Enzyme catalysis system with function of targeted inhibition of growth of streptococcus mutans as well as preparation and application of enzyme catalysis system

Abstract

The invention belongs to the technical field of synthesis and antibiosis of nano-enzyme catalysis systems, and discloses an enzyme catalysis system with a function of targeted inhibition of growth of streptococcus mutans as well as preparation and application of the enzyme catalysis system. The enzyme catalysis system with the targeted inhibition function on the growth of the streptococcus mutans is formed by loading AuNPs and PCN-22 (Fe) on a CMD molecular chain through in-situ growth, and the mass ratio of the AuNPs to the PCN-22 (Fe) to the CMD is (0.22%-0.33%) to (12.34%-21.79%) to (77.88%-87.44%). The cascade catalytic effect of the nano-enzyme is adjusted by regulating and controlling the carboxymethyl substitution degree of CMD, so that glucose in a decayed tooth pathological environment is efficiently catalyzed in one step to generate ROS, and damage to probiotics is reduced while the inhibition efficiency of pathogenic bacteria is improved.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme catalytic system synthesis and antibacterial technology, and specifically relates to an enzyme catalytic system that targets and inhibits the growth of Streptococcus mutans, its preparation method and application. Background Technology

[0002] Biofilms, composed of microorganisms embedded in an extracellular polymeric substance (EPS) matrix, form a protected community that binds to surfaces that enhance antibiotic resistance and can cause infectious diseases in humans. Dental caries is a biofilm-induced disease driven by dietary and microbial interactions. During the formation and development of caries, the biofilm acts as a barrier against external mechanical erosion and host defense, preventing the penetration of antimicrobial agents, protecting internal bacteria from harm, and increasing bacterial resistance. If left untreated, pathogenic microorganisms can lead to tooth mineralization and subsequent acid dissolution. If the bacteria are absorbed and spread throughout the body, they can increase the risk of sepsis, heart disease, nephritis, arthritis, and other organ diseases. It has been reported that over 30% of children (2-11 years old) and 91% of adults (20-60 years old) worldwide are affected by this disease, with treatment costs exceeding $120 billion in the United States alone. Therefore, the prevention and treatment of dental caries, namely, developing effective means to destroy and remove the biological film and kill pathogens in the process of dental caries, has become a key technical issue that must be considered in protecting dental health and reducing the systemic diseases caused by dental caries. It is also one of the key bottleneck issues that urgently need to be solved in the prevention of dental caries.

[0003] Among the effective methods for preventing tooth decay, the most commonly used are mechanical cleaning or fluoride, chlorhexidine, and antibacterial agents with broad-spectrum antibacterial effects. However, these methods have limited effectiveness in treating tooth decay and may pose serious health risks, such as uterine cancer and bladder cancer. Nanomaterials, due to their excellent biocompatibility, do not trigger adverse reactions or rejection responses when in contact with living organisms. This makes nanotechnology highly safe in the treatment of tooth decay. Furthermore, nanotechnology can improve drug delivery efficiency and bioavailability, enhance antibacterial properties, improve the performance of dental materials, provide personalized treatment plans, and ensure safety and long-term effectiveness. These advantages make nanotechnology a promising field with broad prospects and potential for future development in dentistry. Nanozymes, a novel material combining high efficiency, stability, controllability, and multi-enzyme activity, exhibit significant application potential and value in multiple fields. Nanozymes exhibit unique advantages in their designable catalytic activity and targeting: on the one hand, their enzyme-like properties can regulate ROS release, achieving highly efficient bactericidal effects at low concentrations; on the other hand, through surface functionalization modification, they can precisely target cariogenic bacteria, reducing damage to other bacteria and normal oral tissues. This integrated "catalysis-targeting" strategy provides a new approach for developing highly efficient and low-toxicity oral health intervention materials.

[0004] Streptococcus mutans, a cariogenic bacterium, thrives in sugar-rich conditions, promoting the formation of biofilms on tooth surfaces and leading to tooth decay. Conversely, probiotics can interfere with pathogen establishment; for example, Streptococcus mitis (such as oral streptococci) can produce H2O2, exhibiting antibacterial activity against pathogens and maintaining tooth health. However, H2O2 production is localized and at relatively low concentrations, and Streptococcus mutans can overcome the damaging effects of H2O2 through growth, metabolism, and enhanced acidification under sugar-rich conditions. Researchers have discovered that iron oxide nanoparticles with peroxidase-like activity at acidic pH can catalyze the production of reactive oxygen species (ROS) from exogenously introduced H2O2, thereby disrupting pathogenic bacteria and pathogenic biofilms. However, exogenous H2O2 presents a challenge for the clinical translation of locally orally administered nanocatalysis. Therefore, constructing a nanoenzyme catalytic system capable of continuously generating ROS based on the characteristics of the carious dental environment to efficiently inhibit cariogenic bacteria while reducing damage from oral streptococci is a critical technical problem that urgently needs to be solved. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing technologies in targeting and inhibiting Streptococcus mutans, a pathogenic bacterium of dental caries, the primary objective of this invention is to provide an enzyme catalytic system that targets and inhibits the growth of Streptococcus mutans. A three-in-one nanozyme is constructed by rationally utilizing the polar group of carboxymethyl dextran to combine gold nanoparticles with a metal-organic framework PCN-222(Fe). By regulating the degree of carboxymethyl substitution of carboxymethyl dextran, the cascade catalytic effect of the nanozyme is adjusted, enabling the efficient one-step catalysis of glucose to produce ROS in the pathological environment of dental caries. This improves the inhibition efficiency of pathogenic bacteria while reducing damage to beneficial bacteria.

[0006] Another objective of this invention is to provide a method for preparing the above-mentioned enzyme catalytic system that targets and inhibits the growth of Streptococcus mutans.

