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

Effects of ferrocene position isomerization on the self-assembly behavior and antibacterial effect of coupled cationic peptides

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(C_(8))FFHK、C_(8)-K(Fc)FFHK]以及对照多肽[C_(8)-K(C_(8))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(C_(8))FFHK、C_(8)-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(C_(8))FFHK、C_(8)-K(Fc)FFHK分别自组装成扭曲纳米带和短纳米纤维结构,C_(8)-K(C_(8))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(C_(8))FFHK在pH6.0时的ROS生成效率高于C_(8)-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50μg/mL,远低于对照多肽的MIC值(400μg/mL);对于大肠杆菌,Fc-K(C_(8))FFHK的抗菌活性优于C_(8)-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。

OBJECTIVE To design the two isomers of ferrocene(Fc)-coupled cationic peptides(hereinafter referred to as“peptides”)[Fc-K(C_(8))FFHK and C_(8)-K(Fc)FFHK]and the control peptide[C_(8)-K(C_(8))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(C_(8))FFHK and C_(8)-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(C_(8))FFHK and C_(8)-K(Fc)FFHK were self-assembled to form twisted nanoribbons and short nanofibers,respectively;C_(8)-K(C_(8))FFHK was assembled into cylindrical nanofibers.The optical spectrum results showed that there were certain differences in the content of structures such asβ-sheet andα-helix.The in vitro ROS generation test results showed that ROS generation efficiency of Fc-K(C_(8))FFHK at pH 6.0 was higher than C_(8)-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 50μg/mL which were far lower than the control peptide(400μg/mL).To Escherichia coli,Fc-K(C_(8))FFHK had better antibacterial activity than C_(8)-K(Fc)FFHK.Finally,cytotoxicity assay and hemolysis test results showed that both isomeric peptides had good biocompatibility.CONCLUSIONS By coupling Fc,the antibacterial activity of cationic selfassembled peptides can be improved.Regulating the position of Fc in the peptide sequence could regulate the self-assembly behavior and antibacterial effect of the self-assembled peptides.