Preparation method of multifunctional composite nano-enzyme with triple cooperative regulation

Abstract

The invention is applicable to the technical field of biomedicine, provides a preparation method of a multifunctional composite nano-enzyme with triple cooperative regulation, and constructs an intelligent nano-enzyme system AgAu C-L based on a triple environment self-adaptive regulation mechanism. According to the system, the catalytic reaction temperature is accurately optimized through the photothermal effect of an AgAu nanocage, meanwhile, the local pH is actively reduced to the optimal activity range of CeO2 in combination with the probiotic metabolism characteristic of lactobacillus reuteri, and the charge transfer efficiency is enhanced in cooperation with an AgAu-coated CeO2 heterojunction interface electron transfer optimization strategy; under the combined action of the three, the catalytic efficiency of the CeO2 nano-enzyme in a physiologically neutral / weakly alkaline environment, especially the peroxidase-like activity of the CeO2 nano-enzyme is remarkably improved. The intelligent system not only can efficiently and physically remove a periodontal pathogenic bacterium biological membrane, but also can disintegrate a microorganism collaborative network by accurately interfering key paths such as pathogenic bacterium nucleotide synthesis and arginine metabolism, and finally remodeling of oral cavity micro-ecological balance is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a method for preparing a triple-coordinated multifunctional composite nanozyme. Background Art

[0002] Nanozymes, especially those with peroxidase-like activity, have shown potential in the biomedical field. However, their clinical translation faces a core obstacle: physiological conditions (approximately 37°C body temperature, neutral or weakly alkaline) are seriously mismatched with the environment required for most nanozymes to exert optimal catalytic activity (usually requiring higher temperatures and acidic pH). Existing materials have significant defects: although nanozymes such as Fe3O4 have strong POD (peroxidase) activity, they are prone to oxidative inactivation in physiological environments and lack stability; although CeO2 nanozymes have excellent environmental stability, their POD activity is highly dependent on efficient CeO2. 3 + / Ce 4+ The redox cycle, a process in which electron transfer is slow under physiological conditions, leads to significantly insufficient catalytic efficiency. Therefore, effectively controlling two key parameters, local temperature and pH, in vivo is a core challenge that needs to be addressed to improve the catalytic efficiency of nanozymes (especially CeO2) and promote their clinical application.

[0003] In response to the above challenges, existing single regulatory methods have obvious limitations. On the one hand, the use of photothermal materials can achieve local temperature increase under near-infrared light irradiation and optimize the catalytic temperature of nanozymes, but this strategy cannot solve the mismatch between the physiological neutral pH environment and the acidic environment required by nanozymes (such as CeO2). On the other hand, although the introduction of chemical acidifiers can reduce the local pH value, they often lack target specificity, may disrupt the normal physiological pH balance, and are accompanied by potential side effects. More importantly, existing technologies have difficulty in simultaneously, accurately and synergistically regulating the three key factors of temperature, pH and promoting interfacial electron transfer in a complex physiological environment, and cannot fully realize the potential of nanozymes (such as CeO2) in disease treatment. Therefore, there is an urgent need to develop a new method that can integrate multiple regulatory mechanisms to overcome the bottlenecks of existing technologies. Summary of the Invention

[0004] The purpose of the embodiments of the present invention is to provide a method for preparing a multifunctional composite nanozyme with triple synergistic regulation, aiming to solve the problems raised in the above background technology.

[0005] The embodiment of the present invention is achieved by a method for preparing a triple-coordinated multifunctional composite nanozyme, comprising the following steps: Step 1: Preparation of AgAu@CeO2 composite material; Step 1.1: Synthesis of silver nanocubes (Ag NCs); Step 1.2: silver-gold nanocage conversion; Step 1.3: core-shell structure construction; Step 2: AgAu CL composite nanozyme assembly; Step 2.1: Amination modification; Step 2.2: Phenylboronic acid coupling; Step 2.3: Probiotic integration.

[0006] According to a further technical solution, step 1.1 includes the following steps: 50 mL of ethylene glycol was placed in a three-necked flask with magnetic stirring and heated to 150°C. 0.6 mL of sodium sulfide solution, 5 mL of 3 mM hydrochloric acid solution, and 12.5 mL of 20 mg / mL polyvinylpyrrolidone solution were rapidly injected in sequence. After 2 minutes of reaction, 4 mL of 282 mM silver trifluoroacetate solution was slowly added and the reaction was kept at a constant temperature for 30 minutes. The reaction system was quenched in an ice-water bath and centrifuged at 8000 rpm for 10 minutes. The product was washed three times with acetone and deionized water in sequence and finally dispersed in 40 mL of deionized water for storage.

[0007] According to a further technical solution, step 1.2 includes the following steps: Take 10 mL of silver nanocube dispersion and add it to 100 mL of deionized water containing 5 mg / mL polyvinyl pyrrolidone, and heat it to 100°C. Then add 10 mL of 1 mM chloroauric acid solution at a constant rate of 40 mL / h. After the solution turns dark blue and the color stabilizes, add saturated sodium chloride solution to remove by-products. Centrifuge at 10,000 rpm for 15 min, collect the product, wash it three times, and vacuum dry it to obtain AgAu nanocages (AgAu NCs).

