Trivalent Titanium-doped Titanium Dioxide Nanoparticles For Combating Periodontitis-related Pathogens

Background:Periodontal disease,caused by bacterial biofilm and its toxic products,is a common oral disease that results in chronic inflammation.It destroys the supporting tissues surrounding teeth and,in severe cases,can lead to tooth mobility and loss,which is a major cause of tooth loss in adults and has serious impacts on both oral and systemic health.Clinical treatment of periodontitis typically involves scaling,root planing,and antibiotic therapy.However,instrument cleaning can cause damage,and antibiotic use over a lengthy period of time can cause bacterial resistance,which affects treatment efficacy.Therefore,it is crucial to find a minimally invasive and effective new treatment method for antibacterial and biofilm removal.In recent years,photodynamic therapy has gradually been applied in the treatment of periodontitis.This method uses photosensitizers to generate reactive oxygen species under light irradiation,which can kill pathogenic microorganisms and avoid the disadvantages of traditional treatment methods,and simple to use and highly effective.Titanium dioxide has gotten a lot of interest as a photosensitizer with clinical potential over the past 20 years.Under ultraviolet light irradiation,titanium dioxide can generate electron-hole pairs,which further react with oxygen or water to produce reactive oxygen species（ROS）,thereby exerting the effect of photodynamic therapy.However,ultraviolet light is harmful to the human body as an excitation light source for titanium dioxide,and its penetration depth in biological tissues is limited,making it difficult to exert photodynamic effects on deep tissues.In addition,the wide bandgap of titanium dioxide leads to relatively poor photocatalytic performance,making it difficult to achieve ideal photodynamic therapy results.Studies have shown that the application of upconversion nanoparticles can effectively solve the problems of harmful ultraviolet excitation light and inadequate penetration depth.After being excited by near-infrared light,upconversion nanoparticles can excite titanium dioxide under ultraviolet light.Moreover,it has been found that doping appropriate amounts of trivalent titanium into titanium dioxide can enhance its photocatalytic efficiency,and trivalent titanium can be used as an efficient photothermal agent and peroxidase-like enzyme for photothermal and chemical dynamic therapy.The above-mentioned properties have achieved relatively ideal effects in the treatment of tumors or infections.Currently,there is little research on antibacterial therapy using self-doped trivalent titanium-dioxide nanomaterials,and it has not yet been applied to the treatment or research of periodontal disease.Therefore,the development of near-infrared light stimulated self-doped trivalent titanium-dioxide materials for sustained and efficient combating of periodontal pathogens is of great clinical significance.Objective:The research aims to prepare NIR-triggered core-shell structured nanoparticles,which consist of upconversion nanoparticles（UCNPs）as the luminescent core and self-doped Ti O₂ with different ratios of Ti³⁺as the excitable shell(UCNPs@Ti O_2-x-x,UT-x).The efficiency of photodynamic therapy（PDT）and chemotherapy（CDT）using UT-x NPs with different ratio of Ti³⁺will be compared.The inhibitory effect of UT-x on periodontal bacteria will also be investigated.Methods:1.Upconversion nanoparticles（UCNPs）were prepared by thermal decomposition,and UCNPs@Ti O2（UT）were synthesized by hydrothermal modification of their surface with titanium dioxide.Ti³⁺was introduced to the Ti O₂ surface by pulsed laser irradiation at 355 nm,producing UT-x.The Ti³⁺content was adjusted by varying the duration of pulsed laser irradiation（0,7.5,15,30,and 60 minutes）to prepare five types of UT-x nanoparticles:UT,UT-7.5,UT-15,UT-30,and UT-60.2.The successful synthesis of UT-x core-shell structure nanoparticles was confirmed by transmission electron microscopy and high-resolution transmission electron microscopy.The optical properties of different UT-x nanoparticles were analyzed using UV-visible absorption spectra and upconversion emission spectra,while the Ti³⁺content in different UT-x nanoparticles was analyzed using zeta potential and X-ray photoelectron spectroscopy techniques.The Ti³⁺content and crystal phase change of UT-x nanoparticles were characterized using Raman spectroscopy.3.The photocatalytic efficiency of different UT-x was compared using photoluminescence spectroscopy,linear sweep voltammetry,transient photocurrent response,and reactive oxygen species detection.The catalytic efficiency of different UT-x in promoting the decomposition of hydrogen peroxide and promoting Fenton-like reactions under thermal conditions was compared by testing the thermal effect and hydroxyl radical detection of the nanoparticles.4.The cell compatibility of UT-x nanoparticles was evaluated using the CCK-8method,cell staining method,and hemolysis test.5.The antibacterial effect of UT-x-mediated photodynamic and chemical therapy was investigated against two bacteria related to periodontitis:Porphyromonas gingivalis（P.gingivalis）and Fusobacterium nucleatum（F.nucleatum）under 980 nm near-infrared light irradiation.The antibacterial effect of UT-x on monospecies biofilms was evaluated using live/dead staining,colony-forming unit（CFU）counting,bacterial metabolic activity detection,and bacterial scanning electron microscopy.Results:1.As the pulse laser irradiation time on UT-x increased,the absorption of UV and visible light and the upconversion luminescence intensity decreased,while the surface potential and Ti³⁺content gradually increased,and the crystal phase also changed accordingly.The photocatalytic efficiency of UT-x showed a first increase and then decrease trend with the irradiation time,and the samples treated with pulse laser exhibited peroxidase-like activity,which also showed a first increase and then decrease trend with the irradiation time.2.When the nanoparticle concentration was less than 1.0 mg/m L,all five kinds of UT-x nanoparticles demonstrated excellent biocompatibility,and the cell survival rate was greater than 80%.3.The CFU of the biofilm was decreased by about three orders of magnitude by NIR-excited UT-15 mediated photodynamic therapy and chemical dynamic therapy,which was higher than that of the control group and other UT-x treatment groups（p<0.05）.The findings of the bacteria live/dead staining revealed that the majority of the bacteria in the control group were mostly green and alive,whereas the bacteria in the UT-15 and UT-30 mediated photodynamic therapy and chemical dynamic therapy groups were mostly red,suggesting a higher percentage of dead bacteria.The bacterial scanning electron microscope results showed that the bacterial cell membranes in the treatment group wrinkled and ruptured,and the bacterial contents leaked out.Photodynamic therapy and chemodynamic therapy of UT-15 also significantly reduced the metabolic activity of the biofilm（p<0.05）.4.Compared with the UT treatment group,the chemical dynamic therapy mediated by UT-15 could continuously reduce the number of bacteria in the biofilm,and the CFU of the biofilm continued to decrease by about 0.5 orders of magnitude after the light exposure ended（p<0.05）.Conclusions:In this study,we successfully synthesized UT-x nanoparticles with a core/shell structure doped with different proportions of Ti³⁺by high-energy pulse laser irradiation.The UT-x nanoparticles showed good biocompatibility.Compared with UT,the photocatalytic efficiency and peroxidase-like activity of UT-x doped with Ti³⁺changed,and both abilities showed a first increase and then decrease trend with the pulse laser treatment time.Among them,UT-15 showed the best photocatalytic efficiency and peroxidase-like activity and had a strong inhibitory effect on two types of periodontal bacteria.This study demonstrates the potential application of Ti O₂ nanoparticles doped with self-doped Ti³⁺in periodontal disease treatment.