H7K(R2)2修饰的pH敏感脂质体对U87-MG细胞的靶向作用

doi:10.5246/jcps.2015.10.084

中国药学(英文版) ›› 2015, Vol. 24 ›› Issue (10): 660-665.DOI: 10.5246/jcps.2015.10.084

H7K(R2)2修饰的pH敏感脂质体对U87-MG细胞的靶向作用

赵阳¹, 任伟¹, 钟婷¹, 王超¹, 张卫强¹, 黄丹¹, 张爽¹, 郭阳¹, 姚鑫¹, 张烜^1*, 张强^1,2

1. 北京大学医学部药学院分子药剂学与新释药系统北京市重点实验室, 北京 100191
2. 北京大学医学部天然药物及仿生药物国家重点实验室, 北京 100191

收稿日期:2015-05-22 修回日期:2015-06-22 出版日期:2015-10-21 发布日期:2015-07-12
通讯作者: Tel./Fax: 86-10-82805928, E-mail: xuanzhang@bjmu.edu.cn
基金资助:
Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20120001110012) and the National Basic Research Program of China (973 Program, Grant No. 2013CB932501) and National Natural Science Foundation of China (Grant No. 81172992).

The targeting effect of H7K(R2)2-modified pH-sensitive liposomes on U87-MG cells

Yang Zhao¹, Wei Ren¹, Ting Zhong¹, Chao Wang¹, Weiqiang Zhang¹, Dan Huang¹, Shuang Zhang¹, Yang Guo¹, Xin Yao¹, Xuan Zhang^1*, Qiang Zhang^1,2

1. Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
2. State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China

Received:2015-05-22 Revised:2015-06-22 Online:2015-10-21 Published:2015-07-12
Contact: Tel./Fax: 86-10-82805928, E-mail: xuanzhang@bjmu.edu.cn
Supported by:
Specialized Research Fund for the Doctoral Program of Higher Education (Grant No. 20120001110012) and the National Basic Research Program of China (973 Program, Grant No. 2013CB932501) and National Natural Science Foundation of China (Grant No. 81172992).

摘要/Abstract

摘要：

为了探讨H₇K(R₂)₂修饰的pH敏感脂质体对U87-MG细胞的靶向作用, 本文选择香豆素-6(coumarin-6)为荧光探针构建了包载香豆素-6的H₇K(R₂)₂修饰的pH敏感脂质体(coumarin-6-PSL-H₇K(R₂)₂)。采用流式实验分别对H₇K(R₂)₂在coumarin-6-PSL-H₇K(R₂)₂中的比例以及coumarin-6-PSL-H₇K(R₂)₂在U87-MG细胞的摄取途径进行研究。采用CD技术对H₇K(R₂)₂在pH 7.4和pH 6.8条件下的二级结构进行考察。实验结果显示, H₇K(R₂)₂在coumarin-6-PSL-H₇K(R₂)₂中占2.5%时, 其靶向作用优于1%和3.5%。细胞摄取途径实验表明, coumarin-6-PSL-H₇K(R₂)₂进入U87-MG细胞不受菲律平、甲基-β-CD和氯丙嗪等抑制剂的影响。H₇K(R₂)₂在pH 6.8时的二级结构多为β-turn。本研究结果表明, 在H₇K(R₂)₂修饰的pH敏感脂质体中, H₇K(R₂)₂的最佳比例可确定为2.5%。H₇K(R₂)₂修饰的pH敏感脂质体进入肿瘤细胞的途径主要通过低pH条件下H₇K(R₂)₂所产生的穿膜肽样作用。H₇K(R₂)₂在pH 6.8条件下所出现的发卡样二级结构是其产生靶向和穿透作用的可能机制。

关键词: H7K(R2)2, pH敏感脂质体, 细胞摄取途径, 二级结构, U87-MG细胞

Abstract:

The present study aimed to investigate the targeting effect of H₇K(R₂)₂-modified pH-sensitive liposomes on U87-MG cells. Using coumarin-6 as a fluorescence probe, we prepared H₇K(R₂)₂-modified pH-sensitive liposomes (designated as coumarin-6-PSL-H₇K(R₂)₂). The flow cytometry assay was used to evaluate the effect of H₇K(R₂)₂proportions on the cellular uptake and endocytosis pathways of coumarin-6-PSL-H₇K(R₂)₂ on U87-MG cells_.The circular dichroism (CD) spectroscopy assay was used to investigate the secondary structures of H₇K(R₂)₂ peptide at pH 7.4 and pH 6.8, respectively. Our results indicated that the 2.5% proportion of H₇K(R₂)₂ in the coumarin-6-PSL-H₇K(R₂)₂ was superior to those of 1% and 3.5% of H₇K(R₂)₂. The uptake of coumarin-6-PSL-H₇K(R₂)₂ on U87-MG cells was not inhibited by filipin, M-β-CD or chlorpromazine. The secondary structure of H₇K(R₂)₂at pH 6.8 was mostly presented as β-turn. In conclusion, we suggested that the appropriate proportion of H₇K(R₂)₂ in the H₇K(R₂)₂-modified pH-sensitive liposomes could be set at 2.5%. The cellular uptake pathway for H₇K(R₂)₂-modified pH-sensitive liposomes was via the cell penetrating capacity of H₇K(R₂)₂ which responded to acidic condition. The secondary structure of H₇K(R₂)₂at pH 6.8, which was presented as the shape of hairpin, might be mainly responsible for its targeting and cell penetrating effect.

Key words: H7K(R2)2-modified, pH-sensitive liposomes, Cellular uptake pathway, Secondary structure, U87-MG cells

中图分类号:

R945

Supporting:

赵阳, 任伟, 钟婷, 王超, 张卫强, 黄丹, 张爽, 郭阳, 姚鑫, 张烜, 张强. H7K(R2)2修饰的pH敏感脂质体对U87-MG细胞的靶向作用[J]. 中国药学(英文版), 2015, 24(10): 660-665.

Yang Zhao, Wei Ren, Ting Zhong, Chao Wang, Weiqiang Zhang, Dan Huang, Shuang Zhang, Yang Guo, Xin Yao, Xuan Zhang, Qiang Zhang. The targeting effect of H7K(R2)2-modified pH-sensitive liposomes on U87-MG cells[J]. Journal of Chinese Pharmaceutical Sciences, 2015, 24(10): 660-665.

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H7K(R2)2修饰的pH敏感脂质体对U87-MG细胞的靶向作用

The targeting effect of H7K(R2)2-modified pH-sensitive liposomes on U87-MG cells

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