[0007] Another object of the present invention is to provide the application of the above-mentioned enzyme catalytic system that targets and inhibits the growth of Streptococcus mutans.

[0008] The objective of this invention is achieved through the following solution: An enzyme catalytic system for targeted inhibition of Streptococcus mutans growth is provided, which is formed by in situ growth of AuNPs with glucose oxidase-like activity and PCN-222(Fe) with catalase-like activity onto a carboxymethyl dextran (CMD) molecular chain. The mass ratio of AuNPs, PCN-222(Fe) and CMD is (0.22%–0.33%): (12.34%–21.79%): (77.88%–87.44%).

[0009] The degree of carboxymethyl substitution of the carboxymethyl dextran (CMD) is 0.22 to 0.81. By adjusting the degree of carboxymethyl substitution of CMD within the range of 0.22 to 0.81, the loading rate of AuNPs and PCN-222(Fe) and the catalytic activity of the AuNPs / PCN-222(Fe) / CMD nanozyme cascade can be regulated.

[0010] The molecular weight of the carboxymethyl dextran (CMD) is 11 × 10⁻⁶. 3 g / mol ~14×10 3 g / mol; A method for preparing the above-mentioned enzyme catalytic system with targeted inhibition of Streptococcus mutans growth includes the following steps: (1) Carboxymethyl dextran was prepared into a carboxymethyl dextran dispersion with acetate buffer, and then chloroauric acid trihydrate was added to react to obtain a gold nanoparticle-carboxymethyl dextran nanozyme (AuNPs / CMD) solution. Then, the solution was dialyzed until there were no chloride ions in the dialysate. The dialyzed solution was freeze-dried to obtain AuNPs / CMD nanozyme. (2) The obtained AuNPs / CMD nanozyme was stirred and reacted with ZrCl4 in a solvent. Then the resulting precipitate was reacted with a solution containing 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-ferric chloride (III) (FeTCPPCl) and benzoic acid to obtain an enzyme catalytic system with targeted inhibition of Streptococcus mutans growth, namely AuNPs / PCN-222(Fe) / CMD nanozyme cascade catalytic material.

[0011] The acetate buffer mentioned in step (1) refers to an acetate buffer with a concentration of 0.1M and a pH of 4.5-6.5 prepared using anhydrous sodium acetate and glacial acetic acid.

[0012] The concentration of the carboxymethyl dextran dispersion mentioned in step (1) is 0.15 g / mL; The mass ratio of carboxymethyl dextran to chloroauric acid trihydrate in step (1) is 19.0:1 to 152.4:1, preferably 38.1:1 to 114.3:1.

[0013] The reaction described in step (1) refers to reacting at 70-100℃ for 15-65 min, preferably at 100℃ for 65 min. After the reaction is completed, the mixture is cooled to room temperature before dialysis. The molecular weight cutoff of the dialysis bag is 3.5 kDa. In step (2), the mass ratio of AuNPs / CMD to ZrCl4 is 4.42:1 to 13.6:1; the solvent is an aqueous solution of DMF, wherein the volume ratio of DMF to water is 8:1. The amount of solvent used is sufficient to ensure uniform dispersion of the reactants.

[0014] The reaction of AuNPs / CMD nanozyme with ZrCl4 in solvent in step (2) refers to stirring at 25-30℃ for 6-12 hours, preferably at 25℃ for 12 hours. Stirring is only to ensure thorough mixing of the raw materials. After the reaction is complete, the precipitate is washed with DMF, centrifuged, dried, and then reacted with a solution containing FeTCPPCl and benzoic acid.

[0015] The amount of solution containing 5,10,15,20-tetra(4-carboxyphenyl)porphyrin-ferric chloride (III) and benzoic acid in step (2) is such that when 1g of AuNPs / CMD nanozyme is used in step (2), a solution containing 1.25-2.25g of 5,10,15,20-tetra(4-carboxyphenyl)porphyrin-ferric chloride (III), 12.5-67.5g of benzoic acid, and 0.8-1.2L of DMF solvent is used.

[0016] The reaction of the precipitate in step (2) with a solution containing 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-ferric chloride (III) (FeTCPPCl) and benzoic acid refers to a reaction in a reactor at 100-120°C in a sealed environment for 12-48 hours, preferably a reaction in a sealed reactor at 120°C for 24 hours.

[0017] Step (2) includes a purification step after the reaction in the reactor is completed. Specifically, the reaction product is cooled to room temperature and centrifuged to collect the product. Then, it is washed with DMF until the supernatant is colorless. Then, it is washed with acetone to replace DMF and dried to obtain AuNPs / PCN-222(Fe) / CMD nanozyme cascade catalytic material.

[0018] The enzyme catalytic system described in step (2) that targets and inhibits the growth of Streptococcus mutans, namely AuNPs / PCN-222(Fe) / CMD nanoenzyme cascade catalytic material, is applied in the form of oral health products (such as mouthwash) to target and inhibit caries-causing bacteria.

[0019] The enzyme catalytic system described in step (2) that targets and inhibits the growth of Streptococcus mutans, namely the AuNPs / PCN-222(Fe) / CMD nanoenzyme cascade catalytic material, has a particle size range of 399.14 ~ 1718.46 nm.

[0020] The targeted inhibitory effect of the AuNPs / PCN-222(Fe) / CMD nanozyme cascade catalytic material in step (2) on cariogenic bacteria increases as the degree of carboxymethyl substitution of CMD decreases.

