Nano-enzyme with sterilization, tooth desensitization and remineralization functions as well as preparation method and application of nano-enzyme

Abstract

The invention provides a nano-enzyme with sterilization, tooth desensitization and remineralization functions as well as a preparation method and application thereof, and relates to the technical field of nano-enzymes. Berberine, hemin and calcium fluoride are used as raw materials and are self-assembled into the multifunctional BHCF nano-enzyme with sterilization, tooth desensitization and remineralization functions through multi-dimensional interaction of ionic bonds, Ca < 2 + > coordination bonds, pi-pi accumulation and hydrogen bonds. The BHCF nano-enzyme has a clear peroxidase-like and superoxide dismutase-like double-enzyme catalytic capability and a photodynamic effect, the antibacterial activity of the BHCF nano-enzyme is obviously activated under an illumination condition, and the sterilization rate can reach 99.995%; meanwhile, the BHCF nano-enzyme has a good immediate and lasting sealing effect, and has excellent acid resistance, wear resistance and remineralization capacity. The BHCF nano-enzyme provided by the invention has huge application potential in preparation of drugs for treating periodontitis.

Description

Technical Field

[0001] The present invention relates to the field of nanozyme technology, and in particular to a nanozyme with sterilization, tooth desensitization and remineralization functions, and a preparation method and application thereof. Background Art

[0002] Nanozymes, a class of nanomaterials with enzymatic properties, represent a new field pioneered and pioneered by Chinese scientists. In 2022, nanozymes were named one of the top ten emerging chemical technologies by the International Union of Pure and Applied Chemistry (IUPAC). Combining the advantages of enzymes and chemical catalysis, nanozymes can catalyze enzyme substrates under mild conditions. They possess high catalytic activity, stable properties, low cost, and ease of modification and large-scale production. As a substitute for natural enzymes, they are being used in disease diagnosis and treatment, chemical analysis, biosensing, environmental treatment, and other fields.

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

[0004] Periodontitis is a chronic infectious disease caused by periodontal pathogens. These bacteria react with the host, leading to inflammation and destruction of periodontal tissues such as the gums, periodontal ligament, and alveolar bone. Because subgingival plaque and tartar are important local factors in the development of periodontitis, subgingival scaling combined with antibiotics is the most effective treatment for controlling its progression. Its primary goal is to remove irritants from the tooth surface that may cause gingival inflammation, such as plaque, tartar, and endotoxins, thereby re-establishing a local environment and microbial system that is compatible with periodontal health. However, periodontal patients are prone to gum recession and root exposure after periodontal scaling, which can lead to dentin exposure and tooth sensitivity, manifested as soreness and pain after exposure to cold, hot, sour, sweet, or mechanical stimuli. Currently, there is no commercially available periodontal treatment that can simultaneously kill bacteria and alleviate the symptoms of dentin sensitivity. Traditional nanozymes are easy to rationally design, so they can selectively exhibit peroxidase-like activity, catalyzing hydrogen peroxide to produce reactive oxygen species, thereby achieving good bactericidal effects. However, the components of traditional nanozymes lack calcium and fluorine elements, and cannot achieve the requirements of tooth desensitization and remineralization. Traditional desensitization drugs do not have good antibacterial effects, resulting in a technical gap in antibacterial and tooth desensitization and remineralization.

[0005] Based on the above technical background, the present invention aims to provide a multifunctional nanozyme with sterilization, tooth desensitization and remineralization functions, so as to achieve the purpose of improving the catalytic treatment effect of nanozymes in periodontitis. Summary of the Invention

[0006] (1) Technical problems solved

[0007] In response to the shortcomings of the existing technology, the present invention provides a multifunctional nanozyme with sterilization, tooth desensitization and remineralization functions to solve the technical problem that the existing nanozymes cannot simultaneously achieve sterilization, tooth desensitization and remineralization in the periodontitis microenvironment, thereby limiting the application of nanozymes in the catalytic treatment of periodontitis.

[0008] (2) Technical solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0010] In the first aspect, the present invention provides a multifunctional nanozyme (BHCF nanozyme for short) with sterilization, tooth desensitization and remineralization functions, wherein the nanozyme is prepared by self-assembly of berberine, hemin and calcium fluoride.

[0011] In a second aspect, the present invention provides a method for preparing the multifunctional BHCF nanozyme, wherein the BHCF nanozyme is prepared according to the following steps:

[0012] (1) Dissolve hemin and berberine in DMSO for later use;

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

[0014] (3) adding the solution obtained in step (1) to the solution obtained in step (2) at room temperature under stirring to obtain a mixed system;

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

[0016] (5) dialyzing the suspension obtained in step (4) in ultrapure water to remove unassembled molecules and ions;

[0017] (6) The system after dialysis in step (5) is centrifuged, the supernatant is removed, and the precipitate is dispersed and freeze-dried to obtain the BHCF nanozyme.

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

[0019] Furthermore, 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] Furthermore, 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 present invention also provides the use of the multifunctional BHCF nanozyme in the preparation of drugs for treating periodontitis.

[0022] (3) Beneficial effects

[0023] The present invention uses berberine, hemin chloride and calcium fluoride as raw materials, and the three are bonded by ionic bonds, Ca 2+ The multi-dimensional interactions of coordination bonds, π-π stacking, and hydrogen bonds self-assemble into a multifunctional BHCF nanozyme with antibacterial, tooth desensitization, and remineralization functions. The BHCF nanozyme has great potential for the preparation of drugs for the treatment of periodontitis.

