Targeted nano-enzyme antibacterial agent with oral flora level self-adaptive regulation and control function and preparation method of targeted nano-enzyme antibacterial agent

Abstract

Relates to the technical field of oral medicine and oral medical products, in particular to a targeted nano-enzyme antibacterial agent with an oral flora level self-adaptive regulation function and a preparation method of the targeted nano-enzyme antibacterial agent. Specifically, a nitrogen-doped zeolite imidazate framework material is used as a carrier, and the bimetallic monatomic nano-enzyme is prepared through a pyrolysis method; the antibacterial peptide is used as a surface anchoring body and is modified on the surface of the nano-enzyme, so that the multifunctional nano-enzyme with high targeting colonization capability, efficient antibacterial biofilm and antibacterial effect is obtained. The invention also relates to a pharmaceutical composition, an antibacterial agent and an oral care product containing the nano-enzyme.

Description

A targeted nanozyme antibacterial agent with adaptive regulation function at the oral flora level and its preparation method Field of the Invention

[0001] The present invention relates to the technical field of oral medicine and oral medical products, and in particular to a targeted nanozyme antibacterial agent with an adaptive regulation function at the oral flora level and a preparation method thereof. Background Art

[0002] Dynamic imbalances in the oral microbiome are a major cause of various oral diseases. For example, in periodontal disease, the microbiome on the surface of teeth or roots is unbalanced, leading to the overgrowth of aggressive Gram-negative bacteria such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, which form multispecies biofilms and secrete virulence factors, causing irreversible damage to the periodontal system. Bacterial biofilms provide a solid matrix barrier for bacteria, hindering the penetration of antibiotics. These antibiotics can only kill surface-free bacteria but are ineffective against bacteria within the matrix, resulting in extremely high drug resistance. Effectively inhibiting biofilm formation and regulating the dynamic balance of the oral microbiome is key to ensuring oral health.

[0003] Previous research on bacterial biofilms has focused on post-treatment methods such as physical removal and chemical disruption of membrane structures. However, these methods are inefficient and fail to eliminate the harmful effects of established virulence factors. While highly effective antimicrobial agents are constantly being developed, their harmful effects on normal tissues are unavoidable and they can also be aggressive towards the normal bacterial flora of the oral microenvironment, resulting in various oral health risks.

[0004] Bacterial biofilm formation relies on its unique quorum sensing (QS) system, which regulates colony aggregation, virulence factors, and metabolite production through the secretion and recognition of signal molecules by bacterial cells. Cutting off the transmission of signal molecules between bacteria can inhibit bacterial biofilm formation at the source, avoiding the toxic effects of quorum metabolites, and free bacteria are more vulnerable to antimicrobial agents. Acylhomoserine lactones (AHLs) are the main signaling molecules of Gram-negative bacteria QS. Biological studies have shown that enzyme-catalyzed inhibition of AHL synthesis, degradation, or obstruction of AHL binding to receptors is an effective strategy to quench the QS system. However, biological enzymes are expensive to produce and have poor stability, making them difficult to widely use.

[0005] In recent years, the research on nanozymes has developed rapidly. They can not only simulate the efficient catalytic activity of biological enzymes, but also have mild conditions, simple synthesis, good stability, and easy structural modification. They have been widely used in the treatment of cancer, cardiovascular diseases and various inflammations.

[0006] Summary of the Invention

[0007] Designing nanozymes with efficient lactone bond hydrolysis function, catalyzing the hydrolysis and inactivation of AHLs, and cutting off the communication process between bacterial groups is an effective means to inhibit the formation of bacterial biofilms from the source. Inhibiting bacterial biofilm formation reduces the toxic effects of bacterial groups, and free attacking bacteria can continue to reproduce. In order to further and efficiently eliminate the effects of attacking bacteria, the present invention constructs a dual-active center single-atom nanozyme functionalized with antimicrobial peptides. The antimicrobial peptides have specific binding and destructive effects on the bacterial outer membrane, while having less effect on human cells; the dual active centers have lactone-like and peroxidase activities respectively, one of which catalyzes the hydrolysis of AHLs to inhibit biofilm formation, and the other catalyzes the decomposition of H2O2 in the bacterial environment into reactive oxygen free radicals (ROS), which has a highly efficient antibacterial effect. Importantly, ROS production varies with the level of the bacterial population and has an adaptive antibacterial effect, which is expected to become an important strategy for regulating the dynamic balance of oral flora.

