摘要
制备载端粒酶反义寡核苷酸的阳离子脂质体并考察其体外癌细胞的抑制作用及转染效率和小鼠体内分布情况。以磷脂-DOPE(二油酰磷脂酰乙醇胺)-十八胺-胆固醇为类脂成分,薄膜超声-吸附-冷冻干燥法制备稳定的载反义寡核苷酸阳离子脂质体。激光粒度仪测定冷冻干燥前后脂质体zeta电位及粒径,透射电镜观察其形态,葡聚糖凝胶柱分离未包封的反义寡核苷酸,紫外法测定冻干前后的载药率。MTT法检测反义寡核苷酸对乳腺癌细胞MCF-7的抑制。荧光显微镜观察不同时间细胞摄取荧光标记反义寡核苷酸(antisenseoligodeoxynucleotides,asODN)的状况,计算转染效率和不同时间在小鼠心、肝、肺、肾、瘤体组织中的分布。海藻糖与甘露醇及甘氨酸为较好的冻干保护剂,制得的阳离子脂质体带正电荷,规则球形,大小较均匀,海藻糖作为保护剂复溶前后平均粒径为175nm和320nm,zeta电位在+32mV,脂质体的载药率复溶后为83.2%。脂质体介导的5′-FITC荧光标记的asODN在乳腺癌MCF-7细胞中的平均转染率在2、4、8h时分别为18%、26%、44%,并且细胞生长明显被抑制,与对照组比较,具有显著性差异(P<0.05)。阳离子脂质体-asODN复合物体内主要分布在肝、肾、肺和瘤体组织,与对照组比较差异显著。采用该处方和工艺可成功制备稳定性大有改善的反义寡核苷酸阳离子脂质体。该脂质体介导的端粒酶asODN能够有效抑制乳腺癌MCF-7细胞生长,体外转染效率较高。该给药系统可明显增加组织特别是瘤体对asODN的摄入量。
The aim of the paper is to prepare stable antisense oligodeoxynucleotides-loaded cationic liposomes and evaluate the transfection efficiency of asODN to MCF-7 oophoroma cells and study their distribution to different tissues in mice. Antisense oligodeoxynucleotides （asODN）-loaded cationic liposomes were prepared by a thin film-adsorption-lyophilization method which is simple and can overcome crucial pharmaceutical defects （e.g. instability） of liposomes during storage. The morphology was investigated by transmission electron microscope. The size and surface charge of the liposomes were determined by laser particle analyter. The dissociated ligodeoxynucleotides were separated from the liposomes by sephadex column and the entrapment efficiency was determined by using an ultraviolet photometer. Trehalose, mannitol, and glycine were suitable for lyophilization especially trehalose. The resulting liposomes were global microcapsule in a narrow particle size with a mean diameter of 175 nm and 320 nm before and after lyophilization, and a high zeta potentials of ＋32 mV. The dissociated asODN were separated from the liposomes by sephadex G-50 column and the entrapment coefficient of asODN was 88.4% pre and 83.2% post-lyophilization separately for trehalose. The growth of MCF-7 oophoroma cells were inhibited in vitro obviously （P 0.05） and transfection efficiency of asODN was 18%, 26%, 44% after 2 h, 4 h and 8 h, respectively. The formulation and method can be used to prepare stable cationic liposomes which can effectively inhibit the growth of MCF-7 oophoroma cells and obtain a high transfection efficiency. This system can improve distribution amount of asODN to tissues especially tumors in mice.
出处
《药学学报》
CAS
CSCD
北大核心
2009年第11期1273-1277,共5页
Acta Pharmaceutica Sinica
基金
国家自然基金资助项目(30772671)
关键词
反义寡核苷酸
阳离子脂质体
冷冻干燥
转染效率
肿瘤靶向
antisense oligodeoxynucleotide
cationic liposome
lyophilization
transfection efficiency
tumor-targeting
