A nano-enzyme with sterilization, tooth desensitization and remineralization functions, and a preparation method and application thereof

Abstract

The application provides a nano-enzyme with sterilization, tooth desensitization and remineralization functions, and a preparation method and application thereof, and relates to the technical field of nano-enzymes. 2+ The three are self-assembled into multifunctional BHCF nano-enzymes with sterilization, tooth desensitization and remineralization functions through multi-dimensional interactions of ionic bonds, Ca The BHCF nano-enzyme has clear peroxidase-like and superoxide dismutase-like dual-enzyme catalytic capacity and a photodynamic effect. Under light conditions, the antibacterial activity of the BHCF nano-enzyme is significantly activated, and the sterilization rate can reach 99.995 %. Meanwhile, the BHCF nano-enzyme has good immediate and persistent sealing effects, excellent acid resistance and wear resistance, and remineralization capacity. The BHCF nano-enzyme provided by the application has great application potential in the preparation of drugs for treating periodontitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanozymes, in particular to a nanozyme with bactericidal, desensitizing and remineralizing functions, and a preparation method and application thereof. BACKGROUND

[0002] Nanozymes are a class of nanomaterials with enzymatic properties, which is a new field originally created and led by Chinese scientists. In 2022, nanozymes were rated as one of the top ten emerging technologies in chemistry by the International Union of Pure and Applied Chemistry (IUPAC). Nanozymes combine the advantages of enzymes and chemical catalysis, and can catalyze enzyme substrates under mild conditions, with high catalytic activity, stable properties, low cost, easy modification and large-scale production, etc. Therefore, as a substitute for natural enzymes, nanozymes are applied in the fields of disease diagnosis and treatment, chemical analysis, biological sensing, environmental treatment, etc.

[0003] In particular, by utilizing the characteristics of nanozymes in catalyzing and regulating the redox balance in the body, the nanozymes can be rationally designed to exhibit peroxidase-like activity to catalyze the production of ROS to kill bacteria in the bacterial microenvironment, or exhibit superoxide dismutase-like activity to scavenge excess ROS to protect cells in the inflammatory microenvironment, thereby achieving catalytic treatment of inflammatory diseases caused by bacterial infection.

[0004] Periodontitis is a chronic infectious disease caused by periodontal pathogenic bacteria. These bacteria react with the host, leading to inflammation and destruction of periodontal tissues such as gingiva, periodontal membrane and alveolar bone. Since subgingival plaque and dental calculus are important local factors in the development of periodontitis, subgingival scaling combined with antibiotics is the most effective treatment method to control the progression of periodontitis. The main purpose is to remove some irritating factors on the tooth surface that can cause gingival inflammation, such as plaque, calculus, endotoxin, etc., so as to re-establish a local environment and microbial system suitable for periodontal health. However, after scaling, patients with periodontal disease are prone to gingival atrophy and root exposure, which leads to dentin exposure and tooth sensitivity, which manifests as pain after the tooth is exposed to cold, heat, acid, sweet stimuli or mechanical stimuli. At present, there is no periodontal treatment drug on the market that can kill bacteria while relieving dentin sensitivity symptoms. Traditional nanozymes can be selectively designed to exhibit peroxidase-like activity, catalyzing hydrogen peroxide to produce reactive oxygen species, thereby achieving good bactericidal effect. However, traditional nanozymes lack calcium and fluorine elements in their composition, which cannot achieve the requirements of tooth desensitization and remineralization, and traditional desensitizing drugs do not have good antibacterial effect, thereby creating a technical gap between antibacterial and tooth desensitization and remineralization.

[0005] Based on the above technical background, the present application aims to provide a multifunctional nanoenzyme with sterilization, tooth desensitization and remineralization functions to improve the catalytic treatment effect of nanoenzyme on periodontitis. SUMMARY

[0006] (I) Technical problems solved

[0007] In view of the deficiencies of the prior art, the present application provides a multifunctional nanoenzyme with sterilization, tooth desensitization and remineralization functions to solve the technical problem that the existing nanoenzyme cannot simultaneously achieve sterilization, tooth desensitization and remineralization in the microenvironment of periodontitis, thereby limiting the application of nanoenzyme in catalytic treatment of periodontitis.

[0008] (II) Technical solutions

[0009] To achieve the above-mentioned purposes, the present application is implemented by the following technical solutions:

[0010] In a first aspect, the present application provides a multifunctional nanoenzyme (referred to as BHCF nanoenzyme) with sterilization, tooth desensitization and remineralization functions, which is made of berberine, hematin chloride and calcium fluoride by self-assembly.

[0011] In a second aspect, the present application provides a preparation method of the above-mentioned multifunctional BHCF nanoenzyme, which is prepared according to the following steps:

[0012] (1) Dissolve hematin chloride and berberine in DMSO for standby;

[0013] (2) Dissolve calcium fluoride in hydrochloric acid solution for standby;

[0014] (3) Under room temperature and stirring, add the solution obtained in step (1) to the solution obtained in step (2) to obtain a mixed system;

[0015] (4) Adjust the pH of the mixed system obtained in step (3) to neutral with sodium hydroxide solution to obtain a suspension;

[0016] (5) Dialyze the suspension obtained in step (4) in ultrapure water to remove un-assembled molecules and ions;

[0017] (6) Centrifuge the system after dialysis in step (5), remove the supernatant, disperse the precipitate and freeze-dry to obtain the BHCF nanoenzyme.

[0018] Further, the molar ratio of hematin chloride, berberine and calcium fluoride is 0.5-1:1-2:4-6, preferably the molar ratio of hematin chloride, berberine and calcium fluoride is 1:2:6.

[0019] Further, the concentration of the hydrochloric acid solution in step (2) is 0.05-0.15 mol / L, preferably, the concentration of the hydrochloric acid solution is 0.1 mol / L.

[0020] Further, the concentration of the sodium hydroxide solution in step (4) is 0.05-0.15 mol / L, preferably, the concentration of the sodium hydroxide solution is 0.1 mol / L.

