Preparation And In Vivo Study In Rats Of Asiaticoside-loaded Modified Liposomes

Objective: Asiaticoside is one of main active ingredients in Centella asiatica and it’s also an ursane-type triterpenoids.It is said that asiaticoside could ameliorate the process of pulmonary fibrosis to mitigate the inflammation of lung tissues by inhibiting the expression of IL-4,TNF-α,TNF-β1,reduceing Smad-3 and raising the level of A2 AR.However,its poor water solubility,cytotoxicity after injection and short half-life limit its clinical application.Liposoms are a kind of micro-ball formulation which have a bilayer structure and usually composed by phospholipids and cholesterol.There are many advantages when the drugs were loaded in liposomes,such as changing the distribution in vivo,enhancing efficacy,reducing toxicity and extending biologic half-life.However,liposomes have no significant targeting without modified.In order to obtain targeting liposomes,researchers usually modify liposomes according to the characteristics of different organ.D-mannose can be recognised by surfactant proteins A and D in pilmonary,and octadecylamine could change the surface charge of liposomes to obtain cationic liposomes which tend to gather in lung.In this study,the asiaticiside-loaded liposomes were modified by D-mannose and octadecylamine to increase the water-soluble of asiaticosside,extend circulation time in the body,and promote the drug into the lung.Methods: An HPLC method was established to determine the concentration of asiaticoside,while its stability,repeatability and precision were also investigated.Mini-column centrifugation method was established to determine the encapsulation efficiency of liposomes after optimizing the column height and centrifugation speed.Among Film dispersion method,ethanol injection method,reverse-phase evaporation method and calcium acetate gradient method,the best method to prepare asiaticoside-loaded liposomes（ASI-L）was selected by encapsulation efficiency.The modified asiaticoside-loaded liposomes（D-ASI-L）were made by adding D-mannose.The preparation process and composition of administration were investigated by single-factor experiments,and three factors were chosen for further study by entral composite design-response surface method.Encapsulation efficiency and drug loading were combined as overall desirability（OD）.The results were fitted with nonlinear equation to predict the encapsulation efficiency and drug loading of optimal formulation according to OD.The optimized D-ASI-L were prepared to verification the efficiency of regression.The asiaticoside cationic liposomes（C-ASI-L）were prepared by adding octadecylamine on the basis of D-ASI-L.The PBS which containing 1% sodium dodecyl sulfate（SDS）was used as dissolution medium.To determine the asiaticoside in dissolution medium by improved HPLC method.The in vitro release behaviors of asiaticoside solution（ASI-S）,ASI-L,D-ASI-L and C-ASI-L were investigated by dissolution apparatus,and the dissolution profile was obtained.The results fitted with different model to compare the difference among various formulation of asiaticoside.To determine asiaticoside in plasma by improved HPLC method.ASI-S,ASI-L,D-ASI-L and C-ASI-L were injected into rats by tail vein injection.The results were fitted with different models to calculate pharmacokinetic parameter by PK Solver 2.0,and the difference among four formulations in vivo process were compared.Long-term stability was processed at different temperature and light conditions to investigate the change of liposomes by appearance and leakage rate.Results: An accurate and reliable HPLC method was established,which could effectively determine the content of asiaticoside.Micro-column centrifugation method could separate liposomes from free drug,and the recovery rate of liposomes through mini-column could exceed 95%,which was meet the requirement to determine the encapsulation efficiency of liposomes.According to the encapsulation efficiency,film dispersion method was the best method to prepare ASI-L among four preparation methods.Due to the results of single-factor experiments,the factors of entral composite designresponse surface method were the ratio of drug to lipid（m/m,X1）,the ratio of cholesterol to drug（m/m,X2）and the content of D-mannose(g·mL^-1,X3).After optimization,the optimal prescription is that X1= 0.07,X2= 0.17,X3=0.03 g·mL^-1,and the predicted encapsulation efficiency was 75.692% while the drug loading was 2.533%.The encapsulation efficiency was（75.529±1.071）%（n=3）and the drug loading was（2.539±0.029）%（n=3）in the verification experiment,and the deviation from the predicted values were-0.217% and 0.205%.In vitro release results showed that there was a significant sustained release when the asiaticoside was loaded in liposomes.After 72 h,ASI-L released 87.58%,D-ASI-L released 94.12% and C-ASI-L released 94.46%,however,the ASI-S released 89.13% after 12 h.Model fitting results showed that ASI-S was fitted with First-order released model,while ASI-L,D-ASI-L and C-ASI-L were fitted with Weibull model.The td of ASI-S,ASI-L,D-ASI-L and C-ASI-L were 3.17 h,37.17 h,34.05 h and 39.97 h,respectively.The pharmacokinetic behaviors showed an obvious differences between liposomes and solution,while liposomes exhibited a significant sustained release.The results were fitted with various models by PK Solver 2.0.According to AIC,four formulations of asiaticoside were conformed to one compartment model with the weight of 1/C2.Half-life(t_1/2)of ASI-S was 14.52±0.56 min,while ASI-L,D-ASI-L and C-ASI-L were 1.51,1.74,2.77 times against ASI-S.Clearance（CL）of ASI-S was 10.43±0.83 m L·min·μg^-1,while ASI-L,D-ASI-L and C-ASI-L were 0.36±0.05、0.22±0.03、0.31±0.03 mL·min·μg^-1,respectively.Area under curve（AUC）value of ASI-S was 1929.70±159.00 min·μg·mL^-1,while ASI-L,D-ASI-L and C-ASI-L were 29.54,48.58,33.55 times against ASI-S.Among the three kind of liposomes,ASI-L have a lower t_1/2,D-ASI-L have a higher CL and there were no differences in CL.Stability experiments showed that there were no significant impact of light and temperature on the leakage rate of liposomes,however,the light would influence the appearance of liposomes.After long-term storage,the liposomes suspensend would agglomerate and delamination.Conclusions: The established HPLC is accurate and realiable to determine the concentration of asiaticoside.Micro-column centrifugation method could separate liposomes from free drug under selected conditions.The asiaticoside-loaded liposomes have high encapsulation efficiency and drug loading by using single-factor experiments and entral composite design-response surface method.Meanwhile,liposomes have a remarkable sustained-release effects in vitro and in vivo compared with solution.