Preparation Of Low Molecular Weight Heparin Microspheres For Oral Administration By Electrostatic Deposition And Evaluation Of Anticoagulant Effect In Rats

Part I Preparation of low molecular weight heparin microspheres for oral administration by electrostatic depositionObjective: To prepare the low molecular weight heparin loaded microspheres(LMWH-MP)by electrostatic deposition using carboxymethyl chitosan(CMC)and polylysine(PLL)as absorption promoters.Then,the LMWH-MP were studied by process optimization.Methods: Polylactic acid microspheres are used as a template,which is prepared by an emulsion solvent evaporation method.LMWH-MP was prepared by alternately depositing cationic polymer carboxymethyl chitosan and polylysine with low molecular weight heparin on the surface of polylactic acid microspheres.The low molecular weight heparin was determined by Azure A staining and the reproducibility was investigated.The LMWH-MP were optimized by single factor test and orthogonal experiment,and the drug loading was the main evaluation index.The drug loading of LMWH-MP under optimal conditions was determined and the entrapment efficiency was calculated.Results: The optimal conditions of CMC-LMWH-MP were: low molecular weight heparin 91.2 IU,pH 2.5,NaCL 0 mM,drug loading(8.81±0.14)%,encapsulation efficiency(54.80±0.64)%;The optimal conditions of PLL-LMWH-MP was: low molecular weight heparin 91.2 IU,pH 2,NaCL 0 mM;drug loading(7.34±0.12)%;encapsulation efficiency(46.07±0.76)%.Conclusion: Electrostatic deposition technology showed advantages of simple process and mild conditions.The optimum preparation process of LMWH-MP was obtained by process optimization.Part II Quality evaluation of low molecular weight heparin microspheres for oral administrationObjective: To evaluate the quality of two LMWH-MP prepared by electrostatic deposition.Methods: The surface morphology of microspheres was observed by scanning electron microscopy.The particle size and surface potential of microspheres were measured by Malvern particle size analyzer.The LMWH-MP were placed in artificial gastric juice for 3h,and then particle size and surface potential of the microspheres were measured to evaluate the stability of LMWH-MP in artificial gastric juice.In addition,LMWH-MP were store at different conditions(4?,25?),and its particle size and surface potential were measured at 1 month and 3 months respectively to evaluate the storage stability of LMWH-MP.At pH6.8,the drug cumulative release rate of LMWH-MP was determined by slurry method,and then the dissolution rate was calculated.Results: Scanning electron microscopy showed that the polylactic acid microspheres and LMWH-MP were spherical,polylactic acid microspheres and PLL-LMWH-MP were smooth,while CMC-LMWH-MP surface was slightly rough.The diameters of polylactic acid microspheres,CMC-LMWH-MP and PLL-LMWH-MP were(2.03±0.13)?m,(4.58±0.36)?m and(4.43±0.31)?m respectively.The surface potential of the microspheres changed alternately in positive and negative phases during the preparation process,indicating that low molecular weight heparin and carboxymethyl chitosan or polylysine were successfully deposited on the surface of polylactic acid microspheres.The particle size showed not change significantly with slightly decreased of potential,so the LMWH-MP in artificial gastric juice were stable.Although the potential of microspheres decreased somewhat,the particle size showed no obvious change.Whereas,the LMWH-MP preserved at 4? showed a comparatively less change in terms of particle size compared with normal temperature.In phosphate buffer at pH 6.8,CMC-LMWH-MP gradually releases about 90% of low molecular weight heparin,and PLL-LMWH-MP only releases a small.Conclusion: The LMWH-MP prepared by electrostatic deposition technique has the advantages of uniform particle size,good stability and reliable quality.Part III Anticoagulant effect of low molecular weight heparin microspheres oral administrationObjective: To investigate the pharmacodynamics of two oral low molecular weight heparin microspheres prepared by electrostatic deposition.Methods: The activated partial thromboplastin time(APTT)was measured at different time points before and after oral administration of LMWH-MP in rats to evaluate the anticoagulant effects of LMWH-MP on low molecular weight heparin in rats.Results: The LMWH-MP significantly prolonged APTT in rats plasma after oral administration(p<0.05).The anticoagulant effect peaked at approximately 3 h and sustained for 5 h,and the CMC-LMWH-MP showed a higher anticoagulant effect in comparison to PLL-LMWH-MP.Conclusion: The LMWH-MP prepared by electrostatic deposition technique can significantly prolong the APTT in rats plasma and show good anticoagulant effect.