Preparation And Antibacterial Activity Against Periodontal Infection Of Oxygenated-nanodiamond Peroxidase Mimic

Nanodiamond?ND?,especially detonation nanodiamond?DND?has many excellent physicochemical properties.Due to the small size,easy surface functionalization,extremely low cytotoxicity and good biocompatibility,ND has been widely used in drug delivery,biomarkers,biological imaging,surface coating modification of implant materials and other medical fields.Recent studies have found that ND also has the catalytic activity similar to peroxidase,which can catalyze the decomposition of H₂O₂to produce·OH.However,it is necessary to explore the enzymatic activity and the potential antibacterial effects of ND.Periodontitis is a common inflammatory disease caused by periodontal pathogen and dental plaque,which leads to the breakdown of tooth-supporting structures.Biofilm,a well-organized cluster of bacterial community,is firmly adherent to dental surfaces and embed in the protective matrix of extracellular polymeric substances.It is difficulty to remove due to the strong mechanical stability of extracellular matrix in biofilm.At the same time,the virulence factors produced by periodontal pathogen could direct or indirect cause the host-mediated destroy of tissues surrounding the teeth,and eventually results of the tooth loss.At present,the clinical methods against oral biofilms are limited to the conventional antimicrobial agents,such as H₂O₂ and chlorhexidine.As a general and accessible antimicrobial agent,H₂O₂ is widely used in oral infusion and clinical treatment against periodontal inflammation.However,bacterial biofilms are resistant to low concentration H₂O₂,resulting in a less pronounced therapeutic efficacy.High concentration H₂O₂ solutions are beneficial to antimicrobial treatments while would cause adverse reactions to healthy tissues,being unsuitable for daily usage.Thus,a further development of newly antibacterial approaches against biofilms became important on these challenges.In recent years,with the rapid development of nanotechnology,nanomaterials with natural enzyme activity have gradually attracted broad attention.As a kind of nanozymes,peroxidase mimics can catalyze low concentration H₂O₂ to produce hydroxyl radical?·OH?,which has strong antibacterial properties to inhibit the growth of bacteria and resist the generation of biofilms by reducing bacterial adhesion.However,the further biomedical applications of these nanomaterials have been limited by their catalytic efficiency and the potential cytotoxicity,particularly in oral medicine.In this thesis,ND was prepared as oxygen-containing nanodiamonds?O-NDs?with various oxidation functional groups to explore the peroxidase activity and cell biocompatibility of O-NDs,based on their excellent characteristics of application in biomedical field.At the same time,the O-NDs with low concentration of H₂O₂ were applied to eliminate the periodontal pathogens and biofilm.Then,the rat periodontal infection model was used to evaluate the performance of O-NDs in protecting periodontal tissues and eliminating periodontal pathogenic bacteria in vivo,which has provided a new strategy for prevention and treatment of periodontal inflammation in clinical.The main research contents and results are as follows:1.NDs was prepared as oxygen-containing nanodiamonds with various oxidation functional groups via mixed acid-assisted reflux.The synthesized O-NDs has been detected with the characteristic of peroxidase-like activity.The results show that:?1?O-NDs particles with uniform size,good dispersion,good surface morphology and abundant groups of carboxyl and carbonyl.?2?The oxygenated-group-enriched O-NDs has a wider p H value,temperature range and peroxidase-like activity than natural enzymes.The peroxidase-like activity of O-NDs could be attributed to the oxygen-containing groups.?3?O-NDs could catalyze the decomposition of H₂O₂ into·OH,which suggesting the potential ability for antibacterial application.2.To investigate the biocompatibility of O-NDs combined with human gingival fibroblasts?HGFs?in different concentrations.The results showed that:?1?The cellular activity,cell adhesion and cell migration have no significant difference on HGFs cell when the concentration of O-NDs in less than 500?g/m L.?2?The early cell migration of HGFs could be accelerated with the O-NDs'concentration was 100?g/m L.When the concentration of O-NDs up to 500?g/m L,the intracellular aggregation of O-NDs nanoparticles have a certain degree of effect on the morphology of HGFs cells.3.Porphyromonas gingivalis,Fusobacterium nucleatum?Gram-negative bacteria?and Streptococcus sanguis?Gram-positive bacteria?were used as the experimental strains,and H₂O₂/O-NDs system was used for in vitro antibacterial experiments.The results showed that:?1?The antibacterial system of H₂O₂/O-NDs present the significantly enhancement of antibacterial activity of H₂O₂,and the system present broad spectrum of antibacterial effect against both Gram-negative and Gram-negative periodontal pathogens.In the presence of O-NDs,H₂O₂ can be catalyzed to decompose into·OH with high antimicrobial properties.?2?The antibacterial properties of H₂O₂/O-NDs system proved highly effective in both suspended bacteria,single-stain biofilm and multispecies biofilm.The antibacterial ability of H₂O₂/O-NDs system against multispecies biofilm was slightly lower than that of the single-stain biofilm.?3?From the above results,O-NDs could serve as a promoter to enhance the activity of low concentration H₂O₂ through converting H₂O₂ into·OH for the inhibiting and destruction formation of biofilm;It could be that the O-NDs adhered on bacterial membrane in-situ producing·OH,and the generated·OH has higher activity to oxidize the nucleic acids,proteins and polysaccharides of bacteria and biofilm,which significantly improved the conversion efficiency and bactericidal ability of H₂O₂.As for the formed plaque biofilm,O-NDs could penetrate into biofilm and promote the production of·OH by H₂O₂,and further damage the bacterial biofilm.4.A rat model of periodontal pathogen infection was established to study the effect of H₂O₂/O-NDs antibacterial system on periodontal inflammation in vivo and the biological safety of O-NDs.The results showed that:?1?O-NDs?100?g/m L?could catalyze the decomposition of low concentration H₂O₂?10 m M?to break down the adhesion of periodontal pathogens in rats'oral cavity and promote the elimination of periodontal inflammation,which proved highly effective in the prevention and the treatment of periodontal disease.?2?The concentration of O-NDs below 500?g/m L has showed no significant hemolysis,and there was no body weight loss in the rat of O-NDs groups;The hematological parameters,serum biochemistry and visceral histomorphology of rats in O-NDs groups also proved a good biological safety and their potential application in clinical antibacterial treatment.