[0021] In a third aspect, the application further provides a use of the multifunctional BHCF nanoscale enzyme in the preparation of a drug for treating periodontitis.

[0022] (III) Beneficial Effects

[0023] The application uses berberine, hematin chloride and calcium fluoride as raw materials, which are self-assembled into multifunctional BHCF nanoscale enzymes with bactericidal, desensitizing and remineralizing functions through ionic bonds, Ca 2+ Coordination bonds, pi-pi stacking and hydrogen bonds. The BHCF nanoscale enzyme has great application potential in the preparation of drugs for treating periodontitis.

[0024] The BHCF nanoscale enzyme provided by the application has the following advantages:

[0025] 1. The BHCF nanoscale enzyme is irregularly map block-shaped, which can increase the specific surface area and provide more exposed sites for the enzyme active center, thereby providing conditions for enhancing the activities of peroxidase (POD) and superoxide dismutase (SOD). At the same time, the BHCF nanoscale enzyme has good adaptability in size and morphology to the microstructure of dentin tubules, and when acting on the dentin surface, the irregular edges and undulations can tightly fit the tubule openings, thereby achieving efficient physical closure. In addition, the BHCF nanoscale enzyme has a negative charge on the surface and has good stability, which can effectively prevent aggregation by means of electrostatic repulsion between particles, thereby maintaining a relatively stable dispersed state.

[0026] 2. The BHCF nanoscale enzyme provided by the application exhibits strong dual-enzyme catalytic ability of peroxidase-like activity and superoxide dismutase-like activity; under light, it simultaneously triggers type I photosensitive reaction (electron transfer to produce superoxide anion) and type II photosensitive reaction (energy transfer to generate singlet oxygen) of the BHCF nanoscale enzyme, has a photodynamic effect, and provides direct evidence for realizing the antibacterial activity of light excitation in the treatment of periodontitis.

[0027] 3、Long-acting antibacterial experiments show that the BHCF nanometer enzyme provided by the application has a MIC of 12.5 mu g / mL for P.g suspended bacteria, compared with berberine, calcium fluoride and chlorinated hematin, and the BHCF nanometer enzyme significantly improves the antibacterial activity of P.g through the synergistic effect of the ternary component, and the BHCF nanometer enzyme has excellent antibacterial activity at a low concentration. At the same time, the BHCF nanometer enzyme has a significant destructive effect on P.g bacterial biofilm, can efficiently kill bacteria in the P.g biofilm, significantly destroy the active structure of the biofilm, and can effectively disperse the P.g biofilm, thereby providing conditions for biofilm removal in periodontitis treatment.

[0028] 4、Photosensitive cascade antibacterial experiments show that under light conditions, the antibacterial activity of the BHCF nanometer enzyme is significantly activated, and the sterilization rate can reach 99.995%; the BHCF nanometer enzyme can rapidly break through the bacterial antioxidant defense system under light conditions through photosensitive cascade reaction, realize the photodynamic synergistic enzyme activity antibacterial effect, and provide conditions for its application in photodynamic antibacterial treatment.

[0029] 5、The BHCF nanometer enzyme provided by the application has good immediate and long-lasting sealing effect, excellent acid resistance and wear resistance, and excellent dentin tubule deep remineralization ability, thereby realizing the stability of long-acting sealing of the dentin tubule. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a SEM diagram of the BHCF nanometer enzyme.

[0031] Figure 2 It is an EDS diagram of the BHCF nanometer enzyme.

[0032] Figure 3 It is a DLS analysis diagram of the BHCF nanometer enzyme, (A) particle size; (B) Zeta potential.

[0033] Figure 4 It is an infrared spectrum diagram of berberine, chlorinated hematin, calcium fluoride and the BHCF nanometer enzyme.

[0034] Figure 5 It is an XPS analysis diagram of the BHCF nanometer enzyme, (A) XPS spectrum; (B) Ca2p spectrum; (C) N1s spectrum; (D) Fe2p spectrum; (E) C1s spectrum, (F) O1s spectrum; (G) F1s spectrum.

[0035] Figure 6 It is a POD-like detection result of the BHCF nanometer enzyme, wherein (A) catalytic activity diagram of berberine, chlorinated hematin, calcium fluoride and the BHCF nanometer enzyme; (B) POD-like activity of the BHCF nanometer enzyme under different concentration conditions;

[0036] Figure 7 The SOD-like detection results of BHCF nanozyme, (A) SOD inhibition rate of BHCF nanozyme at different concentrations; (B) SOD-like catalytic activity of 25 ug / ml BHCF nanozyme

[0037] Figure 8 The photosensitive performance of BHCF nanozyme, (A) UV-Vis absorption spectra of hemin, berberine, calcium fluoride and BHCF nanozyme; (B) ESR detection of the types of reactive oxygen species produced by BHCF nanozyme under light irradiation.

[0038] Figure 9 The minimum inhibitory concentration (MIC) determination results of hemin, berberine, calcium fluoride and BHCF nanozyme on P.g.

[0039] Figure 10 The antibacterial effect of hemin, berberine, calcium fluoride and BHCF nanozyme on P.g by bacterial plate counting method (A); and the colony forming unit statistics of P.g treated by different drugs (B).

[0040] Figure 11 The SEM images of P.g treated by hemin, berberine, calcium fluoride and BHCF nanozyme.

[0041] Figure 12 The live and dead bacteria staining images of P.g biofilm treated by hemin, berberine, calcium fluoride and BHCF nanozyme.

[0042] Figure 13 The crystal violet staining results of P.g biofilm treated by hemin, berberine, calcium fluoride and BHCF nanozyme (A); and the crystal violet staining statistics of P.g biofilm treated by different drugs (B).