[0021] The above-mentioned enzyme catalytic system with targeted inhibition of Streptococcus mutans growth can be used in the preparation of oral health products such as mouthwash.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The AuNPs / PCN-222(Fe) / CMD nanozymes constructed in this invention possess Streptococcus mutans targeting and glucose oxidase-like and catalase-like activities, and are sequentially assembled into a cascade catalytic system. The nanozymes have a particle size range of 399.14 ~ 1718.46 nm, which can effectively penetrate biofilms to exert antibacterial effects. This cascade catalytic nanozyme system overcomes the drawbacks of natural enzymes, such as easy inactivation, short service life, and high preparation cost.

[0023] (2) The nanozyme constructed in this invention is suitable for application in pathological environments such as the high-sugar environment of dental caries. By regulating the degree of substitution of carboxymethyl dextran, the mass transfer distance between glucose and the subsequently generated substrates such as hydrogen peroxide can be effectively controlled, thereby adjusting the efficiency of AuNPs / PCN-222(Fe) / CMD nanozyme in catalyzing the oxidation of glucose to generate reactive oxygen species in one step, thus achieving targeted inhibition of Streptococcus mutans, a pathogenic bacterium of dental caries. The cascade catalytic system described in this invention can be used as a nanozyme catalytic material for preventing dental caries, possessing the ability to not interfere with the growth of beneficial bacteria and to target and kill pathogenic Streptococcus mutans in biofilms. Attached Figure Description

[0024] Figure 1 The effect of the mass ratio of CMD to chloroauric acid trihydrate on the formation of AuNPs / CMD.

[0025] Figure 2 The particle size (a), crystal structure (b), and cascade catalytic effect (c) of AuNPs / CMD / PCN-222(Fe) nanozyme with a CMD substitution degree of 0.22 are shown.

[0026] Figure 3 The particle size (a), crystal structure (b), and cascade catalytic effect (c) of AuNPs / CMD / PCN-222(Fe) nanozyme with a CMD substitution degree of 0.43 are shown.

[0027] Figure 4 The particle size (a), crystal structure (b), and cascade catalytic effect (c) of AuNPs / CMD / PCN-222(Fe) nanozyme with a CMD substitution degree of 0.81 are shown.

[0028] Figure 5 The inhibitory effect (a), ROS-dependent inhibition (b), targeted antibacterial ability (c), and biofilm degradation (d) of AuNPs / CMD / PCN-222(Fe) nanozyme (ACP) on Streptococcus mutans. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0030] In the examples, the acetate buffer solution was prepared from anhydrous sodium acetate and glacial acetic acid.

[0031] Example 1 With a molecular weight of 11×10 3CMD with a concentration of g / mol and a degree of substitution of 0.22 was prepared into a 0.15 g / mL CMD solution using acetate buffer (0.1M, pH 4.5) and stirred at 300 r / min for 5 min to ensure complete dissolution. Chloroauric acid trihydrate was added to the above CMD solution at CMD to chloroauric acid trihydrate mass ratios of 38.1:1, 76.2:1, 114.3:1, 152.4:1, and 19.0:1, respectively. The mixture was then magnetically stirred at 500 r / min for 30 min at 25 °C to ensure thorough mixing. The reaction was then carried out at 100 °C for 65 min, followed by cooling to 25 °C to obtain a gold nanoparticle-carboxymethyl dextran nanozyme (AuNPs / CMD) solution. The AuNPs / CMD solution was dialyzed using a dialysis bag with a cutoff of 3.5 kDa until no chloride ions were detected in the dialysate using AgNO3. Finally, the dialysis solution was freeze-dried to obtain AuNPs / CMD nanozymes.

[0032] 40 mg of dried AuNPs / CMD (prepared by mixing CMD and chloroauric acid trihydrate at a mass ratio of 76.2:1) was dissolved in DMF aqueous solution (DMF to water volume ratio of 8:1) at a mass ratio of 13.6:1 with ZrCl4. The solution was stirred at 25 °C for 12 h to allow the AuNPs / CMD and ZrCl4 to react. 4+ After thorough cross-linking, the sample was washed three times with DMF solution by centrifugation (10000 × g, 5 min), and then vacuum dried to obtain AuNPs / CMD-Zr. 4+ Composite system. According to the synthesis conditions in Table 2, AuNPs / CMD-Zr... 4+ The composite system, along with TCPPFe and benzoic acid, was sequentially dissolved in DMF solution and stirred at 25°C for 1 h to ensure uniform dispersion. The reaction solution was then sealed in a reaction vessel with a polytetrafluoroethylene liner and reacted at 120°C for 24 h. After cooling to 25°C, the product was collected by centrifugation (10000 × g, 5 min). The product was washed with DMF and centrifuged until the supernatant was colorless. The supernatant was then washed twice with acetone to replace the DMF. After drying at 70°C for 12 h, a brown solid product was obtained, which was the AuNPs / PCN-222(Fe) / CMD nanozyme.

[0033] The UV-Vis absorption spectra and particle sizes of AuNPs / CMD prepared under different conditions were measured, and the results are as follows: Figure 1As shown, when the mass ratio of CMD to chloroauric acid trihydrate was 19.0:1 and 152.4:1, no characteristic UV-Vis absorption peaks belonging to AuNPs were observed in the 500-550 nm wavelength range, indicating that the synthesis of AuNPs failed under these reactant ratios. When the mass ratio of CMD to chloroauric acid trihydrate was 76.2:1, the UV-Vis absorption spectrum of AuNPs at 500-550 nm showed the position of the minimum absorption peak, indicating that the AuNPs were well dispersed and had the smallest AuNPs / CMD particle size of 46.12 nm.

[0034] Table 1. Particle size of AuNPs / CMD under different preparation conditions .

[0035] Table 2. Particle size of AuNPs / PCN-222(Fe) / CMD nanozymes under different preparation conditions .