[0024] The BHCF nanozyme provided by the present invention has the following advantages:

[0025] 1. The BHCF nanozyme is in the shape of an irregular map plate, which can increase the specific surface area, provide more exposure sites for the enzyme active center, and provide conditions for enhancing the activity of peroxidase (POD) and superoxide dismutase (SOD). At the same time, the morphology and size of the BHCF nanozyme are well adapted to the microstructure of the dentinal tubules. When acting on the dentin surface, the irregular edges and undulations can fit tightly to the tubule openings, achieving efficient physical closure. In addition, the surface of the BHCF nanozyme is negatively charged and has good stability. It can effectively prevent each other from agglomerating by relying on the electrostatic repulsion between particles, thereby maintaining a relatively stable dispersion state.

[0026] 2. The BHCF nanozyme provided by the present invention exhibits strong dual-enzyme catalytic ability of POD-like activity and SOD-like activity; under light irradiation, it simultaneously triggers the type I photosensitivity reaction (electron transfer to produce superoxide anions) and type II photosensitivity reaction (energy transfer to generate singlet oxygen) of the BHCF nanozyme, with a photodynamic effect, providing direct evidence for its light-stimulated antibacterial activity in the treatment of periodontitis.

[0027] 3. Long-term antibacterial experiments show that the BHCF nanozyme provided by the present invention has an MIC of 12.5 μg / mL against Porphyromonas gingivalis (Pg) suspension bacteria. Compared with berberine, calcium fluoride and hemin, BHCF nanozyme significantly improves the antibacterial activity against Pg through the synergistic effect of the ternary components, and BHCF nanozyme has excellent antibacterial activity at low concentrations. At the same time, BHCF nanozyme has a significant destructive effect on Pg bacterial biofilms, can efficiently kill bacteria in Pg biofilms, significantly destroy the active structure of the biofilm, and can effectively disperse Pg biofilms, providing conditions for biofilm removal in periodontitis treatment.

[0028] 4. The photosensitized cascade reaction antibacterial experiment showed that under light conditions, the antibacterial activity of BHCF nanozyme was significantly activated, and the bactericidal rate could reach 99.995%; BHCF nanozyme could quickly break through the bacterial antioxidant defense system through photosensitized cascade reaction under light conditions, achieving photodynamic synergistic enzyme activity antibacterial effect, providing conditions for its application in photodynamic antibacterial therapy.

[0029] 5. The BHCF nanozyme provided by the present invention has good immediate and long-lasting sealing effects, excellent acid resistance and wear resistance, and outstanding deep remineralization ability of dentinal tubules, achieving long-term sealing stability of dentinal tubules. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the SEM image of BHCF nanozyme.

[0031] Figure 2 This is the EDS diagram of BHCF nanozyme.

[0032] Figure 3 DLS analysis of BHCF nanozyme, (A) particle size; (B) Zeta potential.

[0033] Figure 4 Infrared spectra of berberine, hemin, calcium fluoride and BHCF nanozyme.

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

[0035] Figure 6 The POD-like detection results of BHCF nanozyme, including (A) catalytic activity diagram of berberine, hemin, calcium fluoride and BHCF nanozyme; (B) POD-like activity of BHCF nanozyme under different concentration conditions;

[0036] Figure 7 The results of the SOD-like detection of BHCF nanozyme are shown in Figure 2. (A) SOD inhibition rate of BHCF nanozyme at different concentrations; (B) SOD-like catalytic activity of 25ug / ml BHCF nanozyme.

[0037] Figure 8 Figure 2 is a diagram of the photosensitivity of BHCF nanozyme, including (A) UV-visible 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 conditions.

[0038] Figure 9 These are the results of the minimum inhibitory concentration (MIC) determination of hemin, berberine, calcium fluoride and BHCF nanozyme against Pg.

[0039] Figure 10 Bacterial plate counting method was used to detect the antibacterial effects of hemin, berberine, calcium fluoride and BHCF nanozyme on Pg (A); and the colony forming unit statistics of Pg treated with different drugs (B).

[0040] Figure 11 SEM images of Pg after treatment with hemin, berberine, calcium fluoride, and BHCF nanozyme.

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

[0042] Figure 13 Crystal violet staining results of Pg biofilm treated with different drugs, including hemin, berberine, calcium fluoride and BHCF nanozyme (A); and crystal violet staining statistics of Pg biofilm treated with different drugs (B).

[0043] Figure 14 Figure 3 shows the photodynamic synergistic antibacterial performance test results of BHCF nanozymes, where (A) is the antibacterial effect of Pg on Pg under different treatment conditions detected by the bacterial plate counting method; (B) is the statistical chart of Pg colony forming units under different treatment conditions; (C) is the SEM image of Pg after treatment with different conditions; (D) is the live and dead bacteria staining image of Pg biofilms under different treatment conditions; (E) is the confocal microscopy observation result of active oxygen staining in Pg bacteria after treatment with different conditions.

[0044] Figure 15 Scanning electron micrographs of dentin samples in the control group and the BHCF nanozyme-treated group after immersion in artificial saliva for 10 minutes.

