鳖甲肽-SSL肝靶向递药系统的制备及初步评价
发布时间：2019-04-13 09:10
【摘要】：目的制备长循环脂质体(SSL)递药系统并检测其表征,在动物水平评价其体内分布及肝靶向效果;采用SSL包封鳖甲肽HGRFG(HGRFG-SSL)并检测其表征,进行最佳药脂比筛选及释放度考察;评价SSL对含5-羧基荧光素(5-FAM)基团的鳖甲肽(HGRFG-5-FAM,HF)药代动力学的影响;考察HGRFG及HGRFG-SSL对CCl4诱导的小鼠急性肝脏损伤的影响。方法1.采用薄膜分散法制备DiR-SSL,检测其粒径及多分散系数(PDI),并观察电镜下形态;2.将DiR-SSL尾静脉注射于正常BALB/c裸鼠,分别于1,2,5,7,24h活体成像并读取其荧光值;3.选取正常SD大鼠尾静脉注射DiR-SSL,24h解剖后取心、肝、脾、肺、肾于荧光成像系统成像并读取其荧光值,将肝组织冷冻切片采用激光共聚焦显微镜观察;4采用HPLC法建立鳖甲肽含量测定方法;5.薄膜分散法制备HGRFG-SSL并检测其表征,并用超滤离心法进行最佳药脂比筛选;6.采用透析法考察不同时间点HGRFG-SSL的体外释放度;7.采用薄膜分散法制备(HF-SSL,HFL),考察HF及HFL分别处理的大鼠不同时间点的血浆HF浓度,计算相关药代动力学参数;8.利用HGRFG、SSL、HGRFG-SSL分别预处理正常小鼠一周,以联苯双酯为对照,考察不同药物对CC14单次给药诱导小鼠急性肝脏损伤的影响。结果1.DiR-SSL粒径在100 nm左右,PDI为0.099,电镜显示呈圆球形,分布均匀;2.DiR-SSL在BALB/c裸鼠中,随着时间增加逐渐被代谢,但活体成像时荧光主要集中于肝脏区域;3.DiR-SSL在SD大鼠内脏中肝脏荧光总量最强,肝脏冷冻切片中DiR-SSL广泛分布;4.鳖甲肽出峰时间在6.389min且峰形良好,标准曲线的线性良好;5.鳖甲肽-SSL粒径在100nm左右,PDI为0.083,形态良好,超滤离心法结果显示最佳药脂比为1:5,包封率为38.80%;6.鳖甲肽在4h时释放度达到最高,其后释放度基本趋于平稳,释放药物接近未包封药物总量;7.HFL相较于HF显著增加了在SD大鼠体内的T1/2,降低了血浆清除率;8.CCl4组相较于Control组显著提高了小鼠血清AST及ALT水平,联苯双酯组显著降低ALT水平,HGRFG、SSL及HGRFG-SSL组在AST及ALT水平与CCl4组相比无显著变化,所有药物处理组血清TNF-α及IL-6水平均有一定程度的升高。肝组织病理分析显示,联苯双酯组肝损伤改善相较其他组最为明显。结论 本研究成功制备了 SSL递药系统,SD大鼠及BALB/c裸鼠实验均证实了 SSL递药系统显著的肝靶向作用;鳖甲肽-SSL的表征满意,在药脂比1:5时具有最高包封率,释放度考察提示脂质体内水相鳖甲肽可能在pH7.4的PBS中不会出现稀释泄漏。药代动力学实验结果表明,SSL提高鳖甲肽的循环半衰期,提高药物的有效作用时间,而HGRFG及HGRFG-SSL未发现其具有改善CCl4所致的急性小鼠肝损伤的显著作用。
[Abstract]:Objective to prepare and characterize the long circulating liposome (SSL) delivery system and evaluate its distribution in vivo and liver targeting effect at animal level. HGRFG (HGRFG-SSL) was encapsulated in soft-shelled turtle (Trionyx sinensis) by SSL and characterized. The optimal drug-lipid ratio was screened and the release rate was investigated. To evaluate the effects of SSL on the pharmacokinetics of 5-carboxylfluorescein (5-FAM)-containing turtle-methyl peptide (HGRFG-5-FAM,HF) and the effects of HGRFG and HGRFG-SSL on the acute liver injury induced by CCl4 in mice. Method 1. DiR-SSL, was prepared by thin film dispersion method to detect the particle size and polydispersity coefficient (PDI),) and observed the morphology under electron microscope. DiR-SSL was injected intravenously into normal BALB/c nude mice at 1, 2, 5, 7 and 24 h, respectively, and their fluorescence values were read in vivo. After DiR-SSL,24h was injected into the tail vein of normal SD rats, the heart, liver, spleen, lung and kidney were imaged by fluorescence imaging system and their fluorescence values were read. The frozen sections of liver tissue were observed by laser confocal microscope. (4) to establish a method for the determination of alpha peptide in soft-shelled turtle by HPLC method; 5. HGRFG-SSL was prepared by thin film dispersion method and characterized by ultrafiltration centrifugation. 6. The in vitro release of HGRFG-SSL at different time points was investigated by dialysis. The plasma HF concentration of rats treated with HF and HFL at different time points was investigated by thin film dispersion method (HF-SSL,HFL), and the pharmacokinetic parameters were calculated. Normal mice were pretreated with HGRFG,SSL,HGRFG-SSL for one week. The effects of different drugs on acute liver injury induced by CC14 in mice were studied by using biphenyl diester as control. Results the particle size of 1.DiR-SSL was about 100 nm and the PDI was 0.099. Electron microscope showed that the particle size was spherical and the distribution was uniform. 