[0036] AuNPs / CMD were prepared according to the method in condition 3 of Table 1, and AuNPs / PCN-222(Fe) / CMD nanozymes were prepared under different conditions in Table 2. The particle size of the nanozymes was measured and the results are shown in Table 2. Nanozymes with a particle size of less than 500 nm were selected to better penetrate the biofilm and reach the deep region where the pathogenic bacteria are located, thereby achieving the purpose of targeted inhibition of the pathogenic bacteria. The application of this nanozyme in subsequent examples will be further illustrated.

[0037] Example 2 A molecular weight of 11 × 10 3 CMD with a concentration of g / mol and a degree of substitution of 0.22 was prepared into a 0.15 g / mL CMD solution using acetate buffer and stirred at 300 r / min for 5 min to ensure complete dissolution. Chloroaurate trihydrate was added to the CMD solution at a CMD to chloroaurate trihydrate mass ratio of 76.2:1. The mixture was then magnetically stirred at 500 r / min for 30 min at 25 °C to ensure thorough mixing. After reacting at 100 °C for 65 min, the mixture was cooled to 25 °C to obtain a gold nanoparticle-carboxymethyl dextran nanozyme (AuNPs / CMD) solution. The AuNPs / CMD solution was dialyzed using a 3.5 kDa dialysis bag until no chloride ions were detected in the dialysate using AgNO3. Finally, the dialyzed solution was freeze-dried to obtain the AuNPs / CMD nanozyme.

[0038] 40 mg of dried AuNPs / CMD was dissolved in an aqueous DMF solution (DMF to water volume ratio of 8:1) at a mass ratio of 13.6:1 to ZrCl4. The solution was stirred at 25 °C for 12 h to allow the AuNPs / CMD to react with ZrCl4. 4+ After thorough cross-linking, the sample was washed three times with DMF solution by centrifugation (10000 × g, 5 min), and then vacuum dried to obtain AuNPs / CMD-Zr. 4+ Composite system. According to synthesis condition 10 in Table 2, AuNPs / CMD-Zr... 4+ The composite system, FeTCPPCl, and benzoic acid were sequentially dissolved in DMF solution and stirred at 25°C for 1 h to ensure uniform dispersion. The reaction solution was then sealed in a reaction vessel with a polytetrafluoroethylene liner and reacted at 120°C for 24 h. After cooling to 25°C, the product was collected by centrifugation (10000 × g, 5 min). The product was washed with DMF and centrifuged until the supernatant was colorless. The supernatant was then washed twice with acetone to replace the DMF. After drying at 70°C for 12 h, a brown solid product was obtained, which was the AuNPs / PCN-222(Fe) / CMD nanozyme. Inductively coupled plasma atomic emission spectrometry (ICP-AES) showed that the loading rates of AuNPs and PCN-222(Fe) were 0.33% and 12.34%, respectively.

[0039] The structure and cascade catalytic activity of the nanozyme were characterized using particle size analysis, X-ray diffraction, and ultraviolet spectrophotometry, respectively. Figure 2 As shown in (a), the AuNPs / PCN-222(Fe) / CMD nanozyme exhibited characteristic diffraction peaks of PCN-222(Fe) at 2θ = 4.90°, 7.17°, and 9.17°, corresponding to its (200), (110), and (210) crystal planes, respectively. Characteristic peaks belonging to AuNPs appeared at 2θ = 38.15° and 44.36°, corresponding to its (111) and (200) crystal planes, respectively. Furthermore, the broad diffraction signal in the 2θ = 10–30° range originated from the amorphous structure of the CMD molecules. The presence of these characteristic peaks indicates that both PCN-222(Fe) and AuNPs were successfully grown in situ on the CMD molecular chain. The particle size of the AuNPs / PCN-222(Fe) / CMD nanozyme was measured using laser particle size analysis. Figure 2 The results in (b) show that the nanozyme has a particle size of 399.14 nm.

[0040] The cascade catalytic effect of AuNPs / PCN-222(Fe) / CMD nanozymes was evaluated based on an enzyme-catalyzed colorimetric reaction combined with ultraviolet spectrophotometry. 1 mg of AuNPs / PCN-222(Fe) / CMD nanozymes was accurately weighed and added to 1 mL of acetate buffer at pH 4.5, 5.5, and 6.5, respectively. The mixture was vortexed for 1 min to ensure uniform dispersion. Then, 10 μL of 25 mM glucose solution and 10 μL of 10 mg / mL chromogenic agent 3,3',5,5'-tetramethylbenzidine were added to the above reaction solutions. The reaction was carried out in a 37 ℃ water bath for 1 h. The absorbance of the reaction system at 652 nm was measured using an ultraviolet spectrophotometer. All experiments were repeated three times. Figure 2 As shown in (c), at reaction pH values ​​of 4.5, 5.5, and 6.5, the absorbance of the AuNPs / CMD / PCN-222(Fe) nanozyme at 652 nm after reacting with glucose was 0.28, 0.15, and 0.1, respectively. This indicates that the cascade catalytic effect of the AuNPs / CMD / PCN-222(Fe) nanozyme on glucose decreases with increasing pH of the reaction solution, and exhibits higher catalytic activity at pH 4.5. Given that the pathogenic bacterium Streptococcus mutans in dental caries mainly grows in acidic environments between pH 6.5 and 4.5, and its activity gradually increases with decreasing pH, exhibiting exponential growth at pH 4.5, it can be concluded that the obtained nanozyme is suitable for the effective inhibition of Streptococcus mutans in dental caries environments.

