Antibacterial Effect Study Of ZIF-8-based Photodynamic Nano-antibacterial Materials Against Streptococcus Mutans And Its Biofilms

Background and objectives:Streptococcus mutans is the main pathogenic bacteria of caries and causes caries by forming plaque biofilm.Antimicrobial photodynamic therapy（a PDT）is an effective method for the prevention of bacterial infectious oral diseases.Due to its high porosity,acid responsiveness and biocompatibility,zeolite imidazole-8（ZIF-8）can be used as a drug delivery carrier and has been widely used in antibacterial field.In this study,the excellent properties of ZIF-8 were utilized to prepare a nano-composite material loaded with O₂-Cu/ZIF-8@Ce6/ZIF-8@HA（OCZCH）,and the antibacterial effect and caries-producing effect of THE nano-composite material on Streptococcus mutans and its biofilms were studied by a PDT.In order to provide new ideas for the prevention and treatment of bacterial infectious oral diseases.Method:1.Synthesis of Cu²⁺doped ZIF-8（Cu/ZIF-8）,O₂-Cu/ZIF-8@Ce6/ZIF-8（OCZC）coated with ZIF-8 loaded with small molecule photosensitizer Ce6 after O₂,synthesis of O₂-Cu/ZIF-8@Ce6/ZIF-8（OCZC）,and then biocompatible hyaluronic acid（HA）was assembled on its surface.Finally,OCZCH nanocomposites with high antibacterial ability were synthesized.2.X-ray diffraction（XRD）,transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,Zeta potential,UV-Visible spectrophotometer（UV-Vis）,X-ray photoelectron spectroscopy（XPS）,automatic specific surface area and porosity analyzer（BET）and in vitro reactive oxygen species（ROS）determination were used to determine the physical and chemical properties of the materials structural characterization.3.CCK-8 assay was used to determine the biocompatibility of OCZCH on L929mouse fibroblasts.4.Determine the minimum inhibitory concentration（MIC）of OCZCH against Streptococcus mutans.5.Crystal violet staining,MTT assay,bacterial LIVE/DEAD staining and SEM were used to study the inhibitory effects of Control group,Ce6 group,CZCH group and OCZCH group on Streptococcus mutans biofilms under 650 nm laser（5 min,120m W/cm~2,denoted as"+"）and dark treatment（denoted as"-"）,respectively.6.The effects of Control group,Ce6 group,CZCH group and OCZCH group on the cariosis of Streptococcus mutans under 650 nm laser and dark treatment were determined by acid production experiment and extracellular polysaccharide experiment.Results:1.Material characterization experiments showed that OCZCH was successfully synthesized.TEM and SEM images showed that OCZCH nanoparticles were about170 nm in size and dispersed evenly without agglomeration.HAADF-STE proved that C,N,Cu,Zn and O elements were uniformly distributed in OCZCH nanoparticles.XRD shows that OCZCH has good ZIF-8 crystal shape.Zeta potential proved that the material modified HA showed obvious negative charge,indicating that HA was successfully modified.The peak values at 932.4e V and 952.1e V belong to Cu(2p_3/2)and Cu(2P_1/2)electron spectra of Cu²⁺,respectively.Oxygen adsorption isotherms showed that the oxygen carrying capacity of Cu/ZIF-8 was twice that of ZIF-8.The UV-Vis absorption spectra showed that the characteristic absorption peaks of Ce6 appeared at 410 nm and 640 nm after loading ZIF-8 shell containing Ce6.According to the Ce6 concentration standard curve,the load rate of Ce6 in OCZCH was 3.2 wt%.BET analysis showed that the surface area,pore volume and pore size of Cu/ZIF-8@Ce6/ZIF-8（CZC）decreased compared with that of ZIF-8,which proved that Ce6 was coated in ZIF-8.Acid response drug release experiments showed that OCZCH skeleton collapsed and the encapsulated Ce6 could be released quickly in a slightly acidic environment,with good acid response.ROS generation test showed that OCZCH produced singlet oxygen under 650 nm red light irradiation.2.CCK-8 assay showed that OCZCH had good biocompatibility and no obvious cytotoxicity.3.The MIC of OCZCH against Streptococcus mutans was 50μg/m L.4.Inhibition effects of Control,Ce6,CZCH and OCZCH on the formation of Streptococcus mutans biofilm under 650 nm laser（5 min,120 m W/cm~2）and dark treatment respectively:（1）Biofilm formation amount:under dark treatment,biofilm formation amount of each group was basically unchanged;under light,the inhibition effect of OCZCH+group>CZCH+group>Ce6+group>Control+group.（2）Biofilm metabolic activity:under dark treatment,biofilm metabolic activity of each group was basically unchanged;under light,the inhibitory effect on biofilm metabolic activity was OCZCH+group>CZCH+group>Ce6+group>Control+group.（3）Bacteria alive/dead staining:The biofilm of the Control+group and the dark treatment group were dense and showed a large area of green fluorescence（live bacteria）;CZCH+and Ce6+groups had a certain antibacterial effect,and the biofilm was sparse and orange.OCZCH+group was dominated by red fluorescence and almost all were dead bacteria.（4）Biofilm morphology:When SEM is lowered to the same multiple,both dark treatment group and Control+group show relatively complete and dense biofilm;In Ce6+group,the density and integrity of biofilm were significantly damaged,and the thickness of biofilm was reduced,and the biofilm was separated into fragments.CZCH+group showed obvious inhibition of bacterial aggregation.OCZCH+group showed no biofilm morphology and only a few dispersed bacteria.5.Effects of Control,Ce6,CZCH and OCZCH on caries of Streptococcus mutans under 650 nm laser and dark treatment respectively:（1）Inhibition of acid production:Inhibition ability of Streptococcus mutans in each group under dark treatment;under illumination,the inhibition rate of acid production of Streptococcus mutans was OCZCH+group>CZCH+group>Ce6+group>Control+group.The inhibition rate of OCZCH+group was87.4%.（2）Extracellular polysaccharide experiment:Under dark treatment,extracellular polysaccharide production of each group had little change;the exopolysaccharide production in CZCH+and OCZCH+groups was lower than that in Control+group.Conclusion:1.OCZCH,a novel high-efficiency photodynamic nano-antibacterial composite,was successfully prepared.2.OCZCH has good acid response,reactive oxygen generation ability and biocompatibility.3.OCZCH can significantly inhibit the growth of Streptococcus mutans and the formation of biofilms.4.OCZCH could significantly reduce cariosis toxicity of Streptococcus mutans.