Ir-titanate nanobiocatalyst and preparation method and application thereof

Abstract

The application belongs to the technical field of catalyst materials, and particularly relates to an iridium-strontium titanate nanobiocatalyst, a preparation method and application thereof. Specifically, the iridium-strontium titanate nanobiocatalyst comprises strontium titanate and iridium nanoclusters loaded on the strontium titanate. The application further provides a preparation method of the iridium-strontium titanate nanobiocatalyst, which comprises preparing an iridium-strontium titanate precursor through a hydrothermal ion exchange reaction of strontium titanate and soluble iridium salt, and then performing calcination treatment on the iridium-strontium titanate precursor to obtain the iridium-strontium titanate nanobiocatalyst. The iridium-strontium titanate nanobiocatalyst has pH-dependent peroxidase-like and catalase-like activities, and has a good application prospect in antibacterial, biofilm removal, and prevention or treatment of dental caries and periodontitis.

Description

Technical Field

[0001] This invention belongs to the field of catalyst materials technology, specifically relating to an iridium-strontium titanate nanobiocatalyst, its preparation method, and its application. Background Technology

[0002] The human oral cavity contains a diverse array of microbial species, forming a mutually beneficial relationship. Oral health depends on the interaction between the host's immune status and the oral microbiota. Imbalances in the oral microbiota can trigger a range of oral diseases, the most common being dental caries and periodontitis. However, the use of routine antibiotics often disrupts the oral ecological balance, increasing the risk of drug resistance. Dental caries is essentially a biofilm-mediated demineralization of hard tissues caused by an imbalance in the oral microecology. Under physiological conditions, the oral symbiotic microbiota maintains enamel surface homeostasis through dynamic equilibrium. However, frequent intake of fermentable carbohydrates leads to the metabolic dominance and proliferation of cariogenic bacteria, which produce organic acids such as lactic acid and acetic acid through glycolysis, acidifying the oral microenvironment and lowering the pH of the dental plaque biofilm microenvironment below 5.5 (the critical demineralization threshold), thus damaging the tooth's mineralized structure. Cariogenic bacteria are acid-resistant, surviving and continuing to multiply in acidic environments, further exacerbating tooth decay. These mechanisms collectively drive the occurrence and progression of dental caries. The progression of periodontitis is more complex. Periodontitis is a chronic inflammatory disease caused by subgingival anaerobic bacterial dysbiosis, characterized by periodontal pocket formation and progressive alveolar bone resorption. During the inflammatory progression phase, the subgingival microenvironment exhibits low oxygen pressure (pO2 < 10 mmHg) and an alkaline microecology (pH 7.5-8.5), conditions that give *Porphyromonas gingivalis* a significant proliferative advantage. *Porphyromonas gingivalis*, by regulating the interaction network of symbiotic flora, not only disrupts microbial homeostasis but also drives the abnormal proliferation of pro-inflammatory bacteria, thereby strengthening the inflammatory cascade and accelerating the periodontal tissue destruction process. Currently, treatments for periodontitis, such as antibiotic therapy and mechanical debridement, mainly focus on plaque control, but their clinical efficacy is usually limited. Furthermore, treatment relying solely on antioxidants and antibacterial drugs fails to effectively break the vicious cycle between bacterial destruction and inflammatory damage, making it difficult to fundamentally alleviate the damage, thus limiting efficacy. Due to the difficulty of bone regeneration caused by inflammation, clinical treatment of periodontitis often focuses on symptom control rather than achieving a cure. Therefore, the occurrence of periodontal disease is a cascade process, and a comprehensive approach that addresses antibacterial, anti-inflammatory, and tissue repair is crucial. Among these approaches, eliminating bacterial biofilms without disrupting the homeostasis of the microbiome and reshaping immune homeostasis is the most critical aspect of treatment.

[0003] In recent years, nanomaterials with natural enzyme-like activities have attracted much attention due to their ease of preparation, storage, and separation, as well as their tunable activity and lower cost compared to natural enzymes. Various nanomaterials have been shown to mimic the activities of oxidases, peroxidases, catalases, and superoxide dismutases, encompassing metals, metal oxides, metal coordination complexes, and carbon-based nanomaterials. The ability of these nanomaterials to substitute for specific enzymes offers new opportunities for enzyme-based applications. For example, nanozymes with oxidase-like or peroxidase-like activities have shown potential applications in antibacterial properties, biosensors, and immunoassays. Nanomaterials possessing superoxide dismutase-like or catalase-like activities exhibit antioxidant activity, protecting aerobic cells from oxidative stress and showing potential applications in inflammation treatment. Furthermore, nanozymes with high catalase activity can generate oxygen at hypoxic tumor sites, becoming effective agents for cancer treatment.

[0004] Iridium nanoparticles (Ir-NPs) possess both peroxidase and antioxidant enzyme-like activities. Due to their abundant d electrons and high reduction and oxidation stability of oxygen intermediates, Ir-NPs can mimic natural enzyme systems, overcoming multi-electron reactions of oxygen species by accelerating electron transfer, thereby achieving efficient redox balance. However, the biotoxicity of iridium nanoparticles limits their concentration as biocatalysts and affects their activity (concentrations exceeding 10 μg / mL exhibit toxic effects on cell proliferation). Strontium titanate (SrTiO3 / STO) is a perovskite oxide with wide applications, such as piezoelectricity, photocatalysis, and electronic devices. Studies have shown that strontium (Sr) mainly coexists with calcium in the mineral phase of bone. Due to their chemical similarity and nearly identical metabolic pathways, strontium can enhance biological activity and positively influence the adhesion, proliferation, and differentiation of pre-osteoblasts. Summary of the Invention

[0005] In view of the above-mentioned existing technology, the present invention provides an iridium-based strontium titanate material, which is found to have pH-dependent peroxidase-like and catalase-like activities, and has good application prospects in antibacterial, biofilm removal and prevention or treatment of dental caries and periodontitis.

