文章摘要
张 钟,黄彦虎,隆 梅,张 攀,陆 琴,方 超.用于核酸递送的GSH响应型纳米粒构建[J].,2022,(6):1001-1006
用于核酸递送的GSH响应型纳米粒构建
Construction of GSH Responsive Nanoparticles for Nucleic Acid Delivery
投稿时间:2021-08-31  修订日期:2021-09-28
DOI:10.13241/j.cnki.pmb.2022.06.001
中文关键词: 核酸递送  介孔硅  GSH响应  MDA-MB-231细胞
英文关键词: Nucleic acid delivery  Mesoporous silica  GSH response  MDA-MB-231 cells
基金项目:国家自然科学基金项目(81773274;82073379)
作者单位E-mail
张 钟 上海交通大学医学院药理学与化学生物学系 上海200025 zhonga977@163.com 
黄彦虎 上海交通大学医学院药理学与化学生物学系 上海200025  
隆 梅 上海交通大学医学院药理学与化学生物学系 上海200025  
张 攀 上海交通大学医学院药理学与化学生物学系 上海200025  
陆 琴 上海交通大学医学院药理学与化学生物学系 上海200025  
方 超 上海交通大学医学院药理学与化学生物学系 上海200025  
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中文摘要:
      摘要 目的:核酸治疗近年来越来越受到关注,但是核酸药物易被快速清除、易被核酸酶降解、非特异性生物分布、以及不易被细胞摄取的缺点使其在体内难以发挥效果。本文提供了一种具有谷胱甘肽(GSH)响应性释放的纳米粒,能够进行有效核酸药物递送。方法:使用十六烷基三甲基氯化铵(CTAC)制备介孔硅纳米粒,在介孔硅纳米粒表面进行巯基修饰并活化,使其与巯基修饰的聚丙烯亚胺和聚乙二醇反应,形成具有GSH响应的介孔硅纳米粒,通过静电吸附进行核酸荷载。马尔文粒度仪测量表面电位、粒径,透射电镜观察纳米粒形态。核酸电泳检测其核酸负载效率,通过体外检测GSH响应释放聚乙烯亚胺(PEI)情况,共聚焦显微镜观察细胞摄取以及溶酶体逃逸情况。结果:成功构建了具有GSH响应的纳米粒,粒径为76.44±1.68 nm,表面电位为33.93±0.59 mV;通过透射电镜观察到纳米粒呈圆形带孔颗粒状;琼脂糖核酸负载试验观察到当氮磷比大于20时,能够有效进行核酸负载。共聚焦显微镜显示该纳米粒能够成功被MDA-MB-231乳腺癌细胞摄取。在溶酶体逃逸试验中观察到纳米粒进入细胞后3 h,Cy5-siRNA与溶酶体的荧光分离,证明构建的纳米粒成功从溶酶体逃脱。结论:成功构建了具有GSH响应的介孔硅纳米粒,能够有效用于核酸递送。
英文摘要:
      ABSTRACT Objective: Nucleic acid therapy has received more and more attention in recent years, but nucleic acid drugs are easily cleared quickly, easily degraded by nucleases, non-specific biodistribution and not easily taken up by cells, making it difficult to exert effects in the body. This article provides glutathione(GSH) responsive release nanoparticles capable of effective nucleic acid drug delivery. Methods: Use hexadecyltrimethylammonium chloride(CTAC) to prepare mesoporous silicon nanoparticles, modify and activate sulfhydryl groups on the surface of mesoporous silicon nanoparticles, and make them react with sulfhydryl modified polypropylene imine and polyethylene glycol to form mesoporous silicon nanoparticles with GSH response. The particles are loaded with nucleic acid by electrostatic adsorption. The Malvern particle size analyzer measures the surface potential and particle size, and observes the morphology of the nanoparticles with a transmission electron microscope. Nucleic acid electrophoresis is used to detect the nucleic acid loading efficiency, the GSH response to the release of PEI is detected in vitro, and the cell uptake and lysosome escape are observed by confocal microscope. Results: The nanoparticles with GSH response were successfully constructed, with a particle size of 76.44±1.68 nm and a surface potential of 33.93±0.59 mV. The nanoparticles were observed to be round and porous particles through transmission electron microscopy. The agarose nucleic acid loading experiment demonstrated when the ratio of nitrogen to phosphorus is greater than 20, nucleic acid can be loaded efficiently. Confocal microscopy showed that the nanoparticles could be successfully taken up by MDA-MB-231 breast cancer cells. In the lysosomal escape experiment, it was observed that the fluorescence of Cy5-siRNA separated from the fluorescence of the lysosome after 3 h entering the cell, which proved the established nanoparticles successful escaped from the lysosome. Conclusion: The mesoporous silicon nanoparticles with GSH response have been successfully constructed, which can be effectively used for nucleic acid delivery.
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