盐酸阿霉素和知母皂苷AⅢ共载脂质体的制备及包封率的测定
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摘要
目的建立盐酸阿霉素和知母皂苷AⅢ共载脂质体包封率的测定方法。方法采用薄膜分散法制备盐酸阿霉素和知母皂苷AⅢ共载脂质体,分别用透析法、微柱离心法和超高速离心法分离脂质体和游离药物,采用HPLC法测定游离药物含量和脂质体中的药物含量,计算得出盐酸阿霉素和知母皂苷AⅢ的包封率。结果确定盐酸阿霉素和知母皂苷AⅢ共载脂质体处方为DPPC-DSPE-PEG2000-TAⅢ-DOX比例为5∶1∶1∶1,采用薄膜分散法制备的脂质体大小适中,粒径为(55.4±0.40)nm,PDI为0.20±0.02,电位为(-17.4±0.6)mV;在所选的色谱条件下,脂质体的其他组分不干扰样品的测定,盐酸阿霉素在24.9～498.0μg/mL(r=0.999 9),知母皂苷AⅢ在50.55～1 011.00μg/mL(r=0.999 6)线性关系良好。研究结果表明微柱离心法能有效地将盐酸阿霉素和知母皂苷AⅢ双载脂质体与游离药物分离,测得的包封率分别为(85.12±1.27)%、(76.51±0.46)%。结论薄膜分散法可用于盐酸阿霉素和知母皂苷AⅢ共载脂质体的制备;微柱离心法准确度高、重复性好并快捷方便,可用于包封率的测定。
Objective To develop a method to determine the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes. Methods In this paper, the thin-film rehydration method was used to prepare doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes. Liposomes and free drugs were separated by dialysis, gel microcolumn centrifugation, and ultra-high speed centrifugation. The content of free drugs and drugs in liposomes was determined by HPLC, and the entrapment efficiency of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes was calculated. Results The optimal formulation of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes was DPPC-DSPE-PEG2000-TAⅢ-DOX with a molar ratio of 5∶1∶1∶1. The liposomes prepared using thin-film rehydration method had a well-defined spherical shape with a size of(55.4 ± 0.40) nm, a PDI of(0.20 ± 0.02), and a weakly negative zeta potential of(-17.4 ± 0.6) mV. The excipients in the liposomal formulation can be well separated from doxorubicin hydrochloride and timosaponin AⅢ in the selected chromatographic conditions. The calibrated linear curve of doxorubicin hydrochloride was within 24.9—498.0 μg/m L(r = 0.999 9) and that of timosaponin AⅢ was within 50.55—1 011.0 μg/m L(r = 0.999 6). Free doxorubicin hydrochloride and timosaponin AⅢ were well separated from liposome by gel microcolumn centrifugation, and the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AⅢ was(85.12 ± 1.27)% and(76.51 ± 0.46)% respectively. Conclusion The thin-film dispersion-method can be used for the preparation of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes. The method of gel microcolumn centrifugation is accurate, reproducible, simple, and suitable for determination of the encapsulation efficiency of co-loaded liposomes.
Objective To develop a method to determine the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes. Methods In this paper, the thin-film rehydration method was used to prepare doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes. Liposomes and free drugs were separated by dialysis, gel microcolumn centrifugation, and ultra-high speed centrifugation. The content of free drugs and drugs in liposomes was determined by HPLC, and the entrapment efficiency of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes was calculated. Results The optimal formulation of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes was DPPC-DSPE-PEG2000-TAⅢ-DOX with a molar ratio of 5∶1∶1∶1. The liposomes prepared using thin-film rehydration method had a well-defined spherical shape with a size of(55.4 ± 0.40) nm, a PDI of(0.20 ± 0.02), and a weakly negative zeta potential of(-17.4 ± 0.6) mV. The excipients in the liposomal formulation can be well separated from doxorubicin hydrochloride and timosaponin AⅢ in the selected chromatographic conditions. The calibrated linear curve of doxorubicin hydrochloride was within 24.9—498.0 μg/m L(r = 0.999 9) and that of timosaponin AⅢ was within 50.55—1 011.0 μg/m L(r = 0.999 6). Free doxorubicin hydrochloride and timosaponin AⅢ were well separated from liposome by gel microcolumn centrifugation, and the encapsulation efficiency of doxorubicin hydrochloride and timosaponin AⅢ was(85.12 ± 1.27)% and(76.51 ± 0.46)% respectively. Conclusion The thin-film dispersion-method can be used for the preparation of doxorubicin hydrochloride and timosaponin AⅢ co-loaded liposomes. The method of gel microcolumn centrifugation is accurate, reproducible, simple, and suitable for determination of the encapsulation efficiency of co-loaded liposomes.
引文
[1]李飞阳,崔纯莹,王玉记,等.阿霉素脂质体的制备及抗肿瘤活性研究[J].首都医科大学学报,2015,36(2):157-160.
[2]Kaname N,Zhang J,Meng Z,et al.Effect of six steroidal saponins isolated from anemarrhenae rhizoma on platelet aggregation and hemolysis in human blood[J].Clin Chim Acta,1999,289(1/2):79-88.
[3]王宁.知母皂苷AIII抑制血小板聚集的分子机制研究[D].大理:大理学院,2009.
[4]Lee B,Jung K,Kim D H.Timosaponin AIII,a saponin isolated from Anemarrhena asphodeloides,ameliorates learning and memory deficits in mice[J].Pharmacol Biochem Be,2009,93(2):121-127.
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[6]Miura T,Ichiki H,Iwamoto N,et al.Antidiabetic activity of the Rhizoma of Anemarrhena asphodeloides and active components,mangiferin and its glucoside[J].Bio Pharm Bull,2001,24(9):1009-1011.
