摘要
通过超速离心法提取大鼠血浆外泌体(plasma exosomes,Pla-Exos),150000×g,离心2.5 h。比较超速离心法、超滤法、磁珠捕获法对Pla-Exos的纯化效果。使用超声共孵育的方法制备载葛根素外泌体(plasma exosomes carrying puerarin,Pue-Exos),以包封率为指标采用响应面法筛选最优处方并测定表征、评估稳定性和体外释放情况。纯化的Pla-Exos和Pue-Exos的粒径均在30~150 nm内;蛋白质免疫印迹法结果显示,纯化的Pla-Exos和Pue-Exos均含有标志蛋白TSG101、CD63、CD81。透射电镜观察纯化后的Pla-Exos和Pue-Exos均具有良好的双层膜囊泡结构。Pue-Exos制备的最优处方为孵育1 h,超声功率39 W,载药比10∶1。制备得到的Pue-Exos有较好的稳定性和缓释作用,提高了葛根素体外血脑屏障的透过效率(P<0.01)。本研究经河北北方学院实验动物伦理审查委员会批准,伦理审批号:HBNV202307012103。
Plasma exosomes(Pla-Exos)were extracted from rats by ultracentrifugation.The ultracentrifuga‐tion method extracted rat Pla-Exos at a speed of 150000×g for 2.5 h.Various purification methods including ultracentrifugation,magnetic bead capture method,and ultrafiltration would be employed to purify Pla-Exos.The Pue-Exos were prepared via sonication-assisted and co-incubation methods.The influence factors and levels of the preparation of plasma exosomes carrying puerarin(Pue-Exos)were investigated by RSM plus CCD method with encapsulation rate as an index.Then the characterization,the stability,and in vitro release of Pue-Exos were determined.The particle sizes of exosomes purified by ultracentrifugation,ultrafiltration,or magnetic bead capture method were all within the range of 30−150 nm.The Western blot results showed that purified Pla-Exos and PueExos contained marker proteins TSG101,CD63,and CD81.The TEM showed that purified Pla-Exos and Pue-Exos exhibited well-defined double-layered membrane vesicle structures.The optimal prescription conditions for preparing Pue-Exos were finally determined as follows:1 h ultrasound time,39 W ultrasound power,mass ratio of 10∶1.The prepared Pue-Exos exhibit good stability and sustained release,significantly enhancing the in vitro transpermeability of puerarin across the blood-brain barrier(P<0.01).This study was approved by the Experimental Animal Ethics Review Committee of Hebei North University,and ethics approval number was HBNV202307012103.
作者
张奇
郝路格
张鑫
张胜霄
张健东
赵振奥
李炜
ZHANG Qi;HAO Lu-ge;ZHANG Xin;ZHANG Sheng-xiao;ZHANG jian-dong;ZHAO Zhen-ao;LI Wei(Department of Pharmacy,Hebei North University,Zhangjiakou 075000,China;Hebei Key Laboratory of Neuropharmacology,Zhangjiakou 075000,China;Institute of Microcirculation,Hebei North University,Zhangjiakou 075000,China)
出处
《药学学报》
北大核心
2025年第5期1534-1542,共9页
Acta Pharmaceutica Sinica
基金
国家自然科学基金资助项目(82370508)
河北省自然科学基金资助项目(H2020405298)。
关键词
葛根素
血浆外泌体
超速离心法
磁珠捕获法
血脑屏障
puerarin
plasma exosome
ultracentrifugation
magnetic bead capture method
blood-brain barrier
