摘要
目的制备混合胶束(PHM),考察其负载小干扰RNA(siRNA)的能力及所形成的复合物在树突状细胞(DC)上摄取、溶酶体逃逸及沉默的能力。方法采用溶剂注入法制备PHM,胶束亲水端带正电的聚乙烯亚胺与通过静电吸附作用复合siRNA形成载药胶束,考察载药胶束的粒径、多分散系数(PDI)和Zeta电位;通过核酸凝胶电泳确定PHM对siRNA的包载能力;考察复合物在小鼠树突状细胞系DC2.4上摄取、溶酶体逃逸和沉默的能力。结果PHM-siRNA的平均粒径为123.7±3.4 nm,PDI为0.212±0.052,Zeta电位为13.4±2.3 mV。核酸凝胶电泳显示:氮磷比(N/P)为8:1时,PHM能完全包载siRNA。制剂在DC2.4细胞上的摄取率为50.79%±5.71%(N/P为8:1)和95.22%±1.27%(N/P为16:1),选择N/P为16:1进行后续实验。共聚焦显微镜观察结果显示:制剂可以在给药后15 h从溶酶体中逃逸出来。实时荧光定量PCR检测到PHM-siRNA对目标基因的沉默效率为66.9%±8.8%。结论PHM-siRNA均一稳定,在DC2.4细胞上具有较高摄取、溶酶体逃逸及沉默的能力。
OBJECTIVE To prepare the polymeric hybrid micelle(PHM)and to investigate its ability to encapsulate small interfering RNA(siRNA)as well as the cellular uptake,lysosomal escape and silencing ability of the formed complex in dendritic cell(DC).METHODS Solvent injection method was used to prepare PHM,which were subsequently complexed with siRNA via electrostatic interaction.Particle size,polydispersity index(PDI),and Zeta potential were determined to characterize the formed PHM-siRNA complex.The encapsulation efficiency was determined by gel electrophoresis.The uptake ability,lysosomal escape ability and silencing ability of the complex were investigated on DC2.4 cells.RESULTS The average particle size of the PHM-siRNA was 123.7±3.4 nm,the PDI of 0.212±0.052 and the Zeta potential of 13.4±2.3 mV.Gel electrophoresis indicated that the siRNA was fully encapsulated by PHM at an N/P ratio of 8:1.50.79%±5.71%(N/P=8:1)and 95.22%±1.27%(N/P=16:1)of DC2.4 cells internalized PHM-siRNA within 2 hours,respectively.The N/P ratio of 16:1 was adopted for subsequent experiments.Confocal microscopy showed that PHM-siRNA escaped from the lysosome within 15 h after dosage.The silencing efficiency of PHM-siRNA determined by real-time fluorescent quantitative PCR was 66.9%±8.8%.CONCLUSION PHM-siRNA forms uniform micelles and shows high cellular uptake,lysosomal escape and silencing capacity in vitro.
作者
唐瑶
孙逊
TANG Yao;SUN Xun(West China School of Pharmacy,Sichuan University,Chengdu,Sichuan,610041 P.R.China)
出处
《华西药学杂志》
CAS
CSCD
2020年第6期595-598,共4页
West China Journal of Pharmaceutical Sciences
基金
国家自然科学基金资助项目(批准号:81872814、81673362)。
关键词
混合胶束
小干扰RNA
疫苗
树突状细胞
溶剂注入法
多分散系数
摄取能力
溶酶体逃逸能力
沉默能力
荧光定量
PHM
siRNA
Vaccine
Dendritic cell
Solvent injection method
Polydispersity index
Uptake ability
Lysosomal escape ability
Silencing ability
Real-time fluorescent quantitative
