Preparation Of PLGA Porous Microspheres By HA Stabilized Pickering Emulsion Template Method And Its Drug Loading Application

Objective:To prepare porous microspheres using a Pickering emulsion prepared from HA（hydroxyapatite）and PLGA（polylactic acid-glycolic acid copolymer）as a template with one step emulsification,oil-soluble and water-soluble drugs were respectively loaded,and their ability to load drugs was examined.The sustained release effect was evaluated.Methods:1.Preparation of HA:2.56 g of（NH₄）₂HPO₄and 7.52 g of Ca（NO₃）₂were weighed separately in a beaker,and 400 ml of water was added for ultrasonication and stirred until completely dissolved.The（NH₄）₂HPO₄solution was slowly added to the Ca（NO₃）₂solution,and the whole process was continuously stirred,and the p H of the solution was maintained at about10 with aqueous ammonia.After the solution was completely mixed,it was allowed to stand for 12 hours,the supernatant was removed,and the remaining precipitate was centrifuged and dried.2.Preparation of blank porous microspheres:Dissolve in methylene chloride as an oil phase with PLGA,F-68（poloxamer）,HA suspended in water as an aqueous phase,and mix the two phases by hand to emulsify into Pickering the emulsion,put it in a shaker,distill off the dichloromethane,filter and dry.Scanning electron microscopy was used to observe the spherical shape,and the ball rate was used as an index.The type and amount of PLGA and the amount of F-68 were investigated by single factor.3.Preparation of drug-loaded porous microspheres:The vancomycin hydrochloride porous microspheres were prepared by embedding method,double emulsion method and adsorption method respectively,and the ibuprofen porous microspheres were prepared by embedding method,and the structure was observed by scanning electron microscopy.4.Characterization of porous microspheres:The prepared blank porous microspheres and drug-loaded porous microspheres were characterized by infrared,differential scanning calorimetry and particle size determination,respectively.The specific surface area of blank porous microspheres was measured by surface area analyzer.5.Determination of drug loading and encapsulation efficiency:Vancomycin hydrochloride porous microspheres was dissolved by dichloromethane.The content of vancomycin hydrochloride was measured by ultraviolet spectrophotometry after extracted with water,and calculate the drug loading and encapsulation efficiency.The ibuprofen porous microspheres were dissolved in sodium acetate-acetonitrile（40:60）,and the solution was subjected to HPLC to determine the drug loading and encapsulation efficiency.6.In vitro release test:a certain amount of drug-loaded microspheres were placed in a 50 ml centrifuge tube,20 ml of PBS buffer was added,shaken at 37℃with a shaker,and samples were taken at different time points for detection.7.In vitro bacteriostatic test:a 6 mm diameter filter paper disc was placed on an agar plate to which the bacterial solution had been applied,and a certain amount of vancomycin hydrochloride microspheres were placed on a circular disc and dispersed with a small amount of water to make a white porous.The microspheres and the vancomycin hydrochloride solution were used as controls.Results:1.HA has been prepared successfully.2.A blank porous microsphere was successfully prepared by using a Pickering emulsion formed by emulsifying HA suspension and PLGA dichloromethane solution as a template.The morphology of the microspheres was observed by scanning electron microscopy.The model and dosage of PLGA and the amount of F-68 were optimized by the spherical rate and porosity index.The best prescription for blank porous microspheres was determined as follows:Oil phase:5 ml of dichloromethane,0.25 g of PLGA（LA:GA=50:50,viscosity:0.38 d L/g）,0.0125 g of F-68;aqueous phase:20 ml of water,0.1 g of HA.The microspheres prepared by the method have a relatively regular spherical shape with a diameter of about 5-25μm and a rich pore structure.3.The vancomycin hydrochloride microspheres prepared by embedding method and double emulsion method were failed.Finally,three kinds of vancomycin hydrochloride porous microspheres with different dosages were prepared by adsorption method.The preparation was successfully carried out by embedding method.3 different doses of ibuprofen porous microspheres.4.The results of infrared and differential scanning calorimetry showed that there was no new chemical bond between the raw material HA and PLGA for preparing microspheres and the drug vancomycin hydrochloride and ibuprofen.the particle diameters of blank porous microspheres,vancomycin hydrochloride microspheres and ibuprofen porous microspheres are 9.77,9.53,and 11.04μm,the specific surface area of the blank porous microspheres is22.16643 m²/g,and there is a rich mesoporous structure inside the microspheres,the average pore size is 25.3755nm.5.The average drug loading of the three groups of vancomycin hydrochloride microspheres from low to high was 4.67±0.06（%）,6.62±0.20（%）,and 7.56±0.22（%）.There were significant differences between the groups;the average encapsulation rates were 7.50±0.10（%）,5.99±0.18（%）,4.32±0.12（%）,respectively,there were significant differences between the groups;the dose of low-to-high three groups of ibuprofen porous.The average drug loading of the microspheres was 2.78±0.01（%）,5.38±0.03（%）,and 9.00±0.04（%）,respectively.There were significant differences among the groups;the average encapsulation efficiency was 38.38±0.08（%）,34.16±0.18（%）,45.12±0.20（%）,there were significant differences between the groups.6.The cumulative release of vancomycin hydrochloride microspheres at64h was 64.94%,reaching 92.28%at 5h,most of the drugs had been released into the solution;the cumulative release of ibuprofen porous microspheres at0.5h was 54.59%.The drug was then slowly released,reaching 94.74%at 48h.Both microspheres have obvious burst release phenomenon.The vancomycin hydrochloride microspheres have a very weak sustained release effect within5 hours,and the ibuprofen microspheres have a certain sustained release effect within 48 hours.7.In the case of the same drug content,the average length of the inhibition zone of the vancomycin hydrochloride microsphere group was22.1±3.3 mm,and the average length of the inhibition zone of the vancomycin hydrochloride solution group was 28.3±2.7 mm,there was a significant difference between the two groups（P<0.01）.Conclusions:1.Porous microspheres can be prepared by one-step process using HA and PLGA as raw materials.2.The porous microsphere prepared is suitable for loading a water-soluble drug by an adsorption method and loading a fat-soluble drug b y an embedding method.3.The microspheres loaded with water-soluble drugs have a weak sustained-release effect and are more suitable for loading fat-soluble drugs.4.In this paper,the preparation method of porous microspheres was determined and its properties were studied,which provided valuable experience for the post-production of composite microsphere biological scaffold.