An Preliminary Experimental Study Of Bone Loaded Rifampin-PLGA Microspheres As An Implantable Composite

Objective:①To achieve the optimal method of preparation of RFP-PLGA microspheres(Ms) by O/W emulsion-solvent evaporation techniques through observation of the morphology, particle size and distribution,encapsulation and loading efficiency of Ms.(2)To observe the filling state and releasing of sustained-release Ms when combining it with allografted bone scaffold in vitro.③To observe the distribution pharmacokinetics and compatibility of RFP-PLGA-allograft bone complex in animal model to provide experimental basis for antituberculosis therapy after spinal surgery.Methods:①The O/W emulsion solvent evaporation method was used to prepare sustained-release Ms. Particle size, drug loading and entrapment efficiency were used as the evaluation index.The PLGA concentration,O/W rate,and the type and concentration of surfactant were investigated as single factor respectively.Some important influencing factors were chose to go through orthogonal experiment to figure out their impact for optimal procedure.②The RFP-PLGA Ms was placed into allografted bone by using ultrasonic vibration,covered with a chitosan membrane.The bone densitometer and electron scanning microscope were used to observe the filling state and the morphology of the Ms as well as the change of BMD (Bone mineral density)after vacuum drying and metal spraying.The vitro releasing experiments were divided into three groups: Group A:RFPcrystal,Group B:RFP Ms;and Group C:RFP-PLGA-allograft bone complex.Three groups were investigated with bag filter method.③The pharmacokinetics in vivo experiments were divided into3groups:Group A:RFP oral dosing,Group B:RFP-PLGA-allograft bone complex and,Group C:RFP crystal-allograft bone.Group B and C were embedded into the rabbit’s lumbar vertebral defect site and compared them in six aspects including observation of general condition,imaging feature,BMD,tissue distribution,biochemistry index and histomorphology.HPLC-UV method was used to detect the drug concentration in blood plasma and other tissues.The results were showed as the(x±S)and multiple statistical methods including the T test, ANOVA, SNK-q test of group comparison and LSD-t test by using the SPSS17.0.The setting, size of test was a=0.05and,p<0.05was considered the significant difference.Results:①On the basis of the single factor exploration we knew the important influential factor and grading-up the method, the conclusion of effect intensity of each factor was:Drug/Copolymer composition> PVA concentration> PLGA concentration> O/W rate.Optimal method was:PLGA concentration15mg·ml-1,RFP/PLGA1:2,PVA concentration2%, O/W rate1:5.The mean diameter of Ms was12.33±0.98μm,with the drug loading and entrapment efficiency was (19.63±0.62)%and (59.49±2.62)%,repectively.After the cryodesiccate of Ms,the redispersibility and appearance was well.②The release in vitro showed: the RFP crystal of Group A was almost released completely in24hours;the Ms of Group B and Ms-bone complex of Group C was sustainsably released. The releasing rate of Group B was approximately up to95%totally in31days,consistent with the zero-order elimination kinetics equation:F=0.168*t.RFP-PLGA-allograft bone complex rate was approximately up to92.31%totally in31days without apparent burst release.With the zero-order elimination kinetics equation:F=0.15*t;the BMD test showed:the BMD of Group B and C showed no apparently statistical difference after filling in three groups,(p>0.05).③In vivo kinetics showed group A(oral dosing) had high drug concentration in both local lumbar vertebra and other organs like heart, liver and spleen. Group C (RFP crystal-allograft bone),although maintaining the effective concentration in target tissue and diminishing the drug concentration in other organ,its duration was short,under the minimal detectable concentration after15d.By comparison,Group B:after the RFP-PLGA-allograft bone complex inserted into New Zealand white rabbits,RFP concentration was11±0.7μg/ml-1in the3rd day,RFP concentration was13.3±0.4μg/ml-1in the7th day and RFP concentration was8.5±1.6μg/ml-1in the15th day,RFP concentration was1.6±0.4μ/ml-1in the30th day,RFP concentration was0.45±0.21μg/ml-1in the40th day,which were apparently higher than MIC.Slowly releasing in vivo tissue,the drug concentration was lower in other organ but could reach the effective sterilization concentrations in lumbar vertebra in the40th day.Compared to Group C,it maintained longer effective sterilization concentrations in local lumbar vertebra.BMD test showed:a.There were no statistical significance after the surgery for7days in three groups: three groups have no statistical difference:P>0.05;b.30days after the surgery:Group A compared with Group B and C:P<0.05;Group B compared with Group C:P>0.05;c.7d and30d after the surgery:A’group compare with A30group:P>0.05;B7group compared with B30group:P<0.05;C group compared with C30group:P<0.05; Hepatotoxicity test:the3groups showed no apparently statistical difference after the dose; After the RFP-PLGA-allograft bone complex had been embedded into lumbar vertebra,lumbar vertebra and psoas tissue didn’t show any apparently inflammatory reaction and no normal tissue damage or paramorphia.Conclusion:①A variety of factors in O/W emulsion solvent evaporation method have significant influence on morphology and quality of Ms. The optimal scheme was screened that the concentration of PLGA was15mg-mL-1;concentration of PVA was2%;RFP/PLGA1:2and O/W rate was1:5.②The RFP Ms was well shaped and,particle diameter distributed evenly.Ultrasonic oscillation worked with the poriferous allograft bone could make up RFP-PLGA-allograft bone complex,to be filled well.The BMD show no significant statistical difference after the filling.Among the three groups and with good slowly-releasing function in vitro. The drug concentration in different time point was above the MIC.③After the RFP-PLGA-allograft bone complex was implanted into para-vertebrae, the RFP concentration in local tissue could maintain above MIC over40d without apparent toxicity and no drug accumulation was found in heart,liver and spleen.No abnormality was found in BMD and histocompatibility.