[0008] According to a further technical solution, step 1.3 includes the following steps: AgAu NCs were dispersed in 40 mL of a mixed solution of water / ethanol (volume ratio 1:1) and sonicated for 30 min. 0.05 mM cerium nitrate and 0.1 mM hexamethylenetetramine were added sequentially, and the mixture was refluxed in an oil bath at 60°C for 2 h. After the reaction, the AgAu@CeO2 core-shell material was collected by centrifugation at 12,000 rpm for 10 min, washed, and vacuum-dried for storage.

[0009] According to a further technical solution, step 2.1 includes the following steps: 50 mg of AgAu@CeO2 was dispersed in 50 mL of anhydrous ethanol, ultrasonicated for 30 minutes, and then 10 wt% of 3-aminopropyltriethoxysilane (APTES) was added. The pH was adjusted to 8.0 and the mixture was reacted at 70°C for 4 hours. The mixture was centrifuged at 10,000 rpm for 10 minutes. The amination product (AgAu@CeO2-NH2) was collected and washed three times with ethanol and deionized water.

[0010] According to a further technical solution, step 2.2 includes the following steps: 10 mg of carboxyphenylboronic acid (CPBA) was dissolved in 10 mL of PBS buffer (pH 7.4) and activated with 20 mg of EDC and 15 mg of NHS at room temperature for 30 minutes. The activated solution was mixed with AgAu@CeO2-NH2 and reacted at room temperature for 12 hours. The CPBA-functionalized product (AgAu@CeO2-PBA) was obtained by centrifugation at 10,000 rpm for 10 minutes and then washed three times with PBS.

[0011] The step 2.3 includes the following steps: 50 μg / mL of AgAu@CeO2-PBA was mixed with 1×10 7 The suspension of Lactobacillus reuteri with CFU / mL was mixed and incubated at 37°C with shaking for 30 minutes for directional coupling through boronate bonds; the AgAu CL composite nanozyme was collected by centrifugation at 5000 rpm for 5 minutes, washed twice in anaerobic medium and then stored.

[0012] The present invention provides a method for preparing a multifunctional composite nanozyme with triple synergistic regulation. This system precisely optimizes the catalytic reaction temperature through the photothermal effect of AgAu nanocages. It also actively reduces the local pH to the optimal CeO2 activity range by combining the probiotic metabolic properties of Lactobacillus reuteri. Furthermore, it synergizes with the electron transfer optimization strategy at the AgAu@CeO2 heterojunction interface to enhance charge transfer efficiency. These three factors work together to significantly enhance the catalytic performance of the CeO2 nanozyme in physiological neutral / weakly alkaline environments, particularly its peroxidase-like activity. This intelligent system not only effectively physically removes periodontal pathogen biofilms but also disrupts microbial cooperative networks by precisely interfering with key pathways such as pathogen nucleotide synthesis and arginine metabolism, ultimately reshaping the oral microecological balance. This research provides an innovative solution to address the key bottleneck of nanozymes' poor adaptability in complex physiological environments and lays an experimental foundation for the development of efficient and safe treatment strategies for periodontitis and other infectious diseases. The established multi-dimensional synergistic regulation strategy of "temperature, pH, and electron transfer" provides a universal methodological reference for the design of next-generation environmentally responsive intelligent therapeutic nanozymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1A flow chart of a method for preparing a triple-coordinated multifunctional composite nanozyme provided in an embodiment of the present invention; Figure 2 TEM images of Ag NCs, AgAu NCs, and AgAu@CeO2 samples coated with CeO2 shells of different thicknesses; Figure 3 EDX image of AgAu@CeO2 sample; Figure 4 UV-Vis spectra of AgAu NCs sample and AgAu@CeO2 sample; Figure 5 is the XPS spectrum of AgAu@CeO2; Figure 6 FTIR spectra of products at each level; Figure 7 TEM and SEM images of AgAu CL; Figure 8 is the Zeta potential diagram of each level of products; Figure 9 This is a photothermal effect diagram of the composite material; Figure 10 Time-dependent absorption spectra of AgAu@CeO2 at different temperatures; Figure 11 is the POD-like activity of AgAu@CeO2 under different H2O2 concentrations; Figure 12 is the effect of pH on the POD-like activity of AgAu@CeO2; Figure 13 EPR spectrum of AgAu@CeO2; Figure 14 FDTD simulation results for different materials; Figure 15 The time-dependent acidified local pH of pure L. reuteri, AgAu@CeO2 and AgAu CL under different simulated environments; Figure 16 is the relative enzyme activity of AgAu CL after different acidification times; Figure 17 is the effect of temperature on the POD-like activity of AgAu CL; Figure 18 is the relative enzyme activity of different materials; Figure 19 To evaluate the cytotoxicity of nanomaterials for CCK-8 assay; Figure 20 The blood compatibility of AgAu@CeO2; Figure 21H&E staining of major organs (including heart, liver, lung, spleen, and kidney) after various treatments; Figure 22 For CFU experiments, the antibacterial effects of different treatments on F. nucleatum and mixed bacterial biofilms were evaluated; Figure 23 Live / dead staining was used to detect bacterial status and film thickness of bacterial biofilm after different treatments; Figure 24 SEM images of F. nucleatum and S. gordonii biofilms after different treatments; Figure 25 Metabolite correlation analysis of Fusobacterium nucleatum biofilm after AgAu CL + NIR treatment; Figure 26 Metabolic pathway analysis of Fusobacterium nucleatum biofilm after AgAu CL + NIR treatment; Figure 27 3D reconstruction and statistical results of maxillary molars of rats after different treatments using Micro CT; Figure 28 The therapeutic effects of periodontitis were evaluated at the histological level after different treatments in rats. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0015] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0016] like Figure 1 As shown, a method for preparing a triple-coordinated multifunctional composite nanozyme provided by one embodiment of the present invention comprises the following steps: Step 1: Preparation of AgAu@CeO2 composite material; Step 1.1: Synthesis of silver nanocubes (Ag NCs); Ethylene glycol (50 mL) was placed in a three-necked flask with magnetic stirring and heated to 150°C. Sodium sulfide solution (0.6 mL), hydrochloric acid solution (3 mM, 5 mL), and polyvinyl pyrrolidone solution (20 mg / mL, 12.5 mL) were then rapidly added sequentially. After 2 minutes of reaction, silver trifluoroacetate solution (282 mM, 4 mL) was slowly added and the reaction was continued at constant temperature for 30 minutes. The reaction system was quenched in an ice-water bath and centrifuged (8000 rpm, 10 min). The product was washed three times with acetone and then deionized water, and finally dispersed in 40 mL of deionized water for storage.

