Study on the Antibacterial Activity of New Silver-based Nanocomposites
Author:Liu Shi Ma
Supervisor:luo li qiang
Bacterial resistance caused by antibiotic abuse has become a major threat to human health and safety.The discovery and application of new antibacterial agents has become a hot spot.Silver nanoparticles(AgNPs)are favored for their broad-spectrum,high-efficiency and low-resistance antibacterial properties,as well as good biocompatibility and heat-resistance properties of synthetic processing.However,the disadvantages of silver nanoparticles,including easy agglomeration,dissociation,and oxidization in air,greatly limit their wide applications in biomedical,textile,water treatment and coatings.In order to overcome these shortcomings,two novel silver-based nanocomposites,namely graphene oxide-silver nanoparticles composites(GO-AgNPs)and Y-DNA-silver nanocluster complexes(Y-AgNCs),are presented in this thesis.Their physicochemical properties,antibacterial properties,biocompatibility and applications in wound healing were systematically studied.The main results are summarized as follows:(1)Preparation and characterization of GO-AgNPs composites and GO-AgNPs substrates.A modified Hummers’method was used to prepare graphene oxide(GO)suspension with good water dispersibility.Atomic force microscopy(AFM)showed that GO was a single-layer nanosheet with a thickness of about 1 nm and a lateral size in a range from hundreds of nanometers to three micrometers.Sodium borohydride was used as the reducing agent to synthesize GO-AgNPs by in-situ chemical reduction.Transmission electron microscopy(TEM)showed that the average particle size of AgNPs on the surface of GO-AgNPs was 7.06±2.54 nm.The glass surface was coated with GO-AgNPs film by a drop coating method,forming GO-AgNPs substrates.A series of glass substrates with different coverage of GO-AgNPs were prepared by adjusting the concentration of GO-AgNPs.(2)Anti-biofilm property of GO-AgNPs suspension.Pseudomonas aeruginosa is selected as the model to investigate the effect of GO-AgNPs suspension on the development and structure of the biofilm.After co-culture for 12 h,the inhibit effect of GO-AgNPs suspension on the formation of Pseudomonas aeruginosa biofilm was concentration-dependent.The higher the concentration of GO-AgNPs suspension was,the stronger the anti-biofilm capacity became.When the concentration of GO-AgNPs suspension was up to 25μg/mL,GO-AgNPs suspension can completely inhibit the formation of biofilm.Moreover,the antibiofilm effect of the GO-AgNPs suspension was superior to individual GO or AgNPs,which was derived from the synergistic effect of GO and AgNPs.In addition,there was a threshold concentration of GO-AgNPs suspension on the growth kinetics of the biofilm,i.e.,15μg/mL.When the concentration was lower than that,the GO-AgNPs suspension delayed the biofilm growth,and mature stage of biofilm after 24 h.When the concentration above that,the biofilm formation was completely inhibited.Meanwhile,we found that the low concentration of GO-AgNPs suspension hardly affected the total biomass of the mature biofilm,but affected the structure of the biofilm.Compared to control group,the biofilm exposure to GO-AgNPs at a low concentration possessed significantly reduced bacterial proliferation and enhanced extracellular polymeric substances(EPS).In summary,these results provided a simple method for regulating the biofilm formation and structure,and also proposed a new idea for preventing bacterial infection caused by biofilm and treating the sewage based on the characteristics of biofilm.(3)Antibiofilm property of GO-AgNPs substrate.When Pseudomonas aeruginosa was grown on GO-AgNPs substrate for 2 h,the number of the bacteria adhered to the substrate was obviously reduced by plate counting.In other words,GO-AgNPs substrate hindered the bacterial adhesion in the initial stage of biofilm development by killing the planktonic bacteria.The amount of GO-AgNPs adsorbed on the substrate surface directly affected the formation of the biofilm.The higher the adsorption amount was,the more difficult it was to form biofilm.When the adsorption amount of GO-AgNPs reached 20μg/cm~2,the formation of biofilm was prevented effectively.Surprisingly,we found that the GO-AgNPs substrate did not affect the growth and differentiation of mammalian cells,nor did they induce hemolysis,which demonstrated the good biocompatibility of GO-AgNPs substrate.The study illustrated the feasibility of GO-AgNPs to be used as a coating for conventional implant materials to inhibit bacterial infections in clinic.(4)Preparation and characterization of Y-AgNCs nanostructure and hydrogels.We constructed a Y-DNA nanostructure(Y-DNA)rich in cytosine(C)rings and sticky ends,based on the programmable and precise self-assembly properties of DNA molecules.Due to the high affinity between silver ions(Ag~+)and cytosine,silver nanoclusters(AgNCs)was prepared by in-situ reduction at the C-ring of Y-DNA,which led to the formation of Y-AgNCs nanostructures.UV-Vis spectra showed that Y-AgNCs nanostructures had the characteristic absorption peak of AgNCs at 408 nm.Fluorescence analysis showed that Y-AgNCs nanostructures glowed red fluorescence,and the excitation and emission wavelengths were 565 nm and 631 nm,respectively.The sticky ends of the Y-DNA nanostructure recognize its complementary sequence,forming the DNA-AgNCs hydrogel.The rheological analysis showed that the storage modulus(G’)of the 250μM DNA-AgNCs hydrogel was greater than its loss modulus(G’’),which was consistent with the hydrogel characteristics.SEM images showed that DNA-AgNCs hydrogel had a typical three-dimensional network structure of hydrogel.In summary,we successfully prepared Y-AgNCs structure and DNA-AgNCs hydrogel.(5)Biological effects of Y-AgNCs nanostructure and hydrogels.The Y-AgNCs nanostructure had a good antibacterial activity against Gram-negative(Escherichia coli)and positive bacteria(Bacillus subtilis).When the concentration was 200 nM,the antibacterial efficiency is close to 100%.Moreover,this Y-AgNCs nanostructures had no significant cytotoxicity against mouse embryonic fibroblasts(NIH-3T3).In the co-culture experiments of bacteria and cells,Y-AgNCs nanostructures maintained an excellent antibacterial property and good biocompatibility.In addition,Y-AgNCs nanostructures effectively promoted the wound healing of mice,which could be used as a potential wound healing spray.DNA-AgNCs hydrogel maintained the broad-spectrum antibacterial property of Y-AgNCs nanostructures,killing Gram-negative bacteria and positive bacteria significantly,no matter the bacteria were on the surface or inside the hydrogel.When Y-AgNCs nanostructures concentration was 200 nM,the antibacterial efficiency of the hydrogel to both bacteria was close to 100%.DNA-AgNCs hydrogel could also be used as a cell culture substrate.The cells were distributed in the three-dimensional hydrogel and grow well.Therefore,DNA-AgNCs hydrogel showed great potential for application in tissue engineering.In summary,two new antibacterial nanomaterials including GO-AgNPs and Y-AgNCs were prepared in my thesis.The study showed that they had excellent antibacterial property and good biocompatibility,which resulted in the broad application in biomedicine.Additionally,these findings can not only broaden our understanding of silver-based antibacterial nanomaterials,but also provide an insight into the potential applications of silver-based materials in other fields.