[0045] Figure 16Scanning electron micrographs of cross-sections (A) and longitudinal sections (B) of dentin samples from the control group and the BHCF nanozyme-treated group after immersion in artificial saliva for 3 weeks.

[0046] Figure 17 XRD patterns of the dentin sample surfaces of the control group and the BHCF nanozyme-treated group after immersion in artificial saliva for 3 weeks. DETAILED DESCRIPTION

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

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

[0049] Example 1

[0050] 1. Preparation of BHCF nanozymes

[0051] 0.05 mol hemin and 0.1 mol berberine (BBr) were dissolved in 1 mL DMSO for later use; 0.3 mol CaF2 was dissolved in 20 mL 0.1 mol / L hydrochloric acid solution for later use; under slow stirring, the pre-mixed DMSO solution containing Hemin and Bbr was added to the CaF2 solution; then the pH was immediately adjusted to about 7.0 with 0.1 mol / L NaOH; the resulting suspension was immediately dialyzed in ultrapure water for 5 days to remove unassembled molecules and ions; the dialyzed liquid was centrifuged at 12000 rpm for 10 minutes, the supernatant was removed, and the precipitate was redispersed with 5 mL ultrapure water; the dispersed nanozyme was pre-cooled at -80°C for 30 minutes and then freeze-dried in a freeze dryer. The powdered solid obtained after freeze-drying was the BHCF nanozyme.

[0052] 2. Characterization of BHCF nanozymes

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

[0054] Scanning electron microscopy results showed that ( Figure 1 ), the BHCF nanozyme provided by the present invention is in the shape of irregular map plates, with uniform plate size distribution and a size between 100-200nm, which is significantly different from the morphology of traditional spherical and rod-shaped nanozymes. This irregular shape greatly increases the specific surface area, can provide more exposure sites for the enzyme active center, and provide conditions for enhancing the activity of peroxidase (POD) and superoxide dismutase (SOD). At the same time, the morphology and size of the BHCF nanozyme are well adapted to the microstructure of the dentinal tubules. When acting on the dentin surface, the irregular edges and undulations can fit tightly to the tubule openings, enabling efficient physical sealing. EDS electronic image ( Figure 2 ) showed that HCFe nanozyme contained elements such as C, N, O, Fe, Ca, and F, and the elements were evenly distributed.

[0055] like Figure 3 The results showed that the particle size of BHCF nanozyme was about 100-200 nm ( Figure 3 A), which is consistent with the results of scanning electron microscopy. The Zeta potential of the BHCF nanozyme surface is -20mV ( Figure 3 B) is negatively charged and has good stability. It can effectively prevent each other from agglomerating by virtue of the electrostatic repulsion between particles, thus maintaining a relatively stable dispersion state.

[0056] like Figure 4 The results showed that the carboxylic acid group of hemin (1698.72 cm -1 ) disappears and splits into COO - Asymmetry (1612.00cm -1 ) and symmetry (1377.99cm -1 ) vibration peak, indicating that the carboxylic acid is bound to Ca2+ ) or ionic bond (with berberine quaternary ammonium salt); the CN peak of berberine quaternary ammonium salt (1225→1223.95cm -1 ) and methylenedioxy CO peak (1033→1036.96 cm -1 ) shifted, revealing that it participated in the assembly through electrostatic interaction and hydrogen bonding; the lattice vibration peak of calcium fluoride (1383→1377.99cm -1 ) and hydroxyl peak (3433→3397.28cm -1 ) significantly shifted, confirming that Ca 2- Out of the lattice and with COO - or berberine oxygen atom coordination; porphyrin ring (1603→1612cm -1 ) and berberine aromatic ring (1505→1496.75cm -1 ) blue shift / red shift, indicating π-π stacking. The coordinated changes of these characteristic peaks prove that the three 2+ The self-assembly of BHCF nanozyme through multidimensional interactions of coordination bonds, π-π stacking and hydrogen bonds laid the foundation for its functional structure.

[0057] like Figure 5 The results show that through systematic analysis of XPS data, the evidence of the interaction between hemin, berberine and calcium fluoride is as follows:

[0058] (1) Calcium (Ca) bridging coordination: Ca 2p3 / 2 peak (347.29 eV) combined with F1s doublet (684.96 eV) lattice F - 、688.94eV coordinated F - ) indicates that some Ca 2+ Detached from the CaF2 lattice, while coordinating the COO of hemin - (O 1s 531.72eV) and free F - , forming Ca 2+ -COO - -F - Ternary coordination network;

[0059] (2) Ionic bond and charge compensation: berberine quaternary ammonium salt N + (N 1s 401.46eV, 0.5–1.0eV negative shift compared to the free state) and COO - and F - Through electrostatic interaction, N + -F - Charge compensation reduces the F-electron cloud density (F1s 688.94eV);

[0060] (3) Coordination expansion of iron (Fe): Fe 3+The main peak (710.25 eV) and satellite peak (718.27 eV) of Fe 2p3 / 2 were enhanced, indicating that the methoxyl / hydroxyl oxygen of berberine participated as ligands in the formation of hexacoordinated [FeN4O2] active centers;

[0061] (4) Carbon-oxygen synergy: 288.62 eV (COO - ) and 286.58 eV (CO) to verify the deprotonation of carboxylic acid and the participation of berberine oxygen atoms in coordination / hydrogen bonding (O 1s 533.38 eV);

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

[0063] The chemical state changes of the above elements fully reveal the interaction of hemin, berberine and calcium fluoride through ionic bonds, Ca 2+ / Fe 3+ Cooperative assembly mechanism of coordination bonds, hydrogen bonds and π-π stacking.