[0017] Step 1.2: silver-gold nanocage conversion; A 10 mL dispersion of silver nanocubes was added to 100 mL of deionized water containing polyvinylpyrrolidone (5 mg / mL) and heated to 100°C. A 1 mM chloroauric acid solution (10 mL) was added dropwise at a rate of 40 mL / h. After the solution turned dark blue and stabilized, saturated sodium chloride solution was added to remove byproducts. The product was collected by centrifugation (10,000 rpm, 15 min), washed three times, and dried under vacuum to obtain AgAu nanocages (AgAu NCs).

[0018] Step 1.3: core-shell structure construction; AgAu NCs were dispersed in a water / ethanol mixture (1:1 v / v, 40 mL) and sonicated for 30 minutes. Cerium nitrate (0.05 mM) and hexamethylenetetramine (0.1 mM) were then added sequentially, and the mixture was refluxed in an oil bath at 60°C for 2 hours. After the reaction, the AgAu@CeO2 core-shell material was collected by centrifugation (12,000 rpm, 10 minutes), washed, and vacuum-dried for storage.

[0019] Step 2: AgAu CL composite nanozyme assembly; Step 2.1: Amination AgAu@CeO2 (50 mg) was dispersed in anhydrous ethanol (50 mL) and sonicated for 30 minutes. 3-Aminopropyltriethoxysilane (APTES, 10 wt%) was added, the pH was adjusted to 8.0, and the mixture was reacted at 70°C for 4 hours. The amination product (AgAu@CeO2-NH2) was collected by centrifugation (10,000 rpm, 10 minutes) and washed three times with ethanol and deionized water.

[0020] Step 2.2: Phenylboronic acid coupling; Carboxyphenylboronic acid (CPBA, 10 mg) was dissolved in PBS buffer (pH 7.4, 10 mL). EDC (20 mg) and NHS (15 mg) were added and activated at room temperature for 30 minutes. The activated solution was mixed with AgAu@CeO2-NH2 and reacted at room temperature for 12 hours. The phenylboronic acid-functionalized product (AgAu@CeO2-PBA) was obtained by centrifugation (10,000 rpm, 10 min) and washed three times with PBS.

[0021] Step 2.3: Probiotic integration; AgAu@CeO2-PBA (50 μg / mL) was mixed with Lactobacillus reuteri suspension (1×10 7The AgAu CL nanozyme was mixed with 100 μg of ...

[0022] As a preferred embodiment of the present invention, 1. The prepared samples were characterized by transmission electron microscopy (TEM). Hollow AgAu NCs were synthesized using Ag NCs as templates. Ag NCs are cubes with a uniform side length of 50 nm, and AgAu NCs present a hollow cage structure. Subsequently, ultrafine CeO2 shells of different thicknesses (6, 10 nm) were coated on the surface of AgAu NCs. Each AgAu NCs was uniformly coated with CeO2, forming an AgAu@CeO2 core-shell composite structure. See the specific TEM image. Figure 2 As shown (where Figure 2 A is the TEM image of Ag NCs, Figure 2 B is the TEM image of AgAu NCs. Figure 2 C is AgAu NCs coated with 6 nm ultrafine CeO2 shell, Figure 2 D is AgAu NCs coated with 10 nm ultrafine CeO2 shell).