[0006] Specifically, in a first aspect, the present invention provides an iridium-strontium titanate nanobiocatalyst (also referred to in this specification as iridium-based strontium titanate material, or simply Ir-STO), the iridium-strontium titanate nanobiocatalyst comprising strontium titanate and iridium nanoclusters supported on the strontium titanate.

[0007] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits a nanocoral-like structure.

[0008] Furthermore, the iridium nanoclusters are uniformly dispersed on a strontium titanate substrate.

[0009] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits electron transfer from the strontium titanate substrate to the iridium species.

[0010] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits pH-responsive enzyme-like activity.

[0011] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits peroxidase-like activity under acidic conditions and catalase-like activity under neutral conditions.

[0012] In a second aspect, the present invention provides a method for preparing iridium-strontium titanate nanobiocatalyst as described herein, comprising: preparing an iridium-strontium titanate precursor by reacting strontium titanate with a soluble iridium salt via a hydrothermal ion exchange reaction, and then calcining the iridium-strontium titanate precursor to obtain the iridium-strontium titanate nanobiocatalyst.

[0013] Furthermore, the preparation method of the iridium-strontium titanate precursor includes the following steps:

[0014] (1) Strontium titanate is dispersed in water or a water-alcohol mixture to obtain a strontium titanate dispersion;

[0015] (2) Add a soluble iridium salt to the strontium titanate dispersion to form a mixed reaction system containing iridium ions and strontium titanate;

[0016] (3) The mixed reaction system is subjected to a hydrothermal ion exchange reaction;

[0017] (4) After the reaction is complete, cool, wash and dry to obtain the iridium-strontium titanate precursor.

[0018] Furthermore, the reaction temperature of the hydrothermal ion exchange reaction is 100-300℃, preferably 180℃, and the reaction time is 2-48 hours.

[0019] Furthermore, the calcination process is carried out in an inert gas or nitrogen atmosphere, the calcination temperature is 200-600℃, preferably 300℃, and the calcination time is 0.5-6 hours.

[0020] Furthermore, the inert gas is argon.

[0021] Furthermore, the molar ratio of the soluble iridium salt to the mass of strontium titanate is 1:10-40, for example, 1:35, 1:25, 1:15, preferably 1:25.

[0022] Furthermore, the soluble iridium salt is selected from IrCl3, H2IrCl6, Ir(NO3)3, Ir(acac)3 or their hydrates.

[0023] Furthermore, the washing is performed using deionized water and ethanol to remove residual ions.

[0024] As used herein, strontium titanate can be prepared using methods well known in the art, which are within the capabilities of those skilled in the art. However, in a preferred embodiment, the method for preparing strontium titanate includes the following steps:

[0025] (1) Dissolve the titanium-containing compound in an alcohol solvent and add an alkaline source to obtain a titanium source solution;

[0026] (2) Dissolve strontium salt in water to obtain a strontium source aqueous solution;

[0027] (3) The strontium source aqueous solution is mixed with the titanium source solution and subjected to a hydrothermal reaction. The reaction product is washed and dried to obtain strontium titanate.

[0028] Furthermore, the molar ratio of the titanium-containing compound to the strontium salt is 4-6:7-8, preferably 5:7.5.

[0029] Furthermore, the titanium-containing compound is Ti[O(CH2)3CH3]4.

[0030] Furthermore, the strontium salt is strontium hydroxide or its hydrate.

[0031] Furthermore, the alcohol solvent is ethanol.

[0032] Furthermore, the alkali source is ammonia water, such as 25% ammonia water.

[0033] Furthermore, the hydrothermal reaction temperature is 120-250℃, preferably 200℃, and the reaction time is 12-60 hours.

[0034] Furthermore, the reaction product is washed with acetic acid and ethanol.

[0035] In a third aspect, the present invention provides the application of iridium-strontium titanate nanobiocatalysts as described herein as peroxidase-like and catalase-like applications.

[0036] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits pH-responsive enzyme-like activity.

[0037] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits peroxidase-like activity under acidic conditions and catalase-like activity under neutral conditions.

[0038] As used herein, the terms “catalase-like activity” and “catalase-mimicking activity” are used interchangeably.

[0039] In a fourth aspect, the present invention provides the use of the iridium-strontium titanate nanobiocatalyst as described herein in the preparation of reagents for antibacterial, biofilm removal, and prevention or treatment of dental caries and periodontitis.

[0040] Furthermore, the antibacterial activity includes resistance to Streptococcus mutans and Porphyromonas gingivalis.

[0041] Furthermore, the biofilm includes caries-related biofilms.

[0042] Furthermore, the biofilm is a biofilm derived from Streptococcus mutans and / or Porphyromonas gingivalis.

[0043] Furthermore, the iridium-strontium titanate nanobiocatalyst exhibits enhanced antibacterial, biofilm-clearing, and caries-preventing or periodontitis-treating activities under NIR light irradiation.

