Solvent Effect During Fabrication Of Glycyrrhetinic Acid-loaded PLGA Microspheres

Objective Mechanism studies about solvent effect on microsphere properties during fabrication process are insufficient.Therefore in the present study,the emulsion-evaporation method and a novel hydrogel template method were applied to prepare glycyrrhetinic acid?GA?-loaded PLGA microspheres under various solvent conditions.The obtained microspheres were characterized and compared to clarify the correlationship between solvent effect and microsphere properties during the formation of drug-loaded microspheres.Method1.HPLC method was established and validated for drug content analysis of GA in different samples.The equilibrium solubility?S?of GA in different media were determined by shake flask method.The flowing time?T₀ and T?and intrinsic viscosity???of different organic solvents and their PLGA solutions were measured by Ubbelohde viscometer.The volatilization rate?V₁ and V₂?of variouss solvent systems has been measured in pure solvent and in the aqueous phase.2.GA-PLGA microspheres were prepared by emulsions-evaporation method and hydrogel template method using different solvent systems?single solvent and different proportion of mixed solvent,the proportion of different solvent conditions?,respectively.Then the influence of solvents on the structure,drug loading?D-L?and drug release of microspheres were investigated.The particle size,D-L,encapsulation efficiency?E-E?and in vitro release of the microspheres were determined.The formation of microspheres was investigated under microscope,and then surface and cross-sectional morphology of microspheres were observed under scanning electron microscope?SEM?.The dynamic process of microsphere formation and drug distribution inside microspheres were observed by single droplet forming technique combined with fluorescence tracer and laser Scanning confocal microscope?CLSM?,then the shrinkage rate and drug distribution parameters were calculated.X-ray diffraction?XRD?and differential scanning calorimetry?DSC?were used to analyze the physical state of drugs in the microspheres and the interaction between drug and PLGA.3.Important parameters?particle size,E-E,shrinkage rate,drug distribution and burst release?were extracted or transformed by aid of various softwares,and then correlated with the solvent characteristic parameters?S???T₀?T?V₁ or V₂,etc.?to describe their correlation quantitatively.Result1.The established HPLC detection method has the advantages of high degree of separation,good precision and strong specificity.The solubility of GA in organic solvent was much higher than that in aqueous phase.V₁ is related to the boiling point of the solvent,while V₂ is also related to the solubility of the solvent in water.2.The GA-PLGA microspheres prepared by oil-in-water?O/W?method were spherical and showed a good sustained-release performance.There were significant differences among their internal structure,D-L and E-E of microspheres prepared by a single solvent,and the E-E of microspheres prepared by ethyl acetate?Et-Ac?was the highest?88.67±2.90%?.The D-L and E-E of dichloromethane/dioxane?DCM/DIO?microspheres were significantly higher than that of single DCM microspheres,but the proportion of DIO in the mixed solvent was not conducive to the improvement of D-L.Among them,the E-E of DCM/DIO?2/1?microspheres was the highest?85.07±1.42%?,the differences in the internal structure and particle size of the obtained microspheres also lead to different behavior of drug release.Both DCM and Et-Ac as the main solvent could give spherical microspheres.The drug in DCM system microspheres were mostly based on a small spherical shape distribution,while the microspheres in Et-Ac system had more pore structure,but the latter microspheres had larger particle size and could effectively improve the drug loading and drug release behavior of microspheres.3.The shape and size of the microparticles prepared by hydrogel template method is uniform and controllable.However,the microparticles prepared by this method exhibited a large burst release,and the E-E increased obviously with the improvement of filling ioeration.Among them,the microspheres prepared with acetone?Ace?as the main solvent showed the best filling effect,the highest E-E as well,but the inner structure of the microparticles was loose and sustained-release performance was less preferable than those prepared by DCM.4.The results of correlation analysis showed that the microspheres prepared by O/W method were more affectted by the solvent effect than those by the hydrogel template method.The particle size,E-E,burst release and in vitro release of the microspheres prepared by O/W method with different proportion of mixed solvent were significantly affected by solvent effect.When the microspheres were prepared by DCM as the main solvent,the solvent had a certain influence on the burst release and slow release properties;the solvent properties such as T₀?T??and S were significantly correlated with the particle size,E-E and release properties of the obtained microspheres when using Et-Ac as the main solvent.The E-E and release properties of the drug loaded microparticles prepared by the hydrogel template method with the single solvent were only controlled by V₁ and V₂;In addition to the V₁ and V₂,E-E,burst release and sustained release behavior of the prepared microparticles were also affected by S and T₀.The mixed solvents showed the most significant effect on microparticles.When DCM was used as the main solvent,the release of the microparticles was mainly controlled by the solvent effect,the correlation between the drug loading and release properties of the microparticles prepared with Ace-based solvent and the solvent parameters was significantly.The curve was estimated,the above correlations were consistent with first or quadratic function,and these correlation between the solvent effect and the microsphere properties was quantified by the function.Conclusion Microspheres with favorable morphology can be gained by both emulsion-evaporation method and hydrogel template method under different solvent systems.However,the influence of solvents on the structure and properties of the obtained microspheres was significant,and a predetermined feature of microspheres can be achieved by selecting proper solvent system.The present study was of great value for revaluate the importance of solvent effect during the fabrication of drug-loaded microspheres,and also was beneficial for improving the optimization efficiency of formulation and process study of microspheres.