硬脂醇半乳糖苷修饰阿西替尼脂质体的制备及体外抗肝癌活性的初步研究
发布时间：2018-08-12 16:50
【摘要】：目的本实验采用阿西替尼为模型药物,硬脂醇半乳糖苷为靶向分子,制备阿西替尼脂质体,优化制备工艺,考察药效。以便进一步增加脂质体的肝靶向性,降低药物的毒副作用。通过冷冻干燥法达到增加脂质体本身稳定性的目的。促使药物达到最好的治疗效果。方法采用葡聚糖凝胶微柱离心-HPLC测定硬脂醇半乳糖苷修饰阿西替尼脂质体的包封率,采用Box-Behnken响应面设计法优化制备工艺。采用CCK-8法测定硬脂醇半乳糖苷修饰阿西替尼脂质体对人肝癌细胞株SMMC-7721和人肺癌细胞株A549的生长抑制情况,采用Annexin V/PI流式细胞分析法检测细胞凋亡的情况,采用western blot检测凋亡蛋白Bax、Bcl-2和P53的情况。结果冷冻干燥法制备的硬脂醇半乳糖苷修饰阿西替尼脂质体平均包封率为69.34%,平均粒径为(145±4.6)nm。采用了Box-Behnken响应面设计法优化制备工艺,所得的最优处方为,胆固醇与磷脂的比例为1:7.67,阿西替尼原料药与磷脂的比例为1:21.50,水合温度为52.75°C,磷脂与硬脂醇半乳糖苷的比例为1:16.69。CCK-8法检测相同浓度的硬脂醇半乳糖苷修饰阿西替尼脂质体对人肝癌SMMC-7721增殖的抑制比人肺癌A549细胞株强,且冷冻干燥法制备的硬脂醇半乳糖苷修饰阿西替尼脂质体对人肝癌细胞株SMMC-7721增殖的抑制强于阿西替尼普通脂质体及阿西替尼原料药,且具有时间及浓度的依赖性。Annexin V/PI流式细胞分析法检测硬脂醇半乳糖苷修饰阿西替尼脂质体对人肝癌细胞株SMMC-7721的抑制率,硬脂醇半乳糖苷修饰阿西替尼脂质体对人肝癌细胞株SMMC-7721的抑制率优于阿西替尼普通脂质体及阿西替尼原料药,进一步证明了硬脂醇半乳糖苷修饰阿西替尼脂质体的肝靶向性。western blot检测随着硬脂醇半乳糖苷修饰阿西替尼脂质体的浓度的增加,Bax、P53的蛋白表达逐渐增高,Bcl-2的蛋白表达逐渐减少。结论本实验制备的硬脂醇半乳糖苷修饰阿西替尼脂质体包封率较高,在一定的浓度范围内,可以抑制人肝癌细胞株SMMC-7721的增殖并诱导其凋亡。初步证明了具有肝靶向性,其靶向性优于阿西替尼普通脂质体。
[Abstract]:Aim to prepare azetinib liposomes with acitinib as model drug and stearic galactoside as target molecule. In order to further increase liposome liver targeting, reduce drug side effects. The stability of liposomes can be increased by freeze-drying. Promote the drug to achieve the best therapeutic effect. Methods the encapsulation efficiency of stearin modified azetinib liposomes was determined by Dextran gel microcolumn centrifugation and HPLC. The preparation process was optimized by Box-Behnken response surface design. The growth inhibition of human hepatoma cell line SMMC-7721 and human lung cancer cell line A549 by stearin galactoside modified acitinib liposome was determined by CCK-8 assay. Apoptosis was detected by Annexin V/PI flow cytometry. Western blot was used to detect the expression of Bcl-2 and p53. Results the average entrapment efficiency of stearol galactoside modified acitinib liposomes was 69.34 and the average diameter was (145 卤4.6) nm. The Box-Behnken response surface design method was used to optimize the preparation process. The ratio of cholesterol to phospholipid was 1: 7.67, the ratio of acitinib to phospholipid was 1: 21.50, the hydration temperature was 52.75 掳C. the ratio of phospholipid to stearic galactoside was determined by 1:16.69.CCK-8 method. The inhibition of human hepatoma SMMC-7721 proliferation was stronger than that of human lung cancer A549 cell line. The antiproliferative effect of stearol galactoside modified acitinib liposomes on the proliferation of human hepatoma cell line SMMC-7721 was stronger than that of azetinib liposomes and azetinib crude drugs. Annexin V/PI flow cytometry was used to detect the inhibition rate of acitinib liposomes modified with stearin on human hepatoma cell line SMMC-7721. The inhibition rate of stearin modified acitinib liposomes on human hepatoma cell line SMMC-7721 was better than that of azetinib liposome and azetinib crude drug. It was further proved that the liver targeting ability of stearic galactoside modified acitinib liposomes was determined by western blot. With the increase of the concentration of stearin modified acitinib liposomes, the protein expression of Baxtinib p53 increased gradually and the protein expression of Bcl-2 decreased gradually. Conclusion the stearic acid galactoside modified acitinib liposome has a high encapsulation efficiency and can inhibit the proliferation and induce apoptosis of human hepatoma cell line SMMC-7721 in a certain concentration range. It was preliminarily proved that liposome has liver targeting, and its targeting is superior to that of azetinib liposomes.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