[0043] Figure 14 The photodynamic synergistic antibacterial performance detection results of BHCF nanozyme, (A) the antibacterial effect on P.g under different treatment conditions by bacterial plate counting method; (B) the colony forming unit statistics of P.g treated by different conditions; (C) the SEM images of P.g treated by different conditions; (D) the live and dead bacteria staining images of P.g biofilm treated by different conditions; (E) the confocal microscope observation results of the active oxygen staining in P.g bacteria after different conditions treatment.

[0044] Figure 15 The scanning electron microscope images of dentin samples after soaking in artificial saliva for 10 minutes, control group and BHCF nanozyme treatment group.

[0045] Figure 16The scanning electron microscope images of the cross-section (A) and longitudinal section (B) of the dentin samples of the control group and BHCF nanozyme treatment group after soaking in artificial saliva for 3 weeks.

[0046] Figure 17 The XRD images of the surface of the dentin samples of the control group and BHCF nanozyme treatment group after soaking in artificial saliva for 3 weeks. DETAILED DESCRIPTION

[0047] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0048] Unless otherwise specifically indicated, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0049] Embodiment 1

[0050] 1. Preparation of BHCF nanozyme

[0051] 0.05 mol of hemin (Hemin) and 0.1 mol of berberine (BBr) were dissolved in 1 mL of DMSO for standby; 0.3 mol of CaF2 was dissolved in 20 mL of 0.1 mol / L hydrochloric acid solution for standby; under slow stirring, the pre-mixed DMSO solution containing Hemin and Bbr was added to the solution containing CaF2; then immediately adjust the pH to about 7.0 with 0.1 mol / L NaOH; then immediately dialyze the obtained suspension in ultrapure water for 5 days to remove un-assembled molecules and ions; after dialysis, the supernatant was removed after centrifugation of the liquid at 12000 rmp for 10 minutes, and the precipitate was re-dispersed with 5 mL of ultrapure water; the dispersed nanozyme was pre-cooled at -80℃ for 30 min and then placed in a freeze dryer for freeze-drying; the powder solid obtained after freeze-drying was the BHCF nanozyme.

[0052] 2. Characterization of BHCF nanozyme

[0053] The morphology of BHCF nanozymes was characterized by scanning electron microscopy (SEM); the distribution of C, N, O, Fe, Ca, and F elements on the surface of BHCF nanozymes was detected by energy dispersive spectroscopy (EDS); the hydration size and zeta potential of BHCF nanozymes were detected by dynamic light scattering (DLS); the chemical bonds of BHCF nanozymes were characterized by Fourier transform infrared spectroscopy (FTIR); and the elemental composition and chemical bond information of BHCF nanozymes were analyzed by X-ray photoelectron spectroscopy (XPS).

[0054] Scanning electron microscopy results show ( Figure 1 The BHCF nanozyme provided by this invention is in the form of irregularly shaped, plate-like structures with a uniform size distribution, ranging from 100 to 200 nm. This morphology differs significantly from traditional spherical and rod-shaped nanozymes. This irregular shape greatly increases the specific surface area, providing more exposure sites for the enzyme's active center and creating conditions for enhanced peroxidase (POD) and superoxide dismutase (SOD) activity. Simultaneously, the morphology and size of the BHCF nanozyme are well-suited to the microstructure of dentinal tubules. When applied to the dentin surface, the irregular edges and undulations tightly adhere to the tubule openings, achieving efficient physical closure. (EDS electronic image) Figure 2 The results show that HCFe nanozymes contain elements such as C, N, O, Fe, Ca, and F, and the elements are evenly distributed.

[0055] like Figure 3 The results show that the particle size of BHCF nanozymes is approximately 100-200 nm. Figure 3 A), consistent with the results of scanning electron microscopy. The Zeta potential on the surface of the BHCF nanozyme is -20 mV ( Figure 3 B) It carries a negative charge and has good stability. It can effectively prevent particles from agglomerating by means of electrostatic repulsion, thus maintaining a relatively stable dispersion state.

[0056] like Figure 4 The results showed that the carboxylic acid group of heme chloride (1698.72 cm⁻¹) -1 ) disappeared and split into COO - The asymmetry (1612.00cm) -1 ) and symmetry (1377.99cm) -1 The vibrational peaks indicate that the carboxylic acid interacts with Ca through a coordinate bond (with Ca).2+ It is either bonded by ionic bonds (with berberine quaternary ammonium salt); the CN peak of berberine quaternary ammonium salt (1225→1223.95cm) -1 ) and the methylenedioxyCO peak (1033→1036.96cm) -1 The shift in the lattice vibration peaks of calcium fluoride (1383→1377.99cm) reveals its participation in assembly through electrostatic interactions and hydrogen bonding; -1 ) and hydroxyl peak (3433→3397.28cm) -1 The significant shift confirms Ca 2- Detached from the lattice and with COO - Or berberine oxygen atom coordination; porphyrin ring (1603→1612 cm) -1 ) and berberine aromatic ring (1505→1496.75cm) -1 The blue / red shift of the peaks indicates π-π stacking. The synergistic changes in these characteristic peaks demonstrate that the three interact through ionic bonds and Ca²⁺. 2+ The multidimensional interactions of coordination bonds, π-π stacking, and hydrogen bonds self-assemble into BHCF nanozymes, laying the foundation for their functionalized structure.

[0057] like Figure 5 The results show that, through systematic analysis of XPS data, the following evidence of the interaction between heme chloride, berberine, and calcium fluoride is presented:

[0058] (1) Bridging coordination of calcium (Ca): The Ca 2p3 / 2 peak (347.29 eV) combines with the F1s doublet (684.96 eV lattice F - 688.94 eV coordination F - This indicates that some Ca 2+ COO that detaches from the CaF2 lattice and is simultaneously coordinated with heme chloride - (O 1s 531.72eV) and free F - , forming Ca 2+ -COO - -F - Ternary coordination networks;

[0059] (2) Ionic bonds and charge compensation: Berberine quaternary ammonium salt N + (N 1s 401.46eV, negatively shifted by 0.5–1.0eV compared to the free state) and COO - and F - N combines through electrostatic interactions + -F - Charge compensation reduces the F-electron cloud density (F1s 688.94 eV);

[0060] (3) Coordination extension of iron (Fe): Fe 3+The enhanced Fe 2p3 / 2 main peak (710.25 eV) and satellite peak (718.27 eV) indicate that the methoxy / hydroxy oxygen of berberine participates as a ligand in the formation of the six-coordinate [FeN4O2] active center;

[0061] (4) Synergistic effect of carbon and oxygen: 288.62 eV (COO) in C 1s - The deprotonation of carboxylic acid and the participation of berberine oxygen atoms in coordination / hydrogen bonding (O 1s 533.38eV) were verified by 286.58eV (CO).

