| Objective1.There were a large number of Glycyrrhetinic acid receptor on the surface of liver parenchymal cells.Based on the principle,synthesis of two kinds of hepatocytes targeting ligand with Glycyrrhetinic acid be the nucleus-18-GA-Gly and 18-GA-Ala.Salvianolic acid B(Sal B)and Tanshinone IIA(TSN)were used as model drugs,Preparaing two kinds of hepatic parenchymal cells active targeting liposomes,study the pharmacokinetics and tissue distribution of drug targeting,calculation of Re,Ce,Te.evaluated the effect of two kinds of ligands.2.Preparation of liposome-X,from the physical and chemical point of view to select the type and concentration of new stability materials,in order to improve the stability of liposome-X.MethodThis experiment is divided into two parts as following.The first part is the preparation of two kinds of liver active targeting liposomes and targeted investigation.With glycyrrhetinic acid as the nucleus,connected with stearyl alcohol on the one side,and connected with succinic anhydride by acylation reaction on the other side and followed by 18-GA-Gly and 18-GA-Ala respectively,then we well get 18-GA-Gly and 18-GA-Ala.Sal B and TSN were used as model drugs,the two kinds of targeting liposomes Gly-TS-Lip and AlaTS-Lip were prepared by film dispersion method-high pressure homogenization.TS-Lip was prepared for comparison.The particle size,potential,EE%,drug loading were detected.Prepared freeze-drying liposomes and determined the Particle size,zeta potential,EE%and drug loading.According to the types of liposomes,the mice were divided into three groups,via tail vein injections to administration,obtained plasma samples and the heart,liver and spleen,lung and kidney tissue samples after administration of 0.083,0.167,0.33,0.5,0.75,1,1.5,2,4 and 6h,determination the Sal B and TSN in each sample with UPLC methods,using DAS 3.2 software processing data according to the non-compartment model.calculate the pharmacokinetic parameters,draw the organization distribution graph and calculate the relative uptake rate(Re),peak concentration ratio(Ce),targeting efficiency(Te),evaluated the effect of two kinds of ligands.The second part:A preliminary study on the stability materials of liposome-X.From the physical and chemical point of view to detected the effection of liposome-X of the initiate of new stability materials.Research method were centrifugal spectrophotometry method on the physical point,calculate the KE before and after adding materials,the changes of KE reflect the effect of stable materials.The materials included:nSiO2,xanthan,hydroxypropyl methyl cellulose,polyvinyl alcohol(PVA18-88),Polyacrylamide(APAM1000 and NPAM1400)and the combination of nSiO2 and Polyacrylamide.On the chemical point,TBA methods was used.Vitamin E was dectected as the antioxidant materials to calculate the MD A before and after water-bath.By means of the above experimental methods,the influence of the added of stability materials were detected and the type and concentration of new stability materials were preliminary determined,in order to improve the stability of liposome-X.ResultsPart I:two new types of hepatic parenchymal cell ligands 18-GA-Gly and 18-GA-Ala were preparaed.UPLC chromatography method was established for the determination of Sal B,TSN and two kinds of ligands.Two kinds of active liver targeting liposomes with ligands 18-GAGly and 18-GA-Ala modified were preparaed by thin film dispersion-high pressure homogenization method.It was found that the Sal B was added after high pressure homogenization and incubated with 30min.The size of Ala-TS-Lip,Gly-TS-Lip and TS-Lip were 169.1±8.13 nm,205.6±19.51 nm.178.6±7.34 nm,potential results were-30.60±1.31 mV,-24.7±0.15 mV,-27.7±0.95 mV.The encapsulation rate of Sal B was 95.33±0.04%,83.09±1.75%,82.45±1.64%.The encapsulation rate of TSN was71.33±0.07%,75.49±2.23%,79.65±10.49%.Two ligands was 18-GA-Ala 93.67±0.08%,18-GA-Gly 97.95±1.85%.Preparation of freeze-drying liposomes by 9%sucrose solution.The method for the determination of plasma and tissue in mice already established.It can determine the content of Sal B and TSN with high efficiency and accuracy.Because of instrument failure,Ala-TS-Lip plasma datas was missing.The plasma pharmacokinetic parameters of Gly-TS-Lip and TS-Lip groups were compared after injected into the tail vein.From the results,the AUCsal B and AUCTSN of Gly-TS-Lip group were 1.17 times and 1.28 times to the TS-Lip group respectively.T1/2Sal B and T1/2TSN of Gly-TS-Lip group were 0.87 times and 1.68 times to the TS-Lip group.Statistical results showed no significant difference between GlyTS-Lip and TS-Lip.Comparison of drug distribution in the organization,The AUCGly-TS-Lip,AUCAla-TS-Lip and AUCTS-Lip of Sal B and TSN in liver were 8.943±2.73,198.583±182.85,198.468±380.124μg·h·ml-1 and 2.312±0.215,11.54±1.698,0.457±0.198μg·h·ml-1 respectively.After two ligand modification,The peak concentration of Sal B and TSN in liver increased than before.Two ligand showed the hepatic targeting effect on TSN.Compared the distribution of liposomes in various tissues,after modification of the 18-GA-Ala,the AUC of Sal B in liver was 1.19 times,1.39 times,63.67 times than spleen,lung,and kidney,the AUC of TSN in liver was 4.64 times,0.36 times,14.12 times than spleen,lung,and kidney.The results showed that 18-GA-Ala ligand modification could increase the distribution of Sal B and TSN in liver tissue.According to the preliminary study,The TSN solution has aobvious lung targeting,it would affect the Te result of TSN in lung.The second part:the stability factor KE was used to assess stability.The screening results showed that the best concentration of hydrophilic nSiO2 was 0.3%(g/g).The best concentration of hydrophobic nSiO2 was 0.2%(g/g).The optimum concentration of xanthan gum was 0.05%(g·ml-1).The initial concentration of hydroxy propyl methyl cellulose was 0.02%(g·ml-1).The optimum concentration of polyvinyl alcohol was 0.03%(g·g-1).Anionic polyacrylamide was added at an optimum concentration of 400 mg·L-1.The optimum concentration of nonionic polyacrylamide was 300mg·L-1.It was found that the combination of polyacrylamide and nSiO2 did not show that the stability of the combination was better than single.The effect of nonionic polyacrylamide was the best,and the concentration was 300 mg·L-1.The effect of vitamin E on the antioxidant and stability of liposome-X was investigated by TBA assay.The optimal dosage of vitamin E was 1 mg·L-1.ConclusionThe above experiments showed that two kinds ligand of liver parenchyma cells to actively target with glycyrrhetinic acid as the mother nucleus had some influence on the distribution and metabolism of the drug in mice.Both ligands had certain liver targeting effect on Sal B and TSN,the two ligands can increase the Ce of Sal B and TSN in liver,and showed the liver targeting effect of TSN.Compared Re of two kind of ligands modified liposomes,18-GA-Ala showed a more significantly effect in liver targeting of Sal B and TSN.The effect of different stabilizers on the stability of liposome-X was different.The optimum concentration of each kind of excipients has been preliminarily selected.It had certain reference value for the subsequent research on liposome-X stabilization system. |
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