2.DiR-SSL was metabolized gradually with the increase of time in BALB/c nude mice, but the fluorescence was mainly concentrated in the liver region during in vivo imaging. The total fluorescence of 3.DiR-SSL was the strongest in the viscera of SD rats, and DiR-SSL was widely distributed in the frozen sections of the liver. The peak time of turtle A peptide was in 6.389min and the peak shape was good, and the linearity of standard curve was good. The particle size of 伪-SSL was about 100nm, PDI was 0.083, and the shape was good. The results of ultrafiltration centrifugation showed that the best drug-fat ratio was 1? 5 and the encapsulation efficiency was 38.80%? After 4 h, the release rate of turtle-A peptide reached the highest level, and then the release rate tended to be stable, and the drug release was close to the total amount of unencapsulated drugs. Compared with HF, 7.HFL significantly increased the T _ (1) _ (2) in SD rats and decreased the plasma clearance rate (P < 0.05). Compared with the Control group, the serum AST and ALT levels in the 8.CCl4 group significantly increased, and the ALT level significantly decreased in the biphenyl diester group, while the AST and ALT levels in the HGRFG,SSL and HGRFG-SSL groups did not change significantly compared with that in the CCl4 group. The levels of serum TNF- 伪 and IL-6 in all drug-treated groups were increased to a certain extent. Pathological analysis of liver tissue showed that the improvement of liver injury in biphenylene diester group was more obvious than that in other groups. Conclusion SSL drug delivery system has been successfully prepared in this study. Both SD rats and BALB/c nude mice have confirmed the significant liver targeting effect of SSL drug delivery system. The characterization of turtle alpha-SSL was satisfactory, and the highest entrapment efficiency was obtained at the ratio of drug to lipid 1: 5. The release degree indicated that there might be no dilution leakage in the PBS of pH7.4 in the aqueous phase of the lipid. The results of pharmacokinetics showed that SSL increased the half-life of turtle-A peptide circulation and increased the effective time of the drug. However, HGRFG and HGRFG-SSL did not find that it could improve the acute liver injury induced by CCl4 in mice.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943
[Abstract]:Objective to prepare and characterize the long circulating liposome (SSL) delivery system and evaluate its distribution in vivo and liver targeting effect at animal level. HGRFG (HGRFG-SSL) was encapsulated in soft-shelled turtle (Trionyx sinensis) by SSL and characterized. The optimal drug-lipid ratio was screened and the release rate was investigated. To evaluate the effects of SSL on the pharmacokinetics of 5-carboxylfluorescein (5-FAM)-containing turtle-methyl peptide (HGRFG-5-FAM,HF) and the effects of HGRFG and HGRFG-SSL on the acute liver injury induced by CCl4 in mice. Method 1. DiR-SSL, was prepared by thin film dispersion method to detect the particle size and polydispersity coefficient (PDI),) and observed the morphology under electron microscope. DiR-SSL was injected intravenously into normal BALB/c nude mice at 1, 2, 5, 7 and 24 h, respectively, and their fluorescence values were read in vivo. After DiR-SSL,24h was injected into the tail vein of normal SD rats, the heart, liver, spleen, lung and kidney were imaged by fluorescence imaging system and their fluorescence values were read. The frozen sections of liver tissue were observed by laser confocal microscope. (4) to establish a method for the determination of alpha peptide in soft-shelled turtle by HPLC method; 5. HGRFG-SSL was prepared by thin film dispersion method and characterized by ultrafiltration centrifugation. 