Preparation And Basic Reaearsh Of Rifapentine-Loaded Poly(Lactic-Co-Glycolic Acid) Microspheres-Calcium Phosphate Cement Composites

PART 1 PREPARATION AND CHARACTERIZATION OF RIFAPENTINE-LOADED POLY(LACTIC-CO-GLYCOLIC ACID) MICROSPHERESObjectiveTo prepare poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with rifapentine to sustainably sterilize in the lesions of osteoarticular tuberculosis. The physicochemical characteristics of rifapentine-loaded PLGA microspheres have been studied in details.MethodsRifapentine-loaded PLGA microspheres have been prepared by using single-emulsion solvent evaporation (water-in-oil, O/W) method, their morphology was observed by light microscope and scanning electron microscopy (SEM). Rifapentine-loaded PLGA microspheres were analyzed about size with laser particle size analyzer, composition structure with fourier infrared spectrometer (FT-IR). The drug-loading rate and encapsulation rate of drug-loaded microspheres were determined by ultraviolet spectrophotometry.ResultsMost of the PLGA microspheres were spherical with excellent dispersibility. Surfaces of the unloaded PLGA were smooth. However, addition of rifapentine changed the surface morphology observably, the more drug in formula, the deeper and more concaves in the surface. The FT-IR showed that, compared with the characteristic absorption peaks of unloaded PLGA microspheres, these peaks did not have a significant shift in rifapentine-loaded microspheres. Meanwhile, there were peaks of carboxyl stretching vibration characteristic located in the 2192 cm-1 in the FT-IR of rifapentine-loaded PLGA microspheres. When 0 mg,20 mg,50 mg and 100 mg of rifapentine were added in the formula, the average size of the microspheres was 12.422±0.372 μm,16.737±0.52 μm,25.267±0.318 μm and 27.833±0.485 μm, respectively. With the increasing of rifapentine in the formula, the microspheres enlarged (p< 0.05). With different rifapentine in the formula, the drug loading of the microspheres were 8.04±0.29%,17.16±0.40% and 23.93±0.48%, the entrapment efficiency were 88.49±3.23%,85.78±2.00% and 71.80±1.45%, respectively. With an increase of rifapentine in formula, the drug loading increased gradually (p <0.05), while the entrapment efficiency decreased (p<0.05).ConclusionThe single-emulsion solvent evaporation (water-in-oil, O/W) method is suitable for preparation of rifapentine-loaded PLGA microspheres. The microspheres were spherical with excellent dispersibility. With an increase of rifapentine in formula, average size and drug loading of the microspheres increased gradually, and the entrapment efficiency decreased. The rifapentine-loaded PLGA microspheres, which were prepared by 200 mg of PLGA and 50 mg of rifapentine in the formula, have an appropriate drug loading (17.16±0.40%) and entrapment efficiency (85.78±2.00%).PART 2 IN VITRO RELEASE AND ANTIBACTERIAL ACTIVITY OF RIFAPENTINE-LOADED POLY(LACTIC-CO-GLYCOLIC ACID) MICROSPHERESObjectiveTo prepare rifapentine-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres with different drug loading, and investigate their in vitro release and antibacterial activity, hoping to get experiment data for the following study and clinical application.MethodsRifapentine-loaded PLGA microspheres with different drug loading were prepared by using single-emulsion solvent evaporation method. The in vitro release tests were carried out in phosphate buffer solution (0.2M, pH7.4), the release amount of rifapentine was detected by UV spectrophotometry and release curve was mapped. S. aureus(ATCC 6538) was used in this study to investigate the in vitro antibacterial performance of rifapentine-loaded PLGA microspheres. The in vitro antibacterial properties of the microspheres were detected by agar diffusion test (ADT) and minimal inhibitory concentration (MIC).ResultsRifapentine-loaded PLGA microspheres with drug loading of 8.04±0.29%,17.16±0.40% and 23.93±0.48% were successfully prepared. All the three kinds of rifapentine-loaded PLGA microspheres exhibited a similar in vitro biphasic release profiles, burst release phase and sustained slow release phase. The cumulative percent release of the three rifapentine-loaded PLGA microspheres were 9.07±0.11%,13.33±0.04% and 15.5±0.09% during the burst release phase.72.10±0.26%,80.22 ± 0.56% and 78.60±0.63% drugs were released during the whole release periods. The release characterization of the three microsphere fit Higuichi discipline, since their correlation coefficients were more close to 1. The inhibition zone diameter and MIC value of rifapentine against S. aureus were 21.88 ±0.40 μm and 5 μg/ml. The inhibition zone diameters of rifapentine -loaded PLGA microspheres with drug loading of 8.04±0.29%, 17.16±0.40% and 23.93±0.48% were 7.62±1.02 μm,11.27±0.58 μm and 13.38±1.01 μm, respectively. And the MIC values