[0008] Therefore, the present application provides the following invention:

[0009] In one aspect, the present application provides a multifunctional nanozyme functionalized with an antimicrobial peptide, wherein the nanozyme is a bimetallic single-atom nanozyme with a nitrogen-doped zeolite imidazolate framework material as a carrier, wherein the metal is selected from two of zinc, iron, and cobalt, and an antimicrobial peptide is anchored on the surface of the nanozyme.

[0010] In this context, nanozymes refer to enzyme mimics that combine the unique properties of nanomaterials with catalytic functionality. "Enzyme-like" can be used to describe nanozymes that share the same substrates and products, while "enzyme-mimicking" is used to describe nanozymes that mimic the structure and function of biological enzymes. Compared to natural enzymes, nanozymes offer advantages such as high stability, reusability, low cost, ease of manufacture, and versatility.

[0011] Single-atom nanozymes are a type of nanozyme that uses metal atoms as enzyme-like catalytic active sites. They have atomically dispersed active sites. Single-atom nanozymes typically consist of a carrier and a metal atom anchored on the carrier as the active center.

[0012] In the present invention, bimetallic single-atom nanozymes refer to single-atom nanozymes with two metal atoms as the active centers. Compared with single-atom nanozymes, bimetallic components exhibit surprising synergistic effects in catalytic activity, or endow nanozymes with multiple catalytic activities.

[0013] The present invention adopts nitrogen-doped zeolite imidazolate framework material as a carrier to form a bimetallic single-atom nanozyme.

[0014] Metal-Organic Frameworks (MOFs) refer to porous crystalline materials formed by the self-assembly of metal ions or metal clusters and multidentate organic ligands. Zeolitic imidazolate frameworks (ZIFs) are a subclass of MOFs, which are composed of divalent metal ions (such as Zn 2+ 、Co 2+ The nanoporous crystalline material is assembled by complexation with imidazole ligands in a four-coordinate manner, and its molecular formula can be expressed as M(IM) 2， Where M represents a metal ion and IM represents an imidazolyl ligand. ZIFs have a tetrahedral three-dimensional network structure, similar in structure to zeolites. By coordinating metal ions with different organic ligands, ZIFs with varying structural properties can be formed. Substituting the metal ions within these materials can yield ZIFs with the same topology but different properties. Common zeolite imidazolate framework materials include but are not limited to: ZIF-1, ZIF-2, ZIF-4, ZIF-5, ZIF-7, ZIF-8, ZIF-9, ZIF-10, ZIF-11, ZIF-12, ZIF-14, ZIF-20, ZIF-23, ZIF-60, ZIF-61, ZIF-62, ZIF-64, ZIF-65, ZIF-67, ZIF-68, ZIF-69, ZIF-70, ZIF-71, ZIF-72, ZIF-73, ZIF-74, ZIF-75, ZIF-77, ZIF-78, ZIF-90, ZIF-95, ZIF-100, ZIF-224, ZIF-268, Zn / Co-ZIF, nZIF-8, etc.

[0015] In some embodiments, the nitrogen-doped zeolitic imidazolate framework material used as a support is ZIF-8.

[0016] In some embodiments, the bimetallic single-atom nanozyme comprises a metal selected from Fe(II), Zn(II), and Co(II).

[0017] In some embodiments, the metals comprised by the bimetallic single-atom nanozyme are Fe(II) and Zn(II), or Co(II) and Zn(II).

[0018] Antimicrobial peptides serve as surface anchors and are modified on the surface of the bimetallic single-atom nanozyme. The antimicrobial peptides selectively bind to the bacterial outer membrane to target the bacteria, directly and efficiently colonize the nanozyme on the bacterial surface, and reduce the damage to normal oral tissues caused by reactive oxygen free radicals.

