| Antibiotics are commonly used to treat bacterial infections,but the misuse and abuse of antibiotics have given rise to a severe problem of the drug resistance of bacteria.Solving this problem has been a vitally important task in the modern medical arena.Antibacterial peptide(AMPs)has become a promising candidate drug to replace antibiotics because of their broad-spectrum antibacterial activity and their difficulty in making bacteria resistant.However,its wider clinical application is limited by the shortcomings of high cytotoxicity and low antibacterial efficiency.In this paper,we constructed an antibacterial peptide(Cu-GGH-KKLRKIAFK,abbreviated as Cu-GGH-AMP)with a DNA cleavage function.The peptide has two functional regions,the C-terminal antibacterial peptide Pa DBS1R6F10(KKLRLKIAFK)and the N-terminal Cu-GGH complex.Pa DBS1R6F10 is a unique antibacterial peptide,which shows a lower tendency to produce bacterial resistance than traditional antibiotics.Cu-GGG complexes are formed by chelating Cu with the classical amino terminal Cu(II)-and Ni(II)-Binding(ATCUN)motif GGH.In the presence of ascorbic acid,Cu-GGH can efficiently catalyze the oxidative cleavage of bacterial DNA,thus playing a synergistic antibacterial role with antibacterial peptides.This work mainly includes the following aspects:1.Synthesis and characterization of Cu-GGH-AMP.First,the three required peptides are synthesized by solid-phase synthesis.The peptides were validated by mass spectrometry(MS)and then purified using high-performance liquid chromatography(HPLC)to produce peptides with a purity of more than 85%.Cu-GGH-AMP was prepared by the classical method of ATCUN motif metal ion complexes.The charge transfer(MLCT)from metal to ligand was observed by UV-vis absorption spectra,indicating the successful synthesis of Cu-GGH-AMP.The resulting Cu-GGH-AMP can act not only on bacterial cell membranes but also on bacterial nucleic acid,which has strong bactericidal performance and good biocompatibility.2.Antibacterial effect of Cu-GGH-AMP.Firstly,ROS production was detected by fluorescence experiment,and then by agarose gel electrophoresis experiment,it was proved that Cu-GGH-AMP can cause DNA damage.Then,Cu-GGH-AMP was incubated with gram-positive bacteria(Staphylococcus aureus and Bacillus subtilis)and Gram-negative bacteria(Escherichia coli).Through agarose-gel electrophoresis experiments,coated plates,and live/dead fluorescence staining of bacteria,it is proved that Cu-GGH-AMP can cause DNA damage and has strong broad-spectrum bactericidal properties.3.Cu-GGH-AMP in the treatment of living wound infection.To establish a mouse model of skin wound infection and evaluate the therapeutic effect of Cu-GGH-AMP on wound infection.The experimental results showed that Cu-GGH-AMP could effectively prevent wound suppuration and promote wound healing in mice.H&E staining,Masson three-color staining,and immunohistochemical experiments have again demonstrated that Cu-GGH-AMP provides a healing-promoting microenvironment for wound treatment and eradication of bacterial infections.In conclusion,functional antimicrobial peptides have good antibacterial and anti-skin infection abilities,as well as the activity of promoting wound healing,which provides a new way for the treatment of bacterial infection and has a wide application prospect in biomedical materials. |
