乳酸-羟乙酸共聚物微球中rhCu,Zn-SOD稳定性研究

Stability of rhCu,Zn-SOD encapsulated in poly(DL-lactide-co-glycolide acid) microspheres

目的 以rhCu ,Zn SOD为模型蛋白 ,包裹入可生物降解聚合物PLGA内制备微球缓释制剂 ,详细研究制备过程中各因素以及体外释放对Cu ,Zn SOD活力和结构完整性的影响。方法 连苯三酚自氧化法测定酶活性 ,凝胶电泳考察蛋白质结构完整性的变化。结果 单独的超声或和二氯甲烷有机溶剂长期接触对Cu ,Zn SOD的活性影响不大 ,不破坏蛋白质的结构 ;体外释放时聚合物降解引起的微酸环境使rhCu ,Zn SOD二聚体解离形成亚基 ,微球中添加的赋形剂影响聚合物的水解速度。微球的体外释放呈现两个阶段 ,首次突释后 ,介质中蛋白质含量有所下降 ,约 1周后释放量才快速递增 ,属于不完全释放。结论 制备过程中对rhCu ,Zn SOD活力和结构完整性的影响不大 ,可在较大范围内改变工艺条件制备高载药量且稳定性好的rhCu ,Zn

OBJECTIVE: To investigate the stability of recombinant human copper and zinc superoxide dismutase(rhCu, Zn-SOD), encapsulated into biodegradable poly(lactide-co-glycolide) microspheres. Various factors during preparation and in vitro release were examined. METHODS: Enzyme activity was monitored by pyrogallol autooxidation and protein structural integrity by PAGE. RESULTS: SDS-PAGE results showed that there was no apparent effect of the drastic encapsulation conditions (contact with dichloromethane, probe sonification, and vigorous shaking) on the structural integrity of Cu, Zn-SOD. On the other hand, it was found that after 22 days of incubation the protein released from the microspheres were untied to smaller subunit, probably due to small environment pH decrease within microspheres as result of the degradation of polymer. The release assay revealed that the amount of protein in the medium started to decrease after burst release till 1 week, probably resulted from the surface absorption of polymer. CONCLUSION: High drug loading efficiency and stable PLGA microspheres loaded with Cu, Zn-SOD could be manufactured by the large range choice of preparation factors.