[0044] Beneficial effects of the invention

[0045] This invention provides a highly efficient, multifunctional, and precise iridium-strontium titanate (Ir-STO) system for the microbial community and immune system in the oral microenvironment, exhibiting strong redox capabilities, good physicochemical stability, and excellent biocompatibility. The innovation of this system lies in the following three aspects: 1) pH-responsive Ir-STO generates reactive oxygen species (ROS) under acidic conditions through peroxidase activity, effectively killing cariogenic bacteria such as *Streptococcus mutans*; while under neutral conditions, it generates oxygen through the enzyme-mimicking activity of catalase, thereby inhibiting the growth of *Porphyromonas gingivalis*, maintaining the balance of the oral flora, and thus preventing and monitoring periodontitis and dental caries. Simultaneously, under stimulation by an 808 nm laser, the system also generates singlet oxygen (…). 11) Ir-STO enhances antibacterial activity against pathogens; 2) Ir-STO's reactive oxygen species scavenging ability reduces intracellular oxidative stress, thereby increasing the ratio of regulatory T cells (Tregs) to helper T cells 17 (Th17s) and the ratio of anti-inflammatory macrophages to pro-inflammatory macrophages in an inflammatory environment to maintain the balance of the immune system; 3) The release of strontium ions promotes the differentiation of mesenchymal stem cells into osteoblasts and angiogenesis, further promoting tissue repair. Notably, experimental and theoretical studies of this invention show that due to the chemical coupling between iridium nanoclusters and strontium titanate substrates, and the strong interfacial charge transfer from strontium titanate substrates to iridium sites, Ir-STO-based nanobiocatalysts exhibit excellent multifunctional pH-controlled antioxidant activity and reactive oxygen species catalytic performance. In an acidic environment, Ir-STO's peroxidase-like action can effectively kill cariogenic bacteria and possesses self-regulating antibacterial properties: that is, after reactive oxygen species are generated in an acidic environment, as the number of bacteria decreases, the local pH automatically increases, thereby weakening the effect of reactive oxygen species generation. Furthermore, Ir-STO exhibits excellent catalase-mimicking activity, capable of scavenging reactive oxygen species and generating oxygen, thus playing a role in combating anaerobic bacteria and inflammation during the treatment of periodontitis. Simultaneously, it promotes bone formation and angiogenesis by releasing strontium ions. The synthesized Ir-STO demonstrates remarkable and unexpected biocatalytic properties, emphasizing its self-regulating and dynamic nature, effectively regulating the imbalance of the oral system and providing a new approach for the comprehensive treatment of periodontitis. Attached Figure Description

[0046] Figure 1 The X-ray diffraction patterns of Ir-STO with different doping ratios are shown.

[0047] Figure 2 Scanning electron microscope images of Ir-STO with different doping ratios before and after argon treatment are shown.

[0048] Figure 3 Transmission electron microscope images of Ir-STO at different magnifications are shown, with the scale bar in Figure a being 10 nm and the scale bar in Figure b being 5 nm.

[0049] Figure 4 The images show aberration-corrected high-angle annular dark-field scanning transmission electron microscope images of Ir-STO at different magnifications. The scale bars in Figures a and b are 2 nm, and the scale bars in Figures c and d are 1 nm. The dashed circles represent Ir clusters, and the straight lines show the STO lattice at 0.276 nm and 0.390 nm, which correspond to the (110) and (100) crystal planes of STO, respectively, indicating that the STO substrate contains Ir clusters.

[0050] Figure 5The energy spectrum of Ir-STO is shown, where Figure a represents Sr, Figure b represents Ti, Figure c represents O, and Figure d represents Ir.

[0051] Figure 6 High-resolution X-ray photoelectron spectra of (a) Ti 2p and (b) Ir 4f in different materials are shown.

[0052] Figure 7 The following are shown: (a) peroxidase-mimicking activities of different materials; (b) peroxidase-mimicking activities of Ir-STO (1:25) at different pH values; (c) a comparison of kinetic parameters of peroxidase-mimicking activities of different materials, including maximum reaction rate, Michaelis constant, and turnover number; (d) halogen peroxidase-mimicking activities of different materials; (e) peroxidase-mimicking activities of different materials, tested at pH 4.5; and (f) catalase-mimicking activities of different materials, tested at pH 7.4.

[0053] Figure 8 SEM images of bacteria after different treatments are shown.

[0054] Figure 9 The minimum inhibitory concentrations of Streptococcus mutans after different treatments are shown.

[0055] Figure 10 The results show (a) Streptococcus mutans plate assays after different treatments, (b) statistical hemolysis rates, (c) bacterial live / dead staining (SYTO9 staining: live bacteria; PI staining: dead bacteria), and (d) live / dead rates.

[0056] Figure 11 The results show (a) live and dead staining of biomembranes treated with Ir-STO combined with NIR photodynamic therapy and (b) statistical live and dead rates; (c) penetration performance of biomembranes treated with Ir-STO combined with NIR photodynamic therapy and (d) statistics.

[0057] Figure 12 The results of scanning electron microscopy show the effects of Ir-STO on gingival porphyrin airborne bacteria.

[0058] Figure 13 The minimum inhibitory concentrations of Porphyromonas gingivalis after different treatments are shown.

[0059] Figure 14 The images show the live and dead staining of Porphyromonas gingivalis after different treatments (left image) and the statistical live rate (right image).

[0060] Figure 15 The results of live and dead staining of Porphyromonas gingivalis biofilms after different treatments are shown.

[0061] Figure 16The time-viable bacteria killing effect of *Porphyromonas gingivalis* biofilm after different treatments is shown. Figure a is a three-dimensional image of the biofilm immunofluorescence staining, and Figure b is the biofilm thickness.

[0062] Figure 17 The release rate of strontium ions from Ir-STO is shown, where Figure a is the cumulative release curve of strontium ions and Figure b is the average release curve of strontium ions.