[0023] 2. In order to verify the composition of the final product AgAu@CeO2 core-shell composite structure sample, the AgAu@CeO2 sample was subjected to energy dispersive X-ray spectroscopy (EDX) analysis. The specific EDX diagram is shown in the figure below. Figure 3 As shown, from Figure 3 The anisotropic structure of AgAu@CeO2 can be determined. The results show that CeO2 is evenly covered on the surface of AgAu NCs, and the distribution of Ag, Au, Ce and O elements is clear and obvious.

[0024] 3. The prepared AgAu NCs samples and AgAu@CeO2 samples were characterized by UV-Vis (Ultraviolet and visible spectrophotometry). The specific UV-Vis spectra are shown in Figure 4UV-visible spectroscopy reveals that the LSPR absorption peak of AgAu NCs is located at 630 nm, but after CeO2 coating, this peak red-shifts to 808 nm. This red-shift is attributed to the increase in the refractive index of the surrounding medium and the coupling effect between the plasmon and semiconductor dielectric constants, further indicating that CeO2 coating changes the local electric field environment of the nanocage.

[0025] 4. X-ray photoelectron spectroscopy (XPS) was performed on AgAu@CeO2 and pure cerium dioxide. XPS analysis showed that the surface elemental composition and chemical state of AgAu@CeO2 changed significantly. Compared with pure CeO2, Ce in AgAu@CeO2 3+ The relative proportion of AgAu@CeO2 increases. The binding energy spectrum analysis shows that the Ce 3d peak in AgAu@CeO2 shifts to a higher binding energy direction, confirming the existence of an electron transfer effect at the interface. Specific XPS results are shown in Figure 5 ( Figure 5 A is the XPS full spectrum image of AgAu@CeO2, Figure 5 B is the XPS image of Ce 3d of AgAu@CeO2 and pure ceria).

[0026] 5. The prepared samples were characterized by FTIR (Fourier Transform Infrared Spectrometer). The specific FTIR spectrum is shown in Figure 6 As shown. The stable combination of AgAu@CeO2 and lactic acid bacteria was achieved by chemical modification. First, the surface of AgAu@CeO2 was modified with APTES to introduce surface amino groups (-NH2). FTIR results showed that the modified AgAu@CeO2-APTES had a wavelength of 1050-1150 cm -1 A characteristic absorption peak appears at 1600-1700 cm, corresponding to the CO stretching vibration in the APTES structure, indicating that APTES has been successfully modified on the surface of the nanomaterial. Subsequently, under the action of NHS / EDC, the amino group (-NH2) modified by APTES undergoes an amidation reaction with the carboxyl group (-COOH) on the surface of CPBA to form a stable amide bond (-CONH-), coupling CPBA to the nanozyme surface. The spectrum of AgAu@CeO2-APTES / CPBA is at 1600-1700 cm -1A new absorption peak appeared at 1350-1450 cm-1, indicating that an amide bond was formed between the amino group and the carboxylphenylboronic acid. Therefore, the phenylboronic acid group was successfully introduced into the AgAu@CeO2 structure. Finally, the phenylboronic acid group underwent a reversible covalent coupling reaction with the hydroxyl group (-OH) on the surface of the lactic acid bacteria through click chemistry to form a stable borate ester bond, thereby achieving stable binding of AgAu@CeO2 to lactic acid bacteria. This binding process was further confirmed by FTIR spectroscopy, where AgAu@CeO2-APTES / CPBA had a peak at 1350-1450 cm-1. -1 The characteristic absorption peak of BO stretching vibration appears at , indicating that the phenylboronic acid group successfully participates in the reaction.

[0027] 6. The prepared AgAu CL was characterized and analyzed by TEM and Scanning Electron Microscope (SEM). Figure 7 As shown ( Figure 7 A is the TEM image of AgAu CL, Figure 7 (B) SEM image of AgAu CL), the distribution of AgAu@CeO2 nanoparticles on the surface of probiotics can be seen, indicating that the nanoparticles are evenly attached while maintaining the integrity of the bacterial morphology.

[0028] 7. The prepared AgAu NCs samples, AgAu@CeO2 samples and AgAu CL samples were analyzed for Zeta potential. The specific Zeta potential diagram is shown in Figure 8 As shown, the zeta potential of the sample to be tested is measured using a Zetasizer series potentiostat. Figure 8 It can be seen that the Zeta potential of AgAu@CeO2 is positively shifted compared with that of AgAu NCs, confirming the successful encapsulation of CeO2, while the negative potential of AgAu CL confirms the effective combination of probiotics and the material.