[0041] Example 3 A molecular weight of 12×10 3 CMD with a carboxymethyl substitution degree of 0.43 g / mol was prepared into a 0.15 g / mL CMD solution using acetate buffer and stirred at 300 r / min for 5 min to ensure complete dissolution. Chloroaurate trihydrate was added to the CMD solution at a CMD to chloroaurate trihydrate mass ratio of 76.2:1. The mixture was then magnetically stirred at 500 r / min for 30 min at 25 °C to ensure thorough mixing. After reacting at 100 °C for 65 min, the mixture was cooled to 25 °C to obtain a gold nanoparticle-carboxymethyl dextran nanozyme (AuNPs / CMD) solution. The AuNPs / CMD solution was dialyzed using a 3.5 kDa dialysis bag until no chloride ions were detected in the dialysate using AgNO3. Finally, the dialyzed solution was freeze-dried to obtain the AuNPs / CMD nanozyme.

[0042] 40 mg of dried AuNPs / CMD was dissolved in an aqueous DMF solution (DMF to water volume ratio of 8:1) at a mass ratio of 7.46:1 to ZrCl4. The solution was stirred at 25 °C for 12 h to allow the AuNPs / CMD to react with ZrCl4.4+ After thorough cross-linking, the sample was washed three times with DMF solution by centrifugation (10000 × g, 5 min), and then vacuum dried to obtain AuNPs / CMD-Zr. 4+ Composite system. According to synthesis condition 10 in Table 2, AuNPs / CMD-Zr... 4+ The composite system, along with TCPPFe and benzoic acid, was sequentially dissolved in DMF solution and stirred at 25°C for 1 h to ensure uniform dispersion. The reaction solution was then sealed in a reaction vessel with a polytetrafluoroethylene liner and reacted at 120°C for 24 h. After cooling to 25°C, the product was collected by centrifugation (10000 × g, 5 min). The product was washed with DMF and centrifuged until the supernatant was colorless. The supernatant was then washed twice with acetone to replace the DMF. After drying at 70°C for 12 h, a brown solid product was obtained, which was the AuNPs / PCN-222(Fe) / CMD nanozyme. The loading rates of AuNPs and PCN-222(Fe) were 0.25% and 16.75%, respectively.

[0043] The structure and cascade catalytic activity of the nanozyme were characterized using particle size analysis, X-ray diffraction, and ultraviolet spectrophotometry, respectively. Figure 3 As shown in (a), the AuNPs / PCN-222(Fe) / CMD nanozyme exhibited characteristic diffraction peaks of PCN-222(Fe) at 2θ = 4.90°, 7.17°, and 9.17°, corresponding to its (200), (110), and (210) crystal planes, respectively. Characteristic peaks belonging to AuNPs appeared at 2θ = 38.15° and 44.36°, corresponding to its (111) and (200) crystal planes, respectively. Furthermore, the broad diffraction signal in the 2θ = 10–30° range originated from the amorphous structure of the CMD molecules. The presence of these characteristic peaks indicates that both PCN-222(Fe) and AuNPs were successfully grown in situ on the CMD molecular chain. The particle size of the AuNPs / PCN-222(Fe) / CMD nanozyme was measured using laser particle size analysis. Figure 3 The results in (b) show that the nanozyme has a particle size of 647.51 nm.

[0044] The cascade catalytic effect of AuNPs / PCN-222(Fe) / CMD nanozymes was evaluated based on an enzyme-catalyzed colorimetric reaction combined with ultraviolet spectrophotometry. 1 mg of AuNPs / PCN-222(Fe) / CMD nanozymes was accurately weighed and added to 1 mL of acetate buffer at pH 4.5, 5.5, and 6.5, respectively. The mixture was vortexed for 1 min to ensure uniform dispersion. Then, 10 μL of 25 mM glucose solution and 10 μL of 10 mg / mL chromogenic agent 3,3',5,5'-tetramethylbenzidine were added to the above reaction solutions. The reaction was carried out in a 37 ℃ water bath for 1 h. The absorbance of the reaction system at 652 nm was measured using an ultraviolet spectrophotometer. All experiments were repeated three times. The blank control group contained no glucose solution. Figure 3 As can be seen from (c), at reaction pH values ​​of 4.5, 5.5, and 6.5, the absorbance values ​​of the reaction solution of AuNPs / CMD / PCN-222(Fe) nanozyme at 652 nm were 0.21, 0.12, and 0.07, respectively. This indicates that the cascade catalytic effect of AuNPs / CMD / PCN-222(Fe) nanozyme on glucose decreases with increasing pH value of the reaction solution, and the catalytic activity is higher under pH 4.5 conditions.

[0045] Example 4 A molecular weight of 14×10 3 CMD with a carboxymethyl substitution degree of 0.81 g / mol was prepared into a 0.15 g / mL CMD solution using acetate buffer and stirred at 300 r / min for 5 min to ensure complete dissolution. Chloroaurate trihydrate was added to the CMD solution at a CMD to chloroaurate trihydrate mass ratio of 76.2:1. The mixture was then magnetically stirred at 500 r / min for 30 min at 25 °C to ensure thorough mixing. After reacting at 100 °C for 65 min, the mixture was cooled to 25 °C to obtain a gold nanoparticle-carboxymethyl dextran nanozyme (AuNPs / CMD) solution. The AuNPs / CMD solution was dialyzed using a 3.5 kDa dialysis bag until no chloride ions were detected in the dialysate using AgNO3. Finally, the dialyzed solution was freeze-dried to obtain the AuNPs / CMD nanozyme.

