微柱离心结合胆固醇浓度变化测定水溶性药物脂质体包封率

Determination of encapsulation efficiency of water-soluble drug in liposomes by minicolumn centrifugation combined with the changes of cholesterol concentration

目的建立微柱离心法结合胆固醇含量变化来考察水溶性药物脂质体包封率的测定条件。方法采用HPLC法测定脂质体混悬液中胆固醇的浓度,以微柱离心法分离游离药物和脂质体,并用过柱前后脂质体混悬液中胆固醇浓度之比考察脂质体的过柱回收率,进一步测定脂质体的包封率。结果所建立的HPLC法可以准确测定脂质体破乳后溶液中的胆固醇浓度。采用筛选的微柱离心条件洗脱,1、2管空白脂质体的柱回收率为（98.49±0.29）%（n=3）,而药物不被洗脱下来,第3~15管游离药物的平均柱回收率为（99.59±0.28）%（n=3）。测定水溶性药物苯妥英钠脂质体的包封率为（25.46±0.27）%（n=3）。结论微柱离心法适用于水溶性药物苯妥英钠脂质体包封率的测定,游离药物和脂质体被有效分离,通过结合过柱前后胆固醇浓度变化考察脂质体的过柱回收率,可获得更加准确的包封率测定结果。

Objective To establish a method of minicolumn centrifugation combined with the changes of cholesterol concentration in order to study the encapsulation efficiency of water-soluble drug in liposomes.Methods The method for measuring the concentration of cholesterol in the liposome suspension were established by high performance liquid chromatography（ HPLC）. The recovery of liposomes through mini-column was calculated by the ratio of cholesterol concentration in the liposomes to that in eluent to define the conditions of minicolumn centrifugation in order to achieve an effective separation between free drug and liposomes. Results The concentration of cholesterol in the liposome solution after demulsification could be accurately measured by the established HPLC method. When applying the method,the 1- 2 tube recovery of blank liposome through minicolumn was（ 98. 49 ± 0. 29） %（ n = 3） with no drug eluted,while the 3- 15 tube recovery of free drug was（ 99. 59 ± 0. 28） %（ n = 3）,which implied that an effective separation between drug and liposome was achieved. The encapsulation efficiency（ EE） of the water-soluble drug phenytoin-sodium（ PHT-Na） liposome determined by this method was（ 25. 46 ± 0. 27） %（ n = 3）. Conclusions The method of minicolumn centrifugation is applicable to determine the EE of water-soluble drug PHT-Na liposome.By combining the changes of cholesterol concentration before and after passing through the minicolumn can investigate the recovery of liposomes through minicolumn more accurately and safely,which means the obtaining of more accurate results of EE.