壳聚糖-明胶/聚乳酸-羟基乙酸联合载药水凝胶的体外抗结核作用

In vitro anti-tuberculosis effect of chitosan-gelatin/poly(lactic acid co-glycolic acid)combined with drug-loaded hydrogel

背景:抗结核化疗是目前治疗骨关节结核的主要手段,然而全身给药难以维持病灶区的有效浓度,治疗效果欠佳。目的:制备一种原位、长期释放抗结核药物且兼备促成骨作用的壳聚糖-明胶/聚乳酸-羟基乙酸联合载药水凝胶。方法:将亲水性的抗结核药物异烟肼和疏水性的基质细胞衍生因子通过复乳法负载到聚乳酸-羟基乙酸中,制备聚乳酸-羟基乙酸载药微球,共混至壳聚糖-明胶水凝胶支架中,制备壳聚糖-明胶/聚乳酸-羟基乙酸联合载药水凝胶。检测聚乳酸-羟基乙酸载药微球、壳聚糖-明胶/聚乳酸-羟基乙酸联合载药水凝胶的体外释药与抗结核杆菌的能力。将成骨前体细胞MC3T3-E1分别接种于载药微球与联合载药水凝胶表面,CCK-8法检测细胞活力,碱性磷酸酶活性检测细胞的成骨性能。结果与结论:①载药微球中异烟肼1 h内的突释约为23.3%,2 d内的释放率约为42.6%,随后进入缓释期,25d后进入平台期;基质细胞衍生因子1在1h内的累积释放率约为19.8%,2d内的释放率约为44.7%,随后进入缓释期,25 d后进入平台期;联合载药水凝胶中异烟肼和基质细胞衍生因子1最初1 h的释放分别为8.3%和8.5%,第2天的累计释放率分别为15.2%和17.6%,远低于聚乳酸-羟基乙酸微球;②体外4周后,联合载药水凝胶的抑菌直径大于载药微球,抑菌率高于载药微球(P<0.05);③联合载药水凝胶与载药微球均具有良好的细胞相容性,细胞活力均约为100%;④培养5,10d后,联合载药水凝胶表面的细胞碱性磷酸酶活性与载药微球比较差异无显著性意义(P>0.05);⑤结果表明,原位壳聚糖-明胶/聚乳酸-羟基乙酸联合载药水凝胶有作为治疗骨关节结核及其他骨关节感染的潜力。

BACKGROUND:Anti-tuberculous chemotherapy is the main method for treating bone and joint tuberculosis.However,systemic administration hardly maintains the effective drug concentration in the focus area,and the therapeutic efficacy is unsatisfactory.OBJECTIVE:To prepare a chitosan-gelatin/poly(lactic-co-glycolic acid)combined with drug-loaded hydrogel,which can release anti-tuberculosis drugs in situ for a long time and promote osteogenesis.METHODS:Isoniazid,a hydrophilic anti-tuberculosis drug,and a hydrophobic stromal cell derived factor-1 were loaded into poly(lactic-co-glycolic acid)by double emulsion method to prepare drug-loaded poly(lactic acid co-glycolic acid)microspheres,which were then mixed into chitosan gelatin/poly(lactic acid co-glycolic acid)combined with drug-loaded hydrogel.The ability of drug delivery and anti-tuberculosis of poly(lactic acid co-glycolic acid)microspheres and chitosan gelatin/poly(lactic acid co-glycolic acid)combined with drug-loaded hydrogels in vitro were tested.MC3T3-E1 cells were inoculated on the surface of microspheres and hydrogel respectively.The biocompatibility was detected by cell counting kit-8 assay.The osteogenetic activity was detected by alkaline phosphatase activity.RESULTS AND CONCLUSION:(1)The burst release of isoniazid in the microspheres was about 23.3%in 1 hour,42.6%in 2 days,and then it entered the sustained-release stage in the later 25 days.The burst release of stromal cell derived factor was about 19.8%in 1 hour,44.7%in 2 days,and then it entered the sustained-release stage in the next 25 days.The release of isoniazid and stromal cell-derived factor in the combined drug-loaded hydrogel was 8.3%and 8.5%in the first hour,respectively.The cumulative release rates on the second day were 15.2%and 17.6%,respectively,which were much lower than that of poly(lactic acid co-glycolic acid)microspheres.(2)After 4 weeks in vitro,the antibacterial diameter of the combined drug-loaded hydrogel was much larger than that of the drug-loaded microspheres,and the antibacterial rate was higher than that of the drug-loaded microspheres(P<0.05).(3)The combined drug-loaded hydrogel and the drug-loaded microspheres had good cytocompatibility and cell viability was about 100%.(4)After 5 and 10 days of culture,there was no significant difference in the activity of alkaline phosphatase on the surface of drug-loaded hydrogel and drug-loaded microspheres.(5)These results show that the in situ chitosan-gelatin/poly(lactic acid co-glycolic acid)combined with drug-loaded hydrogel can be used for treating tuberculosis and other bone and joint infections.