[0029] 8. The prepared AgAu NCs samples and AgAu@CeO2 samples were tested for photothermal effects. The results are as follows: Figure 9 As shown ( Figure 9 A is the photothermal image of different nanomaterials under 808 nm laser excitation. Figure 9 B is the corresponding heating curve, Figure 9 C is different light intensities (0.5, 1.0, 2 W / cm 2 ) characterization of the photothermal performance of AgAu@CeO2, Figure 9D is the temperature change of AgAu@CeO2 after five cycles of "on-off" irradiation under 808 nm laser. The temperature of the AgAu@CeO2 solution increases with time, reaching 50°C after 10 minutes, much higher than that of the AgAu NCs and phosphate-buffered saline (PBS) control groups. The temperature distribution at different laser power densities shows a strong correlation between laser power density and temperature rise. As the power density increases from 0.5 W / cm 2 Increased to 2 W / cm 2 , the temperature rise significantly increased. Increasing the laser power density further enhanced the temperature rise, demonstrating the material's efficient photothermal conversion capability, which can be effectively controlled by adjusting the laser power density. AgAu@CeO2 maintained stable photothermal performance after five consecutive irradiation-cooling cycles, demonstrating excellent photothermal stability.

[0030] 9. The time-dependent absorption spectra of the prepared AgAu@CeO2 at different temperatures were measured, such as Figure 10 As shown in the figure, under the same conditions, when the temperature increases, the enzymatic reaction rate of AgAu@CeO2 is faster and more ROS can be produced to oxidize TMB.

[0031] 10. The POD-like catalytic activity of AgAu@CeO2 was evaluated using TMB assay, e.g. Figure 11 and Figure 12 As shown, AgAu@CeO2 efficiently catalyzes the generation of •OH radicals, oxidizing TMB to blue ox-TMB, which exhibits a distinct absorption peak at 652 nm. The absorbance increases with increasing H2O2 concentration, indicating the generation of more •OH radicals. The catalytic activity of AgAu@CeO2 is significantly dependent on pH, reaching its peak at pH 4. Under near-neutral or alkaline conditions, the catalytic activity decreases significantly.

[0032] 11. The prepared AgAu@CeO2 samples were subjected to Electron Paramagnetic Resonance (EPR) analysis to further confirm the types and concentrations of ROS generated by AgAu@CeO2 under dark and near-infrared irradiation. Figure 13 As shown in Figure 2, under dark conditions, AgAu@CeO2 only produces hydroxyl radicals (•OH), indicating that it has intrinsic catalytic activity. However, under 808 nm near-infrared irradiation, the EPR signal is significantly enhanced, the generation of •OH increases, and singlet oxygen ( 1 O2) and superoxide anion ( )Signal.

[0033] 12. To clarify the catalytic enhancement mechanism of AgAu@CeO2 composite materials, the Finite-Difference Time-Domain method (FDTD) simulation was used to reveal the regulation of the local electric field distribution by the material geometry and shell thickness. Figure 14 As shown ( Figure 14 A is the FDTD simulation result of AgAu NCs under 808 nm laser irradiation, Figure 14 B is the FDTD simulation result of CeO2 under 808 nm laser irradiation, Figure 14 C is the FDTD simulation result of AgAu@CeO2 with a CeO2 shell thickness of 6 nm under 808 nm laser irradiation, Figure 14 (D) FDTD simulation results of AgAu@CeO2 with a 10 nm CeO2 shell thickness under 808 nm laser irradiation. Due to the sharp corners and vertices of the cubic geometry, AgAu NCs significantly enhance the local electric field intensity through the tip effect, providing a critical driving force for electron transfer in subsequent catalytic reactions. Notably, the formation of such high-field regions is impossible using nanospheres or non-angular structures (such as nanorods), demonstrating the necessity of designing a cubic configuration for precious metals.

[0034] The introduction of a CeO2 shell further revealed a non-monotonic variation in the electric field enhancement. When the CeO2 thickness increased to 6 nm, the electric field strength decreased compared to that of the bare nanocage. This is attributed to the dielectric shielding effect of the thin semiconductor layer, which partially suppresses the plasmon resonance. However, the 10 nm shell, by optimizing the band matching at the heterojunction interface, promotes directional electron migration while maintaining plasmon coupling, resulting in an electric field strength at the corners exceeding that of the bare nanocage. This phenomenon suggests that a CeO2 shell of appropriate thickness can both mitigate charge recombination on the noble metal surface and, through geometric constraints, reshape the electromagnetic field distribution, extending the strongest electric field region from the metal surface to the semiconductor-solution interface, directly affecting the catalytic active sites.

[0035] 13. To evaluate the acidification ability of probiotics in the local environment, a pH meter was used to detect the pH changes of unmodified probiotics (L. reuteri), AgAu@CeO2 alone, and AuAg CL in normal physiological environment and periodontitis simulation environment. Figure 15 As shown ( Figure 15 A is the time-dependent acidified local pH of pure L. reuteri, AgAu@CeO2, and AgAu CL under normal physiological conditions. Figure 15(B) Time-dependent local pH acidification by L. reuteri alone, AgAu@CeO2, and AgAu CL under a simulated periodontitis environment. In this simulated periodontitis environment, both unmodified probiotics and AgAu CL significantly lowered pH, an effect likely related to the production of acetic and lactic acids. Notably, AgAu CL exhibited ultrasensitive pH regulation, lowering pH by an additional 0.15 units compared to unmodified L. reuteri. In a normal physiological environment, no significant difference in the acidification capacity of the modified probiotics was observed, indicating that AgAu CL is specific for enhancing acidification in the periodontitis microenvironment. Of particular note, the AgAu@CeO2 group alone did not cause a significant decrease in pH compared to the control group, suggesting that the enhanced acidification was primarily due to the metabolic activity of the probiotics, rather than the direct action of the nanozyme.