[0063] Figure 18 This study demonstrated the role of Ir-STO in promoting ALP and calcium nodules, early markers of osteogenic differentiation in BMSCs. Detailed Implementation

[0064] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0065] Example 1: Synthesis of Ir-STO (1:25)

[0066] Synthesis of STO substrate: STO was prepared using strontium hydroxide (Sr(OH)₂•H₂O) and the titanium butoxy compound (Ti[O(CH₂)₃CH₃]₄) as starting materials. Briefly, 25 mmol of Ti[O(CH₂)₃CH₃]₄ was mixed with 10 mL of ethanol, and then 3.5 mL of ammonia solution (25% ammonia) was added to the mixture. Next, in another container, 37.5 mmol of Sr(OH)₂•H₂O was dissolved in 12.5 mL of deionized water to prepare a clear Sr(OH)₂•H₂O solution. This Sr(OH)₂•H₂O solution was then added to the mixed solution. Finally, the resulting suspension was transferred to a 100 mL Teflon-lined stainless steel autoclave and heat-treated at 200 °C for 48 hours. After the reaction, the product was repeatedly washed with acetic acid and ethanol, and then dried in an oven at 60 °C for 24 hours.

[0067] Synthesis of Ir-STO: First, 200 mg of synthesized STO was weighed and added to 40 mL of deionized water. The mixture was sonicated for 30 minutes to ensure thorough dispersion. Then, iridium chloride (IrCl3•xH2O) was added, with a molar ratio of IrCl3•xH2O to STO of 1:25. Sonication was repeated for another 30 minutes to ensure uniform dispersion. Subsequently, the solution was heated to 180°C in a Teflon-lined stainless steel autoclave and maintained for 24 hours. After the reaction was complete, the autoclave was cooled to room temperature. The resulting product (named Ir-STO-untreated) was washed with deionized water and ethanol to remove residual ions, and then dried overnight in an oven at 50°C. To synthesize Ir-STO, the previously synthesized untreated Ir-STO was heated to 300°C in a tube furnace under an argon atmosphere and held for 2 hours. Finally, it was naturally cooled to room temperature to obtain Ir-STO (1:25). 1:25 refers to the molar ratio of IrCl3•xH2O to the mass ratio of STO.

[0068] Example 2: Synthesis of Ir-STO (1:15)

[0069] Synthesis of STO substrate: STO was prepared using strontium hydroxide (Sr(OH)₂•H₂O) and the titanium butoxy compound (Ti[O(CH₂)₃CH₃]₄) as starting materials. Briefly, 20 mmol of Ti[O(CH₂)₃CH₃]₄ was mixed with 10 mL of ethanol, and then 3.5 mL of ammonia solution (25% ammonia) was added to the mixture. Next, in another container, 35 mmol of Sr(OH)₂•H₂O was dissolved in 12.5 mL of deionized water to prepare a clear Sr(OH)₂•H₂O solution. This Sr(OH)₂•H₂O solution was then added to the mixed solution. Finally, the resulting suspension was transferred to a 100 mL Teflon-lined stainless steel autoclave and heat-treated at 150 °C for 48 hours. After the reaction, the product was repeatedly washed with acetic acid and ethanol, and then dried in an oven at 60 °C for 24 hours.

[0070] Synthesis of Ir-STO: First, 200 mg of synthesized STO was weighed and added to 40 mL of deionized water. The mixture was sonicated for 30 minutes to ensure thorough dispersion. Then, iridium chloride (IrCl3•xH2O) was added, with a molar ratio of IrCl3•xH2O to STO of 1:35. Sonication was repeated for another 30 minutes to ensure uniform dispersion. Subsequently, the solution was heated to 150°C in a Teflon-lined stainless steel autoclave and maintained for 24 hours. After the reaction was complete, the autoclave was cooled to room temperature. The resulting product (named Ir-STO-untreated) was washed with deionized water and ethanol to remove residual ions, and then dried overnight in an oven at 50°C. To synthesize Ir-STO, the previously synthesized untreated Ir-STO was heated to 200°C in a tube furnace under an argon atmosphere and held for 2 hours. Finally, it was naturally cooled to room temperature to obtain Ir-STO (1:15). 1:15 refers to the molar ratio of IrCl3•xH2O to the mass ratio of STO.

[0071] Example 3: Synthesis of Ir-STO (1:35)

[0072] Synthesis of STO substrate: STO was prepared using strontium hydroxide (Sr(OH)₂•H₂O) and the titanium butoxy compound (Ti[O(CH₂)₃CH₃]₄) as starting materials. Briefly, 30 mmol of Ti[O(CH₂)₃CH₃]₄ was mixed with 10 mL of ethanol, and then 3.5 mL of ammonia solution (25% ammonia) was added to the mixture. Next, in another container, 40 mmol of Sr(OH)₂•H₂O was dissolved in 12.5 mL of deionized water to prepare a clear Sr(OH)₂•H₂O solution. This Sr(OH)₂•H₂O solution was then added to the mixed solution. Finally, the resulting suspension was transferred to a 100 mL Teflon-lined stainless steel autoclave and heat-treated at 180 °C for 48 hours. After the reaction, the product was repeatedly washed with acetic acid and ethanol, and then dried in an oven at 60 °C for 24 hours.

[0073] Synthesis of Ir-STO: First, 200 mg of synthesized STO was weighed and added to 40 mL of deionized water. The mixture was sonicated for 30 minutes to ensure thorough dispersion. Then, iridium chloride (IrCl3•xH2O) was added, with a molar ratio of IrCl3•xH2O to STO of 1:15. Sonication was repeated for another 30 minutes to ensure uniform dispersion. Subsequently, the solution was heated to 200°C in a Teflon-lined stainless steel autoclave and maintained for 24 hours. After the reaction was complete, the autoclave was cooled to room temperature. The resulting product (named Ir-STO-untreated) was washed with deionized water and ethanol to remove residual ions, and then dried overnight in an oven at 50°C. To synthesize Ir-STO, the previously synthesized untreated Ir-STO was heated to 400°C in a tube furnace under an argon atmosphere and held for 2 hours. Finally, it was naturally cooled to room temperature to obtain Ir-STO (1:35). 1:35 refers to the molar ratio of IrCl3•xH2O to the mass ratio of STO.

