Study On Glycyrrhetinic Acid Liposomes Containing Ligand Of Mannose-diester Lauric Diacid-cholesterol Conjugate For Liver-targeting Delivery

Objective:Glycyrrhetinic acid（GA）,serving as an active metabolite of glycyrrhizic acid,has been found the effects of hepatoprotection,anti-hepatitis and anti-hepatocellular carcinoma.However,GA is a lipophilic drug with a very low solubility in water,which may result in poor bioavailability.In addition,GA may cause unwanted sodium retention and potassium loss.To resolve the problems existed and improve the therapeutic effect of GA,the researchers are concentrating on the drug delivery based on nanoparticles which could decrease side effects and maintain an essential concentration of the drug.Compared with other drug delivery based on nanoparticles,liposomes have many advantages,such as the high cell affinity,tissue compatibility and without immunosuppressive effects.However,liposomes as a kind of passive target of drug carrier,are vulnerable to be uptook by reticuloendothelial system.If we are consider on the special modification of liposomes,for example,modifying liposomes with liver-targeting ligand could improve GA concentration in hepatocellular cell.The method would achieve the effect of active target.Some literatures reported that mannose receptors are highly expressed on the liver endothelial cells and dendritic cells.The carbohydrate-recognition domains of mannose receptor have the ability to recognize compounds containing terminal residues of mannose,N-acetyl-glucosamine or fucose.Thus,based on the characteristics of mannose receptor and liposomes,we design a liver-target ligand which could be recognized by mannose receptor to modify liposomes.According to the theory of hepatic targeting drug delivery system,there are three purposes of this study:Firstly,the conjugate named mannose-diester lauric diacid-cholesterol（Man-DLD-Chol）is synthesized by lipase-catalysis in organic phase.The technology of enzyme catalysis is used to seek for a kind of synthetic method with high efficiency,low energy and high regioselectivity.The purpose of Man-DLD-Chol containing a hydrophilic part and hydrophobic part is to obtain a target ligand which highly combined with liposomes.Secondly,GA is embedded into the lipid bilayer and Man-DLD-Chol is anchored on the surface by liposomes as drug carrier.The purpose of a drug carrier of liposomes and a target ligand of Man-DLD-Chol is to improve GA bioavailability and liver-targeting.Thirdly,the active target GA liposomes named Man-DLD-Chol-GA-Lp are prepared.The purpose of this is to achieve liver-targeting delivery of GA and decrease side effects in normal tissues and cells by mannose receptor mediated endocytosis,thus enhancing GA effects on the treatment of hepatitis and hepatocellular carcinoma.This study would provide ideas for the development of traditional Chinese medicine of GA and the clinical application of hepatic diseases.Methods:1.The liver-targeting ligand of Man-DLD-Chol was synthesized by lipase catalysis.Its chemical structure was confirmed by mass spectrometry and nuclear magnetic resonance.The relatively simple and high-effective method for separation and purification and the methodology for content measure of Man-DLD-Chol were established.The reactional conditions affecting synthesis were studied and the parameters of the synthesis were optimized by the method of Central Composite Design.2.The preparation of Man-DLD-Chol was carried out.The HPLC method for determination of GA concentration in liposomes was established.The encapsulation rate was a criterion of judgment in comparison of three kinds of liposomes preparation methods.The single-factor experiments in prescription process,preparation process and lyophilization process affecting entrapment efficiency and particle size of liposomes were investigated to obtain the optimal parameters for preparation of liposomes.3.The quality control of Man-DLD-Chol-GA-Lp was studied.The physicochemical properties of Man-DLD-Chol-GA-Lp were evaluated in detail.The morphology of liposomes were observed by scanning electronic microscope.The particle size,polydispersity index and Zeta potential of liposomes were determined by nanometer granulometry.Sephadex G-50was used to separate uncombined GA and liposomes and HPLC method was used to measure the entrapment efficiency,drug loading capacity and the rate of drug leakage.The oxidation product value of liposomes was determined by the method of thiobarbital acid reaction.The dialysis method was used to detect the drug release of GA preparations in vitro.The stability study of liposomes were investigated,including influencing factor experiment,accelerated experiment and long-term experiment.4.The preliminary safety of Man-DLD-Chol-GA-Lp was evaluated.HepG2 cells were chose