脑靶向基因转运免疫脂质体的制备

Preparation of brain targeted immunoliposomes

构建一种高效的可以携带外源基因通过静脉注射的方式、透过血脑屏障进入脑内特异性表达目的蛋白的非病毒载体。通过不同比例的配比,对免疫脂质体合成过程中油相种类和油水比例、磷脂和胆固醇比例、旋转蒸发温度、超声温度和时间进行筛选优化,测定免疫脂质体的包封率和抗体偶联率。将包裹LacZ基因的免疫脂质体经尾静脉注射至大鼠体内,通过组织化学染色检测LacZ基因在大鼠体内的表达情况,验证合成的免疫脂质体是否可以转运外源基因跨过血脑屏障进入脑内表达。免疫脂质体合成的最佳比例为磷脂-胆固醇为1∶1;脂药比为100∶1;油相种类为二氯甲烷,油水比例为4∶1;旋转蒸发温度为30℃;超声温度为10℃;超声时间为5min,10%海藻糖可以增加免疫脂质体的稳定性。脂质体的包封率为87.24%,抗体偶联率为69%。将免疫脂质体通过尾静脉注射到大鼠体内后观察到LacZ基因在脑内及周围器官的表达。因此,通过工艺优化可以得到携带外源基因的免疫脂质体,其可以通过静脉系统跨过血脑屏障进入脑内并且表达,为实现颅内疾病的基因治疗奠定了基础。

To prepare a kind of effective non-viral transduction vector, which can deliver exogenous gene into the brain, this vector can be injected through vein system and has the ability to penetrate blood brain barrier. Several groups of materials proportion, type of oil phase, water-oil ratio, phosphatides-cholestrol ratio, temperature of steaming, ultrasonic temperature and time were compared for optimization. Well-constructed immunoliposomes encapsuling LacZ gene were infused into rats through tail vein. 48 h after injection, expression product betagalactosidase of LacZ gene was detected by histochemistry staining to convince the validity of immunoliposomes as non-viral vectors. The best proportion of synthesis immunoliposomes is as following： phosphatides- cholestrol ratio is 1∶1, lipids/drug is 100∶1, the type of oil phrase is dichloromethane, oil-water ratio is 4∶1, temperature of steaming is 30 ℃, ultrasonic temperature and time is 10 ℃ and 5 min. At last, 10% trehalose was added as a stabilizer. The entrapment rate is 87.24% and antibody coupling rate is 69%. When immunoliposomes were infused into rats, the expression of LacZ gene could be observed in the brain and periphery organs. Through the best proportion of materials, gene delivering immunoliposomes had been synthesized successfully. This non-viral vector can deliver exogenous gene penetrating blood brain barrier and express in the brain, and will be well-used in the field of gene therapy of cerebral diseases.