基于介孔碳纳米粒包载胰岛素实现口服递药的研究

Study on achieve oral drug delivery based on insulin encapsulated by mesoporous carbon nanoparticles

目的利用介孔碳纳米粒(MCN)包载胰岛素从而实现口服缓释递药。方法 (1)制备与表征:制备包载胰岛素的介孔碳纳米粒(MCN-I),通过扫描电镜和透射电镜进行表征,高效液相色谱测定包封率和载药量,激光粒度仪测定粒径、多分散系数及Zeta电位,考察MCN-I的体外释放性能。(2)体内分布实验:按照体重将SD大鼠随机分为4组:荧光标记胰岛素(FI)灌胃组(10 mg·kg^(-1))、介孔碳纳米粒包载荧光标记胰岛素(MCN-FI)灌胃组(10 mg·kg^(-1))、尾静脉注射FI组(10 mg·kg^(-1))和空白组(灌胃0.9%的生理盐水),每组3只。用荧光显微镜观察并统计大鼠心、肝、脾、肺、肾和脑的平均荧光强度。(3)体内药效实验:将糖尿病模型大鼠随机分4组:皮下注射胰岛素组(5 U·kg^(-1)),MCN-I灌胃组(50 U·kg^(-1)),MCN灌胃组(5 mg·kg^(-1))和胰岛素灌胃组(50 U·kg^(-1)),每组6只。用血糖仪测定12 h内的血糖变化。结果 (1)成功制备MCN-I,包封率为(65.18±1.59)%,载药量为(20.19±1.18)%,平均粒径为(195.00±2.80) nm,多分散系数为0.24±0.01,Zeta电位为(-26.47±1.28)mV,MCN-I在盐酸溶液(pH 1.0)中2 h和磷酸盐缓冲液(pH 6.8)中6 h的累积释放量分别为(36.0±2.0)%和(70.3±3.6)%。(2)MCN-FI灌胃组和尾静脉注射FI组的心、肝、脾、肺、肾组织平均荧光强度与空白组比较,差异均有统计学意义(均P<0.0001),说明MCN可以保护FI通过口服吸收进入血液循环。(3)MCN-I灌胃给予糖尿病模型大鼠在2 h后开始缓慢降低血糖,8 h时达到最低血糖值,为初始血糖值的(28.5±3.6)%,具有缓释降糖效果。结论介孔碳纳米粒可保护胰岛素口服吸收进入血液循环发挥药效,有望开发成为一种多肽、蛋白质类药物口服递药的载体。

Objective To achieve oral sustained release drug delivery by encapsulating insulin mesoporous carbon nanoparticles(MCN).Methods(1)Preparation and characterization:Insulin-encapsulated mesoporous carbon nanoparticles(MCN-I)were prepared,and characterized by scanning electron microscope(SEM)and transmission electron microscope(TEM).The entrapment efficiency and drug loading were determined by high performance liquid chromatography(HPLC).The particle size,polydispersity coefficient and Zeta potential were determined to use laser particulate size analyzer.The in vitro release performance of MCN-I was investigated.(2)In vivo distribution experiment:SD rats were randomly divided into 4 groups according to weight:intragastric fluorescence labeled insulin(FI)group(10 mg·kg^(-1)),intragastric administration of mesoporous carbon nanoparticles encapsulated fluorescent labeled insulin(MCN-FI)group(10 mg·kg^(-1)),tail vein injection of FI group(10 mg·kg^(-1))and blank group(intragastric administration of 0.9%normal saline),with 3 rats in each group.Statistical average fluorescence intensity of rat heart,liver,spleen,lung,kidney and brain was observed by inverted fluorescence microscope.(3)Pharmacodynamic experiment in vivo:Diabetic model rats were randomly divided into 4 groups:subcutaneous insulin injection group(5 U·kg^(-1)),MCN-I gavage group(50 U·kg^(-1)),MCN gavage group(5 mg·kg^(-1))and insulin gavage group(50 U·kg^(-1)),with 6 rats in each group.The changes of blood glucose within 12 h were measured by the blood glucose meter.Results(1)MCN-I was successfully prepared.The entrapment efficiency was(65.18±1.59)%.While the drug loading was(20.19±1.18)%.The average particle size was(195.00±2.80)nm.The polydispersity coefficient was 0.24±0.01,and the zeta potential was(-26.47±1.28)mV.The cumulative release of MCN-I in pH 1.0 hydrochloric acid solution for 2 h was(36.0±2.0)%,and the cumulative release of MCN-I in pH 6.8 phosphate buffer for 6 h was(70.3±3.6)%.(2)The significant difference in the average fluorescence intensity of heart,liver,spleen,lung and kidney between MCN-FI intragastric administration group as well as tail vein injection FI group and that of the blank group(all P<0.0001),indicating that the absorption of oral-administrated FI into the blood circulation could be protected by MCN.(3)The blood glucose of diabetic rats began to decrease slowly after 2 h by intragastric administration of MCN-I,and reached the lowest value at 8 h,which was(28.5±3.6)%of the initial blood glucose value,showing a slow-release hypoglycemic effect.Conclusion MCN can protect the oral absorption of insulin into the blood circulation and exert its efficacy.It is expected to be developed as a carrier for oral administration of polypeptide and protein drugs.