[0064] Example 2

[0065] Study on the catalytic properties of BHCF nanozymes

[0066] 1. POD-like activity detection

[0067] Nanozymes with peroxidase-like activity can catalyze H2O2 to produce hydroxyl radicals, which can convert colorless TMB into blue, showing a characteristic absorption peak at 652nm. In order to study the enzyme-like activity of BHCF nanozymes, we used TMB as a substrate and explored its peroxidase-like activity by monitoring the changes in absorbance at 652nm on a UV spectrophotometer. This experiment also examined the strength of the peroxidase-like catalytic activity of BHCF nanozymes with different drugs and different concentrations. The specific method is:

[0068] (1) Using an ultraviolet spectrophotometer, the peroxidase-like activity of BHCF nanozyme was kinetically analyzed by measuring the absorbance change at 652 nm within a reaction time of 300 s. The experiment was carried out at 37 ° C, using 10 μg / mL of BBR, Hemin, CaF2 and BHCF nanozyme, respectively, in a pH 4.5 buffer solution, with a H2O2 concentration of 30 mM and a substrate TMB concentration of 1 mM. A blank control system was set up with a H2O2 concentration of 30 mM, a substrate TMB concentration of 1 mM, and a pH 4.5 buffer solution.

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

[0070] like Figure 6 As shown in A, the blank control group, BBR group and CaF2 group had no obvious absorbance at 652nm, hemin had a weak absorbance at 652nm, and BHCF nanozyme had a strong absorbance at 652nm. The above results show that BBR and CaF2 have no peroxidase-like activity, Hemin can exert weak enzyme activity, and the BHCF nanozyme provided by the present invention exhibits a strong peroxidase-like activity, which can catalyze H2O2 to produce ROS and oxidize colorless TMB into blue. As the concentration of BHCF nanozyme increases, the absorption intensity at 652nm increases, indicating that this enzyme activity is positively correlated with the drug concentration ( Figure 6 B).

[0071] 2. SOD-like activity detection

[0072] Method: The SOD-like enzyme activity of BHCF nanozyme can be studied by using the SOD detection kit-WST, which uses the highly water-soluble tetrazolium salt WSTR6-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid phenyl)-2-hydrogen-tetrazolium salt, disodium salt) to react with superoxide anions (O2 - ) reacts to generate a water-soluble dye WST-1fomazan, which has an absorption peak at 440nm. The change in absorbance at 440nm corresponds to the change in superoxide anion concentration, so the SOD activity can be calculated by detecting the absorbance at 440nm. Using an ultraviolet spectrophotometer, the superoxide dismutase-like activity of BHCF nanozyme was kinetically analyzed by measuring the change in absorbance within a 20-min reaction time at 450nm. The experiment was carried out at 37°C, and the reaction system of BHCF nanozyme with different concentrations (100, 80, 60, 40, 20, 4, 0.8ug / ml) was tested, and the inhibition rate of SOD of different concentrations in each group was calculated.

[0073] based on The results showed that BHCF nanozyme could effectively inhibit the superoxide anion and The reduction reaction inhibition rate reached 50% ( Figure 7AB), calculated based on the unit definition (20 μL sample solution required to inhibit 50% of the reaction corresponds to 1 U SOD activity), its specific activity is 166.67 U / mg, indicating that the nanozyme has a clear superoxide dismutase-like catalytic ability.

[0074] 3. UV-visible absorption spectroscopy: BBR, Hemin, CaF2, and BHCF nanozymes were dissolved in double-distilled water to prepare a test solution with a concentration of 100 μg / ml. The absorption peak was measured using a UV spectrophotometer with a wavelength scanning range of 200-800 nm to observe the absorption peak position, shape, and intensity changes of each component.

[0075] like Figure 8 As shown in A, the absorption peaks of BBR are near 345nm and 421nm, the characteristic absorption peaks of Hemin are near 385nm and 600nm, and there is no obvious absorption peak of CaF2. However, there are two continuous characteristic absorption peaks between 350-400nm for BHCF nanozyme. Compared with BBR, the maximum absorption peak moves from 345nm to 352nm, which is red-shifted, which may be related to the π-π interaction between BBR molecules.

[0076] 4. ESR detects the types of free radicals produced under light

[0077] Superoxide radical test: Take 30μL 1mg / ml BHCF nanozyme and 30μL DMPO methanol, mix them evenly, illuminate them with 500W mercury lamp for 5 minutes, use a capillary to draw a certain amount of the mixture, place it in a quartz tube, and place it in the EPR resonant cavity to test superoxide radicals.

[0078] Singlet oxygen free radicals: Take 30μL 1mg / ml BHCF nanozyme and 50μL 100mM TEMP, mix them evenly, and illuminate them with 500W mercury lamp for 5 minutes. Use a capillary to draw a certain amount of the mixture, put it into a quartz tube, put it into the EPR sample cavity, and place it in the quartz tube. Then put it into the EPR resonant cavity to test singlet oxygen free radicals.

