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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。
Preparation of cationic liposomes carrying telomerase antisense oligonucleotide and investigate its inhibition of cancer cells in vitro and transfection efficiency and in vivo distribution in mice. Antisense oligodeoxynucleotide cationic liposomes were prepared by the membrane ultrasound-adsorption-freeze drying method using phospholipid-DOPE (dioleoylphosphatidylethanolamine) -octadecylamine-cholesterol as lipid components. The zeta potential and particle size of the liposomes before and after lyophilization were measured by laser particle size analyzer. The morphologies of the liposomes were observed by transmission electron microscopy. The unencapsulated antisense oligonucleotides were separated on a Sephadex column. The drug loading rate . The inhibition of antisense oligonucleotide on breast cancer cell line MCF-7 was detected by MTT assay. Fluorescence microscopy was used to observe the uptake of antisense oligodeoxynucleotides (asODN) by cells at different time points. The transfection efficiency and the distribution in mouse heart, liver, lung, kidney and tumor tissues at different time points were calculated. Trehalose, mannitol and glycine were the best lyoprotectants. The prepared cationic liposomes were positively charged, regularly spherical and of relatively uniform size. The average particle size before and after the solubilization of trehalose as the protective agent was 175 nm and 320 nm, The zeta potential was at +32 mV, and the drug loading rate of liposomes was 83.2% after reconstitution. The average transfection rates of liposome-mediated 5’-FITC fluorescently labeled asODN in breast cancer MCF-7 cells were 18%, 26%, 44% at 2,4,8 h, respectively, and cell growth was significantly Inhibition, compared with the control group, with significant difference (P <0.05). The cationic liposomes-asODN complex mainly distributed in the liver, kidney, lung and tumor tissues, which was significantly different from the control group. Antisense oligonucleotide cationic liposomes with greatly improved stability can be successfully prepared by adopting the prescription and the process. The liposome-mediated telomerase asODN can effectively inhibit the growth of breast cancer MCF-7 cells, with high transfection efficiency in vitro. The drug delivery system can significantly increase tissue uptake of asODN, especially the tumor.