[0074] Comparative Example: Synthesis of Ir-TO

[0075] In this invention, an Ir-doped TiO2 substrate (Ir-TO) was synthesized as a control sample. Its preparation method is the same as that of Ir-STO, except that strontium hydroxide (Sr(OH)2•H2O) is not added in the synthesis of the TiO2 substrate.

[0076] Test Example 1: Structural Characterization of Ir-STO

[0077] As shown in Examples 1-3, in the typical synthesis process of this invention, Ir-doped strontium titanate (STO) precursors were prepared via a hydrothermal ion exchange reaction between STO and IrCl3. The precursors were then calcined at 200-400°C under an argon atmosphere to generate Ir-STO-based artificial enzymes. By doping with different amounts of Ir, Ir-doped STO samples with different ratios were obtained, labeled Ir-STO (1:15), Ir-STO (1:25), and Ir-STO (1:35). As demonstrated below, Ir-STO (1:25) exhibits the best performance; therefore, unless otherwise specified, Ir-STO refers to the Ir-STO (1:25) sample in the experiments described below.

[0078] First, the crystal structure of the sample was obtained using X-ray diffraction. Figure 1 The X-ray diffraction patterns of all samples were similar, and the main diffraction peaks matched well with STO (PDF#35-0734). No diffraction peaks of large Ir particles were detected.

[0079] We used scanning electron microscopy and transmission electron microscopy to study the morphology of STO and Ir-STO at different ratios. All samples showed a nanocoral-like structure. Figure 2 , Figure 3 (a and 3b). We further employed high-angle annular dark-field imaging to observe the spatial location and size of the Ir nanoclusters. (e.g., ...) Figure 4 As shown in the diagram, uniformly dispersed Ir nanoclusters, approximately 0.94 nm in size, are distributed across the STO substrate. Notably, high-angle annular dark-field scanning transmission electron microscopy (HISM) images at atomic resolution provide detailed lattice fringes with lattice spacings of 0.276 nm and 0.390 nm, corresponding to the (110) and (100) crystal planes of the STO nanocrystals in the Ir-STO. Elemental mapping by energy-dispersive X-ray spectroscopy further confirms that the Ir nanoclusters are uniformly dispersed on the STO substrate. Figure 5 ad).

[0080] After confirming the crystal structure, X-ray photoelectron spectroscopy was performed to investigate the electronic structure of the Ir nanoclusters in Ir-STO (1:25). Compared with the original STO, Ir-STO (1:25) showed a positive core level shift at the Ti 2p binding energy. Figure 6 a) This indicates that electron transfer occurred from the STO substrate to the Ir species during the calcination process. The X-ray photoelectron spectrum of Ir 4f is as follows: Figure 6 As shown in b, the binding energies of 60.4 eV and 62.1 eV correspond to the metallic state Ir, respectively. 0 and oxidized Ir 4+ In Ir-STO (1:25), the relative percentage of metallic Ir is higher than that of Ir. 4+ Based on the above results, the chemical coupling between the Ir nanoclusters and the STO substrate, as well as the strong interfacial charge transfer from the STO substrate to the Ir site, may promote the stability and catalytic reduction properties of these Ir-STO-based nanobiocatalysts.

[0081] Test Example 2: Ir-STO Enzyme-Mimetic Activity Assay

[0082] After successfully characterizing the preparation, chemical composition, and electronic structure of Ir-STO nanobiocatalysts, we further tested and compared their peroxidase-mimicking catalytic activities. Experiments were performed using a colorimetric method based on 3,3',5,5'-tetramethylbiphenyl. Figure 7As shown in a and 7b, Ir-STO (1:25) exhibited the best peroxidase-like performance and displayed pH-dependent activity. We then calculated the maximum reaction rate, Michaelis constant, and turnover number (i.e., the maximum amount of substrate converted per unit of active catalytic center). Compared to Ir-STO (1:35) and Ir-STO (1:15), Ir-STO (1:25) showed a higher maximum reaction rate and turnover number, as well as a relatively lower Michaelis constant. Figure 7 c), indicating its highly efficient catalytic kinetics and affinity for H2O2. In addition to its excellent peroxidase-like activity, we also assessed the halogen peroxidase-like activity of the Ir-STO-based nanocatalyst for the generation of hypochlorous acid using Bruce Blue reagent (see c). Figure 7 d). Ir-STO (1:25) exhibited the best halogen peroxidase mimicry activity.

[0083] To verify the pH-responsive bifunctional properties of Ir-STO, we synthesized Ir-doped TiO2 (Ir-TO) as a control sample. Under acidic conditions, Ir-STO exhibited excellent peroxidase-mimicking activity, capable of generating reactive oxygen species (ROS). Figure 7 e), while in a neutral environment, the activity of Ir-STO undergoes a smart switching ( Figure 7 f), exhibiting excellent H2O2 scavenging activity (decomposing H2O2 to produce O2 and H2O). While Ir-TO shows some peroxidase-like activity under acidic conditions, its H2O2 scavenging activity is poor under neutral conditions. Figure 7 (e, f) indicates that the combination of Ir element and STO substrate is a necessary condition for the intelligent switching function of reactive oxygen generation and scavenging.

[0084] The specific testing method for this test case is as follows.