[0046] 40 mg of dried AuNPs / CMD was dissolved in an aqueous DMF solution (DMF to water volume ratio of 8:1) at a mass ratio of 4.42:1 to ZrCl4. The solution was stirred at 25 °C for 12 h to allow the AuNPs / CMD to react with ZrCl4. 4+ After thorough cross-linking, the sample was washed three times with DMF solution by centrifugation (10000 × g, 5 min), and then vacuum dried to obtain AuNPs / CMD-Zr. 4+Composite system. According to synthesis condition 10 in Table 2, AuNPs / CMD-Zr... 4+ The composite system, along with TCPPFe and benzoic acid, was sequentially dissolved in DMF solution and stirred at 25°C for 1 h to ensure uniform dispersion. The reaction solution was then sealed in a reaction vessel with a polytetrafluoroethylene liner and reacted at 120°C for 24 h. After cooling to 25°C, the product was collected by centrifugation (10000 × g, 5 min). The product was washed with DMF and centrifuged until the supernatant was colorless. The supernatant was then washed twice with acetone to replace the DMF. After drying at 70°C for 12 h, a brown solid product was obtained, which was the AuNPs / PCN-222(Fe) / CMD nanozyme. The loading rates of AuNPs and PCN-222(Fe) were 0.22% and 21.79%, respectively.

[0047] The structure and cascade catalytic activity of the nanozyme were characterized using particle size analysis, X-ray diffraction, and ultraviolet spectrophotometry, respectively. Figure 4 As shown in (a), the AuNPs / PCN-222(Fe) / CMD nanozyme exhibited characteristic diffraction peaks of PCN-222(Fe) at 2θ = 4.90°, 7.17°, and 9.17°, corresponding to its (200), (110), and (210) crystal planes, respectively. Characteristic peaks belonging to AuNPs appeared at 2θ = 38.15° and 44.36°, corresponding to its (111) and (200) crystal planes, respectively. Furthermore, the broad diffraction signal in the 2θ = 10–30° range originated from the amorphous structure of the CMD molecules. The presence of these characteristic peaks indicates that both PCN-222(Fe) and AuNPs were successfully grown in situ on the CMD molecular chain. The particle size of the AuNPs / PCN-222(Fe) / CMD nanozyme was measured using laser particle size analysis. Figure 4 The results in (b) indicate that the nanozyme has a particle size of 825.00 nm.

[0048] The cascade catalytic effect of AuNPs / PCN-222(Fe) / CMD nanozymes was evaluated based on an enzyme-catalyzed colorimetric reaction combined with ultraviolet spectrophotometry. 1 mg of AuNPs / PCN-222(Fe) / CMD nanozymes was accurately weighed and added to 1 mL of acetate buffer at pH 4.5, 5.5, and 6.5, respectively. The mixture was vortexed for 1 min to ensure uniform dispersion. Then, 10 μL of 25 mM glucose solution and 10 μL of 10 mg / mL chromogenic agent 3,3',5,5'-tetramethylbenzidine were added to the above reaction solutions. The reaction was carried out in a 37 ℃ water bath for 1 h. The absorbance of the reaction system at 652 nm was measured using an ultraviolet spectrophotometer. All experiments were repeated three times. The blank control group contained no glucose solution. Figure 4As can be seen from (c), at reaction pH values ​​of 4.5, 5.5, and 6.5, the absorbance values ​​of the reaction solution of AuNPs / CMD / PCN-222(Fe) nanozyme at 652 nm were 0.17, 0.09, and 0.05, respectively. This indicates that the cascade catalytic effect of AuNPs / CMD / PCN-222(Fe) nanozyme on glucose decreases with increasing pH value of the reaction solution, and the catalytic activity is higher under pH 4.5 conditions.

[0049] Example 5: In vitro targeted antibacterial activity and biofilm degradation of AuNPs / PCN-222(Fe) / CMD nanozymes In the process of inhibiting cariogenic bacteria, controlling the particle size of antibacterial agents is crucial. This is because cariogenic bacteria (such as Streptococcus mutans) are usually found in dental plaque biofilms, and the structure of biofilms is dense and complex. Large-particle antibacterial agents have difficulty effectively penetrating into the biofilm, thus limiting their antibacterial effect. Studies have shown that nanoparticles with a particle size of less than 500 nm can better penetrate biofilms and reach the deeper regions where pathogenic bacteria reside, thereby achieving targeted inhibition of pathogenic bacteria. Therefore, nanoparticles with a molecular weight of 11 × 10⁻⁶ were selected. 3 AuNPs / PCN-222(Fe) / CMD nanozymes (i.e., the AuNPs / PCN-222(Fe) / CMD nanozymes prepared in Example 2) constructed with g / mol and CMD with a degree of substitution of 0.22 were analyzed for their targeted antibacterial effect against Streptococcus mutans and their anti-biofilm activity using plate counting, laser confocal microscopy, and ELISA reader detection techniques.

[0050] The minimum inhibitory concentration (MIC) of AuNPs / PCN-222(Fe) / CMD nanozymes against Streptococcus mutans was determined using the plate count method. The experimental method was referenced in “Zhang, C., et al. Cu(II)@ZIF-8 nanoparticles with dual-enzyme-like activity bound to bacteria specifically for efficient and durable bacteria inhibition. Applied Surface Science. 2023, 611.” The specific steps were as follows: The MIC of AuNPs / PCN-222(Fe) / CMD nanozymes against Streptococcus mutans was determined using the microdilution method. Using tryptone-tryptone-yeast extract culture medium containing 1% glucose and pH 4.5 (Shanghai Ruichu Biotechnology Co., Ltd.), solutions of AuNPs / PCN-222(Fe) / CMD nanozymes with concentrations of 1%, 0.5%, and 0.25 mg / mL were prepared, along with single-component AuNPs / CMD solutions and single-component PCN-222(Fe) solutions with concentrations of 1 mg / mL and 1 mg / mL respectively (synthesized according to the reference, Feng, Dawei, et al. "Zirconium‐Metalloporphyrin PCN‐222: Mesoporous Metal–Organic Frameworks with Ultrahigh Stability as Biomimetic Catalysts." #i{Angewandte Chemie International Edition} (2012).). These solutions were then added to sterile shake tubes. Subsequently, a 10% solution of [unclear text - likely a specific compound or formula] was added to each tube. 9 A CFU / mL suspension of *Streptococcus mutans* (ATCC 700610) was co-incubated at 37 °C and 5% CO2 for 24 hours. 20 μL of the sample was spread onto sterile brain heart infusion agar plates (BHI) and incubated at 37 °C and 5% CO2 for 48 h. The minimum bactericidal concentration of the AuNPs / PCN-222(Fe) / CMD nanozyme was defined as the concentration at which no bacterial growth was observed on the BHI agar.

