【摘要】：目的制備長循環(huán)脂質體(SSL)遞藥系統(tǒng)并檢測其表征,在動物水平評價其體內分布及肝靶向效果;采用SSL包封鱉甲肽HGRFG(HGRFG-SSL)并檢測其表征,進行最佳藥脂比篩選及釋放度考察;評價SSL對含5-羧基熒光素(5-FAM)基團的鱉甲肽(HGRFG-5-FAM,HF)藥代動力學的影響;考察HGRFG及HGRFG-SSL對CCl4誘導的小鼠急性肝臟損傷的影響。方法1.采用薄膜分散法制備DiR-SSL,檢測其粒徑及多分散系數(PDI),并觀察電鏡下形態(tài);2.將DiR-SSL尾靜脈注射于正常BALB/c裸鼠,分別于1,2,5,7,24h活體成像并讀取其熒光值;3.選取正常SD大鼠尾靜脈注射DiR-SSL,24h解剖后取心、肝、脾、肺、腎于熒光成像系統(tǒng)成像并讀取其熒光值,將肝組織冷凍切片采用激光共聚焦顯微鏡觀察;4采用HPLC法建立鱉甲肽含量測定方法;5.薄膜分散法制備HGRFG-SSL并檢測其表征,并用超濾離心法進行最佳藥脂比篩選;6.采用透析法考察不同時間點HGRFG-SSL的體外釋放度;7.采用薄膜分散法制備(HF-SSL,HFL),考察HF及HFL分別處理的大鼠不同時間點的血漿HF濃度,計算相關藥代動力學參數;8.利用HGRFG、SSL、HGRFG-SSL分別預處理正常小鼠一周,以聯苯雙酯為對照,考察不同藥物對CC14單次給藥誘導小鼠急性肝臟損傷的影響。結果1.DiR-SSL粒徑在100 nm左右,PDI為0.099,電鏡顯示呈圓球形,分布均勻;2.DiR-SSL在BALB/c裸鼠中,隨著時間增加逐漸被代謝,但活體成像時熒光主要集中于肝臟區(qū)域;3.DiR-SSL在SD大鼠內臟中肝臟熒光總量最強,肝臟冷凍切片中DiR-SSL廣泛分布;4.鱉甲肽出峰時間在6.389min且峰形良好,標準曲線的線性良好;5.鱉甲肽-SSL粒徑在100nm左右,PDI為0.083,形態(tài)良好,超濾離心法結果顯示最佳藥脂比為1:5,包封率為38.80%;6.鱉甲肽在4h時釋放度達到最高,其后釋放度基本趨于平穩(wěn),釋放藥物接近未包封藥物總量;7.HFL相較于HF顯著增加了在SD大鼠體內的T1/2,降低了血漿清除率;8.CCl4組相較于Control組顯著提高了小鼠血清AST及ALT水平,聯苯雙酯組顯著降低ALT水平,HGRFG、SSL及HGRFG-SSL組在AST及ALT水平與CCl4組相比無顯著變化,所有藥物處理組血清TNF-α及IL-6水平均有一定程度的升高。肝組織病理分析顯示,聯苯雙酯組肝損傷改善相較其他組最為明顯。結論 本研究成功制備了 SSL遞藥系統(tǒng),SD大鼠及BALB/c裸鼠實驗均證實了 SSL遞藥系統(tǒng)顯著的肝靶向作用;鱉甲肽-SSL的表征滿意,在藥脂比1:5時具有最高包封率,釋放度考察提示脂質體內水相鱉甲肽可能在pH7.4的PBS中不會出現稀釋泄漏。藥代動力學實驗結果表明,SSL提高鱉甲肽的循環(huán)半衰期,提高藥物的有效作用時間,而HGRFG及HGRFG-SSL未發(fā)現其具有改善CCl4所致的急性小鼠肝損傷的顯著作用。
[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 偽-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- 偽 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.
【學位授予單位】：浙江中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943

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