[0019] Antimicrobial peptides refer to a class of basic polypeptide substances with antimicrobial activity, composed of a few to dozens of amino acids. Common antimicrobial peptides can be divided into five families: ① α-helical antimicrobial peptides (α-antimicrobial peptides); ② β-pleated and small protein peptide antimicrobial peptides (β-antimicrobial peptides); ③ antimicrobial peptides with a thioether ring; ④ random coil antimicrobial peptides; and ⑤ cysteine-rich antimicrobial peptides. The present invention can use α-antimicrobial peptides, β-antimicrobial peptides, and random coil antimicrobial peptides.

[0020] In one aspect, the present application provides a method for preparing the nanozyme of the present invention, the method comprising:

[0021] Using nitrogen-doped zeolite imidazolate framework as a carrier, bimetallic single-atom nanozymes were prepared by thermal decomposition; and

[0022] The antimicrobial peptide is used as a surface anchor and modified on the surface of the bimetallic single-atom nanozyme.

[0023] Nanozymes can be prepared based on metals, metal oxides, non-metals (such as carbon nanotubes, graphene, etc.), metal-organic frameworks, etc., through methods such as impregnation, coprecipitation, deposition-precipitation, and hydrothermal / solvothermal methods. The present invention uses a nitrogen-doped zeolite imidazolate framework material as a carrier to prepare bimetallic single-atom nanozymes.

[0024] Common methods for preparing zeolitic imidazolate frameworks include stirring, solvothermal, microwave-assisted, ionothermal, ultrasonic, vapor-phase, and microfluidic methods. Stirring involves dispersing or dissolving the metal ion and ligand separately in a solvent, mixing them, and stirring at room temperature. Solvothermal synthesis involves dissolving the metal ion and ligand separately in a solvent and heating the solution to synthesize ZIFs.

[0025] In the present invention, a stirring method is used to obtain a nitrogen-doped zeolitic imidazolate framework material. In some embodiments, the stirring method comprises: dispersing or dissolving a hydrated metal nitrate and 2-methylimidazole (MeIM) in a solvent, mixing the two and stirring at room temperature to obtain a solid and separating the solid.

[0026] In some embodiments, the metal may be selected from iron, zinc, and cobalt. The hydrated nitrate of the metal includes, but is not limited to, Fe(NO3)2·9H2O, Zn(NO3)2·6H2O, and Co(NO3)2·6H2O. In some embodiments, the salt is selected from two of the above salts, such as Fe(NO3)2·9H2O and Zn(NO3)2·6H2O, such as Co(NO3)2·6H2O and Zn(NO3)2·6H2O. In some embodiments, the two salts are fed in equimolar amounts. In some embodiments, the two salts are dissolved or dispersed separately.

[0027] In some embodiments, the solvent used to dissolve or disperse the salt is the same as the solvent used to dissolve or disperse 2-methylimidazole, and both are alcohols, such as methanol.

[0028] In some embodiments, the stirring method comprises the following steps:

[0029] (1) dissolving or dispersing two of Fe(NO3)2·9H2O, Zn(NO3)2·6H2O, and Co(NO3)2·6H2O in a solvent (e.g., methanol), stirring to form a salt solution or salt dispersion, injecting a solution or dispersion of 2-methylimidazole into the salt solution or salt dispersion, and vigorously stirring at room temperature for a certain period of time;

[0030] (2) separating and washing the precipitate with a solvent (eg, methanol), and drying the precipitate under heating (eg, 100-120° C.) and vacuum conditions to obtain a nitrogen-doped zeolite imidazolate framework material (ZIF-8).

[0031] In some embodiments, the feed ratio of the two salts and 2-methylimidazole is 1 / 1 / 4 to 1 / 1 / 20 (e.g., 1 / 1 / 4 to 1 / 1 / 6, 1 / 1 / 4 to 1 / 1 / 8, 1 / 1 / 4 to 1 / 1 / 10, 1 / 1 / 4 to 1 / 1 / 12, 1 / 1 / 4 to 1 / 1 / 14, 1 / 1 / 4 to 1 / 1 / 16, 1 / 1 / 4 to 1 / 1 / 18 or 1 / 1 / 4 to 1 / 1 / 20).