[0062] (5) Chemical bonding of fluorine (F): F - After detaching from the lattice, through Ca 2+ Bridge or directly with N + By combining synergistic π-π stacking (shift of porphyrin ring and berberine aromatic peak in FTIR) and hydrogen bond network, a multidimensional and stable nanozyme interface is constructed.

[0063] The above-mentioned changes in the chemical states of each element fully reveal that heme chloride, berberine, and calcium fluoride are linked by ionic bonds and Ca... 2+ / Fe 3+ Cooperative assembly mechanisms involving coordination bonds, hydrogen bonds, and π-π stacking.

[0064] Example 2

[0065] Catalytic properties of BHCF nanozymes

[0066] 1. Detection of POD-like activity

[0067] Nanozymes with peroxidase-like activity can catalyze the generation of hydroxyl radicals from H₂O₂. These radicals can convert colorless TMB into blue, exhibiting a characteristic absorption peak at 652 nm. To investigate the peroxidase-like activity of BHCF nanozymes, we used TMB as a substrate and monitored the change in absorbance at 652 nm using a UV spectrophotometer to explore its peroxidase-like activity. This experiment also examined the peroxidase-like catalytic activity of BHCF nanozymes with different drugs and concentrations. The specific methods are as follows:

[0068] (1) The kinetics of BHCF nanozyme activity were analyzed by measuring the absorbance change over a 300s reaction time at 652nm using a UV spectrophotometer. The experiment was conducted at 37℃, using 10μg / mL of BBR, Hemin, CaF2, and BHCF nanozyme in a buffer solution at pH 4.5, with an H2O2 concentration of 30mM and a substrate TMB concentration of 1mM. A blank control system was also set up with an H2O2 concentration of 30mM, a substrate TMB concentration of 1mM, and a buffer solution at pH 4.5.

[0069] (2) In order to study the relationship between the enzyme catalytic activity of BHCF nanozyme and the concentration of drugs, 0, 2.5, 5, 7.5, 10 μg / mL of BHCF nanozyme were set for testing, the pH of the buffer solution was 4.5, the concentration of H2O2 was 30 mM, and the concentration of the substrate TMB was 1 mM.

[0070] As shown in Figure 6 A, the blank control group, the BBR group and the CaF2 group have no obvious absorbance at 652 nm, hemin has weak absorbance at 652 nm, and BHCF nanozyme has strong absorbance at 652 nm, which shows that BBR and CaF2 have no peroxidase-like activity, hemin has weak enzyme activity, and the BHCF nanozyme provided by the application has strong peroxidase-like activity, can catalyze H2O2 to produce ROS, and can oxidize colorless TMB into blue. With the increase of the concentration of BHCF nanozyme, the absorption intensity at 652 nm increases, which shows that the enzyme activity is positively correlated with the concentration of drugs Figure 6 B).

[0071] 2, SOD-like activity detection

[0072] Method: The SOD-like enzyme activity of BHCF nanozyme can be studied by SOD detection kit-WST, which is a highly water-soluble tetrazolium salt WST6-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2-hydrogen-tetrazolium, disodium salt), which can react with superoxide anion (O2 - ) to generate a water-soluble dye WST-1fomazan. The change of absorbance at 440 nm corresponds to the change of superoxide anion concentration, so the SOD activity can be calculated by detecting the absorbance at 440 nm. The ultraviolet spectrophotometer is used to measure the change of absorbance at 450 nm within 20 min of reaction time, and the kinetics analysis of SOD-like enzyme activity of BHCF nanozyme is carried out. The experiment is carried out at 37℃, and the reaction system of BHCF nanozyme with different concentrations (100, 80, 60, 40, 20, 4, 0.8 ug / ml) is tested, and the inhibition rate % of SOD of each group with different concentrations is calculated.

[0073] Based on Method detection shows that the inhibition rate of BHCF nanozyme on the reduction reaction of superoxide anion and is 50% at a concentration of 25 μg / mL Figure 7A-B), according to the unit definition (1 U SOD activity required for 20 μL sample solution to inhibit 50% of the reaction), the specific activity is 166.67 U / mg, indicating that the nanoscale enzyme has clear superoxide dismutase-like catalytic ability.

[0074] 3、UV-Vis absorption spectrum: dissolve BBR, Hemin, CaF2 and BHCF nanoscale enzyme with double distilled water respectively, prepare the test solution with a concentration of 100 ug / ml, use UV spectrophotometer to measure the absorption peak, the wavelength scanning range is 200-800 nm, and observe the absorption peak position, shape and intensity change of each component.

[0075] As shown in Figure 8 A, the BBR absorption peak is near 345 nm and 421 nm, the characteristic absorption peak of Hemin is near 385 nm and 600 nm, CaF2 has no obvious absorption peak, and BHCF nanoscale enzyme has two continuous characteristic absorption peaks between 350-400 nm, compared with BBR, the maximum absorption peak moves from 345 nm to 352 nm, which may be related to the π-π interaction between BBR molecules.

[0076] 4、ESR detection of free radical species generated under light

[0077] Superoxide radical test: take 30 μL of 1 mg / ml BHCF nanoscale enzyme and 30 μL of DMPO methanol, mix uniformly, irradiate with a 500 W mercury lamp for 5 min, use a capillary to suck a certain amount of mixed solution, place it in a quartz tube, and put it into the EPR resonance cavity for superoxide radical test.

