Synthesis And Antibacterial Activity And Mechanisms Of Iturin-AgNPs

Drug resistant bacteria and fungal infections have become an important cause of human death in clinical cases.It is of great medical significance to develop new drugs with broad antibacterial spectrum and strong inhibition of drug-resistant bacteria.Iturin has excellent antifungal activity but weak antibacterial activity.AgNPs（silver nanoparticles）have good antibacterial activity but weak antifungal activity.Moreover,AgNPs can induce bacterial resistance.In order to develop a broad antibacterial spectrum drug that can inhibit both fungi and drug-resistant bacteria,iturin was used to synthesize AgNPs by reducing Ag⁺.The mechanisms for the synthesis of iturin-AgNPs was revealed.The capability of iturin-AgNPs to inhibit a variety of fungi and antibiotic resistant bacteria were investigated,as well as its mechanisms.The molecular mechanisms for iturin-AgNPs to inhibit chemical synthesized AgNPs resistant bacteria were also revealed.Iturin-AgNPs was also used to prepare chitosan hydrogel dressings and used to treat Escherichia coli infected wounds in mice.The obtained main results are as follows:（1）Preparation and characterization of iturin-AgNPs.The effects of different cyclic lipopeptide produced by Bacillus subtilis in the synthesis of AgNPs under ultraviolet irradiation were investigated.It was found that iturin was the main component for efficient synthesis and stable existence of AgNPs.The iturin-AgNPs synthesized under different conditions was investigated,and it was found that the optimal condition was p H9.8,UV_365nm,and AgNO₃ concentration of 100μg/m L.Under these conditions,the iturin-AgNPs showed good lattice structure,with spherical shape and size of 10-30 nm.The outer surface of the AgNPs was wrapped by iturin,and the surface charge was-37 mv,indicating iturin-AgNPs had good stability.（2）Mechanisms for the synthesis of iturin-AgNPs.To explore the role of composition of the iturin in the synthesis process,a chain peptide with the same amino acid composition as iturin（CP）was used as the comparison.It was found that the tyrosine residues,amino groups,and fatty acid chain are the main factors involved in the synthesis of AgNPs.The ring structure and fatty acid chain of iturin play important roles in the speedup of iturin-AgNPs by iturin.Both of the synthesis process of iturin-AgNPs and CP-AgNPs fitted the second-order kinetic model.The synthesis process was identified as the following.Under ultraviolet irradiation,the dissolved oxygen reacts with the phenolic hydroxyl group of iturin and generates superoxide anion radicals（O₂.^-）,and then reduce Ag⁺to Ag,and finally form AgNPs.It was also found that the formed iturin-AgNPs and CP-AgNPs were mainly spherical and wrapped by iturin and CP,respectively.Compared with CP-AgNPs,iturin-AgNPs has better stability and lower activation energy for the synthesis process（19.710 k J/mol vs 47.382 k J/mol）.（3）The antimicrobial activity of iturin-AgNPs and its mechanisms.Compared with the chemically synthesized AgNPs（PVP-AgNPs）and iturin,iturin-AgNPs showed higher activity to inhibit Candida albicans,Escherichia coli,Staphylococcus aureus,and other tested fungi and bacteria.The form of iturin-AgNPs effectively improved the antibacterial activity of iturin and the antifungal activity of AgNPs.The mechanisms were identified as the following:iturin–AgNPs enter the bacteria and fungi cell within 1 h,induce the accumulation of active oxygen,destroy the cell structure,and cause a large amount of content to leak from the bacteria and fungi cells.（4）The capability and mechanisms of iturin-AgNPs to inhibit chemical AgNPs resistant E.coli..Compared with chemically synthesized AgNPs（PVP-AgNPs）,iturin-AgNPs had good antibacterial activity against E.coli that have developed drug resistance to PVP-AgNPs and caused less drug resistance.The E.coli develop resistant capability to PVP-AgNPs by up-regulating the expression of flagellin,accumulating AgNPs outside the cell,and reducing the energy metabolisms in bacteria cells.However,iturin-AgNPs could decrease the secretion of flagellin in AgNPs-resistant bacteria and enhance the energy metabolism of bacteria,thereby inhibit the PVP-AgNPs resistant E.coli.（5）Preparation of iturin-AgNPs based chitosan dressing and its application in wound healing.A chitosan dressing loaded with iturin-AgNPs was prepared and used to test its antibacterial activity against E.coli in vitro and in vivo on wound infection mice model.As results,the prepared chitosan dressing loaded with iturin-AgNPs showed the good bacterial inhibitory effect on E.coli in vitro and efficient promotion of wound healing in speed.The application of the chitosan dressing loaded with iturin-AgNPs did not cause damage and Ag residue in mice organs.Compared with the current reports,this study has novelty in the following aspects:it established an efficient system to synthesize iturin-AgNPs,and revealed its mechanism at the molecular level;enhanced the antibacterial activity of iturin and the antifungal activity of AgNPs via the synthesis of iturin AgNPs;revealed the inhibitory effect and mechanisms of iturin-AgNPs against Candida albicans,bacteria,and the chemosynthetic AgNPs resistant E.coli.The study provides important theoretical support for the development of new,efficient,and broad-spectrum antifungal and antibacterial drugs,especially the new antibacterial agents which can effectively inhibit multi-antibiotic resistant bacteria and chemically synthesized AgNPs resistant bacteria.In addition,the results of the application of chitosan-dressing with iturin-AgNPs in wound healing of mice provides practical basis for the development of clinical medical materials.