【摘要】：目的本實(shí)驗(yàn)采用阿西替尼為模型藥物,硬脂醇半乳糖苷為靶向分子,制備阿西替尼脂質(zhì)體,優(yōu)化制備工藝,考察藥效。以便進(jìn)一步增加脂質(zhì)體的肝靶向性,降低藥物的毒副作用。通過(guò)冷凍干燥法達(dá)到增加脂質(zhì)體本身穩(wěn)定性的目的。促使藥物達(dá)到最好的治療效果。方法采用葡聚糖凝膠微柱離心-HPLC測(cè)定硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體的包封率,采用Box-Behnken響應(yīng)面設(shè)計(jì)法優(yōu)化制備工藝。采用CCK-8法測(cè)定硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體對(duì)人肝癌細(xì)胞株SMMC-7721和人肺癌細(xì)胞株A549的生長(zhǎng)抑制情況,采用Annexin V/PI流式細(xì)胞分析法檢測(cè)細(xì)胞凋亡的情況,采用western blot檢測(cè)凋亡蛋白Bax、Bcl-2和P53的情況。結(jié)果冷凍干燥法制備的硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體平均包封率為69.34%,平均粒徑為(145±4.6)nm。采用了Box-Behnken響應(yīng)面設(shè)計(jì)法優(yōu)化制備工藝,所得的最優(yōu)處方為,膽固醇與磷脂的比例為1:7.67,阿西替尼原料藥與磷脂的比例為1:21.50,水合溫度為52.75°C,磷脂與硬脂醇半乳糖苷的比例為1:16.69。CCK-8法檢測(cè)相同濃度的硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體對(duì)人肝癌SMMC-7721增殖的抑制比人肺癌A549細(xì)胞株強(qiáng),且冷凍干燥法制備的硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體對(duì)人肝癌細(xì)胞株SMMC-7721增殖的抑制強(qiáng)于阿西替尼普通脂質(zhì)體及阿西替尼原料藥,且具有時(shí)間及濃度的依賴性。Annexin V/PI流式細(xì)胞分析法檢測(cè)硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體對(duì)人肝癌細(xì)胞株SMMC-7721的抑制率,硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體對(duì)人肝癌細(xì)胞株SMMC-7721的抑制率優(yōu)于阿西替尼普通脂質(zhì)體及阿西替尼原料藥,進(jìn)一步證明了硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體的肝靶向性。western blot檢測(cè)隨著硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體的濃度的增加,Bax、P53的蛋白表達(dá)逐漸增高,Bcl-2的蛋白表達(dá)逐漸減少。結(jié)論本實(shí)驗(yàn)制備的硬脂醇半乳糖苷修飾阿西替尼脂質(zhì)體包封率較高,在一定的濃度范圍內(nèi),可以抑制人肝癌細(xì)胞株SMMC-7721的增殖并誘導(dǎo)其凋亡。初步證明了具有肝靶向性,其靶向性優(yōu)于阿西替尼普通脂質(zhì)體。
[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 鹵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 擄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.
【學(xué)位授予單位】：錦州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R943;R96

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