[0036] Furthermore, the dynamic monitoring of nanozyme activity at different time points (0 h, 12 h, 24 h) was used to explore the correlation mechanism between acidification effect and enzyme activity. Figure 16 As shown in the figure, the activity of the nanozyme showed a time-dependent enhancement trend, among which AuAg CL showed the most significant activity improvement over time. This suggests that probiotic-mediated acidification may further enhance the catalytic efficiency of the nanozyme through a pH-dependent activation mechanism.

[0037] 14. The effect of temperature on the catalytic activity of the system was evaluated. Figure 17 As shown in Figure 2, the catalytic activity of AgAu@CeO2 reaches its highest at 40℃.

[0038] 15. Comparative analysis of the enzyme activities of different nanozyme formulations, e.g. Figure 18 As shown in the results, the AuAg C-L+NIR group exhibited the highest catalytic activity among all experimental groups, which was significantly better than the CeO2, AgAu@CeO2 and AgAu@CeO2+NIR groups.

[0039] 16. The in vitro cytotoxicity of AgAu@CeO2 was evaluated by CCK-8 cell viability assay. Figure 19As shown, L929 fibroblasts were treated with various concentrations of AgAu@CeO2. After 24 hours, cell viability remained above 91.53% at a nanoparticle concentration of 100 μg / mL. At a concentration of 125 μg / mL, cell viability decreased slightly to 82.36%, but remained above 80%, demonstrating good biocompatibility within this timeframe. However, cell viability gradually decreased with increasing material concentration and prolonged treatment time. Notably, after 72 hours of treatment, cell viability remained above 83.45% at a concentration of 100 μg / mL, while at 125 μg / mL, cell viability dropped below 80%, indicating potential toxicity from long-term exposure at higher concentrations. Based on these results, a concentration of 100 μg / mL was determined to be relatively safe and suitable for subsequent experiments.

[0040] 17. The blood compatibility of AgAu@CeO2 was further evaluated by hemolysis test. Figure 20 As shown in the figure, no significant hemolysis of erythrocytes occurred when treated with different concentrations of AgAu@CeO2 (0, 25, 50, 75, 100, and 125 μg / mL). Quantitative analysis further confirmed that the hemolysis rate was less than 5% at all tested concentrations, indicating that AgAu@CeO2 has good blood compatibility.

[0041] 18. Perform histological analysis on major organs (heart, liver, spleen, lungs, and kidneys). Figure 21 As shown in Figure 3, no obvious pathological damage or abnormalities were found under H&E staining after 30 days of treatment. These results further confirmed the excellent biosafety of AgAu@CeO2 in vivo.

[0042] 19. The antibacterial effects of different treatments on F. nucleatum and mixed bacterial biofilms were evaluated by CFU experiments. Figure 22 As shown ( Figure 22 A is an image of F. nucleatum colonies. Figure 22 B is the statistical information corresponding to the image of F. nucleatum colonies, Figure 22 C is an image of mixed bacterial colonies. Figure 22(D is the statistical information corresponding to the image of the mixed bacterial colony.) Compared with the control group, in the absence of NIR, the CFU counts of the single-bacterial biofilm and mixed biofilm in the AgAu@CeO2 group decreased by approximately 2 logs and 1 log, respectively. After adding NIR irradiation (AgAu@CeO2 + NIR group), the antibacterial activity was significantly enhanced, with the CFU reductions for single-bacterial and mixed biofilms increasing to 3 logs and nearly 2 logs, respectively. Further introduction of probiotic-mediated regulation (AgAuC-L group, no NIR) reduced the CFU counts of single-bacterial and mixed biofilms by 3 logs and 2 logs, respectively. The antibacterial effect reached its highest level when AgAu CL was combined with NIR irradiation (AgAu CL + NIR group), with CFU counts for single-bacterial and mixed biofilms reduced by 4 logs and 3 logs, respectively.

[0043] 20. In order to evaluate the degree of damage to the biofilm structure and the survival status of deep-layer bacteria, three-dimensional imaging analysis of F. nucleatum single-bacterial biofilm and S. gordonii and F. nucleatum mixed biofilm was performed using live / dead staining. Figure 23 As shown ( Figure 23 A is a representative three-dimensional live / dead image of F. nucleatum biofilm. Figure 23 (B) Representative 3D live / dead images of a mixed biofilm of S. gordonii and F. nucleatum.) In the absence of NIR, the AgAu@CeO2 + NIR and AgAu CL groups significantly outperformed the control group in biofilm removal, as demonstrated by reduced biofilm thickness and an increased proportion of dead bacteria within the biofilm. The AgAu CL + NIR group exhibited the strongest biofilm disruption ability, with significantly reduced biofilm thickness compared to the control group. Furthermore, the biofilm matrix integrity was severely compromised in this group, demonstrating its significant clearance of deeply resistant bacteria.