[0078] Singlet oxygen radical: take 30 μL of 1 mg / ml BHCF nanoscale enzyme and 50 μL of 100 mM TEMP, mix uniformly, irradiate with a 500 W mercury lamp for 5 min, use a capillary to suck a certain amount of mixed solution, put it into the EPR sample cavity, and place it in the EPR resonance cavity for singlet oxygen radical test.

[0079] The ESR detection results show that Figure 8 B), under dark conditions, no superoxide anion (O2 - ) and singlet oxygen ( 1 O2) signals were detected in the BHCF nanoscale enzyme system; while under light conditions, characteristic signals of superoxide anion and singlet oxygen were significantly present in the system. This result fully proves that BHCF nanoscale enzyme has a photodynamic effect, which simultaneously triggers type I photosensitive reaction (electron transfer to produce superoxide anion) and type II photosensitive reaction (energy transfer to generate singlet oxygen) under light, providing direct evidence for its light-activated antibacterial activity in the treatment of periodontitis.

[0080] Example 3

[0081] Study on long-acting antibacterial performance of BHCF nanozyme

[0082] Experimental methods

[0083] 1. Determination of minimum inhibitory concentration (MIC)

[0084] BBR, Hemin, CaF2 and BHCF nanozyme were prepared into stock solutions with concentrations of 1000, 500, 250, 125 and 62.5 μg / mL respectively using sterilized water. The P.g bacteria solution was diluted to an OD600 of 0.2. In the 96-well plate, 20 μL of the drug stock solution and 180 μL of the diluted bacteria solution were added to each well to make the final concentration of each drug group 100, 50, 25, 12.5 and 6.25 μg / mL. The liquid medium without bacteria was used as the negative control, and the mixture of 180 μL bacteria solution and 20 μL ddH2O without drugs was used as the positive control. Each well had three replicate wells. After the addition of the sample, 100 μL was taken out of each well to measure the initial OD value. After 24 h of anaerobic incubation at 37°C, the OD value was measured again.

[0085] 2. Bacterial plate count

[0086] To evaluate the inhibitory effect of BBR, Hemin, CaF2 and BHCF nanozyme on P.g suspended bacteria, the diluted bacteria solution was divided into 5 groups, each containing 1 mL. Diluted BBR, Hemin, CaF2 and BHCF nanozyme were added to each group, respectively. The control group was added with the same volume of deionized water as the drugs. The final concentration of the drugs was 12.5 μg / mL. The centrifuge tubes were placed in a 37°C incubator for anaerobic culture for 24 h. Then, the solution was diluted by 10 times continuously. 100 μL of the diluted solution was spread on Columbia agar medium, which was incubated anaerobically at 37°C for 5-7 days. After the incubation, the colony count was performed.

[0087] 3. Scanning electron microscope observation of bacterial morphology

[0088] The diluted bacteria solution was divided into groups and treated as described above (bacterial plate count). After the incubation, the bacteria solution was centrifuged at 5000 rpm for 5 min at 4°C. The supernatant was discarded, and the precipitate was washed with PBS for 3 times. 2.5% glutaraldehyde was added for fixation, and the bacteria were solidified by placing them in a 4°C refrigerator overnight. The bacteria were dehydrated by ethanol gradient, replaced with tert-butyl alcohol, and dried in a freeze dryer. After the surface was sprayed with gold, the bacteria were placed in the scanning electron microscope instrument, and the parameters were adjusted for scanning observation.

[0089] 4. Confocal live-dead staining experiment

[0090] TSB liquid medium was diluted with PBS (PBS:TSB = 2:1). The OD value of the overnight Pg bacterial culture was adjusted to 0.2. 500 μL of this solution was added to each chamber of the slide and anaerobically cultured at 37°C for 24 h. After biofilm culture, the culture was rinsed three times with PBS. Following the grouping method for suspended bacteria as described in the experimental bacterial plate counting section, the culture was anaerobic for another 24 h. The drugs and culture medium were then removed, and the culture was rinsed three times with PBS. Live and dead bacterial dye was added and incubated in the dark for 15 min. Excess dye was then washed away with PBS, and the laser confocal microscope parameters were adjusted for observation. Images were saved using Zenlite software after imaging.

[0091] 5. Crystal Purple Experiment

[0092] Adjust the overnight cultured pg bacterial suspension to an OD value of 0.1 and add 1 mL to each well of a 24-well plate. Incubate at 37°C for 24 h. The next day, aspirate excess bacterial suspension and gently wash twice with PBS. Following the grouping method described in the experimental bacterial plate counting procedure (i.e., 12.5 μg / ml BBR, Hemin, CaF2, and BHCF nanozymes, with deionized water as the control group), add 500 μL of culture medium or drug to each well. Set up 3 replicates for each treatment group. Continue incubation for 24 h. Afterward, aspirate the drug and culture medium from the 96-well plate, gently wash twice with PBS, and dry at room temperature for 30 min. Add 500 μL of 4% paraformaldehyde solution to each well for fixation for 15 min. Then remove the paraformaldehyde solution, wash three times with PBS, dry at room temperature, and add 500 μL of 0.1% crystal violet solution to each well for staining in the dark for half an hour. After crystal violet staining, the sample was rinsed three times with PBS, and 1 mL of 96% anhydrous ethanol was added to each well for dissolution. After being protected from light for 1 hour, the absorbance of each well at 590 nm was measured using a microplate reader.

[0093] Experimental results

[0094] like Figure 9 As shown, the MIC of BHCF nanozyme against suspended Porphyromonas gingivalis (Pg) was 12.5 μg / mL. In comparison, the MIC of berberine was 50 μg / mL, while the MICs of calcium fluoride and hemin chloride both exceeded 400 μg / mL. These results indicate that, compared to single components, BHCF nanozyme, through the synergistic effect of its three components, significantly enhanced its antibacterial activity against Pg, achieving an antibacterial efficacy four times that of berberine, and exhibiting an order-of-magnitude advantage over CaF2 and Hemin, thus providing a basis for its application in the antibacterial treatment of periodontitis.