[0085] In the peroxidase activity assay, the material solution (2 mg / mL, 10 μL) was added to 100 mmol of sodium acetate-acetic acid buffer (pH 4.5), followed by the addition of 25 μL of 3,3',5,5'-tetramethylbiphenyl (10 mg / mL) and 25 μL of hydrogen peroxide (0.1 M). The final volume of the mixture was 2 mL. After ten minutes of reaction, 200 μL of the supernatant was collected and the spectra were measured using a UV-Vis spectrophotometer in the range of 475–800 nm. Halogen peroxidase biomimetic activity was determined using a Bruce Blue-based assay. First, a 200 μmol Bruce Blue solution was prepared and dissolved in phosphate buffer at pH 5.8. Then, 1880 μL of the Bruce Blue solution was mixed with 100 μL of biocatalyst and 20 μL of hydrogen peroxide (3%) to initiate the reaction. The catalytic activity was studied by measuring the change in Bruce Blue absorption from 645 nm to 520 nm after the reaction, with a reaction time of 30 minutes.

[0086] Steady-state kinetics experiments were conducted to assess the activity of peroxidases by varying the concentration of hydrogen peroxide. For each H2O2 group, the initial reaction rate (V0) was calculated from the absorbance change according to the Beer-Lambert Law (Equation (1)) (where the attenuation coefficient ε of oxTMB is 39,000 M). −1 cm −1 (where c represents the concentration of oxTMB and l cm is the length of the solution in the optical path). Then, the reaction rate was plotted against the corresponding H2O2 and fitted using the Michaelis-Menten equation (2). In addition, the maximum reaction rate (V) was determined using a linear double reciprocal plot (Lineweaver-Burk plot, equation (3)). max ) and Michaelis constant (K m Furthermore, the transformation number (TON) is calculated according to equation (4).

[0087] A=εlc (1)

[0088] (2)

[0089] (3)

[0090] TON = V max / [E0] (4)

[0091] [S] represents the concentration of hydrogen peroxide, while [E0] represents the molar concentration of the metal in the nanozyme.

[0092] Test Example 3: Antibacterial Activity Test of Ir-STO

[0093] Studies have shown that when cariogenic bacteria dominate the oral microbiota, they disrupt the oral ecosystem, lower pH levels, and lead to demineralization of tooth hard tissues over time, in conjunction with dietary factors. Previous research indicates that the pH of saliva in the oral cavity is typically between 6.6 and 7.1. Consuming sugary foods promotes acid-producing and acid-tolerant bacteria, especially *Streptococcus mutans* (the main cariogenic bacteria), leading to acidification of the oral microenvironment and causing demineralization of tooth hard tissues and degradation of organic matter, forming organic acids and further lowering the local pH (pH < 5.5). Ultimately, this process results in demineralization of tooth structure and dissolution of organic matter, eventually leading to tooth decay. Therefore, we hypothesize that under acidic conditions, the peroxidase activity of Ir-STO can effectively exert its anti-caries effect by producing reactive oxygen species.

[0094] First, the in vitro antibacterial activity of Ir-STO against Streptococcus mutans was investigated. Streptococcus mutans UA159 (source: Guangdong Microbiological Culture Collection Center) was cultured anaerobically to the logarithmic growth phase using Brain Heart Infusion (BHI) medium at 37°C under mixed atmosphere (5% CO2, 10% H2, 85% N2). Bacterial growth was dynamically monitored using colony count (CFU) and spectrophotometry (OD600). A calibrated OD600 of 0.1 corresponded to a bacterial concentration of 2 × 10⁻⁶. 6 CFU / mL, this concentration was used for subsequent experiments. For morphological observation, bacteria were fixed in 2.5% glutaraldehyde PBS solution at 4°C for 12 h, dehydrated with gradient ethanol, and then analyzed using scanning electron microscopy (SEM). Anti-plankton activity was assessed using the following methods: the minimum inhibitory concentration (MIC) was determined in 96-well plates using the microbroth dilution method to define the lowest drug concentration required to completely inhibit growth; the bacterial culture was co-incubated with a nano-biocatalyst, and samples were taken every 2 h for gradient dilution plating and colony counting; simultaneously, the bacterial culture was treated with LIVE / DEAD® BacLight staining reagent, and bacterial viability was observed using confocal microscopy. For anti-biofilm activity assays, biofilms were cultured in 24-well plates for 48 h, washed with PBS, and stained for both live and dead cells, then imaged using confocal microscopy in three dimensions; additionally, Alexa Fluor 647-labeled dextran was used to trace extracellular polysaccharides (EPS), and the DCFH-DA probe was used to detect reactive oxygen species (ROS). The distribution of EPS and ROS was analyzed using fluorescence and confocal microscopy.

[0095] Scanning electron microscopy revealed significant morphological changes in the treated Streptococcus mutans. In the Ir-STO+NIR (808 nm near-infrared light (1.5 W / cm², 10 min)) treatment group, over 82% of the Streptococcus mutans exhibited membrane structure disintegration and cytoplasmic leakage. In contrast, the bacteria in the Ir-STO-only group maintained a localized streptococcal morphology, indicating that the Ir-STO+NIR group more thoroughly disrupted the bacterial envelope structure. Figure 8 ).

[0096] Next, we treated Streptococcus mutans under planktonic conditions with different methods and tested its antibacterial effects. The planktonic antibacterial experiment showed that the photodynamic therapy group using Ir-STO combined with 808 nm near-infrared light (1.5 W / cm², 10 min) exhibited the lowest inhibitory concentration (MIC) against Streptococcus mutans. 50 The concentration was 100 μg / mL, which was 1-fold lower than that of the Ir-STO group alone (MIC50 = 200 μg / mL). Figure 9 ).