[0044] 21. The morphology of the treated bacteria was further analyzed by SEM. Figure 24 As shown. Bacteria in the AgAu CL + NIR group showed significantly reduced biofilm coverage. The AgAu CL group and the AgAu@CeO2 + NIR group also showed damage or disintegration of the bacterial film. In contrast, the bacterial structure in the control group and the AgAu@CeO2 group remained largely intact, consistent with the CFU count and live / dead staining results. Notably, smooth-surfaced, rod-shaped bacteria were found in both the AgAu CL and AgAu CL + NIR groups in both single-species and dual-species experiments. These bacteria were identified as surviving and proliferating probiotics that exhibited high tolerance to ROS, further emphasizing their potential in biofilm disruption.

[0045] 22. In order to further explore the antibacterial mechanism of AgAu CL complex against F. nucleatum, metabolomics method was used to comprehensively analyze the metabolic changes of F. nucleatum before and after treatment. Figure 25 ( Figure 25 A is the principal component analysis (PCA) of metabolites, Figure 25 B is a volcano plot of up-regulated and down-regulated metabolites in treated biofilms. Figure 25 C is the heat map of differentially expressed metabolites, Figure 25 D is a histogram of significantly differentially expressed metabolites). Figure 26 ( Figure 26 A is the KEGG enrichment pathway diagram, Figure 26 B is the heat map of metabolites related to the D-amino acid metabolic pathway. Figure 26 (C) Schematic representation of the specific antibacterial effect of AgAu CL + NIR. Principal component analysis (PCA) revealed significant separation in metabolite distribution between the two sample groups, indicating that the complex systematically perturbs the bacterial metabolic network. A total of 449 metabolites were identified in both positive and negative ion modes, of which 89 showed significant changes in abundance. Volcano plots and heat maps further revealed unusual fluctuations in nucleotide metabolism-related molecules such as orotic acid, uridine monophosphate (UMP), and guanosine monophosphate (GMP), suggesting possible perturbations in the pyrimidine and purine synthesis pathways. Notably, levels of 2,6-diaminopimelate (DAP) in the D-amino acid metabolic pathway were significantly upregulated, suggesting adaptive changes in bacterial cell wall synthesis-related metabolism. Furthermore, chord plots and correlation heat maps revealed strong correlations between differentially expressed metabolites. Metabolic interaction network analysis revealed a significant decrease in the abundance of key nodes (arginine and ornithine) in the arginine-ornithine-putrescine metabolic axis, potentially affecting metabolic synergy among bacteria. Further KEGG pathway enrichment analysis revealed that the amino acid metabolism (arginine, ornithine), lipid metabolism (dephosphorylation of coenzyme A) and nucleotide metabolism (pyrimidine synthesis) pathways were significantly perturbed.

[0046] 23. Male Wistar rats (weighing 180 ± 10 g) were ligated around the left maxillary second molar with an orthodontic ligature (4-0 silk) and subjected to local pathogen inoculation to establish a periodontitis model. After successful modeling, the rats were randomly divided into six groups (n = 6): a blank control group (no treatment), an inflammation control group (local injection of PBS), an AgAu@CeO2 group (100 μg / mL), an AgAu@CeO2+NIR group (100 μg / mL + 808 nm laser irradiation), an AuAg CL group (100 μg / mL), and an AuAg C-L+NIR group (100 μg / mL + 808 nm laser irradiation). The treatment groups received a local injection of the nanomaterial (0.2 mL / injection) in the periodontal pocket, combined with near-infrared light (NIR; power density 0.5 W / cm2, 10 minutes / injection). Treatment was administered once every other day for a total of 15 days. After treatment, the rats were killed and the maxilla was isolated and fixed with 4% paraformaldehyde for 48 hours. The alveolar bone was scanned using a Micro-CT system, and the distance from the cementoenamel junction to the alveolar crest (CEJ-ABC) was measured after three-dimensional reconstruction to quantify the degree of bone resorption. Figure 27 As shown ( Figure 27 A is the 3D reconstruction of maxillary molars by Micro CT. Figure 27 (B) Statistical results of 3D reconstruction. Compared with the inflammation control group (1.35 ± 0.02 mm), the CEJ-ABC distance was significantly reduced in all treatment groups. In the AgAu CL + NIR group, the CEJ-ABC distance decreased to 0.49 ± 0.02 mm, demonstrating the most significant therapeutic effect.

