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目的:运用DEAE-Sephadex A50微柱离心法结合AgCl胶体溶液散射光强度猝灭法测定BSA脂质体的包封率。方法:以BSA为模型药,采用逆向蒸发法制备脂质体;建立DEAE-Sephadex A50微柱离心分离游离蛋白质和脂质体的方法,并结合AgCl胶体溶液散射光强度猝灭法测定脂质体包封率。结果:AgCl胶体溶液散射光强度猝灭法中能达到散射光强度和散射光猝灭效果的最佳Cl-与Ag+摩尔比为1,选择反应体系为pH值4.8的醋酸-醋酸钠缓冲液,反应时间在50~80min较好,本实验中的反应时间为60min,不同的环境温度对测定结果有不可忽略的差异,应始终保证在同一环境温度下测定。本法的平均柱回收率为97.78%,RSD为0.18%(n=4),柱分离效率>90%,柱分离效果较好,可以适用于游离蛋白与脂质体的分离和检测。结论:用DEAE-Sephadex A50微柱离心结合AgCl胶体溶液散射光强度猝灭法测定包封率的方法,可适用于给药剂量较低的蛋白质类药物脂质体包封率测定过程中痕量蛋白质的检测。
OBJECTIVE: To determine the entrapment efficiency of BSA liposomes by DEAE-Sephadex A50 microcolumn centrifugation combined with AgCl colloidal solution scattering light intensity quenching. Methods: Liposomes were prepared by reverse-evaporation method using BSA as a model drug. The separation of free protein and liposomes by DEAE-Sephadex A50 micro column was carried out. Combined with AgCl colloidal solution scattering light intensity quenching method, Encapsulation rate. Results: The optimum molar ratio of Cl- to Ag + was 1 in the quenching method of AgCl colloidal solution. The optimal reaction conditions were as follows: acetic acid-sodium acetate buffer (pH 4.8) The reaction time is better in 50 ~ 80min, the reaction time in this experiment is 60min, different ambient temperature can not be ignored for the determination results, and should always be guaranteed at the same ambient temperature. The average column recovery of this method was 97.78% with RSD of 0.18% (n = 4). The column separation efficiency was> 90%. The column separation was better and could be applied to the separation and detection of free protein and liposome. Conclusion: The method of DEAE-Sephadex A50 micro-column centrifugation combined with AgCl colloidal solution scattering light intensity quenching method for determination of entrapment efficiency can be applied to the determination of entrapment efficiency of liposomes with lower dosage Protein testing.