[0097] like Figure 10 As shown in a and b, for Streptococcus mutans, a dilution of 10... 4 After concentration reduction, without 808 nm NIR irradiation, the hemolysis rate in the Ir-STO group was 36.27 ± 3.39%. After 808 nm NIR irradiation, the hemolysis rate in the Ir-STO combined with NIR photodynamic therapy group significantly decreased. Further staining of bacteria for live / dead cells was performed. Figure 10 (c, d) SYTO9 is a fluorescent dye that can penetrate the cell membranes of all bacteria and bind to their DNA. When the bacterial cell membrane is intact, SYTO9 can easily enter the cell and bind to the DNA, producing fluorescence. Therefore, SYTO9 staining can label all live bacteria. Propidium iodide (PI) is a fluorescent dye that can only enter bacteria with damaged cell membranes. When the bacterial cell membrane is damaged (e.g., dead or damaged cells), PI can penetrate the cell membrane, bind to the DNA, and produce fluorescence. Because PI staining cannot enter the intact cell membranes of live bacteria, it only labels dead bacteria. Based on SYTO9 / PI dual-staining fluorescence imaging, the proportion of live bacteria in the photodynamic therapy group was only 6.3±1.8% (SYTO9 staining fluorescence), while the proportion of dead bacteria was 93.7±2.1% (PI staining fluorescence), significantly better than the Ir-STO group (64.2±5.4% live bacteria) and the blank control group (98.5±0.7% live bacteria). Experiments showed that the Ir-STO combined with NIR photodynamic therapy group had the lowest bacterial survival rate, demonstrating the most effective inhibitory effect against Streptococcus mutans.

[0098] It is worth noting that dental plaque, as a defensive biofilm adhering to the tooth surface, possesses multiple mechanisms to resist antimicrobial drug responses, and removing this biofilm remains a challenge. Therefore, we further tested the biofilm removal capability of Ir-STO combined with NIR photodynamic therapy. Figure 11 As shown in a and b, without NIR light irradiation, the SYTO9 staining fluorescence proportion of Ir-STO was 43.73±7.27%. After the addition of 808nm NIR light irradiation, the PI staining fluorescence signal significantly increased, occupying most of the biofilm area. The results indicate that the biofilm treated with Ir-STO combined with NIR photodynamic therapy had the highest proportion of dead bacteria, with almost no surviving bacteria. Furthermore, we investigated the ability of Ir-STO to generate reactive oxygen species under near-infrared irradiation to explore its anti-biofilm mechanism. Figure 11 (c, d) In the Ir-STO and NIR-treated biofilms, fluorescence representing reactive oxygen species (ROS) as indicated by DCFH-DA was uniformly distributed throughout the biofilm, while fluorescence representing the biofilm as indicated by dextran dissipated by 77.5%, showing a significant change compared to the Ir-STO group. This suggests that NIR may promote biofilm penetration and dispersion by increasing the generation of ROS.

[0099] After confirming the high efficiency of Ir-STO nanobiocatalysts in scavenging cariogenic biofilms under acidic conditions, we further investigated their performance against *Porphyromonas gingivalis* under neutral conditions. Specifically, *Porphyromonas gingivalis* (ATCC 33277) was used as the experimental subject. It was revived and cultured in BHI medium at 37℃ in an anaerobic environment (80% N2, 10% CO2, 10% H2) for 72 h, and a concentration of 1×10⁻⁶ was prepared. 8 CFU / mL and 2×10 6 CFU / mL bacterial suspensions were prepared for use. The minimum inhibitory concentration (MIC) was determined using the microbroth dilution method. In the dead / live bacterial staining experiment, the bacterial suspension was co-cultured with Ir-STO (MIC concentration) for 24 h, washed, and then double-stained with SYTO 9 and propidium iodide. The biofilm structure was observed using a confocal microscope (CLSM). For scanning electron microscopy (SEM) sample preparation, the bacterial suspension underwent material treatment, washing with distilled water, fixation overnight at 4°C with 2.5% glutaraldehyde, gradient dehydration with ethanol, and drying followed by gold sputtering. The ultrastructure of the bacteria was then observed using SEM.

[0100] Scanning electron microscopy images showed that the cell membranes of bacteria in the Ir-STO+NIR group were shrunken and ruptured, while those in the Ir-STO group were short rod-shaped. The Ir-STO+NIR group was able to more thoroughly disrupt the bacterial envelope structure. Figure 12 ).

[0101] Next, we treated *Porphyromonas gingivalis* under different conditions and tested its antibacterial effects. Minimum inhibitory concentration (MIC) data showed that the MIC of the Ir-STO combined with NIR photodynamic therapy group was significantly lower than that of the Ir-STO group, indicating that the Ir-STO combined with NIR photodynamic therapy group exhibited the most effective inhibitory effect against *Porphyromonas gingivalis*. Figure 13 ).

[0102] Based on SYTO9 / PI double-staining fluorescence imaging, the viable bacteria percentage in the photodynamic therapy group was only 4.7±2.8% (SYTO staining), significantly better than the Ir-STO group (58.2±7.4% viable bacteria) and the blank control group (97.6±0.9% viable bacteria). The experiment showed that the Ir-STO+NIR group exhibited significantly superior antibacterial activity against *Porphyromonas gingivalis* compared to other groups. Figure 14 ).

[0103] Regarding the anti-biofilm effects of different treatment groups, such as Figure 15 As shown, the biofilm in the control group was relatively intact, while the Ir-STO+NIR group showed the lowest level of viable bacteria, almost entirely covered by dead bacteria.

