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目的制备透明质酸修饰的尿酸酶脂质体(hyaluronic acid-uricase liposomes,UHLP),并对尿酸酶(uricase,UC)和UHLP的体外活性及稳定性进行研究。方法采用逆向蒸发法制备UHLP,测定UHLP的包封率、粒径与zeta电位,用透射电镜对UHLP进行观察。考察游离UC和UHLP中UC的最适温度和最适pH,还考察了其热稳定性、贮存稳定性、酸碱稳定性、抗胰蛋白酶水解能力以及抗部分金属离子和有机化合物能力,并对UHLP提高UC活性的机制进行了初步研究。结果 UHLP的平均包封率为(57.27±3.93)%(n=3),平均粒径为(322.6±8.2)nm(n=3),zeta电位为(-19.4±1.7)mV(n=3)。透射电镜下UHLP呈分布均匀的圆形或椭圆形。游离UC和UHLP中UC最适温度均为40℃;游离UC最适pH为8.5,UHLP中UC最适pH为8.0。稳定性结果显示,UHLP中UC的热稳定性、贮存稳定性、酸碱稳定性、抗胰蛋白酶水解能力以及抗部分金属离子和有机化合物能力均优于游离UC。UHLP提高UC活性机制初步研究结果表明,UC经UHLP包裹后其活性发生改变,和UC与UHLP脂质膜的相互作用有关,它们在相互作用的过程中使UC的构象发生翻转,构效发生改变,UC活性中心暴露,从而导致其活性增强。结论 UHLP不仅能提高UC在体外的活性,还能提高UC在体外的稳定性。
Objective To prepare hyaluronic acid-modified uricase liposomes (UHLP) and to study the in vitro activity and stability of uricase (UC) and UHLP. Methods UHLP was prepared by reverse evaporation method. The encapsulation efficiency, particle size and zeta potential of UHLP were determined. UHLP was observed by transmission electron microscopy. The optimal temperature and optimum pH of UC in free UC and UHLP were investigated. The thermal stability, storage stability, acid-base stability, antitrypsin hydrolysis ability and resistance to some metal ions and organic compounds were also investigated. The mechanism of UHLP enhancing UC activity was studied. Results The average encapsulation efficiency of UHLP was (322.2 ± 8.2) nm (n = 3) and the zeta potential was (-19.4 ± 1.7) mV (n = 3 ). Under the transmission electron microscope, UHLP showed a uniformly distributed circle or oval. The optimal temperature of UC in free UC and UHLP was 40 ℃, the optimum pH of free UC was 8.5, and the optimum pH of UC in UHLP was 8.0. The stability results showed that the thermal stability, storage stability, acid-base stability, antitrypsin hydrolysis ability and resistance to some metal ions and organic compounds of UC in UHLP were superior to those of free UC. The results of preliminary studies on the mechanism of UHLP enhancing UC activity showed that the activity of UC changed after being encapsulated by UHLP and the interaction between UC and UHLP lipid membrane. They reversed the conformation of UC and the structure-activity effect changed during the interaction , UC active center exposure, resulting in increased activity. Conclusion UHLP not only can improve the activity of UC in vitro, but also improve the stability of UC in vitro.