[7]Miura T,Ichiki H,Iwamoto N,et al.Timosaponin A-IIIinduces autophagy preceding mitochondria-mediated apoptosis in HeLa cancer cells[J].Cancer Res,2008,68(24):10229-10237.
[8]尤杰,孙兆林,季宇彬,等.知母皂苷AIII药理活性及机制研究进展[J].中国医药导报,2012,9(3):11-13.
[9]Kang Y J,Chung H J,Nam J W,et al.Cytotoxic and antineoplastic activity of timosaponin A-III for human colon cancer cells[J].J Nat Prod,2011,74(4):701-706.
[10]高晓黎,季兴梅.葡聚糖凝胶柱色谱法测定脂质体包封率的条件筛选[J].中国药学杂志,2003,38(7):515-517.
[11]闫丹,江敏瑜,王云红,等.积雪草总苷脂质体的制备及体外透皮研究[J].中草药,2018,49(9):2041-2048.
[12]郑杭生,佐拉·沙肯迪克,王湘林,等.离心沉淀-离心超滤法测定盐酸青藤碱脂质体的包封率[J].中草药,2011,42(8):1523-1527.
[13]邵红霞,奉建芳,龙晓英.脂质体包封率的测定方法[J].中南药学,2009,7(3):212-215.
[14]高晓非,邓英杰,曹金娜,等.微柱离心-HPLC法测定六甲蜜胺脂质体包封率[J].药物分析杂志,2009,29(2):247-249.
[15]于燕燕,赵继会,冯年平,等.微柱离心-HPLC法测定鬼臼毒素醇质体的包封率研究[J].中草药,2010,41(10):1634-1637.
[16]赵海霞,郭兴奎,孔德亮,等.脂质体制备技术[J].山东中医杂志,2000,19(7):435-437.
[17]孙庆雪,邵伟,黄桂华.脂质体制备方法的选择[J].中成药,2010,32(8):1397-1401.
[18]张祺照.脂质体制备方法研究概况[J].亚太传统医药,2013,9(12):71-72.
[19]王汀,李文秀,邓英杰.微柱离心-药脂比测定脂质体药物包封率[J].沈阳药科大学学报,2008,25(1):10-14.
[20]刘利萍,金阳,李毅.微柱凝胶离心分离-HPLC法测定克霉唑脂质体的包封率[J].药物分析杂志,2007,27(11):1812-1815.
[2]Kaname N,Zhang J,Meng Z,et al.Effect of six steroidal saponins isolated from anemarrhenae rhizoma on platelet aggregation and hemolysis in human blood[J].Clin Chim Acta,1999,289(1/2):79-88.
[3]王宁.知母皂苷AIII抑制血小板聚集的分子机制研究[D].大理:大理学院,2009.
[4]Lee B,Jung K,Kim D H.Timosaponin AIII,a saponin isolated from Anemarrhena asphodeloides,ameliorates learning and memory deficits in mice[J].Pharmacol Biochem Be,2009,93(2):121-127.
[5]King F W,Fong S,Griffin C,et al.Timosaponin AIII is preferentially cytotoxic to tumor cells through inhibition of mTOR and induction of ER stress[J].PLoS One,2009,4(9):e7283.
[6]Miura T,Ichiki H,Iwamoto N,et al.Antidiabetic activity of the Rhizoma of Anemarrhena asphodeloides and active components,mangiferin and its glucoside[J].Bio Pharm Bull,2001,24(9):1009-1011.
[7]Miura T,Ichiki H,Iwamoto N,et al.Timosaponin A-IIIinduces autophagy preceding mitochondria-mediated apoptosis in HeLa cancer cells[J].Cancer Res,2008,68(24):10229-10237.
[8]尤杰,孙兆林,季宇彬,等.知母皂苷AIII药理活性及机制研究进展[J].中国医药导报,2012,9(3):11-13.
[9]Kang Y J,Chung H J,Nam J W,et al.Cytotoxic and antineoplastic activity of timosaponin A-III for human colon cancer cells[J].J Nat Prod,2011,74(4):701-706.
[10]高晓黎,季兴梅.葡聚糖凝胶柱色谱法测定脂质体包封率的条件筛选[J].中国药学杂志,2003,38(7):515-517.
[11]闫丹,江敏瑜,王云红,等.积雪草总苷脂质体的制备及体外透皮研究[J].中草药,2018,49(9):2041-2048.
[12]郑杭生,佐拉·沙肯迪克,王湘林,等.离心沉淀-离心超滤法测定盐酸青藤碱脂质体的包封率[J].中草药,2011,42(8):1523-1527.
[13]邵红霞,奉建芳,龙晓英.脂质体包封率的测定方法[J].中南药学,2009,7(3):212-215.
[14]高晓非,邓英杰,曹金娜,等.微柱离心-HPLC法测定六甲蜜胺脂质体包封率[J].药物分析杂志,2009,29(2):247-249.
[15]于燕燕,赵继会,冯年平,等.微柱离心-HPLC法测定鬼臼毒素醇质体的包封率研究[J].中草药,2010,41(10):1634-1637.
[16]赵海霞,郭兴奎,孔德亮,等.脂质体制备技术[J].山东中医杂志,2000,19(7):435-437.
[17]孙庆雪,邵伟,黄桂华.脂质体制备方法的选择[J].中成药,2010,32(8):1397-1401.
[18]张祺照.脂质体制备方法研究概况[J].亚太传统医药,2013,9(12):71-72.
[19]王汀,李文秀,邓英杰.微柱离心-药脂比测定脂质体药物包封率[J].沈阳药科大学学报,2008,25(1):10-14.
[20]刘利萍,金阳,李毅.微柱凝胶离心分离-HPLC法测定克霉唑脂质体的包封率[J].药物分析杂志,2007,27(11):1812-1815.