[0095] Bacterial plate count results showed ( Figure 10A-B), at the concentration of 12.5 μg / mL, there was no significant difference in the number of P.g colonies between the berberine, hemin and calcium fluoride treated groups and the control group, indicating that the single component had no significant inhibitory effect on P.g at this concentration. However, after BHCF nanozyme treatment, the number of P.g colonies was significantly reduced, with a 4.4-log reduction in colony forming units (CFU) and a bactericidal rate of up to 99.996%, indicating that BHCF nanozyme had excellent antibacterial activity at low concentrations.

[0096] Scanning electron microscope (SEM) observation results showed that Figure 11 ), after treatment with berberine, hemin and calcium fluoride, the bacterial cells of P.g still maintained the typical rod-shaped morphology, with rough cell surface and uniform morphology, indicating that the single component did not significantly destroy the bacterial structure. However, the P.g cells treated with BHCF nanozyme showed obvious morphological changes, with increased cell depression and shrinkage, and some cells even showed disintegration, directly revealing the damage of BHCF nanozyme to the structure of P.g cells, and providing direct evidence for its high-efficiency antibacterial activity at the morphological level.

[0097] Laser confocal microscope observation results showed that after treatment with BHCF nanozyme, P.g single-species biofilm showed significant red fluorescence, indicating that most of the bacteria in the biofilm had died; while the biofilms of the control group and other single-component treatment groups (BBR, Hemin, CaF2) showed green fluorescence, indicating that the biofilm contained a large number of living bacteria Figure 12 ), which was consistent with the microscope observation results, indicating that BHCF nanozyme could efficiently kill the bacteria in P.g biofilm and significantly destroy the active structure of the biofilm.

[0098] The dispersing effect of BHCF nanozyme on P.g biofilm was investigated by crystal violet staining method, and the results are shown in Figure 13 A-B, the results showed that 12.5 μg / mL of BHCF nanozyme could significantly destroy the integrity of P.g biofilm, which showed a significant lightening of the biofilm color after staining Figure 13 A). Compared with the control group, and the same concentration of berberine group, hemin group and calcium fluoride group, there was a significant difference in the staining intensity of the biofilm in the BHCF nanozyme treatment group Figure 13 B). This result confirmed that BHCF nanozyme could effectively disperse P.g biofilm at low concentrations, and its dispersing efficiency was significantly better than that of single component, which provided a condition for the removal of biofilm in the treatment of periodontitis.

[0099] Example 4

[0100] Study on the cascade reaction antibacterial performance of BHCF nanozyme

[0101] Experimental method

[0102] In the photodynamic synergistic enzyme activity antibacterial experiment, the treatment condition is to add the drug to the bacterial solution and then irradiate it with a light curing lamp (450-470 nm, 800 mW / cm 2 ) for 40 s, and the total incubation time is 15 min. The remaining experimental conditions (such as temperature, reagent concentration, and reaction system composition) are consistent with those of Example 3. (Note: the treatment condition of Example 3 is without light, and the total incubation time is 24 h. The treatment condition of this example is with light, and the total incubation time is 15 min. This is the difference between the two.) Under this condition, the influence of light excitation on the antibacterial activity and biofilm removal ability of BHCF nanoszyme is explored to reveal the synergistic mechanism of photodynamic effect and rapid antibacterial effect.

[0103] 1. Colony forming unit count

[0104] To evaluate the inhibitory effect of BHCF nanoszyme on P. g suspended bacteria under light conditions, 10 mL of diluted bacterial solution and drug (grouped as control group, control group + light, Bbr, Bbr + light, BHCF nanoszyme, and BHCF nanoszyme + light) were added to a 15 mL centrifuge tube. The drug was gently blown and mixed to a final concentration of 12.5 μg / mL. After irradiation with a light curing lamp (450-470 nm, 800 mW / cm 2 ) for 40 s, the mixture was placed in a 37°C constant temperature incubator for 15 min of incubation. The mixture was serially diluted by 10 times, and 100 μL of the diluted solution was spread on Columbia agar medium. The medium was incubated at 37°C for 5-7 days in an anaerobic incubator. After incubation, colony counting was performed. Another group without light treatment was set as the control group.

[0105] 2. Scanning electron microscope observation of bacterial morphology

[0106] The diluted bacterial solution was treated and cultured as described above. After incubation, the bacterial solution was centrifuged at 5000 rpm for 5 min in a 4°C centrifuge. The supernatant was discarded, and the precipitate was washed with PBS for 3 times. 2.5% glutaraldehyde was added for fixation, and the solution was placed in a 4°C refrigerator overnight for bacterial solidification. Ethanol gradient dehydration, tert-butyl alcohol replacement, and freeze-drying machine drying of the bacterial solution were performed. After gold spraying, the solution was placed in a scanning electron microscope instrument, and the parameters were adjusted for scanning observation.

[0107] 3. Confocal live-dead staining experiment

[0108] The TSB liquid medium was diluted with PBS (PBS:TSB = 2:1), and then the OD value of the overnight cultured P. g bacterial solution was adjusted to 0.2. 500 μL of the solution was added to each chamber of the chamber slide, and the solution was incubated in a 37°C incubator for 24 h in an anaerobic environment. After the biofilm culture was completed, the solution was washed with PBS for 3 times. The drug was added according to the above-mentioned grouping method for suspended bacteria. A light curing lamp (450-470 nm, 800 mW / cm 2After irradiation for 40 seconds, the bacteria were placed in a 7°C incubator for 15 minutes. The drug and culture medium were then removed, and the bacteria were rinsed three times with PBS. Live and dead bacterial dye was added, and the bacteria were incubated in the dark for 15 minutes. Excess dye was then washed away with PBS, and the laser confocal microscope parameters were adjusted for observation. Images were saved using Zenlite software after imaging.