as model cells to investigate the toxicity of Man-DLD-Chol liposomes（Man-DLD-Chol-Lp）and blank liposomes（B-Lp）.According to the method described in Chinese Pharmacopoeia,the pyrogen and hemolysis tests of GA formulations（GA-S,GA-Lp and Man-DLD-Chol-GA-Lp）were carried out in rabbits.The LD₅₀ value of different GA formulations was calculated by administration of a single dose in mice to evaluate the acute toxicity of GA-Lp and Man-DLD-Chol-GA-Lp in vivo.5.The targeting of Man-DLD-Chol-GA-Lp in vitro was studied.Coumarin-6（Cou6）was used as fluorescent indicator.The Cou6 liposomes（Cou6-Lp）and Cou6-Lp modified with Man-DLD-Chol（Man-DLD-Chol-Cou6-Lp）were prepared by the film-dispersion method.We evaluated the characterizations of liposomes.HepG2 cells were as model cells,and we investigated the uptake efficiency of different GA formulations on HepG2 cells by qualitative and quantitative analysis.The MTT method was used to determine the proliferation inhibition effect of different GA formulation on HepG2 cells.The apoptosis effect of different GA formulation on HepG2 cells was evaluated using the fluorochrome of Annexin V-FITC/PI by flow cytometry.6.The methodology of GA in plasma and tissues was carried out by high-performance liquid chromatography-tandem mass spectrometry（LC-MS/MS）.A LC-MS/MS assay was developed for the determination of GA in plasma and tissue samples,and the experiments including specificity,linearity,precision,recovery and stability were investigated.7.The Pharmacokinetics and tissue distribution of Man-DLD-Chol-GA-Lp were studied.The experimental rabbits and mice were divided into three groups,including Man-DLD-Chol-GA-Lp,GA-Lp and GA-S（5.25mg/mL）.Each rabbit was intravenously administered via the ear vein and each group of mice were administered by caudal vein.The GA concentrations in plasma and tissues（heart,liver,spleen,lung and kidney）were determined by LC-MS/MS.The pharmacokinetics characteristics of different GA formulations were evaluated in comparison the difference of pharmacokinetic parameters.To measure the difference of GA concentrations in tissues,the four target parameters including target efficiency（Te）,relative targeting efficiency(R_Te),relative uptake rate（Re）and peak concentration retio（Ce）were compared.Results:1.The chemical structure and properties of divinyl ester lauric diacid,vinyl ester lauric diacid-cholesterol and Man-DLD-Chol were confirmed by thin-layer chromatogram（TLC）,HPLC,mass spectrometry（MS）and nuclear magnetic resonance（NMR）.The methods of separation and purification were silica gel column,recrystallization and carbon-18 column,respectively.The optimal parameters of the synthesis were obtained by the method of Central Composite Design:the amount of vinyl ester lauric diacid-cholesterol was 0.536 mmol,the amount of mannose was 0.100 mmol,the weight 4?molecular sieve was 60 mg,the weight of candida antarctica lipase was 61.2 mg,the volume of dehydrated tetrahydrofuran was 2.5mL and pyridine was 1.5 mL,the reactional temperature was 56.6℃and the reactional time was 27 h.2.The preparation of Man-DLD-Chol-GA-Lp and GA-Lp were prepared by the method of thin film dispersion.The single-factor experiments in prescription process,preparation process and lyophilization process affecting entrapment efficiency and particle size of liposomes were investigated.The optimal parameters for preparation of liposomes were as follows:the proportion of cholesterol to egg phosphatidylcholine（EPC）was 1:4,the molar ratio of Man-DLD-Chol to EPC was 8%,the pH value of buffer was 7.4,the temperature was50℃,the ultrasonic power was 250 W and ultrasonic time was 20 min,the type of lyophilized protective agent was glucose-mannitol and its proportion was 1:1,the proportion of lyophilized protective agent:EPC was 10:1,the joining method of lyophilized protective agent was latter joining and the precool temperature was-60℃.3.The physicochemical properties of GA-Lp and Man-DLD-Chol-GA-Lp were determined in detail.The results were as follows:the morphology of liposomes were spheroids with regular shape by scanning electronic microscope observed,the particle sizes of GA-Lp and Man-DLD-Chol-GA-Lp were 108.60±0.28 nm and 120.80±0.85 nm,the PDI values were 0.107±0.008 and 0.095±0.004,Zeta potentials were-26.15±0.64 mV and-33.15±1.34 mV,the entrapment efficiency was respectively 88.38±1.35%and 85.90±0.73%,drug loading capacities were 6.56±0.10%and 6.38±0.05%,the rate of drug leakage was less than 0.10%within 10 days,the oxidation product values of liposomes were 0.106±0.011 and0.069±0.004,the rate of lysophospholipids was 1.27%and 1.16%,respectively.The drug release of Man-DLD-Chol-GA-Lp in vitro was similar to Higuchi release model,indicated that Man-DLD-Chol-GA-Lp were release at a non-constant rate.The study of stability of