卡培他滨—奥沙利铂复方脂质体的制备及体外细胞毒性研究

发布时间：2018-04-05 19:17

本文选题：卡培他滨　切入点：奥沙利铂　出处：《锦州医科大学》2016年硕士论文

【摘要】：目的建立卡培他滨-奥沙利铂复方脂质体(Cap-Ox复方脂质体)包封率的测定方法,对卡培他滨、奥沙利铂进行处方前实验研究,并建立方法学验证,确定卡培他滨、奥沙利铂的含量测定方法。研究Cap-Ox复方脂质体的制备方法,考察其制剂学性质,通过单因素实验及正交实验进行处方优化,并通过体外细胞毒性实验,来确定最优处方以达到更好的疗效,并初步研究其抗癌效果。方法采用葡聚糖凝胶微柱-HPLC来测定卡培他滨-奥沙利铂复方脂质体的包封率。以包封率作为指标,筛选卡培他滨-奥沙利铂复方脂质体的最优制备方法,选择单因素考察与正交试验,设计优化复方脂质体的制备处方。为了考察该复方脂质体的形态、粒径以及包封率,利用透射电子显微镜(TEM)、激光粒度分析仪测定。通过MTT细胞毒性试验及western blot,研究对象是人肝癌细胞,考察卡培他滨-奥沙利铂复方脂质体对肝癌细胞的抑制率及影响细胞凋亡的蛋白的表达,对该复方脂质体的药效进行初步研究。结果分别建立了卡培他滨、奥沙利铂含量测定的分析方法,葡聚糖凝胶微柱-HPLC可用于测定卡培他滨-奥沙利铂复方脂质体的包封率。处方优化结果表明:采用薄膜分散法,在药脂比1:15、卡培他滨与奥沙利铂质量比为10:1、磷脂与胆固醇之比为3:1和磷脂浓度为8 g/L的条件下可制得平均粒径为199.1 nm、卡培他滨和奥沙利铂平均包封率为59.8%和52.6%的复方脂质体。体外细胞毒性试验结果表明,卡培他滨与奥沙利铂质量比不同的脂质体中,比例为15:1的复方脂质体抑制肝癌细胞SMMC-7721细胞生长作用最强,并且抑制细胞生长。通过影响细胞凋亡的蛋白表达情况,验证Cap-Ox复方脂质体较其他形式具更好的细胞凋亡作用。结论本实验将可产生协同作用的卡培他滨、奥沙利铂共同包载于脂质体中,制备卡培他滨-奥沙利铂复方脂质体,为使HPLC能够同时测定出两种药物,采用改变流动相与固定相之比、流动相的流速以及C18液相色谱柱的温度等方法,从而达到分离药物的峰型较好以及分离效果明显,通过正交实验进行优化后的处方,制得的卡培他滨-奥沙利铂复方脂质体有较高包封率,均匀的粒径分布,形态均匀。通过体外细胞毒性实验结果证明Cap-Ox复方脂质体能够达到诱导肿瘤细胞凋亡的作用,并且抗癌效果优于其他形式。
[Abstract]:Objective to establish a method for the determination of capecitabine-oxaliplatin compound liposome entrapment efficiency, to study the preprescription of capecitabine and oxaliplatin, and to establish a methodology for the determination of capecitabine.Method for determination of oxaliplatin.The preparation method of Cap-Ox compound liposome was studied and its properties were investigated. The prescription was optimized by single factor experiment and orthogonal experiment, and the optimal prescription was determined by in vitro cytotoxicity test to achieve better curative effect.And its anti-cancer effect was preliminarily studied.Methods the encapsulation efficiency of capecitabine-oxaliplatin compound liposomes was determined by dextran gel microcolumn-HPLC.The optimal preparation method of capecitabine-oxaliplatin compound liposomes was screened by using the encapsulation efficiency as the index. The single factor investigation and orthogonal test were selected to design and optimize the preparation prescription of compound liposomes.In order to investigate the morphology, particle size and encapsulation efficiency of the compound liposome, the transmission electron microscope (TEM) and laser particle size analyzer (LPA) were used.The inhibition rate of capecitabine-oxaliplatin compound liposome on hepatoma cells and the expression of apoptotic proteins were investigated by MTT cytotoxicity test and western blot.The pharmacodynamics of the compound liposome was studied.Results A method for the determination of capecitabine and oxaliplatin was established, and the encapsulation efficiency of capecitabine-oxaliplatin liposomes was determined by dextran gel microcolumn HPLC.The results of prescription optimization showed that the film dispersion method was used.When the ratio of lipids to lipids is 1: 15, the mass ratio of capecitabine to oxaliplatin is 10: 1, the ratio of phospholipid to cholesterol is 3:1 and the concentration of phospholipid is 8 g / L, the average particle size is 199.1 nm, the average entrapment efficiency of capecitabine and oxaliplatin is 59.8% and 52.6%.The results of cytotoxicity test in vitro showed that compound liposome with 15:1 ratio of capecitabine and oxaliplatin had the strongest inhibitory effect on the growth of SMMC-7721 cells and inhibited cell growth.The effect of Cap-Ox compound liposomes on apoptosis was proved to be better than other forms by influencing the expression of apoptotic proteins.Conclusion in this study, the synergistic capecitabine and oxaliplatin were co-encapsulated in liposomes to prepare compound liposomes of capecitabine-oxaliplatin, in order to determine two drugs simultaneously by HPLC.By changing the ratio of the mobile phase to the stationary phase, the flow rate of the mobile phase and the temperature of the C18 liquid chromatographic column, the peak type of the drug was better and the separation effect was obvious. The optimized prescription was obtained by orthogonal experiment.The capecitabine-oxaliplatin compound liposomes have high entrapment efficiency, uniform particle size distribution and uniform morphology.The results of cytotoxicity test in vitro showed that Cap-Ox compound liposomes could induce apoptosis of tumor cells, and the anticancer effect was superior to other forms.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943;R96

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