[0047] To evaluate the effect of AgAu CL + NIR composite on inflammation, histological evaluation was performed. Figure 28 As shown ( Figure 28 A is a typical H&E staining image. Figure 28 B is the statistics of the number of immune cells in the inflammatory area of periodontal tissue, Figure 28 C is a typical Masson staining image, Figure 28D represents the percentage of collagen degradation in the inflammatory site of the periodontal tissue. H&E staining revealed a significant infiltration of neutrophils and lymphocytes in the gingiva and alveolar bone of the positive control group. However, the inflammatory cell infiltration in the AgAu CL + NIR treatment group was significantly reduced, approaching that of the blank control group. Masson trichrome staining was used to assess the effect of treatment on collagen fibers in the inflammatory site. The blue area represents collagen fibers. The blank control group had dense, blue collagen fibers, indicating healthy tissue. In the positive control group, collagen fibers were degraded, with fiber breakage and enhanced red staining. The collagen fiber status in the AgAu CL + NIR group was similar to that of the blank control group, indicating that this treatment strategy effectively protected collagen fiber integrity. This may be due to the multifunctional synergistic effect of AgAu CL, which facilitates periodontal tissue recovery.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a triple-coordinated multifunctional composite nanozyme, characterized in that: The following steps are involved: Step 1: Preparation of AgAu@CeO2 composite material; Step 1.1: Ag NCs synthesis; Step 1.2: silver-gold nanocage conversion; Step 1.3: core-shell structure construction; Step 2: AgAu CL composite nanozyme assembly; Step 2.1: Amination modification; Step 2.2: Phenylboronic acid coupling; Step 2.3: Probiotic integration.

2. The method for preparing the triple synergistically regulated multifunctional composite nanozyme according to claim 1, characterized in that: The step 1.1 includes the following steps: 50 mL of ethylene glycol was placed in a three-necked flask with magnetic stirring and heated to 150°C. 0.6 mL of sodium sulfide solution, 5 mL of 3 mM hydrochloric acid solution, and 12.5 mL of 20 mg / mL polyvinylpyrrolidone solution were rapidly injected in sequence. After 2 minutes of reaction, 4 mL of 282 mM silver trifluoroacetate solution was slowly added and the reaction was kept at a constant temperature for 30 minutes. The reaction system was quenched in an ice-water bath and centrifuged at 8000 rpm for 10 minutes. The product was washed three times with acetone and deionized water in sequence, and finally dispersed in 40 mL of deionized water for storage.

3. The method for preparing the triple synergistically regulated multifunctional composite nanozyme according to claim 2, characterized in that: The step 1.2 includes the following steps: 10 mL of silver nanocube dispersion was added to 100 mL of deionized water containing 5 mg / mL polyvinyl pyrrolidone and heated to 100°C. 10 mL of 1 mM chloroauric acid solution was added dropwise at a rate of 40 mL / h. After the solution turned dark blue and the color stabilized, saturated sodium chloride solution was added to remove by-products. The product was centrifuged at 10,000 rpm for 15 min, collected, washed three times, and vacuum-dried to obtain AgAu NCs.

4. The method for preparing the triple synergistically regulated multifunctional composite nanozyme according to claim 3, characterized in that: The step 1.3 includes the following steps: AgAu NCs were dispersed in 40 mL of a mixed solution of water / ethanol (volume ratio 1:1) and sonicated for 30 minutes. 0.05 mM cerium nitrate and 0.1 mM hexamethylenetetramine were added sequentially, and the mixture was refluxed in an oil bath at 60°C for 2 hours. After the reaction, the mixture was centrifuged at 12,000 rpm for 10 minutes, and the AgAu@CeO2 core-shell material was collected, washed, and vacuum-dried for storage.

5. The method for preparing the triple synergistically regulated multifunctional composite nanozyme according to claim 1, characterized in that: The step 2.1 includes the following steps: 50 mg of AgAu@CeO2 was dispersed in 50 mL of anhydrous ethanol, ultrasonicated for 30 minutes, and then 10 wt% APTES was added. The pH was adjusted to 8.0 and the mixture was reacted at 70°C for 4 hours. The mixture was centrifuged at 10,000 rpm for 10 minutes. AgAu@CeO2-NH2 was collected and washed three times with ethanol and deionized water.

6. The method for preparing the triple-coordinated multifunctional composite nanozyme according to claim 5, characterized in that: The step 2.2 includes the following steps: 10 mg CPBA was dissolved in 10 mL PBS buffer, and 20 mg EDC and 15 mg NHS were added for activation at room temperature for 30 minutes. The activated solution was mixed with AgAu@CeO2-NH2 and reacted at room temperature for 12 hours. The AgAu@CeO2-PBA was obtained by centrifugation at 10,000 rpm for 10 minutes and washed three times with PBS.

7. The method for preparing the triple-coordinated multifunctional composite nanozyme according to claim 6, characterized in that: The step 2.3 includes the following steps: 50 μg / mL of AgAu@CeO2-PBA was mixed with 1×10 7 The suspension of Lactobacillus reuteri with CFU / mL was mixed and incubated at 37°C with shaking for 30 minutes for directional coupling through boronate bonds; the AgAu CL composite nanozyme was collected by centrifugation at 5000 rpm for 5 minutes, washed twice in anaerobic medium and then stored.

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