Man-DLD-Chol-GA-Lp was investigated.The result of influencing factor experiment showed that the storage of Man-DLD-Chol-GA-Lp should be sealed,cool and dark.The result of accelerated experiment found that the particle size and the rate of drug leakage in Man-DLD-Chol-GA-Lp were increased obviously after 6 months.The results of long-term experiment showed that liposomes were abnormal after 12 months.4.The result of cytotoxicity showed that the toxicity of Man-DLD-Chol-Lp was low within the concentration range of 0.5-50μg/mL,which cell viability was more than 90%.Besides,GA-Lp and Man-DLD-Chol-GA-Lp did not cause pyrogen phenomenon.The result of hemolysis test showed that GA-Lp and Man-DLD-Chol-GA-Lp did not cause hemolysis,while GA-S could cause hemolysis.The result of the test by administration of a single dose in mice showed that the LD₅₀ values of GA-Lp and Man-DLD-Chol-GA-Lp were 1.63-fold（90.165 mg/kg）and 1.56-fold（87.717 mg/kg）compared with that of GA-S.5.The preparation and characteristics of Cou6-Lp and Man-DLD-Chol-Cou6-Lp were desirable:the particle size was within 120-180 nm,Zeta potential was stable and the entrapment efficiency was more than 85%.The result of cellular uptake showed that the fluorescence intensity of Man-DLD-Chol-Cou6-Lp was most strong,which the value was1.60-fold in 1 h and 1.45-fold in 4 h compared with that of Cou6-Lp.The result of the proliferation inhibition on HepG2 cells showed that the IC₅₀ values of GA-Lp and Man-DLD--Chol-GA-Lp were 1.29¹.50-fold and 1.82¹.92-fold compared with that of GA-S.The result of apoptosis effect indicated that the apoptosis rate of GA-S on HepG2 cells was12.98%,and GA-Lp could apoptosis about 19.78%,while the apoptosis rate of Man-DLD-Chol-GA-Lp would reach 27.37%.6.The result of methodology showed that there were no significant endogenous interference for determination of GA and ursolic acid in plasma and tissue homogenates,the specificity was desirable.The linear coefficients（r²）of plasma samples and tissue samples were more than 0.9989 within the range of 5-5000 ng/mL.The maximum RSD of intra-day precision and inter-day precision were 10.51%and 11.19%respectively,both were less than15%.The rate of extraction recoveries ranged from 85%to 110%,which RSD was less than15%.The RE between the real concentration and measure concentration was less than 15%when GA were stored at room temperature,frozen-thaw cycle,low temperature（4℃）and frozen（-20℃）condition.7.The result of mean plasma concentration-time was obtained.Compared with GA-S,the elimination half-time(t_1/2)of GA-Lp and Man-DLD-Chol-GA-Lp decreased,whose values were 2.51±0.44 h and 1.78±0.08 h,respectively.Meanwhile,the mean clearance（CL）of Man-DLD-Chol-GA-Lp（5.81±0.30 L/（h·kg））was higher than that of GA-Lp（5.00±0.30L/（h·kg））and GA-S（3.36±0.11 L/（h·kg））.Moreover,the mean residence time(MRT_0-∞)of Man-DLD-Chol-GA-Lp was shortest（1.35±0.05 h）.In addition,the relative value of distribution volume（Vd）of GA-Lp（18.15±3.42 L/kg）and Man-DLD-Chol-GA-Lp（14.90±0.55 L/kg）were higher than that of GA-S（12.83±0.88 L/kg）.The area under the curve of drug concentration(AUC_0-∞)of Man-DLD-Chol-GA-Lp was about 1.16 times less than that of GA-Lp.The results of target parameters in tissue distributions were calculated.The Te of GA-S in the plasma and kidney were 30.27%and 29.14%,respectively,demonstrating the highest selection rate in the plasma and kidney.While Te of Man-DLD-Chol-GA-Lp reached 54.67%to reflect the highest selective rate in the liver.Man-DLD-Chol-GA-Lp possessed outstanding liver-targeting with an R_Te of 3.39 in comparison with GA-S.The Re of GA-Lp and Man-DLD-Chol-GA-Lp was more than 1 in liver,spleen and lung,moreover the Re and Ce of Man-DLD-Chol-GA-Lp in liver were maximum,which value was 4.78 and 3.46,respectively.Conclusion:In conclusion,a liposomal target ligand,Man-DLD-Chol was successfully synthesized by lipase-catalysis.Man-DLD-Chol was anchored on the surface by liposomes as drug carrier to prepare liposomes with liver targeting Man-DLD-Chol-GA-Lp.The characteristics and preliminary safety of Man-DLD-Chol-GA-Lp were desirable.We found that Man-DLD-Chol could improve HepG2 cells uptake of GA in the targeting test on HepG2 cells,demonstrating that Man-DLD-Chol had an effect of liver targeting.In addition,Man-DLD-Chol-GA-Lp could improve the proliferation inhibition effect of GA on HepG2 cells and enhance apoptosis due to the Man-DLD-Chol possessing liver targeting.In the pharmacokinetics and tissue distribution study,the data of target parameters in liver indicated that Man-DLD-Chol-GA-Lp had an excellent effect of liver-targeting.Based on the results supporting our hypothesis,we have reason to believe that Man-DLD-Chol-GA-Lp could be a potential hepatic target drug delivery to improve the therapeutic effect of hepatic diseases.