Chlorin E6 Based Antibacterial Photodynamic Therapy Against Biofilms Of Peri-implantitis-related Pathogens

In recent times,with the widespread application of oral implantology,the incidence of peri?implant inflammation has been increasing.Studies have found that bacterial infection is the main cause of peri?implant inflammation and among them,dental plaque biofilm is the initiation factor of inflammation around implants.Compared with planktonic bacteria,bacteria in plaque biofilm are more virulent and resistant to host immune system.Hence,the study of pathogenic bacteria in free state cannot be used as an evaluation of their inhibitory efficacy on bacteria in plaque biofilm.To evaluate the antibacterial effect more comprehensively,it is essential to study the bacteria in the biofilm state.Antimicrobial Photodynamic Therapy?aPDT?is an effective way to selectively destroy bacteria or other pathogenic microorganisms in recent years.Because of its high targeting,low damage,ability to reach deep tissues eifficacy,and also achieved good results for multi-drug resistant bacteria,aPDT has received widespread attention and been gradually used in the treatment of peri-implant inflammation.The anti-microbial mechanism of aPDT mainly depends on three factors:photosensitizer,light source and oxygen.Most of the photosensitizers absorb light in ultraviolet or visible bands which has poor tissue penetration ability.Lanthanide-doped upconversion nanoparticles?UCNPs?can convert near-infrared light into visible light and ultraviolet light emission.UCNPs have many advantages,such as non-autofluorescence,good antibacterial properties,high chemical stability,high light transmission depth,long life,little tissue damage.Therefore,we envisage that the combination of UCNPs with Chlorin e6?Ce6?,a photosensitizer using red light as excitation source,can upconvert near-infrared light into red light which exhibit strong tissue penetration rate and stimulate Ce6 to administer aPDT.Moreover,this kind of design can enhance its chemical stability and antimicrobial properties.With silylation of Ce6-UCNPs can also increase its hydrophilicity and bioavailability.Finally,we enhanced the ability of aPDT further by doping different ratios of Mn²⁺to improve the up-conversion performance of red light.So we conducted the following experimental studies:Experiment 1:The effect of different concentrations of Ce6 on the antibacterial properties of single strain biofilm.The cytotoxicity of Ce6 on L929 mouse fibroblasts was investigated by CCK8 to determine the concentration of Ce6.The antimicrobial properties of Porphyromonas gingivalis?P.gingivalis?,Prevotella intermedia?P.intermedia?,Fusobacterium nucleatum?F.nucleatum?are tested via dead/live staining of bacterial biofilm,CFU counting,MTT assay,crystal violet biofilm assay and sulfuric acid-phenol assay.The results showed that the three single-strain biofilms were reduced in quantity,activity,and polysaccharide yield,and there were statistical differences?P<0.05?.Experiments showed that Ce6 has bactericidal effect on single-strain biofilm.However,as the concentration of Ce6 increases,its antibacterial properties are no longer enhanced.Because Ce6 is excited by red light which has poor tissue penetration rate and poor water solubility of Ce6 leads to low penetration rate of drug,thus affecting the therapeutic effect of aPDT.Therefore,using Ce6 alone has a certain antibacterial effect,but it has not reached our expectations.Experiment 2:Synthesis and characterization of UCNPsMn@Ce6@silane.U UCNPs@Ce6 was silylated to increase its hydrophilicity,and different ratios of Mn²⁺were added to enhance its luminescent intensity.The new nanoparticle materials UCNPsMn@Ce6@silane were synthesized.The excitation light is converted from red light to near-infrared light to enhance its penetration,enhancing the antibacterial properties.The transmission electron microscopy?TEM?,Fourier transform infrared?FTIR?spectroscopy,the XRD measurement,Zeta potential instrument to test the physical and chemical properties of nanoparticles.The results showed that a thin silane layer is visible on the surface of Mn²⁺doped Mn²⁺silanes with a thickness of about 2-3 nm.When a single nanoparticle is selected and observed by a high power transmission electron microscope,the thin silane layer is clear on the surface of the particle.The new characteristic peaks of Si-O-Si and Si-OH appeared,indicating that the silane modification was successful.In addition,due to the long carbon chain,the intensity of the characteristic peak of-CH2 also indicates the success of silane modification.The structure of UCNPs with and without Mn²⁺doping was detected by XRD.The results confirmed that the pure hexagonal phase NaYF₄ without Mn²⁺doping changed the crystal structure of UCNPs from a hexagonal phase to a cubic phase after introducing Mn²⁺into the nanocrystals.Moreover,with the increase of Mn²⁺doping,the diffraction peak of the crystal plane can be found to move towards a large angle,which further explains the success of Mn²⁺doping.Experiment 3:The effect of UCNPsMn@Ce6@silane on the antibacterial properties of single-bacterial biofilms was examined.The nanocomposites of UCNPsMn@Ce6@silane with different proportion of Mn²⁺doping was synthesized.Compared with UCNPs@Ce6@silane,three different single-species oral biofilms were tested via dead/live biofilm staining assay,Colony-forming units?CFU?assay,MTT assay,crystal violet biofilm assay,sulfuric acid-phenol assay.Results showed that the aPDT effect of pure Ce6 could not be effectively activated by NIR stimulation alone,but with the addition of UCNPs made the bacterial biofilm of three single strains of bacteria in the experimental group significantly lower than that of pure silanized Ce6 in bacterial colony number,activity and polysaccharide yield?P<0.05?.At the same time,with the increase of Mn²⁺loading ratio,the antibacterial properties of UCNPsMn@Ce6@silane were significantly improved,and the 30%Mn²⁺doped group showed the best antibacterial ability.Silanization enhances the hydrophilicity of UCNPs@Ce6,and the addition of Mn²⁺enhances ability of aPDT to resist single-strain biofilm.Experiment 4:Study the effect of UCNPsMn@Ce6@silane on the antibacterial properties of multi-strain biofilms.The antibacterial properties of UCNPsMn@Ce6@silane against single,three and six stain of oral biofilms were studied.Pure silylated Ce6 was selected as control group,while UCNPs@Ce6@silane,and UCNPs@Ce6@silane doped with 30%Mn²⁺were selected as experimental groups.Colony-forming units?CFU?counting,dead/live biofilm staining assay,and metabolic activity of biofilms were carried out to evaluate the aPDT function.The results showed that UCNPs@Ce6@silane showed good antibacterial properties against single-strain biofilm,but its antibacterial properties decreased significantly in three strains and six strains of biofilms.However,UCNPsMn@Ce6@silane,due to the red light enhancement effect of Mn²⁺,leads to an increase of the aPDT effect of Ce6,thus still main a strong antibacterial activity against the multi-strain biofilms.In conclusion,this paper uses UCNPs combining with photosensitizer Ce6 which excited by red light to study its effect on oral biofilms,obtains a good antibacterial effect.At the same time,the luminescence efficiency of up-conversion red light of UCNPs was improved by doping Mn²⁺to enhance the antibacterial effect of Ce6.Finally,the hydrophilicity of the material was increased by silylation surface modification to improve the bio-absorbability of the material.The results of this experiment showed that the emerging antibacterial photodynamic therapy based on up-converting nanoparticles can effectively inhibit the formation and development of inflammation-related bacterial biofilms around oral implants,with low toxicity and high bioavailability.This novel design provides theoretical basis and experimental support using UCNPs to administer aPDT for clinical treatment of peri-implantitis.