[0051] Depend on Figure 5As shown in (a), compared with the blank control group, the inhibitory effects of single-component AuNPs / CMD and single-component PCN-222(Fe) solutions on Streptococcus mutans were negligible, while the AuNPs / PCN-222(Fe) / CMD nanozyme solution with a concentration of 1 mg / mL could achieve complete inhibition of Streptococcus mutans.

[0052] The inhibitory effect of AuNPs / PCN-222(Fe) / CMD nanozymes on Streptococcus mutans and its biofilm degradation activity were observed using laser confocal microscopy. The test method was based on the reference "Huang, Yue, et al. Precision targeting of bacterial pathogen via bi-functional nanozyme activated by biofilm microenvironment. Biomaterials 2021, 268." The specific steps were as follows: 1 mg of AuNPs / CMD / PCN-222(Fe) nanozyme was mixed with logarithmically growing Streptococcus mutans (concentration 10...). 9 CFU / mL was added to 1 mL of tryptone-tryptone-yeast extract culture medium (Shanghai Ruichu Biotechnology Co., Ltd.) containing 1% glucose and pH 4.5, and incubated at 37℃ in a 5% CO2 incubator for 24 h. Live and dead bacteria were labeled with SYTO 60 (652 / 678 nm) and propidium iodide (PI, 535 / 617 nm), respectively, and ROS were labeled with hydroxyphenyl fluorescein (HPF, 490 / 515 nm). Staining was performed at room temperature in the dark for 30 min. The supernatant was discarded after centrifugation at 6000 × g for 10 min, and the bacteria were washed three times with sterile physiological saline. The suspension was resuspended in sterile physiological saline. 20 μL of the bacterial suspension was placed on a coverslip, and the ROS distribution was observed using a laser confocal microscope.

[0053] Depend on Figure 5 (b) It can be seen that after treatment with nanozymes, Streptococcus mutans cells were effectively killed, accompanied by the in-situ generation of a large amount of ROS. The distribution area of ​​dead bacteria highly overlaps with the ROS generation site, indicating that AuNPs / PCN-222(Fe) / CMD nanozymes induce oxidative stress through in-situ ROS generation, leading to loss of cell membrane integrity and bacterial death.

[0054] The concentrations of Streptococcus mutans and Streptococcus oralis (ATCC 35037) in the mid-growth stage were adjusted to 10. 6 CFU / ml and 10 2CFU / mL. Then, 100 μL of each of the two bacterial strains were inoculated into 24-well plates containing cell spreaders to construct biofilms. After biofilm establishment, the culture medium was discarded. Then, 1 mg of AuNPs / PCN-222(Fe) / CMD nanozyme was added to 1 mL of tryptone-tryptone-yeast extract culture medium (Shanghai Ruichu Biotechnology Co., Ltd.) containing 1% glucose and pH 4.5, mixed well, and added to the above 24-well plates for 24 h as the experimental treatment group. Simultaneously, the biofilm without nanozyme served as the control group. The biofilm was washed with PBS and fixed with 4% paraformaldehyde at 4 °C for 4 h. Next, the bacteria were labeled with specific FISH probes: MUT590, 5'-ACTCCAGACTTTCCTGAC-3' and Cy5 labeled Streptococcus mutans; MIT588, 5'-ACAGCCTTTAACTTCAGACTTATCTAA-3' and Cy3 labeled Streptococcus oralis. The changes in *Streptococcus mutans* and *Streptococcus oralis* were observed using laser confocal microscopy (Cy3: 550-653 nm, Cy5: 644-752 nm). Figure 5 As shown in (c), compared with the blank control group, the red fluorescence signal of Streptococcus mutans was significantly weakened after nanozyme treatment, indicating a significant reduction in its number; while the green fluorescence signal of oral streptococci remained at a high intensity, indicating that its growth was not significantly inhibited. This result confirms that the AuNPs / PCN-222(Fe) / CMD nanozyme has a significant targeted sterilization effect on Streptococcus mutans, while having little effect on beneficial oral streptococci.

