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目的:制备稳定的载反义寡核苷酸的阳离子脂质体前体制剂。方法:以磷脂-二油酰磷脂酰乙醇胺(dioleoylphophatidylethanolamine,DOPE)-十八胺-胆固醇为类脂成分,采用薄膜超声-挤压制备空白阳离子脂质体,吸附-冷冻干燥法制备载反义寡核苷酸阳离子脂质体前体。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。结果: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前体带正电荷,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175和320 nm左右,复融前后Zeta电位值在+32和+40 mV左右,脂质体的载药率复溶前后分别为87.6%与83.21%。结论:海藻糖作为冻干保护剂,薄膜超声挤压法与冷冻干燥法结合,可成功制备反义寡核苷酸阳离子脂质体前体制剂,稳定性大大改善。
Objective: To prepare a stable cationic liposome precursor preparation containing antisense oligonucleotides. Methods: Blank cationic liposomes were prepared by membrane ultrasound-extrusion using dioleoylphophatidylethanolamine (DOPE) -octadecylamine-cholesterol as the lipid component. The antisense oligodextracts were prepared by adsorption-freeze drying Nucleotide cationic liposome precursors. 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 . Results: Trehalose, mannitol and glycine were the best lyoprotectants. The cationic liposome precursors were positively charged and regularly spherical in shape. The average particle sizes of trehalose before and after reconstitution were The values of Zeta potential before and after thawing were around +32 and +40 mV at 175 and 320 nm, respectively. The loading rates of liposomes before and after reconstitution were 87.6% and 83.21%, respectively. CONCLUSION: Trehalose as a lyoprotectant can be successfully prepared by ultrathin extrusion and freeze-drying methods. The stability of antisense oligodeoxynucleotide cationic liposome preparation can be greatly improved.