| Over the past few decades,the overuse of antibiotics has led to the emergence of drug-resistant bacteria and even environmental problems.The alarming growth of antibiotic resistance has developed into a serious global health problem,and the development of new drugs that can replace antibiotics is urgent.Both silver nanoparticles(Ag NPs)and antimicrobial peptides(AMPs)have the potential to replace antibiotics,and are recognized as new materials that are expected to replace antibiotics in recent years.Although both Ag NPs and AMPs have antibacterial activities,their mechanisms of action are significantly different.Therefore,if Ag NPs can be combined with AMPs,more excellent antibacterial activity may be obtained.In addition,the polypeptide itself can produce a certain reducibility through sequence design,and can generate a covalent bond-like binding force with precious metals,so through sequence design,the polypeptide can even directly wrap Ag NPs,which is expected to construct a synergistic bactericidal property composite nanostructures.Taking this as a starting point,the present study systematically investigated the synthesis,preparation and antibacterial properties of composite nanostructures with AMPs and Ag NPs as two components,with special focus on synergistic antibacterial activity,reduced cytotoxicity,and enhanced antibacterial properties under physiological conditions,stability,etc.To this end,we first designed and synthesized an antimicrobial peptide composed of 13 amino acids(hereinafter referred to as P-13).P-13 has two fimctional domains: one fiinctional domain mainly has antibacterial activity,while the other fimctional domain adds cysteine(C)residues to wrap and stabilize Ag NPs.Then,we successfully synthesized P-13-encapsulated peptide-silver composite nanostructures(P-13@Ag NPs)via a one-pot reaction.Through a series of physical and chemical properties characterization,we concluded that the average dynamic particle size of the synthesized peptide-silver composite nanostructures is about 11 nm,and has excellent water solubility and stability under physiological conditions.The results of cell experiments showed that the cytotoxicity of Ag NPs was significantly reduced after P-13 protection.Further analysis showed that the peptides increased the interaction distance between silver nanoparticles and cells,and the biocompatibility of the peptides played a key role.Meanwhile,P-13 not only endowed Ag NPs with a positively charged surface potential,thereby enhancing the electrostatic adsorption between P-13@Ag NPs and negatively charged bacteria,but also the antibacterial activity of P-13 itself accelerated the bacterial wall/membrane degradation,enhanced the accumulation and antibacterial properties of Ag NPs in bacteria;on the other hand,the nano-silver particles increased the size of the antibacterial peptide itself,promoted the endocytosis of cells,and finally showed a stronger synergistic antibacterial effect.The results of antibacterial experiments showed that the average minimum inhibitory concentration of P-13@Ag NPs against Gram-positive bacteria such as Staphylococcus aureus and Bacillus pumilus was about 7.8μg/m L,while the average antibacterial concentration against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa was about 15.6 μg/m L and 7.8 μg/m L.Using flow cytometry,the mortality rates of P-13@Ag NPs against Escherichia coli,Staphylococcus aureus,Pseudomonas aeruginosa and Bacillus pumilus were 96%,96%,91% and 90%,respectively.The research results of this work demonstrate the application potential of the synergistic effect of two antibacterial materials in the construction of novel antibacterial materials.Polypeptides,as the building blocks of life functional structures,not only have excellent biocompatibility,but also have sequence-dependent function tunability,which also provides a theoretical and experimental basis for the development of composite nanomaterials wrapped by novel biologically active polypeptide molecules. |
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