Page 56 - 《中国药房》2023年18期

二茂铁位置异构对偶联阳离子多肽自组装行为及其抗菌效果的

影响

1＊
2 #
李奕捷 ，王婧雯 （1.上海交通大学药学院，上海 201100；2.空军军医大学西京医院药剂科，西安 710038）
中图分类号 R978.1+6 文献标志码 A 文章编号 1001-0408（2023）18-2226-07
DOI 10.6039/j.issn.1001-0408.2023.18.10

摘 要 目的 设计二茂铁（Fc）偶联阳离子多肽（以下简称“多肽”）的2种同分异构多肽[Fc-K（C8 ）FFHK、C8-K（Fc）FFHK]以及对
照多肽[C8-K（C8 ）FFHK]，探究Fc位置异构对多肽自组装行为及其抗菌效果的影响。方法 采用标准固相合成法合成位置异构的
多肽，采用反相高效液相色谱法进行纯化；采用紫外可见分光光度法检测紫外吸收光谱，Zeta电位分析仪测定Zeta电位以分析多
肽的稳定性；采用圆二色光谱（CD）、傅里叶红外光谱（FTIR）、硫代黄素T（ThT）荧光光谱、透射电子显微镜（TEM）等进行二级结
构表征；通过体外活性氧（ROS）生成效率实验、生长曲线法测定实验、平板法实验、细胞毒性实验和溶血性实验评价2种同分异构
多肽在抗菌活性和生物相容性上的差异。结果 合成了3种纯度均高于95%的多肽。稳定性实验结果显示，室温下放置24、96 h
时，Fc-K（C8 ）FFHK、C8-K（Fc）FFHK的紫外吸收图谱几乎没有改变，二者的Zeta电位分别减少了0.3、0.5 mV。二级结构表征结果
显示，Fc-K（C8 ）FFHK、C8-K（Fc）FFHK分别自组装成扭曲纳米带和短纳米纤维结构，C8-K（C8 ）FFHK自组装成圆柱状纳米纤维结
构；光学图谱结果显示，3种多肽的β-折叠、α-螺旋等结构含量具有一定差异。体外ROS生成实验结果显示，Fc-K（C8 ）FFHK在pH
6.0时的ROS生成效率高于C8-K（Fc）FFHK。体外抗菌活性结果显示，对于耐甲氧西林金黄色葡萄球菌，2种同分异构多肽的最低
抑菌浓度（MIC）值均为 50 μg/mL，远低于对照多肽的 MIC 值（400 μg/mL）；对于大肠杆菌，Fc-K（C8）FFHK 的抗菌活性优于
C8-K（Fc）FFHK。细胞毒性实验和溶血性实验均结果显示，2种同分异构多肽均具有较好的生物相容性。结论 通过偶联Fc，能够
提高多肽的抗菌活性；调节Fc在多肽序列中的位置，可调控多肽的自组装行为和抗菌效果。
关键词 抗菌肽；自组装；同分异构；芬顿反应

Effects of ferrocene position isomerization on the self-assembly behavior and antibacterial effect of coupled
cationic peptides
2
LI Yijie ，WANG Jingwen（1. School of Pharmacy， Shanghai Jiaotong University， Shanghai 201100， China；
1
2. Dept. of Pharmacy， Xijing Hospital， Air Force Medical University， Xi’an 710038， China）
ABSTRACT OBJECTIVE To design the two isomers of ferrocene （Fc）-coupled cationic peptides （hereinafter referred to as
“peptides”） [Fc-K（C8 ）FFHK and C8-K（Fc） FFHK] and the control peptide [C8-K（C8 ）FFHK]， and to explore the effects of Fc
position isomerization on the self-assembly behavior and antibacterial effect of peptides. METHODS All isomerized peptides were
prepared by standard solid-phase synthesis and purified by reversed-phase high-performance liquid chromatography. The stability of
the peptide was analyzed by using UV spectrophotometry to detect UV absorption spectra， and Zeta potential analyzer to determine
Zeta potential. The secondary structure was characterized by circular dichroism spectrum （CD）， Fourier transform infrared
spectrometer （FTIR）， thioflavin T （ThT） fluorescence spectrum and transmission electron microscopy （TEM）. The differences in
antibacterial activity and biocompatibility of the 2 kinds of isomerized peptides were evaluated by in vitro reactive oxygen species
（ROS） generation test， growth curve determination test， plate method， cytotoxicity assay and hemolysis test. RESULTS Three
peptides with purity higher than 95% were synthesized. The stability test results showed that the UV absorption spectra of
Fc-K（C8 ）FFHK and C8-K（Fc）FFHK remained almost unchanged when placed at room temperature for 24 and 96 hours， and their
Zeta potential were decreased by 0.3 mV and 0.5 mV， respectively. Secondary structure characterization results showed that
Fc-K（C8）FFHK and C8-K（Fc）FFHK were self-assembled to form twisted nanoribbons and short nanofibers， respectively；
C8-K（C8）FFHK was assembled into cylindrical nanofibers. The optical spectrum results showed that there were certain differences
in the content of structures such as β-sheet and α-helix. The in vitro ROS generation test results showed that ROS generation
efficiency of Fc-K（C8 ）FFHK at pH 6.0 was higher than C8-K（Fc）FFHK. The results of in vitro antibacterial activity showed that
for methicillin-resistant Staphylococcus aureus， both the isomeric peptides had similar minimum inhibitory concentration （MIC）
values of 50 μg/mL which were far lower than the control peptide （400 μg/mL）. To Escherichia coli， Fc-K（C8 ）FFHK had better
antibacterial activity than C8-K（Fc）FFHK. Finally， cytotoxicity
＊第一作者 硕士研究生。研究方向：自组装多肽的设计。E-
assay and hemolysis test results showed that both isomeric
mail：936640537@qq.com
# 通信作者 主任药师，硕士。研究方向：临床药物研究。E-mail： peptides had good biocompatibility. CONCLUSIONS By
wangjingwen8021@163.com coupling Fc， the antibacterial activity of cationic self-

· 2226 · China Pharmacy 2023 Vol. 34 No. 18 中国药房 2023年第34卷第18期