[0079] Electron spin resonance (ESR) detection results show that ( Figure 8 B) Under dark conditions, no superoxide anions (O2 - ) and singlet oxygen ( 1 O2) signal; under light conditions, the system showed significant superoxide anion and singlet oxygen characteristic signals. This result fully demonstrates that BHCF nanozyme has a photodynamic effect, and that it triggers both type I photosensitivity reaction (electron transfer to produce superoxide anion) and type II photosensitivity reaction (energy transfer to produce singlet oxygen) under light, providing direct evidence for its light-induced antibacterial activity in the treatment of periodontitis.

[0080] Example 3

[0081] Study on the long-term antibacterial properties of BHCF nanozymes

[0082] Experimental methods

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

[0084] BBR, Hemin, CaF2, and BHCF nanozymes were prepared into stock solutions of 1000, 500, 250, 125, and 62.5 μg / mL with sterile water for later use. Porphyromonas gingivalis (Pg) bacterial solution was diluted to an OD600 of 0.2 for later use. 20 μL of the drug stock solution and 180 μL of the diluted bacterial solution were added to each well of a 96-well plate, respectively, to achieve a final drug concentration of 100, 50, 25, 12.5, and 6.25 μg / mL in each group. Liquid culture medium without bacteria was used as a negative control, and a mixture of 180 μL of drug-free bacterial solution and 20 μL of ddH2O was used as a positive control. Three replicates were set up in each well. After the addition of the sample, 100 μL was taken from each well to measure the initial OD value. The OD value was measured again after incubation at 37°C under anaerobic conditions for 24 hours.

[0085] 2. Bacterial plate counting

[0086] In order to evaluate the inhibitory effect of BBR, Hemin, CaF2 and BHCF nanozymes on Pg suspension bacteria, the diluted bacterial solution was divided into 5 groups, 1 ml each, and then diluted BBR, Hemin, CaF2 and BHCF nanozymes were added respectively. The control group was added with deionized water of the same volume as the drug, and gently blown evenly to make the final drug concentration of 12.5 μg / mL. After anaerobic culture in a 37°C incubator for 24 hours, the centrifuge tube was continuously diluted 10 times, and 100 μL of the dilution was smeared on Columbia agar medium and cultured anaerobically at 37°C for 5-7 days. The colonies were counted after the end of culture.

[0087] 3. Observation of bacterial morphology using scanning electron microscopy

[0088] The diluted bacterial suspension was collected separately, grouped, and cultured as above (bacterial plating and counting). After incubation, the suspension was centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the pellet was washed three times with PBS. Fixation was performed with 2.5% glutaraldehyde and the cells were refrigerated at 4°C overnight to solidify the bacteria. Dehydration was performed using an ethanol gradient, followed by replacement with tert-butanol. The suspension was then freeze-dried and the surface was sprayed with gold. The suspension was then placed in a scanning electron microscope (SEM) and the parameters were adjusted for scanning observation.

[0089] 4. Confocal live-dead bacteria staining experiment

[0090] TSB liquid medium was diluted with PBS (PBS:TSB = 2:1). The overnight Pg culture was then adjusted to an OD value of 0.2 and 500 μL was added to each chamber on a chamber slide. The cells were then anaerobically incubated at 37°C for 24 hours. After the biofilm culture was complete, the cells were rinsed three times with PBS and treated according to the suspension grouping method described for the experimental bacterial plate count. After anaerobically incubating for another 24 hours, the drug and culture medium were aspirated, the cells were rinsed three times with PBS, and live-dead dye was added. The cells were then incubated in the dark for 15 minutes. The excess dye was then washed away with PBS. The laser confocal microscope settings were then adjusted and the cells were observed under a laser confocal microscope. After recording, images were saved using Zenlite software.

[0091] 5. Crystal violet experiment

[0092] The overnight cultured pg bacterial solution was adjusted to an OD value of 0.1 and added to a 24-well plate, 1 mL per well, and cultured in a 37°C incubator for 24 hours. The next day, the excess bacterial solution was aspirated and gently washed twice with PBS. According to the grouping method described in the experimental bacterial plate count (i.e., 12.5 ug / ml of BBR, Hemin, CaF2, and BHCF nanozymes, and deionized water for the control group), 500 μL of culture medium or drug was added to each well. Three replicates were set for each treatment group. After continuing to culture for 24 hours, the drugs and culture medium in the 96-well plate were removed, gently rinsed twice with PBS, dried at room temperature for 30 minutes, and 500 μL of 4% paraformaldehyde solution was added to each well for 15 minutes. The paraformaldehyde solution was then removed, rinsed three times with PBS, dried at room temperature, and 500 μL of 0.1% crystal violet solution was added to each well and stained in the dark for half an hour. After crystal violet staining, the cells were washed three times with PBS, and 1 mL of 96% anhydrous ethanol was added to each well for dissolution. After protection from light for 1 hour, the absorbance of each well at 590 nm was measured with a microplate reader.

[0093] Experimental results

[0094] like Figure 9 As shown in the figure, the MIC of BHCF nanozyme against Porphyromonas gingivalis (Pg) suspension is 12.5μg / mL. In comparison, the MIC of berberine is 50μg / mL, while the MICs of calcium fluoride and hemin are both over 400μg / mL. The results show that compared with a single component, BHCF nanozyme significantly enhances the antibacterial activity against Pg through the synergistic effect of the ternary components. The antibacterial efficacy is 4 times that of berberine, and it has an order of magnitude advantage over CaF2 and Hemin, providing conditions for its application in the antibacterial treatment of periodontitis.

