而在体内抑制热中子辐照下肿瘤的生长。2)利用新型脂质体“Qplex”进行体外肿瘤治疗的脂质体基因传递：阳离子脂质体和阳离子聚合物等非病毒载体的优点是不具有免疫原性，不具有潜在的重组和互补性。利用非病毒载体进行肿瘤基因治疗需要提高转染效率。我们制备了新型质粒DNA阳离子脂质体，称为四链复合体（Qplex），并评估了DNA的传递效率。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-三甲氨酰乙酰基）-二十二烷基-d -谷氨酰胺(TMAG)/二酰光空间胆碱(DLPC)/二酰光空间乙醇胺（DOPE）（1:2:2）的脂质。在Xgal染色中，用Qplex转染lac Z基因的效率从常规阳离子脂质体的4%提高到67%。在血清存在的情况下，转染效率较高。Tob （Erb -2换能器）是一种新发现的肿瘤抑制因子，它可能与酪氨酸激酶受体（包括Erb -2）相互作用和干扰。将tob基因导入NIH3T3细胞后，细胞生长受到抑制。Tob质粒被困在“Qplex”中。AsPC-1胰腺癌细胞在50%的胸腺嘧啶摄取消退中显示出肿瘤生长抑制。这些结果表明，Qplex具有非病毒载体的候选基因治疗癌症的治疗。3)荧光素酶质粒/壳聚糖复合物的转染及转染机制分析：本研究以质粒/壳聚糖复合物转染肿瘤细胞A549、B16和Hela。该复合物比质粒/脂质体复合物具有更高的转染活性。虽然聚半乳糖胺与壳聚糖是同一种氨基酸多糖，但质粒/聚半乳糖胺复合物未表现出任何基因表达。为此，我们分析了壳聚糖与聚半乳糖胺配合物的转染机理。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。用凝胶移位法评价了在Dnase I和阴离子表面活性剂存在下配合物的稳定性。少

项目成果

T.Sato et al: "Quantitative measurement of the interaction between ganglioside monolayers and wheat germ agglutinin (WGA) by a guartzcrystal microbalance."Biochim.Biophys.Acta.. 1380. 82-92 (1998)

T.Sato 等人：“通过石英晶体微天平定量测量神经节苷脂单层和麦芽凝集素 (WGA) 之间的相互作用。”Biochim.Biophys.Acta.. 1380. 82-92 (1998)

K.Kono,H.Yanagie et al: "Novel gene delivery systems : complexes of fusogenic polymer-modified liposomes and lipoplexes"Gene Therapy. (in press). (2000)

K.Kono、H.Yanagie 等人：“新型基因传递系统：融合聚合物修饰的脂质体和脂质复合物的复合物”基因治疗。

Yanagie et al.: "Inhibition of human pancreatic cancer growth by the liposomal delivery of a novel growth suppressing gene "tob" in vitro"Reseach in Experimental Medicine. (on press).

Yanagie 等人：“在体外通过脂质体递送新型生长抑制基因“tob”来抑制人类胰腺癌生长”实验医学研究。

H.Yanagie, et al: "Inhibition of human pancreatic cancer growth by the adenovirus-mediated introduction of a novel growth suppressing gene, tob, in vitro."In P.Walden et al (eds) ; Gene Therapy of Cancer. Plenum Press, New York. 91-96 (1998)

H.Yanagie 等人：“在体外通过腺病毒介导的新型生长抑制基因 tob 的引入来抑制人类胰腺癌的生长。”P.Walden 等人（编）；

H.Yanagie, et al: "Neutron Capture Auto-radiography for the Detection of ^<10>B Distributions and Concentrations in Tumor Bearing Mice."Journal of Nondestructive Testing and Evaluation. (in press). (2000)

H.Yanagie等人：“用于检测荷瘤小鼠中^ 10 B分布和浓度的中子捕获自动放射照相术”。无损检测与评估杂志。