Ⅲ型钠磷共转运子小分子干扰RNA-壳聚糖纳米球的制备及性质分析

Preparation and characterization of type Ⅲ sodium-phosphate cotransporter siRNA-loaded chitosan nanoparticles

目的:制备Ⅲ型钠磷共转运子小分子干扰RNA(NaPi-ⅢsiRNA)-壳聚糖纳米球,并考察其理化性质及体外释放情况。方法:将壳聚糖和NaPi-ⅢsiRNA通过复凝聚方法制备NaPi-ⅢsiRNA-壳聚糖纳米球;透射电镜观察其形态,激光粒度分析仪测定其粒径分布,RNase酶解实验观察其对NaPi-ⅢsiRNA有无保护作用,并计算其包封率、载药量和对NaPi-ⅢsiRNA的体外控释能力。结果:成功制备NaPi-ⅢsiRNA-壳聚糖纳米球。电镜观察发现纳米球呈球形,分布均匀;激光粒度分析仪测定其平均粒径为173 nm;经RNase酶解后,纳米球混悬液D260上升较单纯NaPi-ⅢsiRNA溶液上升缓慢(P<0.05,t=4.32);药剂学分析发现其载药量为28.1%,包封率为73.07%,12 h内NaPi-ⅢsiRNA的体外释放率不足20%。结论:复凝聚方法制备的NaPi-ⅢsiRNA-壳聚糖纳米球包封率高、稳定性好、大小均匀,体外能显著延缓NaPi-ⅢsiRNA释放,可以在一定程度上保护NaPi-ⅢsiRNA免受酶解。

Objective：To prepare type Ⅲ sodium-phosphate eotransporter （NaPi-Ⅲ） siRNA-loaded ehitosan microspheres and to evaluate their physieo-chemieal properties and siRNA release in vitro. Methods： NaPi-Ⅲ siRNA was loaded into chitosan mierospheres by using the complex eoaeervation method. The structure of the nanopartieles was observed under scanning electron microscope and their diameter distribution was measured by a laser grain analyzer. RNase assay was used to detect the efficacy of anopartieles in prevention of siRNA from degradation. Ultraviolet speetrophotometry and HPLC technique were used to determine the entrapment efficiency, loading capacity, and siRNA releasing rate in vitro. Results： The ehitosan nanoparticles loaded with NaPi-Ⅲ siRNA were successfully prepared. The nanoparticles were spherical in shape and were well distributed, with an average diameter of 173 nm. After RNase treatment, D260 rose more slowly in ehitosan nanoparticles-based siRNA suspension than in simple NaPi-Ⅲ siRNA solution（P〈0.05,t= 4.32）. Loading capacity of the nanopartieles was 28.1%, the encapsulation efficiency was 73.07%, and the total releasing rate of NaPi-Ⅲ siRNA was less than 20% within 12 hours. Conclusion： Chitosan nanoparticles loaded with NaPi-Ⅲ siRNA have been prepared through a complex coacervation method. The particles have high encapsulation efficency and good stability and are homogeneous in size. They can obviously delay the release of NaPi-Ⅲ siRNA in vitro and therefore prevent NaPi-Ⅲ siRNA from degradation.