[0095] The bacterial plate count results showed that ( Figure 10AB), at a concentration of 12.5 μg / mL, the bacterial colony counts of Pg treated with berberine, hemin, and calcium fluoride were not significantly different from those in the control group, indicating that the single components had no significant inhibitory effect on Pg at this concentration. However, after treatment with BHCF nanozyme, the number of Pg colonies was significantly reduced, with the colony-forming units (CFU) reduced by 4.4 logarithmic levels, and the bactericidal rate was as high as 99.996%, indicating that BHCF nanozyme has excellent antibacterial activity even at low concentrations.

[0096] Scanning electron microscopy (SEM) observation results showed that ( Figure 11 ), after treatment with berberine, hemin, and calcium fluoride, the Pg bacteria still maintained a typical rod-shaped morphology, with a rough and uniform cell surface, indicating that a single component failed to significantly destroy the bacterial structure. However, the Pg bacteria in the BHCF nanozyme-treated group showed obvious morphological changes, manifested as concavity and increased wrinkling, and some cells even showed disintegration. This intuitively revealed the destructive effect of BHCF nanozyme on Pg cell structure and provided direct morphological evidence for its efficient antibacterial activity.

[0097] Laser confocal microscopy observations showed that after treatment with BHCF nanozymes, the Pg single-species biofilm exhibited significant red fluorescence, indicating that most bacteria in the biofilm had died; while the biofilms of the control group and other single-component treatment groups (BBR, Hemin, CaF2) all exhibited green fluorescence, indicating that the biofilm contained a large number of live bacteria ( Figure 12 ). This is consistent with the results of microscopic observation, indicating that BHCF nanozymes can effectively kill bacteria in Pg biofilms and significantly destroy the active structure of the biofilm.

[0098] Crystal violet staining was used to investigate the discrete effect of BHCF nanozymes on Pg biofilms. Figure 13 As shown in AB, the results showed that 12.5 μg / mL BHCF nanozyme could significantly destroy the integrity of Pg biofilm, which was manifested by the obvious lighter color of the biofilm after staining ( Figure 13 A). Compared with the control group, and the berberine group, hemin group, and calcium fluoride group at the same concentration, the biofilm staining intensity in the BHCF nanozyme treatment group was significantly different ( Figure 13 B) These results confirm that BHCF nanozymes can effectively disperse Pg biofilms at low concentrations, and their dispersion efficiency is significantly better than that of a single component, providing conditions for biofilm removal in periodontitis treatment.

[0099] Example 4

[0100] Study on the antibacterial properties of BHCF nanozymes through cascade reaction

[0101] Experimental methods

[0102] In the photodynamic synergistic enzyme activity antibacterial experiment, the treatment conditions were as follows: the drug was added to the bacterial solution and immediately cured with a light curing lamp (450-470nm, 800mW / cm 2 ) irradiation for 40 seconds, the co-incubation time was 15 minutes, and the other experimental conditions (such as temperature, reagent concentration, and reaction system composition) were consistent with the experiment in Example 3. (Note: The treatment conditions in Example 3 were no light and co-incubation for 24 hours, while the treatment conditions in this example were light and co-incubation for 15 minutes. This is the difference between the two.) Under these conditions, the effect of light excitation on the antibacterial activity and biofilm removal ability of BHCF nanozymes was explored to reveal the synergistic mechanism of its photodynamic effect and rapid antibacterial effect.

[0103] 1. Colony forming unit counting

[0104] To evaluate the inhibitory effect of BHCF nanozyme on Pg suspension bacteria under light conditions, 10 mL of diluted bacterial solution and drug (grouped as control group, control group + light, Bbr, Bbr + light, BHCF nanozyme, BHCF nanozyme + light) were added to a 15 mL centrifuge tube and gently pipetted to make the final drug concentration 12.5 μg / mL. The light curing lamp (450-470 nm, 800 mW / cm 2 After irradiation for 40 seconds, incubate in a 37°C incubator for 15 minutes. Serially dilute 10-fold, spread 100 μL of the dilution onto Columbia agar and incubate anaerobically at 37°C for 5-7 days. Count the colonies after the incubation period. A control group treated without light was also established.

[0105] 2. Observation of bacterial morphology using scanning electron microscopy

[0106] The diluted bacterial suspension was collected separately, grouped, and cultured as above. After incubation, the suspension was centrifuged at 5000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the pellet was washed three times with PBS. Fixation was performed with 2.5% glutaraldehyde and the cells were refrigerated at 4°C overnight to solidify the bacteria. Dehydration was performed using an ethanol gradient, followed by replacement with tert-butanol. The suspension was then freeze-dried and the surface was sprayed with gold. The suspension was then placed in a scanning electron microscope (SEM) and the parameters were adjusted for scanning observation.

