| ObjectiveThe solid-phase synthesis(Fmoc)was used to synthesize RIP,then,the synthesized peptide was purified by the reverse phase high performance liquid chromatography Liquid-phase multiple emulsion method was used to synthesize PLGA/RIP microspherical of diameter 50μm-70μm.The exosyndrome of microspheres were analysed.The microspheres' elution characteristic and bacteriostasis activity were studied vitro.The microspheres' histocompatibility and blood compatibility were estimated.The new rabbit chronically infected model of total knee arthroplasty was established on the base of prophase experimental in order to simulate chronically infected common seen clinically.The effect of prophylaxis for post THA chronically infected of Staphylococcus epidermidis were studied.Methods1.Construction of PLGA/RIP microspheres and study of microspheres' elution characteristic and bacteriostasis activity vitro:The solid-phase synthesis was used to synthesize the peptide according the known amino acid sequence of RIP,then, the synthesized peptide was purified by the reverse phase high performance liquid chromatography,and detected by mass spectroscopy,amino acid composition analysis,amino acid sequence analysis,Fourier transform infrared spectroscopy(FTIR).Liquid-phase multiple emulsion method was used to synthesize PLGA/RIP microspheres of diameter 50μm-70μm.The envelopment ratio ang drug-carried ratio were determined.Using high performance liquid chromatography to determin the pH,velocity,accumulate releaseand density of RIP in the different time and different density of PLGA/RIP eluent,the characteristic of drug release of PLGA/RIP vitro.The bacteriostasis activity was determined by inhibiting Staphylococcus aureus ATCC25923.2.The study of microspheres' histocompatibility and blood compatibility:The microspheres' blood compatibility were estimated by haemolysis test,hemagglutinatin test,experiment of PLGA/RIP effect on PT and APTT,experiment of PLGA/RIP effect on leucocyte,erythrocyte and thrombocyte and experiment of PLGA/RIP effect on platelet aggregation.The microspheres' histocompatibility were estimated by acute general toxicity test,cytotoxiciity test by MTT,intramuscular implanting test,sensitivity test and pyrogen test.3.The study of prophylaxis for post THA chronically infected of Staphylococcus epidermidis:The new rabbit chronically infected model of total knee arthroplasty was established on the base of prophase experimental in order to simulate chronically infected common seen clinically.The leucocyte,C-RP,pathology examination,SEM and LCSM were carried.The effect of prophylaxis for post THA chronically infected of Staphylococcus epidermidis were studied.Results1.Construction of PLGA/RIP microspheres and study of microspheres' elution characteristic and bacteriostasis activity vitro:The stable RIP were synthesised by solid-phase synthesis(Fmoc).The RIP of purity of 96.8%was synthesized by reverse phase high performance liquid chromatography.The amino acid composition and amino acid sequence of RIP synthesized was as same as natural RIP by mass chromatographic analysis,amino acid composition analysis,amino acid sequence analysis and FTIR analysis.The envelopment ratio was(68.22+6.20)%and drug-carried ratio.was(15.35±3.26)%.The kinetic equation of release of PLGA/RIP in the initial 12h is y = 3.8507x + 10.193,and the kinetic equation of release of PLGA/RIP in the30-day is y = 31.016x + 59.611.They were all coincidence with Huguchi equations.The PLGA/RIP was better delayed release drug-loading system.Bacteriostasis experiment vitro showed PLGA/RIP had better bacteriostasis activity.2.The study of microspheres' histocompatibility and blood compatibility:The haemolysis test,hemagglutinatin test,experiment of PLGA/RIP effect on PT and APTT,experiment of PLGA/RIP effect on leucocyte,erythrocyte and thrombocyte and experiment of PLGA/RIP effect on platelet aggregation showed PLGA/RIP has good blood compatibility.The acute general toxicity test,cytotoxiciity test by MTT,intramuscular implanting test,sensitivity test and pyrogen test showed PLGA/RIP has good histocompatibility. 3.The study of prophylaxis for post THA chronically infected of Staphylococcus epidermidis:After 1×10~2CFU Staphylococcus epidermidis ATCC35984 0.25mL injecting into knee joint everyday,all animal were confirmed with chronic infection by leucocyte,C-RP,pathology examination,SEM and LCSM.The chronic infection post THA resulted by persistence existent low dose pathogenic bacterium was prevented by PLGA/RIP sustained release in local region.conclusionThe highly purified RIP were synthesised by solid-phase synthesis(Fmoc)and reverse phase high performance liquid chromatography.The amino acid composition and amino acid sequence of RIP synthesized was as same as natural RIP.The PLGA/RIP microspheres' envelopment ratio and drug-carried ratio was satisfied.The kinetic equation of release of PLGA/RIP was coincidence with Huguchi equations.The PLGA/RIP was better delayed release drug-loading system.Bacteriostasis experiment vitro showed PLGA/RIP had better bacteriostasis activity.The study of microspheres' histocompatibility and blood compatibility showed PLGA/RIP has good blood compatibility,and good histocompatibility and can be used into joint safely.Persistence existent low dose pathogenic bacterium can result in rabbit's chronic infection post THA and this chronic infection can be prevented by PLGA/RIP sustained release in local region.
|
PDF Full Text Request