[0109] 4. Bacterial reactive oxygen species staining experiment

[0110] *Porphyromonas gingivalis* was inoculated into TSB liquid medium and anaerobically cultured at 37°C until the logarithmic growth phase (OD600≈0.5). The cells were collected by centrifugation (3000×g, 5 min), washed twice with sterile PBS (pH 7.4), and resuspended to a final concentration of 1×10⁸ CFU / mL. The suspension was then treated with the aforementioned method of grouping the bacteria, and the culture was cured under a light-curing lamp (450-470 nm, 800 mW / cm²). 2 After irradiation for 40 seconds, the sample was incubated at 7°C for 15 minutes. The drug and culture medium were then removed, and the sample was washed three times with PBS. The DCFH-DA stock solution (10 mM, dissolved in DMSO) was diluted with PBS to a final concentration of 10 μM (in the dark). 1 mL of the bacterial suspension was added to the DCFH-DA solution and incubated at 37°C in the dark for 30 minutes. The sample was centrifuged (3000 × g, 5 minutes) to remove excess probe, washed twice with PBS, and resuspended in 1 mL of PBS. 10 μL of the bacterial suspension was dropped onto a glass slide, covered with a coverslip, and observed using a laser confocal microscope (excitation light 488 nm, image acquisition via the green fluorescence channel).

[0111] Experimental results

[0112] Bacterial plate count results showed that ( Figure 14 In both experiments (AB), under no-light conditions and with a co-incubation time of 15 minutes (clinically acceptable operation time), different drugs did not show significant bactericidal effects against Pg. However, after irradiation with a light-curing lamp for 40 seconds, the number of Pg colonies treated with BHCF nanozyme was significantly reduced, with a decrease in colony-forming units (CFU) of up to 4.32 logarithmic orders, and a bactericidal rate as high as 99.995%. This fully demonstrates that light irradiation significantly activates the antibacterial activity of BHCF nanozyme, providing conditions for its application in photodynamic antibacterial therapy.

[0113] Scanning electron microscopy (SEM) observation results show that ( Figure 14C), without light, the P. g bacteria treated by different drugs maintained typical rod-shaped morphology, the cell surface was rough and the morphology was uniform; while after the light curing lamp irradiation treatment, the P. g bacteria treated by BHCF nanoszyme presented significant morphological changes, showing that the bacterial body was deepened and the degree of shrinkage was intensified, and part of the cell structure was disintegrated and broken. The results show that the light activates the BHCF nanoszyme antibacterial mechanism, realizing the photodynamic synergistic enzyme activity antibacterial effect.

[0114] The laser confocal microscope observation results show that Figure 14 D), without light, the P. g single bacterial biofilm treated by different drugs all presented green fluorescence, indicating that the biofilm contained a large number of living bacteria. While under the light condition, the biofilm treated by BHCF nanoszyme presented significant orange-red fluorescence, indicating that most of the bacteria in the biofilm had died; consistent with the microscope observation results, indicating that BHCF nanoszyme can efficiently kill the bacteria in the P. g biofilm under light conditions, and significantly destroy the active structure of the biofilm.

[0115] The bacterial intracellular reactive oxygen staining confocal microscope observation results show that Figure 14 E), without light, no obvious green fluorescence was detected in the bacteria, indicating that without exogenous ROS induction, the endogenous ROS level in the bacteria was very low, and the DCFH-DA probe did not have obvious oxidation reaction. While the bacteria treated by BHCF nanoszyme + light presented significant green fluorescence, indicating that DCFH was efficiently oxidized to fluorescent product DCF by intracellular ROS, indicating that BHCF nanoszyme can rapidly break through the bacterial antioxidant defense system under light conditions through photosensitive cascade reaction.

[0116] Example 5

[0117] Study on the immediate desensitization performance of BHCF nanoszyme

[0118] Experimental method

[0119] Eighteen freshly extracted caries-free third molars were selected, and a slow cutting machine was used to cut along the enamel-dentin junction perpendicular to the long axis of the tooth under water cooling, remove the crown enamel to expose the middle dentin, and continue to cut to prepare a 1 mm ± 0.1 mm thick dentin disc sample. In turn, use 600, 800, 1000 mesh sandpaper to polish the dentin disc under water flushing for 60 s to make the surface flat and form a standard contamination layer. After polishing, ultrasonic (power 400 W) cleaning for 15 min, then repeatedly rinse with deionized water and dry with air gun. Soak the dentin disc in a 1% citric acid solution for 20 s to open the dentinal tubules to establish a sensitive dentin model, then wash thoroughly with deionized water. According to different treatment methods, the dentin disc samples were randomly divided into six groups (3 pieces per group):

[0120] First group (blank control): Dentin discs were immersed in equal volume of deionized water;

[0121] Second group (blank control + acid etching treatment): The dentin discs of the first group were immersed in 6% citric acid solution for 10 min every day.

[0122] Third group (blank control + abrasion treatment): The dentin discs of the first group were brushed by the same researcher using a Colgate soft toothbrush for 3 min twice a day.

[0123] Fourth group (BHCF nanoszyme): BHCF nanoszyme was ultrasonically dispersed in deionized water at a concentration of 1 mg / mL, and the solution was evenly coated on the surface of the dentin discs using a small brush. After 2 min of drying, the dentin discs were immediately rinsed thoroughly with a large amount of deionized water.

[0124] Fifth group (acid etching treatment): The sealed dentin discs of the fourth group were immersed in 6% citric acid solution for 10 min every day.

[0125] Sixth group (abrasion treatment): The sealed dentin discs of the fourth group were brushed by the same researcher using a Colgate soft toothbrush for 3 min twice a day.

[0126] All dentin discs were placed in artificial saliva daily. After 10 min of immersion, the dentin discs were fixed with 3% glutaraldehyde, dehydrated with gradient ethanol, dried, and vacuum gold sprayed. The dentin discs were placed under a SEM to observe the sealing of the dentin tubule sections.

