The Preparation And Study On Transdermal Characteristics In Vitro Of Kojic Acid-loaded Liposomes And Ninosomes

Part 1 Establish the method for determination of the kojic acid liposomes and ninosomes encapsulation efficiencyObjective: To establish a method for determination of kojic acid,kojic acid HPLC,establish a method for determination of the entrapment efficiency.Methods:1 An HPLC method was established to determine the concentration of kojic acid,while its stability,repeatability and precision were also investigated.2 Mini-column centrifugation method was employed to determine the encapsulation efficiency of kojic acid liposomes after optimizing the column height and centrifugation speed,then the repeatability of this method was investigated.3 Ultracentrifugation method was employed to determine the encapsulation efficiency of kojic ninosomes,then the repeatability of this method was investigated.Results:1 An accurate and reliable HPLC method was established,which could effectively determine the content of asiaticoside.2 Micro-column centrifugation method could separate liposomes from free drug,and the recovery rate and repeatability could meet the requirement to determine the encapsulation efficiency of liposomes.3 Ultracentrifugation method could separate ninosomes from free drug,and the repeatability was meet the requirement to determine the encapsulation efficiency of liposomes.Part 2 Optimization of preparation and formulation of kojic acid liposomesObjective: To determine the optimal kojic acid liposome preparation and prescription.Methods:1 According to the entrapment efficiency of kojic acid liposomes,the optimal preparation method was determined between the reverse phase evaporation,film dispersion method,ammonium sulfate gradient and calcium acetate gradient method.2 The preparation process and composition were investigated by singlefactor 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 experimental results of central composite design were fitted with nonlinear equation to predict the encapsulation efficiency and drug loading of optimal formulation according to OD.The optimized KA-L were prepared to verification the efficiency of regression.Results:1 According to the encapsulation efficiency,calcium acetate gradient method was chosen to prepare KA-L among four preparation methods.2 Due to the results of single-factor experiments,the factors of entral composite design-response surface method were the ratio of drug to lipid?m/m,X₁?,the ratio of cholesterol to drug?m/m,X₂?and the calcium acetate concentration?mol·mL-1,X₃?.After optimization,the optimal prescription is that X₁=0.09,X₂=0.125,X₃=0.15 mol·mL-1,and the predicted encapsulation efficiency was 40.645% while the drug loading was 3.078%.The encapsulation efficiency was?41.19±1.071?%?n=3?and the drug loading was?3.064±0.029?%?n=3?in the verification experiment,and the deviation from the predicted values were 1.341% and-0.877%.Part 3 Optimization of preparation and formulation of kojic acid liposomesObjective: To determine the optimal kojic acid ninosmes preparation and prescription.Methods:1 According to the entrapment efficiency of kojic acid ninosomes,the optimal preparation method was determined between the reverse phase evaporation and film dispersion method were investigated to screen the optimal preparation method.2 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 experimental results of central composite design were fitted with nonlinear equation to predict the encapsulation efficiency and drug loading of optimal formulation according to OD.The optimized kojic acid ninosomes loaded kojic acid were prepared to verification the efficiency of regression.Results:1 According to the encapsulation efficiency,reverse phase evaporation method was chosen to prepare kojic acid ninosomes among the two preparation methods.2 Due to the results of single-factor experiments,the factors of entral composite design-response surface method were the kojic acid?g,X₁?,the cholesterol?g,X₂?and the Span 40?g,X₃?.After optimization,the optimal prescription is that X₁=0.045,X₂=0.200,X₃=0.260,and the predicted encapsulation efficiency was 39.32% while the drug loading was 3.66%.The encapsulation efficiency was?38.95±0.871?%?n=3?and the drug loading was?3.60±0.025?%?n=3?in the verification experiment,and the deviation from the predicted values were-0.940% and-1.630%.Part 4 Study on the in vitro characteristics of kojic acid liposomes and niosomes percutaneous penetrationObjective: To study the vitro characteristics of kojic acid liposome and ninosomes percutaneous permeability,then compared with kojic acid solution.Methods: The kojic acid liposomes and kojic acid niosomes were prepared according to the optimal preparation.The transdermal characteristics of kojic acid liposomes,ninosomes,solution were investigated by transdermal test in vitro,using improved Franze transdermal diffusion instrument.The cumulative release rate time curve was draw based on the transdermal test date in vitro,then the 24 h cumulative penetration and skin retention were calculated.The drug release date was fitted by zero order,first order,RitgerPeppas and Higuchi model to explore the transdermal mechanism of kojic acid in vitro.The effect on the kojic acid percutaneous absorption in vitro of kojic acid solution,liposomes,ninosomes was evaluated by residence time method.Results: In vitro transdermal test,the results showed that: the 24 h cumulative release of KA-S was 2.862 mg·cm-2,24 h cumulative release rate was 90%,the 24 h skin retention per unit area was 0.233 mg·cm-2;the 24 h cumulative release of KA-L was 1.722 mg·cm-2,24 h cumulative release rate was 54%,the 24 h skin retention per unit area was 0.746 mg·cm-2;the 24 h cumulative release of KA-N was 1.722 mg·cm-2,24 h cumulative release rate was 54%,the 24 h skin retention per unit area was 0.534 mg·cm-2.In vitro permeation curve was fitted with Higuchi equation.Penetration rate of KA-L and KA-N were slower than KA-S,it indicated that KA-L and KA-N had slow-release effect,and the slow-release effect of KA-L was the best.At the same time,the skin retention of KA-L and KA-N were bigger than KA-S,indicated that KA-L and KA-N could have better curative effect.Part 5 The preliminary investigation on the stability of kojic acid liposomes and ninosmesObjective: To study preliminarily the stability of kojic acid liposomes and ninosomes.Methods: Influencing factors experiment and long-term stability were processed at different temperature and light conditions to investigate the change of KA-L and KA-N by appearance and leakage rate.Results: The results of influence factors and the long-term stability of the experimental showed that the temperature and illumination both had a effect on liposome and ninosomes leakage rate,and at the same time,the illumination had a bad effect on the liposome appearance.Long time storage will aggravates the leakage of the liposome and ninosomes.Conclusion: The established HPLC is accurate and realiable to determine the concentration of kojic acid.Micro-column centrifugation and ultracentrifugation method could separate liposomes and ninosomes from free drug under selected conditions.The KA-L and KA-N have high encapsulation efficiency and drug loading papered by the optimal paperation.In vitro transdermal test indicated that KA-L and KA-N have a slow-release effect,meanwhile can increase the skin retention of kojic acid.