聚乳酸-羟基乙酸微球内葡激酶突变体(K35R)的稳定性研究

 目的研究微球内葡激酶突变体(K35R,DGR)的存在状态和影响其稳定性的主要因素。方法使用傅里叶变换红外光谱法(FT-IR)研究了微球内DGR的二级结构的变化,并纤溶活性测定法考察了pH和聚乳酸-羟基乙酸(PLGA)表面对DGR稳定性的影响。结果①包封的DGR的二级结构明显发生了改变,但是,这种改变是β-折叠受到微扰的结果,并未影响微球内DGR的稳定性;②体外释放时,微球内PLGA的降解产物产生的酸性微环境会导致DGR变性,在内水相中加入Mg(OH)₂纳米粒可以抑制DGR的变性聚集;③对PLGA吸附DGR的研究表明,PLGA表面对DGR的吸附是离子相互作用和疏水相互作用共同起作用的结果,也是造成DGR失活的重要原因。结论PLGA对DGR的包封会导致蛋白质药物的二级结构发生变化,但是并不影响微球内DGR的稳定性;导致微球内DGR变性失活的主要原因是微球内的酸性微环境,碱性添加物可以抑制这种变性失活。PLGA表面对蛋白质药物的非特异性吸附也会导致部分DGR的失活。

Study on Stability of Staphylokinase Variant (K35R) Loaded Poly (lactic-co-glycolic acid) Microspheres

OBJECTIVE To investigate the status of a staphylokinase variant (K35R,DGR) loaded microspheres and the main factors that influence the stability of encapsulated DGR.METHODS Fourier transform infrared (FT-IR) spectroscopy was used to examine the secondary structure of DGR encapsulated in Poly(lactic-co-glycolic acid) (PLGA) microspheres. The influence of pH and adsorption of PLGA surface on the stability of DGR was evaluated. RESULTS The second structure of encapsulated DGR changed significantly.The stability of DGR was not influenced by the change. SDS-PAGE analysis of PLGA microspheres showed that dimeric and polymeric DGR formed after being released for 20 d. Mg(OH)₂ nanoparticles coencapsulated with DGR inhibited the aggregation of DGR within microspheres during release.Acidic microclimate within microspheres was an important source for the denaturation of encapsulated DGR. Research on adsorption of DGR to PLGA showed that non-specific adsorption of DGR to PLGA by hydrophobic and ionic interaction were also potential sources,which leaded to the denaturation of DGR within microspheres.CONCLUSION The second structure of DGR is significantly changed by the microencapsulation. However,the change of the second structure don′t influence the stability of encapsulated DGR. The main factors resulting in the denaturation of encapsulates DGR included the acidic microenvironment and non-specific adsorption of DGR to PLGA surface.