[0055] The degradation effect of AuNPs / PCN-222(Fe) / CMD nanozymes on biofilms was observed using laser confocal microscopy. SYTO82 (541 / 560 nm; molecular probes) was used to label bacteria, and AlexaFluor647-dextran conjugate (647 / 668 nm; molecular probes) was used to label insoluble EPS. Simultaneously, the formation and degradation of biofilms were detected using the crystal violet method described in the reference "Rumbo, et al. Colonization of Electrospun Polycaprolactone Fibers by Relevant Pathogenic Bacterial Strains. Acs Applied Materials & Interfaces. 2018, 10." The specific experimental steps are as follows: After the biofilm was established, the culture medium was discarded. Then, 1 mg of AuNPs / PCN-222(Fe) / CMD nanozymes was added to 1 mL of a solution containing 1% glucose at pH [missing value]. 4.5% tryptone-tryptone-yeast extract culture medium was mixed and added to the above 24-well plates. The plates were cultured for 24 hours as the experimental treatment group, while a biofilm without nanozymes was used as the control group. After the experiment, the cell suspension in the 24-well plates was discarded, and each well was washed 5 times with PBS buffer to remove airborne bacteria. 300 μL of 0.01% crystal violet solution was added to each well, and staining was performed at room temperature for 20 min. After staining, the crystal violet solution in the wells was discarded, and the 24-well plate was gently immersed in a 5 L beaker filled with water, repeating this process until the water in the beaker no longer changed color. The 24-well plate was inverted on a paper towel and the back of the plate was gently tapped to dry, allowing it to air dry for 15 min. After the wells were dry, 300 μL of 95% ethanol was added to each well, allowing the crystal violet to dissolve and decolorize at room temperature for 15 min. 125 μL of the solution was removed from each well. μL of elution buffer was added to a new transparent 96-well microplate, and the absorbance at 600 nm was measured using a full-wavelength microplate reader. Figure 5 As shown in (d), laser confocal microscopy revealed that, compared to the blank control group, the AuNPs / PCN-222(Fe) / CMD nanozyme could degrade the biofilm and reduce the extracellular polysaccharide matrix (EPS). The absorbance of the biofilm at 600 nm before and after nanozyme treatment was measured using a crystal violet assay; a higher absorbance value indicated a greater amount of biofilm.

[0056] Compared with the blank control group, the biofilm treated with AuNPs / PCN-222(Fe) / CMD nanozymes showed a significant decrease in absorbance at 600 nm after being stained with crystal violet, further confirming that the nanozymes have a good degradation effect on biofilms.

[0057] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An enzyme catalytic system for targeted inhibition of Streptococcus mutans growth, characterized in that... It is formed by in situ growth of AuNPs with glucose oxidase-like activity and PCN-222(Fe) with catalase-like activity onto the CMD molecular chain.

2. The enzyme catalytic system for targeted inhibition of Streptococcus mutans growth according to claim 1, characterized in that: The mass ratio of AuNPs, PCN-222(Fe) and CMD was (0.22%–0.33%): (12.34%–21.79%): (77.88%–87.44%).

3. The enzyme catalytic system for targeted inhibition of Streptococcus mutans growth according to claim 1, characterized in that: The degree of carboxymethyl substitution of the carboxymethyl dextran CMD is 0.22 to 0.

81.

4. The enzyme catalytic system for targeted inhibition of Streptococcus mutans growth according to claim 1, characterized in that: The enzyme catalytic system with targeted inhibition of Streptococcus mutans growth, namely the AuNPs / PCN-222(Fe) / CMD nanoenzyme cascade catalytic material, has a particle size range of 399.14 ~ 1718.46 nm.

5. A method for preparing an enzyme catalytic system with targeted inhibition of Streptococcus mutans growth according to any one of claims 1-4, characterized in that... Includes the following steps: (1) Carboxymethyl dextran was prepared into a carboxymethyl dextran dispersion with acetate buffer, and then chloroauric acid trihydrate was added to react to obtain a gold nanoparticle-carboxymethyl dextran nanozyme solution. Then, the solution was dialyzed until there were no chloride ions in the dialysate. The dialyzed solution was freeze-dried to obtain AuNPs / CMD nanozyme. (2) The obtained AuNPs / CMD nanozyme was stirred and reacted with ZrCl4 in a solvent. Then the resulting precipitate was reacted with a solution containing 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-ferric chloride (III) and benzoic acid to obtain an enzyme catalytic system with targeted inhibition of Streptococcus mutans growth, namely AuNPs / PCN-222(Fe) / CMD nanozyme cascade catalytic material.

6. The method for preparing the enzyme catalytic system with targeted inhibition of Streptococcus mutans growth according to claim 5, characterized in that: The acetate buffer mentioned in step (1) refers to an acetate buffer with a concentration of 0.1M and a pH of 4.5-6.5 prepared using anhydrous sodium acetate and glacial acetic acid; The mass ratio of carboxymethyl dextran to chloroauric acid trihydrate in step (1) is 19.0:1 to 152.4:1, preferably 38.1:1 to 114.3:1; The reaction described in step (1) refers to the reaction at 70-100℃ for 15-65 min; after the reaction is completed, the mixture is cooled to room temperature before dialysis is performed, and the molecular weight cutoff of the dialysis bag is 3.5 kDa.

7. The method for preparing the enzyme catalytic system with targeted inhibition of Streptococcus mutans growth according to claim 5, characterized in that: The mass ratio of AuNPs / CMD to ZrCl4 in step (2) is 4.42:1 to 13.6:1; the solvent is an aqueous solution of DMF. The reaction of AuNPs / CMD nanozyme with ZrCl4 in solvent in step (2) refers to stirring the reaction at 25-30℃ for 6-12 hours.

8. The method for preparing the enzyme catalytic system with targeted inhibition of Streptococcus mutans growth according to claim 5, characterized in that: The amount of solution containing 5,10,15,20-tetra(4-carboxyphenyl)porphyrin-ferric chloride (III) and benzoic acid in step (2) is such that when 1g of AuNPs / CMD nanozyme is used in step (2), a solution containing 1.25-2.25g of 5,10,15,20-tetra(4-carboxyphenyl)porphyrin-ferric chloride (III), 12.5-67.5g of benzoic acid and 0.8-1.2L of DMF solvent is used. The reaction of the precipitate in step (2) with a solution containing 5,10,15,20-tetra(4-carboxyphenyl)porphyrin-ferric chloride (III) and benzoic acid refers to a reaction in a closed reactor at 100-120°C for 12-48 hours.

9. The application of the enzyme catalytic system with targeted inhibition of Streptococcus mutans growth according to any one of claims 1-4 in the preparation of oral health products, especially in mouthwash.

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