were 320 μg/mL,80 μg/ml and 40 μg/ml, respectively. With increase of rifapentine loading in the microspheres, the inhibition zone diameter increased gradually (p< 0.05), and the MIC values decreased (p<0.05).ConclusionRifapentine-loaded PLGA microspheres exhibited a biphasic in vitro release profiles and mostly fit the Higuichi discipline. Increasing of rifapentine loading in the microspheres would gradually increase the inhibition zone diameter and decrease the MIC values. In our studies, PLGA microspheres with 23.93 ± 0.48% rifapentine loading (23.93% RPMs) could release and maitain rifapentine at a higher level than MIC values of rifapentine against Mycobacterium tuberculosis for about 28 days in vitro. The period is longer than that of PLGA microspheres with 8.04 ± 0.29% and 17.16±0.40% rifapentine loading. Taking the in vitro release characterization and antibacterial activity into consideration,23.93% RPMs will be used in the following studies to prepare a focal drug delivery system for the treatment of osteoarticular tuberculosis.PART 3 PREPARATION AND CHARACTERIZATION OF RIFAPENTINE-LOADED POLY(LACTIC-CO-GLYCOLIC ACID) MICROSPHERES-CALCIUM PHOSPHATE CEMENT COMPOSITESObjectiveTo prepare rifapentine loaded poly(lactic-co-glycolic acid) microspheres-calcium phosphate cement (RPMs-CPC) composites and investigate their physicochemical characteristics, in vitro release, in vitro cell compatibility and in vivo osteogenesis, hoping to get experiment data for their clinical application.MethodsThe RPMs-CPC composites were prepared by mix of rifapentine loaded poly(lactic-co-glycolic acid) microspheres (RPMs), CPC and disodium hydrogen phosphate solution. Structures of the composites were analyzed by scanning electron microscopy, fourier infrared spectrometer (FT-IR), X-ray diffraction (XRD). The other physicochemical characteristics such as porosity, seting time and mechanical properties also were detected. In vitro release tests were carried out in phosphate buffer solution. Extracts of RPMs-CPC composites were prepared and cultured with bone mesenchymal stem cells to evaluate the cytotoxicity of the composites. Totally 18 New Zealand white rabbits were randomly divided into two groups:rifapentine-CPC group and 30 wt% RPMs-CPC group,9 rabbits in each group. Defects in femoral condyle were established and filled with materials respectively.4 weeks,8 weeks and 12 weeks after operation, the rabbits were euthanized. The femoral condyles were excised and investigated by histopathological observation.12 weeks after operation, the liver, kidney, lung and spleen tissues of the rabits were excised and investigated.ResultsThe SEM images showed that RPMs evenly distributed in CPC matrix. The characteristic peaks of CPC did not shift, and no novel formed peaks appeared in FT-IR and XRD spectrogram, suggesting RPMs did not disturb the hydration reaction of CPC and there were no chemical bonds formed during the preparation process. However, RPMs changed the physicochemical characteristics of CPC significantly. The porosity, initial seting time, final seting time and compressive strength of pure CPC were 46.72±1.01%,2.0±0.11 min,22.6 ±0.73 min and 47.31±2.83 MPa, respectively. That of 30 wt% RPMs-CPC was 73.56±0.87%,4.6±0.27 min, 36.4±2.14 min and 21.30±3.25 MPa, respectively. No burst release was observed in the release profiles of RPMs-CPC composites. With increase of RPMs loading, the quantity and cumulative release percentage increased. The daily drug release quantity of 10 wt% RPMs-CPC,20 wt% RPMs-CPC and 30 wt% RPMs-CPC was about 8 microgram,19 microgram and 41 microgram. Obviously, only the daily drug release quantity of 30 wt% RPMs-CPC was higher than the minimal inhibitory concentrations of rifapentine against Mycobacterium tuberculosis. So the 30 wt% RPMs-CPC composites were used to investigate the osteogenesis in vivo. All the incisions healed well without infection. Abscess or inflammation was not observed in the neighboring bone or muscle tissues of the implantation site. At the twelfth week after operation, in the rifapentine-CPC group, only a small amount of bone-like tissues formed in the edge of the bone defects, along with little material degraded. While in the RPMs-CPC group, a large amount of composites degraded and bone tissues generated to form bone trabeculas. Some of the bone trabeculas regularly arranged and connected with the host bone, suggesting the bone defects were well repaired.ConclusionRifapentine-loaded poly(lactic-co-glycolic acid) microspheres-calcium phosphate cement composites have excellent biocompatibility, release rifapentine sustainedly in vitro, and could repair bone defect effectively without significantly inflammatory reaction. The composites exhibit a good clinical application prospect for the treatment of osteoarticular tuberculosis.