| Objective In this study,zinc was selected as the raw material of antibacterial agent,which presented broad antimicrobial spectrum and low cytotoxicity,attapulgite-loaded zinc-cobalt layered double hydroxide and attapulgite-loaded zinc-cobalt layered double oxide were successfully prepared after incorporation of human essential trace element cobalt and natural attapulgite for antibacterial application.The effects of the attapulgite addition and molar ratio of zinc to cobalt on the antibacterial properties and biocompatibility of the materials were investigated,while the antibacterial mechanism of the materials was also analyzed to provide a theoretical basis for the later clinical application.Method Zinc-cobalt layered double hydroxide(APT/ZnCo LDH)loaded on attapulgite were prepared by simple method of coprecipitation.The effects of different molar ratios of zinc to cobalt and additions of attapulgite on the antibacterial properties and biocompatibility of the materials were investigated by in vitro antimicrobial experiments and CCK-8 experiments.In order to improve the antibacterial properties of the antibacterial materials,the above series of antibacterial materials were calcined at 300-800 °C to obtain zinc-cobalt layered double oxide loaded on attapulgite(APT/ZnCo LDO).The effects of calcining temperature,molar ratio of zinc to cobalt and the added amount of attapulgite on the antibacterial activity and biocompatibility of APT/ZnCo LDO were also evaluated.The structures and physicochemical properties of all materials were characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM),Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD)and Zeta potential.The morphology and activity of bacteria before and after contact with antibacterial materials were analyzed by transmission electron microscopy(TEM)and fluorescencemicroscopy(FM),and the possible antimicrobial mechanism was discussed.Results1.Attapulgite /zinc-cobalt layered double hydroxide(APT/ZnCo LDH)1.1 From the result of Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),transmission electron microscopy(TEM)and scanning electron microscopy(SEM),the mixed-dimensional antibacterial materials consisted of one-dimensional attapulgite and two-dimensional zinc-cobalt layered double hydroxides were successfully prepared.1.2 The results of in vitro antibacterial experiment showed that APT/ZnCo LDH presented the best antibacterial effect prepared as the added amount of attapulgite was40 wt.% and the molar ratio of zinc to cobalt was 8:1,and the MIC of APT/ZnCo LDH to Staphylococcus aureus and Escherichia coli was 0.25 mg/mL and 1.0 mg/mL,respectively.1.3 The results of CCK-8 experiment showed that APT/ZnCo LDH with the best antibacterial properties exhibited no cytotoxicity after co-culture with Hela cells for24,48 and 72 hours.1.4 The microscopic morphology of bacteria changed after contacting with antibacterial materials according to the results of transmission electron microscopy,and bacteria changed from the complete and regular morphology to irregular morphology due to the destroy of cell structure.The viability of bacteria was observed by fluorescence staining,and it was found that bacteria changed from living bacteria stained with green fluorescence to dead bacteria with red fluorescence after contacting with antibacterial materials.2.Attapulgite/ zinc-cobalt layered double oxide(APT/ZnCo LDO)2.1 A series of characterization methods were used to analyze the structure and physical and chemical characteristics of the materials.The results show that the prepared mixed-dimensional composites consisted of one-dimensional attapulgite and two-dimensional zinc-cobalt layered double oxide were successfully prepared.2.2 Based on in vitro antibacterial experiments,it was found that APT/ZnCo LDO exhibited the excellent antibacterial behavior as the calcining temperature was500 oC,the molar ratio of zinc to cobalt was 8:1 and the content of attapulgite was 40 wt.%.Compared with the control group of APT/ZnCo LDH and without attapulgite and cobalt,its antibacterial performance was obviously improved,and its minimum antimicrobial concentration for Staphylococcus aureus and Escherichia coli was 0.125mg/mL and 0.5 mg/mL,respectively.2.3 The results of CCK-8 experiment showed that APT/ZnCo LDO with the optimum antibacterial properties still had no cytotoxicity after co-culture with Hela cells for 24,48 and 72 hours.2.4 It was found that the cell structure of bacteria was destroyed and the morphology also changed after contacting with antibacterial materials.The change of bacterial activity before and after contacting with antibacterial materials suggested that the bacteria changed from the living bacteria with green fluorescence to the dead one with red fluorescence.In addition,it was observed a large number of dead bacteria were adhered to the surface of antibacterial materials.Conclusions1.Attapulgite/zinc-cobalt layered double hydroxides(APT/ZnCo LDH)and attapulgite/zinc-cobalt layered double oxides(APT/ZnCo LDO)were prepared for antibacterial application by coprecipitation method and coprecipitation method followed by calcining process,respectively.The prepared composites were composed of one-dimensional and two-dimensional mixed structure.2.When attapulgite and cobalt were introduced into the antibacterial material,the relative addition of zinc was reduced,but the antibacterial activity and biocompatibility were maintained.The cell compatibility of the material was positively correlated with the addition of attapulgite and cobalt.3.The antibacterial properties of APT/ZnCo LDO were clearly improved after APT/ZnCo LDH composites were calcined.The minimum inhibitory concentrations of APT/ZnCo LDO to Staphylococcus aureus and Escherichia coli increased from0.25 mg/mL and 1.0 mg/mL to 0.125 mg/mL and 0.5 mg/mL,respectively.4.After contacting with antibacterial materials,bacteria were dead and adhered to the surface of antibacterial materials in large quantities,which was related to the excellent adsorption properties of attapulgite.When bacteria were adhered to attapulgite,which was favor of increasing the contact area with zinc and cobalt on the surface of materials to enhance the antibacterial properties.5.In conclusion,this study provides a feasible strategy for zinc-based antimicrobial agents to improve the antimicrobial activity of materials and reduce their cytotoxicity by combining natural attapulgite and human trace elements. |
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