[0127] Experimental results

[0128] The dentin samples of different treatment groups were observed by scanning electron microscopy, as shown in Figure 15 The dentin tubules of the control group were exposed, with clear lumen and no obvious sealing material attached to the surface. After acid etching and abrasion treatment, the dentin tubules were more open, showing complete exposure. In the nanoszyme group, BHCF nanoszyme could effectively seal the dentin tubules, with most of the tubule lumens tightly filled with nanoszyme particles. Only a few tubules were partially open, and most of the tubules remained closed after acid etching and abrasion treatment, indicating that BHCF nanoszyme had good immediate sealing effect and excellent acid and abrasion resistance.

[0129] Example 6

[0130] Study on the continuous desensitization and remineralization performance of BHCF nanoszyme

[0131] Experimental method

[0132] 1. Electron microscopy observation: 18 freshly extracted, caries-free third molars were selected and dentin sections were prepared. The grouping and processing were the same as in Example 5. The dentin sections of each group were soaked in artificial saliva for 3 weeks (10 min in Example 5). A shallow groove was ground from the center of the pulp surface of each dentin section to the enamel surface using a slow cutting machine. Then, the dentin section was gently broken in half along the shallow groove. One half was used for observation of the cross-sectional morphology of the dentinal tubules, and the other half was used for observation of the longitudinal sectional morphology of the dentinal tubules.

[0133] 2. Crystal Phase Analysis: XRD patterns of the dentin sample surface were recorded using an XRD-6000 X-ray diffractometer to analyze the crystal orientation and mineral phases of the newly formed mineralized layer in each group of samples. Data collection conditions: Cu Kα radiation was generated at 40 kV and 40 mA. The spectra were recorded as 2θ in the range of 10° to 60°, with a step size of 0.021° and a counting time of 0.5 s.

[0134] Experimental results

[0135] Cross-sections of dentin samples from different treatment groups were observed using scanning electron microscopy (e.g., Figure 16 As shown in Figure A, the results indicated that after 3 weeks of immersion in artificial saliva, the dentinal tubules in the control group remained open, with no obvious blockage at the tubule openings; while the BHCF nanozyme-treated group remained completely closed. Even after daily acid etching and abrasion challenges, the dentinal tubules maintained their sealed integrity, demonstrating the significant durability of its sealing effect.

[0136] Observation of longitudinal sections using scanning electron microscopy (e.g.) Figure 16 (As shown in B) In the control group, a small amount of deposits were present in the dentinal tubules, but most tubules remained empty with no obvious mineralization. In contrast, the tubules in the BHCF nanozyme-treated group showed significant mineralization, reaching a depth of over 100 μm. After acid etching and abrasion treatment, a large amount of mineralized deposits remained in the tubules of the treated group, further confirming the stability of its sealing and mineralization capabilities.

[0137] XRD patterns show ( Figure 17 As shown in the figure, the characteristic diffraction peaks of hydroxyapatite (002), (300), (213), and (004) at 2θ = 25.8°, 32.8°, 49.5°, and 53.2° of demineralized dentin were significantly weakened or disappeared. After immersion and culture in artificial saliva, the characteristic diffraction peaks of hydroxyapatite in the BHCF nanozyme treatment group were significantly restored and highly consistent with the characteristic peaks of healthy dentin, indicating that the nanozyme can effectively promote dentin remineralization.

[0138] The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A nanoenzyme with antibacterial, tooth desensitizing, and remineralizing functions, characterized in that, The nanozyme is prepared by self-assembly of berberine, heme chloride, and calcium fluoride, and is prepared according to the following steps: (1) Dissolve heme chloride and berberine in DMSO for later use; (2) Dissolve calcium fluoride in hydrochloric acid solution for later use; (3) At room temperature and under stirring, add the solution obtained in step (1) to the solution obtained in step (2) to obtain a mixed system; (4) Adjust the pH of the mixture obtained in step (3) to neutral with sodium hydroxide solution to obtain a suspension; (5) Dialyze the suspension obtained in step (4) in ultrapure water to remove unassembled molecules and ions; (6) Centrifuge the system after dialysis in step (5), remove the supernatant, disperse the precipitate, and freeze-dry to obtain the nanozyme.

2. The method for preparing a nanoenzyme with bactericidal, tooth desensitizing, and remineralizing functions as described in claim 1, characterized in that, The nanozyme was prepared according to the following steps: (1) Dissolve heme chloride and berberine in DMSO for later use; (2) Dissolve calcium fluoride in hydrochloric acid solution for later use; (3) At room temperature and under stirring, add the solution obtained in step (1) to the solution obtained in step (2) to obtain a mixed system; (4) Adjust the pH of the mixture obtained in step (3) to neutral with sodium hydroxide solution to obtain a suspension; (5) Dialyze the suspension obtained in step (4) in ultrapure water to remove unassembled molecules and ions; (6) Centrifuge the system after dialysis in step (5), remove the supernatant, disperse the precipitate, and freeze-dry to obtain the nanozyme.

3. The method for preparing a nanoenzyme with bactericidal, tooth desensitizing, and remineralizing functions according to claim 2, characterized in that, The molar ratio of heme chloride, berberine, and calcium fluoride in the nanozyme is 0.5-1:1-2:4-6.

4. The method for preparing a nanoenzyme with bactericidal, tooth desensitizing, and remineralizing functions according to claim 2, characterized in that, The concentration of the hydrochloric acid solution in step (2) is 0.05-0.15 mol / L.

5. The method for preparing a nanoenzyme with bactericidal, tooth desensitizing, and remineralizing functions according to claim 2, characterized in that, The concentration of the sodium hydroxide solution in step (4) is 0.05-0.15 mol / L.

6. The application of the nanoenzyme with bactericidal, tooth desensitizing and remineralizing functions as described in claim 1 in the preparation of drugs for treating periodontitis.

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