[0107] 3. Confocal live-dead bacteria staining experiment

[0108] TSB liquid medium was diluted with PBS (PBS:TSB = 2:1), and then the overnight cultured Pg bacterial solution was adjusted to an OD value of 0.2. 500 μL was added to each chamber of the chamber slide and incubated anaerobically in a 37°C incubator for 24 hours. After the biofilm culture was completed, it was rinsed three times with PBS and the suspension was grouped according to the experimental method. The light curing lamp (450-470 nm, 800 mW / cm 2After irradiation for 40 seconds, the cells were placed in a 7°C incubator and incubated for 15 minutes. The drug and culture medium were then removed by aspiration, and the cells were rinsed three times with PBS. Live-kill dye was added and the cells 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. After recording, images were saved using Zenlite software.

[0109] 4. Bacterial reactive oxygen species staining experiment

[0110] Porphyromonas gingivalis (P.gingivalis) was inoculated into TSB liquid medium and cultured anaerobically 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 drugs were added according to the grouping method of the suspended bacteria described above, and the cells were illuminated with a light curing lamp (450-470 nm, 800 mW / cm 2 ) After irradiation for 40 seconds, place the cells in a 7°C incubator and incubate for 15 minutes. After aspiration of the drug and culture medium, rinse with PBS three times, and dilute the DCFH-DA stock solution (10 mM, dissolved in DMSO) with PBS to a final concentration of 10 μM (protect from light). Take 1 mL of the bacterial suspension and add the DCFH-DA solution. Incubate at 37°C in the dark for 30 minutes. Centrifuge (3000 × g, 5 minutes) to remove excess probe, wash twice with PBS, and resuspend in 1 mL of PBS. Take 10 μL of the bacterial suspension and drop it onto a glass slide, add a coverslip, and observe under a laser confocal microscope (excitation light 488 nm, green fluorescence channel acquisition image).

[0111] Experimental results

[0112] The bacterial plate count results showed that ( Figure 14 AB), under the condition of no light and 15 minutes of co-incubation time (clinically acceptable operation time), different drugs showed no significant bactericidal effect on Pg. However, after 40 seconds of irradiation with a light curing lamp, the number of Pg colonies treated with BHCF nanozymes was significantly reduced, with the colony-forming units (CFU) reduced by 4.32 logarithms, and the bactericidal rate was as high as 99.995%. This fully confirms that light significantly activates the antibacterial activity of BHCF nanozymes, providing conditions for its application in photodynamic antibacterial therapy.

[0113] Scanning electron microscopy (SEM) observations showed that ( Figure 14C) In the absence of light, Pg cells treated with different drugs maintained a typical rod-shaped morphology with a rough and uniform cell surface. However, after irradiation with a light-curing lamp, Pg cells treated with BHCF nanozymes exhibited significant morphological changes, manifested by deeper cell depressions, increased shrinkage, and partial cell structural disintegration and fragmentation. This result indicates that light activates the antibacterial mechanism of BHCF nanozymes, achieving photodynamic synergistic antibacterial effects.

[0114] Laser confocal microscopy results showed that ( Figure 14 D) In ​​the absence of light, the Pg biofilms treated with different drugs all exhibited green fluorescence, indicating that the biofilms contained a large number of live bacteria. However, the biofilms treated with BHCF nanozymes under light conditions exhibited significant orange-red fluorescence, indicating that most bacteria within the biofilms were dead. This is consistent with the results of microscopic observations, indicating that BHCF nanozymes can effectively kill bacteria within Pg biofilms under light conditions, significantly disrupting the active structure of the biofilms.

[0115] The results of confocal microscopy of active oxygen staining in bacteria showed that ( Figure 14 E) No significant green fluorescence was detected in the bacteria without light, indicating that in the absence of exogenous ROS induction, the endogenous ROS level in the bacteria was extremely low and the DCFH-DA probe did not undergo significant oxidation. However, bacteria treated with the BHCF nanozyme + light group exhibited significant green fluorescence, indicating that DCFH was efficiently oxidized by intracellular ROS to the fluorescent product DCF, demonstrating that the BHCF nanozyme can rapidly break through the bacterial antioxidant defense system through a photosensitized cascade reaction under light conditions.

[0116] Example 5

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

[0118] Experimental methods

[0119] Eighteen newly extracted caries-free third molars were selected. A slow-speed cutter was used to cut the teeth perpendicular to the long axis of the tooth below the enamel-dentin junction under running water cooling. The crown enamel was removed to expose the middle dentin. The dentin slices were then cut to prepare 1mm±0.1mm thick dentin slices. 600, 800, and 1000 grit sandpaper were used to polish the dentin slices for 60 seconds under running water to make the surface smooth and form a standard smear layer. After polishing, the slices were ultrasonically cleaned (power 400W) for 15 minutes, and then repeatedly rinsed with deionized water and blown dry with an air gun. The dentin slices were soaked in a 1% citric acid solution for 20 seconds to open the dentinal tubules to establish a sensitive dentin model, and then thoroughly rinsed with deionized water. The dentin slice samples were randomly divided into six groups (3 slices per group) according to different treatment methods:

[0120] Group 1 (blank control): the dentin slices were immersed in an equal volume of deionized water;

[0121] The second group (blank control + acid etching treatment): The dentin slices from the first group were taken and soaked in 6% citric acid solution for 10 minutes every day.

[0122] Group 3 (blank control + abrasion treatment): The dentin slices of Group 1 were brushed by the same researcher using a Colgate soft-bristled toothbrush with the same intensity for 3 minutes twice a day.

