| Objective: To prepare rifapentine-loaded local bone-graft drug delivery system(RPT/Bone-graft DDS)for intraoperative application and peripheral bone-target drug delivery system(RPT/Bone-target DDS)for preoperative or postoperative application based on rifapentine(RPT),emulsification technology and Poly-d,l-lactide-co-glycolide(PLGA)based polymer,the prepared RPT/Bone-target DDS and RPT/Bone-graft DDS were evaluated by both in vivo and in vitro experiments.Methods:(1)Preparation of the drug delivery systems using the SPG membrane emulsification technology to prepare polylactic acid microspheres encapulated with rifapentine,the apparent characteristics of the microspheres were observed,the particle size of the microspheres was measured,and RPT/ bone-graft DDS were prepared by integrating the microspheres into hydroxyapatite/tricalcium phosphate scaffold,the apparent characteristic of this system was also observed.(2)Polyethylene glycolated PLGA bone-targeting copolymer was synthesized by amide reaction,and the composition of the copolymer was determined by ultraviolet and infrared spectroscopy.Then,rifapentine was loaded in this copolymer by ultrasound emulsification technology to prepare RPT/ bone-target DDS,and its particle size,surface potential and polydispersity index were measured to observe its apparent characteristics.(3)The ultraviolet detection conditions of rifapentine were determined for in vitro evaluation of the drug delivery systems,the encapsulation efficiency,drug loading,in vitro drug release and in vitro bacteriostasis of PLGA rifapentine microspheres and bone-targeting nanoparticles were measured,respectively,and the bone-targeting ability of the peripheral bone-targeting drug delivery system was also measured in vitro.The rat bone marrow mesenchymal stem cells(r BMSCs)were isolated,cultured and identified.The cytotoxicity of rifapentine microspheres,nanoparticles and scaffold extracts was detected,respectively.The hemolytic toxicity of rifapentine loaded nanoparticles was also detected by using the rat erythrocyte suspension.The obtained stem cells were co-cultured with drug-carrying microspheres or RPT/ bone-graft DDS,and the apparent characteristics were observed,in addition,the cell adhesion rate in scaffold was also calculated.(4)The bone defect model of the lateral femoral condyle in rat was established followed by implanting the RPT/Bone-graft DDS for the purpose of in vivo evaluation of the drug delivery system,at different time points after surgery,the femur specimens were collected,and the X-ray and CT imaging examination was performed,respectively.HE staining,safranin O-green staining,immunohistochemistry and immunofluorescence staining were also performed.(5)The detection conditions of high performance liquid chromatography of rifapentine in rat plasma biological products were established for detecting the drug release from the RPT/ bone-graft DDS in the femoral condyle of rat.(6)The bone-targeting properties of RPT/ bone-target DDS were detected by small animal imager in vivo after administration.(7)Blood samples were collected after surgery to detect the function of liver and kidney at different time points,and the drug concentrations in plasma,organs and bone tissue were determined to evaluate the ability of bone targeting.All organs were finally stained with HE to observe their morphology.Results: The first part of this study,preparation of the drug delivery systems:(1)The rifapentine microspheres were brick red in appearance,with a smooth spherical surface and an average particle size of 36.63 μm.The scanning electron microscopy of RPT/ bone-graft DDS showed that the pores were porous and interconnected,and the dispersed microspheres were visible in the internal pores.(2)Ultraviolet and infrared spectroscopy indicated that tetracycline was successfully grafted onto polyethylene glycol modified PLGA copolymer.The surface of RPT/ bone-target DDS was negatively charged,with an average particle size of 48.83 nm.TEM showed that the nanoparticles were black,with regular spherical and uniformly dispersed.The second part of this study,in vitro evaluation of the drug delivery system :(3)Rifapentine has the maximum absorption peaks at 477 nm and 336 nm under UV detection conditions,and the standard curve equation can be calculated at both wavelengths for the concentration detection,and 336 nm may has a lower detection limit.The encapsulation rate of rifapentine microspheres was 70.28%,the drug loading was36.12%,the initial stage of in vitro release was explosive release,and the cumulative release was 78.3% on the 40 th day.In vitro bacteriostatic experiments showed that the bacteriostatic activity of rifapentine released from the local bone graft system was the same as that of free rifapentine.(4)r BMSCs were identified to conform to the characteristics of mesenchymal stem cells.The rifapentine microspheres and the extracts of its local bone graft drug delivery system were non-toxic to rat stem cells in a certain concentration range,and could be co-cultured with stem cells,and the cells showed adhesion growth.The electron microscopy scanning results of RPT/bone-graft DDS indicated good biocompatibility.(5)The drug loading of RPT/bone-target DDS was 8.1%,and the encapsulation rate was 76.3%.In vitro release showed explosive release within 6hours,followed by the slow release within 64 hours.In a certain concentration range,rifapentine nanoparticles had no toxicity to r BMSCs,and no obvious hemolysis was observed.The antimicrobial experiment showed that the minimum antimicrobial concentration of RPT/bone-target DDS was 0.094 μg/m L,which was lower than that of free drug(P < 0.05).In vitro bone-targeting experiments showed that the bone affinity of this drug delivery system was 60.80%,which was higher than that of the non-bone-targeting drug delivery system(P < 0.05).The third part of this study,in vivo evaluation of drug delivery system:(6)The results of gross observation and imaging examination(X ray and computer tomography)after the construction of rat model of the femur bone defects suggested that drug-loaded group and non drug loaded group compared with the blank control group could promote bone formation,in addition,the new bone volume percentage was higher than the control group at six weeks after surgery(P < 0.05),in addition,the scaffold degraded gradually with the extension of time postoperatively.The histopathological results indicated that the bone repair ability of the drug-loaded scaffold group and the non-drug-loaded scaffold group was better than the control group,and their levels of OCN,BMP-2 and TGFβ1 osteogenic indexes were higher than those of the control group(P < 0.05).The results of high performance liquid chromatography indicated that RPT/ bone-graft DDS could slowly release rifapentine in the local bone of rats.(7)The living tomography scanning results indicated that flurescent NPs(RPT/Bone-target DDS)accumulated in bone tissue after being administered through tail vein of mice.The results of high performance liquid chromatography indicated that RPT/bone-target DDS could change the characteristics of drug distribution,and its drug concentration was significantly higher than that of non-target group with lower drug concentration in liver,furthermore,the liver blood biochemical indexes and the visceral pathological examination showed no obvious toxicity.Conclusion: Both RPT/Bone-graft drug delivery system and RPT/Bone-target drug delivery system showed a sustained-release property of rifapentine and good biocompatibility,which could be used to fill the intraoperative bone defect,improve the drug concentration in the bone grafting area and speed up bone repair(RPT/Bone-graft DDS).RPT/Bone-target DDS could be used to target bone tissue after peripheral administration,increase the drug concentration in bone tissue,change the drug distribution,and may help to reduce the drug dose and dosing frequency during preoperative or postoperative drug therapy. |
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