[0104] To verify the enhanced biofilm permeability, we investigated the biofilm condition after Ir-STO treatment with and without NIR irradiation. Figure 16 As shown, the *Porphyromonas gingivalis* biofilm underwent treatment with Ir-STO and laser irradiation. Figure 16 The results show significant structural damage and a reduction in biofilm biomass over time. Figure 16 b shows that the biofilm thickness decreased significantly with increasing material treatment time. These results confirm that the oxygen generated in the Ir-STO group inhibited the growth of *Porphyromonas gingivalis*, but had a weaker bactericidal effect on mature biofilms, possibly related to poor oxygen permeability. Conversely, the Ir-STO+NIR group exhibited faster and better dispersion of mature biofilms, which may be attributed to the oxygen generated by NIR irradiation. 1 O2 enhances the permeability of biofilms.

[0105] Test Example 4: Osteogenic Performance Test of Ir-STO

[0106] Following periodontal bone loss caused by bacterial damage, bone regeneration is a topic of great interest. Strontium ions play a promoting role in osteogenic processes, enhancing the proliferation, differentiation, and bone mineralization of osteogenic cells (such as osteoblasts and bone marrow mesenchymal stem cells) by mimicking the role of calcium ions in bone. This invention utilizes Ir-STO nanomaterials, aiming to promote periodontal bone formation by releasing strontium ions, thereby enhancing osteogenic processes in periodontal tissues, repairing bone damage, and mitigating bone loss caused by periodontitis.

[0107] This test used bone marrow mesenchymal stem cells (BMSCs) isolated from 2-week-old Sprague Dawley rats, with cells from passages 3-5 (P3-P5) used for the experiment. The main experimental consumables included T25 culture flasks, 24-well plates, cell cryopreservation tubes, centrifuge tubes, pipettes, and sterile pipette tips. Osteogenesis induction medium was prepared freshly by mixing three sterile stock solutions (1 mM dexamethasone, 10 mM L-ascorbic acid, and 1 M β-glycerophosphate) in complete culture medium before use, protected from light. Cells were seeded in 12-well plates and cultured for 8 hours, then the medium was replaced with osteogenic induction medium, with the medium changed every 3 days. Early osteogenic markers were detected on day 7, and late markers were analyzed on day 10. For alkaline phosphatase staining, cells were fixed and incubated in the chromogenic solution for 1 hour in the dark, washed three times with PBS, and then imaged under a stereomicroscope. Calcium nodules were stained with Alizarin Red to observe calcium deposition and acquire microscopic images.

[0108] We further explored whether strontium ions released by Ir-STO could promote osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells. ICP-OES assays showed a relatively high initial release rate (within 3 days), while the release rate slowed down after 7 days. Figure 17 (a, b) Meanwhile, the strontium ion concentration in the 100µg / mL Ir-STO group released several micrograms / mL per day over 2 days. This concentration is within the strontium ion therapy window (0.5-10μg / mL), which can stimulate angiogenesis and osteogenic processes while maintaining cell survival and proliferation.

[0109] Next, we used BMSCs to investigate the effect of Ir-STO extract on osteogenic capacity. With increasing Ir-STO concentration, we observed increased expression of alkaline phosphatase (ALP, an early osteogenic marker) and mineralized nodules (labeled by Alizarin red S, a late osteogenic manifestation). Figure 18 ).

[0110] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The application of an iridium-strontium titanate nanobiocatalyst in the preparation of reagents for antibacterial, biofilm removal, and prevention or treatment of dental caries and periodontitis, characterized in that, The iridium-strontium titanate nanobiocatalyst comprises strontium titanate and iridium nanoclusters supported on the strontium titanate; The preparation method of the iridium-strontium titanate nanobiocatalyst includes: preparing an iridium-strontium titanate precursor by reacting strontium titanate with a soluble iridium salt via a hydrothermal ion exchange reaction, and then calcining the iridium-strontium titanate precursor to obtain the iridium-strontium titanate nanobiocatalyst. The molar ratio of the soluble iridium salt to the mass ratio of strontium titanate is 1:

25. The iridium-strontium titanate nanobiocatalyst exhibits enhanced antibacterial, biofilm removal, and preventative or therapeutic activities against dental caries and periodontitis under NIR light irradiation. The method for preparing the strontium titanate includes the following steps: (1) Dissolve the titanium-containing compound in an alcohol solvent and add an alkaline source to obtain a titanium source solution; (2) Dissolve strontium salt in water to obtain a strontium source aqueous solution; (3) The strontium source aqueous solution and the titanium source solution are mixed and subjected to a hydrothermal reaction. The reaction product is washed and dried to obtain strontium titanate. The molar ratio of the titanium-containing compound to the strontium salt is 4-6:7-8; the titanium-containing compound is Ti[O(CH2)3CH3]4; the strontium salt is strontium hydroxide or its hydrate; the alcohol solvent is ethanol; the alkali source is ammonia; the hydrothermal reaction temperature is 120-250℃, and the reaction time is 12-60 hours. The preparation method of the iridium-strontium titanate precursor includes the following steps: (1) Strontium titanate is dispersed in water or a water-alcohol mixture to obtain a strontium titanate dispersion; (2) Add a soluble iridium salt to the strontium titanate dispersion to form a mixed reaction system containing iridium ions and strontium titanate; (3) The mixed reaction system is subjected to a hydrothermal ion exchange reaction, wherein the reaction temperature of the hydrothermal ion exchange reaction is 100-300℃ and the reaction time is 2-48 hours; (4) After the reaction is complete, cool, wash and dry to obtain the iridium-strontium titanate precursor.

2. The application according to claim 1, characterized in that, The calcination process is carried out in an inert gas or nitrogen atmosphere, at a calcination temperature of 200-600℃, and for a calcination time of 0.5-6 hours.

3. The application according to claim 1, characterized in that, The soluble iridium salt is selected from IrCl3, H2IrCl6, Ir(NO3)3, Ir(acac)3 or their hydrates.

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