本文编号：2179666
[Abstract]:Aim to prepare azetinib liposomes with acitinib as model drug and stearic galactoside as target molecule. In order to further increase liposome liver targeting, reduce drug side effects. The stability of liposomes can be increased by freeze-drying. Promote the drug to achieve the best therapeutic effect. Methods the encapsulation efficiency of stearin modified azetinib liposomes was determined by Dextran gel microcolumn centrifugation and HPLC. The preparation process was optimized by Box-Behnken response surface design. The growth inhibition of human hepatoma cell line SMMC-7721 and human lung cancer cell line A549 by stearin galactoside modified acitinib liposome was determined by CCK-8 assay. Apoptosis was detected by Annexin V/PI flow cytometry. Western blot was used to detect the expression of Bcl-2 and p53. Results the average entrapment efficiency of stearol galactoside modified acitinib liposomes was 69.34 and the average diameter was (145 卤4.6) nm. The Box-Behnken response surface design method was used to optimize the preparation process. The ratio of cholesterol to phospholipid was 1: 7.67, the ratio of acitinib to phospholipid was 1: 21.50, the hydration temperature was 52.75 掳C. the ratio of phospholipid to stearic galactoside was determined by 1:16.69.CCK-8 method. The inhibition of human hepatoma SMMC-7721 proliferation was stronger than that of human lung cancer A549 cell line. The antiproliferative effect of stearol galactoside modified acitinib liposomes on the proliferation of human hepatoma cell line SMMC-7721 was stronger than that of azetinib liposomes and azetinib crude drugs. Annexin V/PI flow cytometry was used to detect the inhibition rate of acitinib liposomes modified with stearin on human hepatoma cell line SMMC-7721. The inhibition rate of stearin modified acitinib liposomes on human hepatoma cell line SMMC-7721 was better than that of azetinib liposome and azetinib crude drug. It was further proved that the liver targeting ability of stearic galactoside modified acitinib liposomes was determined by western blot. With the increase of the concentration of stearin modified acitinib liposomes, the protein expression of Baxtinib p53 increased gradually and the protein expression of Bcl-2 decreased gradually. Conclusion the stearic acid galactoside modified acitinib liposome has a high encapsulation efficiency and can inhibit the proliferation and induce apoptosis of human hepatoma cell line SMMC-7721 in a certain concentration range. It was preliminarily proved that liposome has liver targeting, and its targeting is superior to that of azetinib liposomes.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
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