重组葡激酶脂质体的制备、活性及包封率测定方法的研究

Study on preparation of recombinant staphylokinase liposome and determination of its activity and entrapment efficiency

目的:研究重组葡激酶脂质体的制备、活性及其包封率的测定方法,为重组葡激酶脂质体的研究开发提供实验数据。方法:薄膜分散法制备重组葡激酶脂质体并用溶圈法测定其活性,分别采用超速离心和直接点样2种方法测定重组葡激酶脂质体的包封率。结果:溶圈法测定重组葡激酶活性的板内精密度的 RSD 为8.4%(n=5),板间精密度的 RSD 为12.4%(n=5)。直接点样法测定所制备的3批重组葡激酶脂质体包封率分别为54.36%,51.67%,54.67%;离心法测定所制备的3批重组葡激酶脂质体包封率分别为71.75%,67.43%,68.46%。所制备的重组葡激酶脂质体表面药物吸附量为15.65%。结论:溶圈法可用于重组葡激酶脂质体活性的测定;离心法测定的重组葡激酶脂质体包封率包括脂质体表面吸附药量;离心法与直接点样法测定包封率的差值为脂质体表面吸附药物量。

Objective： To prepare recombinant staphylokinase （ r - Sak） liposome and develop a methods to determinate its activity and entrapment efficiency for providing the experimental basis in the study and development of r- Sak liposome preparation. Methods： The liposomes containing r - Sak were prepared by film dispersion method and their fibrinolytic activities were determined by ＂lytic circle＂ method. Entrapment efficiency was respectively measured by ultracentrifugation spotting method and directly spotting method. Results： The RSDs （ n = 5 ） of intra - plates and inter - plates for assay of fibrinolytic activity with lytic circle method were 8.4% and 12. 4% respectively. The entrapment efficiencies of three batches of r - Sak liposomes were 54. 36% ,51.67% and 54. 67% respectively using directly spotting method and 71.75% ,67.43% and 68.46% respectively using ultracentrifugation spotting assay method. The adsorption percentage of r -Sak on the surface of liposomes was 15.65%. Conclusions：＂ Lytic circle＂ method can be used to assay the activities of r - Sak liposomes, and the entrapment efficiencies of r - Sak in liposomes determined by using uhracentrifugation spotting assay contained the amounts of r - Sak adsorbed on surface of liposome. The difference of two entrapment efficiencies obtained respectively from using ultracentrifugation spotting assay and directly spotting method was the amounts of r - Sak adsorbed on surface of liposome.