Development of gene delivery liposome vector for suicide gene therapy

为了获得高转染效率的可注射型脂质体，对脂质体与质粒DNA复合物（lipoplexes）和脂质体包埋DNA（LED）进行了研究，优化了脂质体的处方和制备工艺。为了提高基因表达的选择性，将中期因子启动子控制的胸苷激酶基因（pMK-tk）用于单纯疱疹病毒胸苷激酶（HSV-tk）基因治疗。采用改进的乙醇注射法制备了3-[N-（N'，N'-二甲氨基乙烷）-氨甲酰基]胆固醇（DC-Chol）、L-二油酰磷脂酰乙醇胺（DOPE）和生物表面活性剂β-谷甾醇-β-D-葡萄糖苷（Sit-G）或甘露糖基赤藓糖醇脂质A（MEL）的脂质体（分别为Sit-G-脂质体和MEL-脂质体）。Sit-G-和MEL-脂质体产生约300-nm大小的脂质复合物。以磷脂酰胆碱、三甲基丙烷二油酰铵（DOTAP）、DOPE、Sit-G和视黄酸（RA）为原料，采用改进的脱水再水化囊泡法制备了RA-LED。通过优化蔗糖与脂质的重量比，可以形成具有66%包封率的313 nm大小的RA-LED。Sit-G-和MEL-脂质体和RA-LED的脂质复合物在细胞中存在血清的情况下显示出更高的荧光素酶标记基因的转染效率和胸苷激酶活性。在实体瘤模型中，在开始转染和注射更昔洛韦后4 - 6天，通过Sit-G-脂质体转染pMK-tk的治疗减少了肿瘤生长。这些脂质体在基因治疗中是很有前途的载体。

For injectable sized liposomes with high transfection efficiency of genes, liposomes complexed with plasmid DNA (lipoplexes) and liposomes-entrapping DNA(LED) were studied to optimize the formulae and preparation. For selectivity of gene expression, the thymidine kinase gene controlled by midkine promoter (pMK-tk) was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine(DOPE) and a biosurfactant such as β-sitosterol β-D-glucoside (Sit-G) or mannosylerythrytol lipid A(MEL)(Sit-G-liposomes or MEL-liposomes, respectively) were prepared by a modified ethanol injection method. Sit-G- and MEL-liposomes produced about 300-nm sized lipoplexes. RA-LED composed of phosphatidylcholine, dioleoyl-3-trimetylammonium propane (DOTAP), DOPE, Sit-G, and retinoic acid(RA), was prepared by a modified dehydration rehydration vesicle method. By optimization of the sucrose : lipid weight ratio, 313-nm sized RA-LED with 66% entrapment efficiency could be formed. Lipoplexes of Sit-G-and MEL-liposomes and RA-LED showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of pMK-tk by Sit-G-liposome reduced tumor growth at 4-6 days after starting transfection and injection of ganciclovir in a solid tumor model. These liposomes are promising vectors in gene-based therapy.