6. The in vitro release of HGRFG-SSL at different time points was investigated by dialysis. The plasma HF concentration of rats treated with HF and HFL at different time points was investigated by thin film dispersion method (HF-SSL,HFL), and the pharmacokinetic parameters were calculated. Normal mice were pretreated with HGRFG,SSL,HGRFG-SSL for one week. The effects of different drugs on acute liver injury induced by CC14 in mice were studied by using biphenyl diester as control. Results the particle size of 1.DiR-SSL was about 100 nm and the PDI was 0.099. Electron microscope showed that the particle size was spherical and the distribution was uniform. 2.DiR-SSL was metabolized gradually with the increase of time in BALB/c nude mice, but the fluorescence was mainly concentrated in the liver region during in vivo imaging. The total fluorescence of 3.DiR-SSL was the strongest in the viscera of SD rats, and DiR-SSL was widely distributed in the frozen sections of the liver. The peak time of turtle A peptide was in 6.389min and the peak shape was good, and the linearity of standard curve was good. The particle size of 伪-SSL was about 100nm, PDI was 0.083, and the shape was good. The results of ultrafiltration centrifugation showed that the best drug-fat ratio was 1? 5 and the encapsulation efficiency was 38.80%? After 4 h, the release rate of turtle-A peptide reached the highest level, and then the release rate tended to be stable, and the drug release was close to the total amount of unencapsulated drugs. Compared with HF, 7.HFL significantly increased the T _ (1) _ (2) in SD rats and decreased the plasma clearance rate (P < 0.05). Compared with the Control group, the serum AST and ALT levels in the 8.CCl4 group significantly increased, and the ALT level significantly decreased in the biphenyl diester group, while the AST and ALT levels in the HGRFG,SSL and HGRFG-SSL groups did not change significantly compared with that in the CCl4 group. The levels of serum TNF- 伪 and IL-6 in all drug-treated groups were increased to a certain extent. Pathological analysis of liver tissue showed that the improvement of liver injury in biphenylene diester group was more obvious than that in other groups. Conclusion SSL drug delivery system has been successfully prepared in this study. Both SD rats and BALB/c nude mice have confirmed the significant liver targeting effect of SSL drug delivery system. The characterization of turtle alpha-SSL was satisfactory, and the highest entrapment efficiency was obtained at the ratio of drug to lipid 1: 5. The release degree indicated that there might be no dilution leakage in the PBS of pH7.4 in the aqueous phase of the lipid. The results of pharmacokinetics showed that SSL increased the half-life of turtle-A peptide circulation and increased the effective time of the drug. However, HGRFG and HGRFG-SSL did not find that it could improve the acute liver injury induced by CCl4 in mice.
【学位授予单位】：浙江中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943
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