[0123] Group 4 (BHCF nanozyme): BHCF nanozyme was ultrasonically dispersed in deionized water at a concentration of 1 mg / mL. The solution was evenly applied to the surface of the dentin slice using a small brush. The solution was blown dry for 2 minutes and then immediately rinsed thoroughly with plenty of deionized water.

[0124] Group 5 (acid etching treatment): The sealed dentin slices from Group 4 were taken and soaked in 6% citric acid solution for 10 minutes every day.

[0125] Group 6 (abrasion treatment): The sealed dentin slices from Group 4 were brushed twice a day for 3 minutes by the same researcher using a Colgate soft-bristled toothbrush with the same intensity.

[0126] All dentin discs were placed in artificial saliva daily. Dentin slices from each group were soaked for 10 minutes, fixed with 3% glutaraldehyde, dehydrated with graded ethanol, dried, and vacuum-sprayed with gold. Dentin slices were then placed under a scanning electron microscope (SEM) to observe the occlusion of dentinal tubules on the slices.

[0127] Experimental results

[0128] Dentin samples from different treatment groups were observed by scanning electron microscopy. Figure 15 As shown in the figure, the dentinal tubules in the control group were exposed, with clear lumen and no obvious blocking material attached to the surface. After acid etching and abrasion treatment, the dentinal tubules were more clearly open and completely exposed. In the nanozyme group, BHCF nanozyme was able to effectively block the dentinal tubules. The lumens of most tubules were tightly filled with nanozyme particles, and only a very small number of tubules were still partially open. Moreover, after acid etching and abrasion treatment, most tubules remained closed, with only a few tubules partially open. This shows that BHCF nanozyme has a good immediate sealing effect and excellent acid and wear resistance.

[0129] Example 6

[0130] Study on the sustained desensitization and remineralization performance of BHCF nanozymes

[0131] Experimental methods

[0132] 1. Electron microscopy observation: Eighteen newly extracted caries-free third molars were selected and dentin slices were prepared. The slices were grouped and treated as in Example 5. After each group of dentin slices was soaked in artificial saliva for 3 weeks (10 min in Example 5), a shallow groove was cut from the center of the pulpal surface of each dentin slice toward the enamel surface using a slow cutting machine. The dentin slice was then gently broken into two pieces along this 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 section of the dentinal tubules.

[0133] 2. Crystalline phase analysis: XRD patterns of the dentin sample surfaces were recorded using an XRD-6000 X-ray diffractometer to analyze the crystal orientation and mineral phase 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 in the 2θ 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 Results (shown in Figure 2A) show that after three weeks of artificial saliva immersion, the dentinal tubules in the control group remained open, with no obstruction observed at the tubule openings; whereas, the BHCF nanozyme-treated group maintained a complete closure. Following daily acid etching and abrasion challenges, the dentinal tubules maintained their closure integrity, demonstrating the remarkable durability of the sealing effect.

[0136] Scanning electron microscopy was used to observe the longitudinal section (e.g. Figure 16 (B) In the control group, a small amount of sediment was present within the dentinal tubules, but the majority of the tubules remained empty, with no apparent mineralized deposits. In contrast, the tubules in the BHCF nanozyme-treated group were significantly filled with mineralized material, reaching a depth exceeding 100 μm. After acid etching and abrasion treatment, a significant amount of mineralized deposits remained within the treated tubules, further confirming the stability of the dentinal tubules' sealing and mineralization capabilities.

[0137] XRD patterns show ( Figure 17 The characteristic diffraction peaks of hydroxyapatite (002), (300), (213) and (004) of demineralized dentin at 2θ = 25.8°, 32.8°, 49.5° and 53.2° were significantly weakened or disappeared. After immersion and culture in artificial saliva, the characteristic diffraction peaks of hydroxyapatite in the BHCF nanozyme-treated 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 embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A nanozyme with sterilization, tooth desensitization and remineralization functions, characterized in that: The nanozyme is prepared by self-assembly of berberine, hemin and calcium fluoride.

2. The method for preparing a nanozyme having sterilization, tooth desensitization and remineralization functions according to claim 1, characterized in that: The nanozyme is prepared according to the following steps: (1) Dissolve hemin and berberine in DMSO for later use; (2) Dissolve calcium fluoride in hydrochloric acid solution for later use; (3) adding the solution obtained in step (1) to the solution obtained in step (2) at room temperature under stirring to obtain a mixed system; (4) adjusting the pH of the mixed system obtained in step (3) to neutral with a sodium hydroxide solution to obtain a suspension; (5) dialyzing the suspension obtained in step (4) in ultrapure water to remove unassembled molecules and ions; (6) The system after dialysis in step (5) is centrifuged, the supernatant is removed, and the precipitate is dispersed and freeze-dried to obtain the BHCF nanozyme.

3. The method for preparing a nanozyme having sterilization, tooth desensitization and remineralization functions according to claim 2, characterized in that: The molar ratio of hemin, berberine and calcium fluoride in the nanozyme is 0.5-1:1-2:4-6.

4. The method for preparing a nanozyme having sterilization, tooth desensitization and remineralization 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 nanozyme having sterilization, tooth desensitization and remineralization 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. Use of the nanozyme having sterilization, tooth desensitization and remineralization functions as claimed in claim 1 in the preparation of drugs for treating periodontitis.

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