[0032] In some embodiments, the total concentration of the two salts in the salt solution or salt dispersion is 0.1 mmol / mL to 1 mmol (e.g., 0.1 mmol / mL to 0.2 mmol / mL, 0.2 mmol / mL to 0.3 mmol / mL, 0.3 mmol / mL to 0.4 mmol / mL, 0.4 mmol / mL to 0.5 mmol / mL, 0.5 mmol / mL to 0.6 mmol / mL, 0.6 mmol / mL to 0.7 mmol / mL, 0.7 mmol / mL to 0.8 mmol / mL, 0.8 mmol / mL to 0.9 mmol / mL, or 0.9 mmol / mL to 1.0 mmol / mL, e.g., 0.133 mmol / mL).

[0033] In some embodiments, the mass of Zn(NO3)2·6H2O is 0.1g to 1g (e.g., 0.1g to 0.2g, 0.2g to 0.3g, 0.3g to 0.4g, 0.4g to 0.5g, 0.5g to 0.6g, 0.6g to 0.7g, 0.7g to 0.8g, 0.8g to 0.9g, or 0.9g to 1g).

[0034] In some embodiments, the mass of 2-methylimidazole is 0.1 g to 1 g (e.g., 0.1 g to 0.2 g, 0.2 g to 0.3 g, 0.3 g to 0.4 g, 0.4 g to 0.5 g, 0.5 g to 0.6 g, 0.6 g to 0.7 g, 0.7 g to 0.8 g, 0.8 g to 0.9 g, or 0.9 g to 1 g).

[0035] In some embodiments, the salt solution or dispersion is stirred for 0.5 to 2 hours.

[0036] In some embodiments, the mixture of salt and 2-methylimidazole is vigorously stirred for 6 to 12 hours.

[0037] After obtaining the nitrogen-doped zeolitic imidazolate framework material, it is converted into the diatomic nanozyme through pyrolysis. The pyrolysis process can include heating the material under an inert atmosphere (e.g., flowing argon) at a high temperature (e.g., 600°C to 1200°C) for a certain time (e.g., 2 hours) and then naturally cooling it to room temperature.

[0038] In some embodiments, pyrolysis is performed in a tube furnace. In some embodiments, heating is performed at a heating rate of 1° C. / min to 10° C. / min (eg, 5° C. / min).

[0039] After pyrolysis, the pyrolysis product is treated with acid to remove metal impurities that are not dispersed in a monoatomic state on the support surface and to improve the dispersion of the catalyst.

[0040] The product obtained by pyrolysis is treated with an acid (eg, hydrochloric acid) for a certain period of time (eg, 6 h to 12 h), and then washed and dried.

[0041] Through the above method, bimetallic single-atom nanozymes can be obtained, such as single-atom nanozymes ZnFe-DAs-C containing two metals, zinc and iron, and single-atom nanozymes ZnCo-DAs-C containing two metals, zinc and cobalt.

[0042] Furthermore, antimicrobial peptides are used as surface anchors and modified on the surface of the bimetallic single-atom nanozyme, and the bacteria are targeted and colonized through the selective affinity of the antimicrobial peptides to the bacterial outer membrane.

[0043] The modification process includes: mixing antimicrobial peptides (such as α-antimicrobial peptides, β-antimicrobial peptides and random coil antimicrobial peptides) with an aqueous solution of a bimetallic single-atom nanozyme, stirring at room temperature in the dark, and dialyzing in the aqueous solution.

[0044] In some embodiments, the molar ratio of the antimicrobial peptide to the bimetallic single-atom nanozyme is 1 / 2 to 1 / 10 (e.g., 1 / 2 to 1 / 3, 1 / 3 to 1 / 4, 1 / 4 to 1 / 5, 1 / 5 to 1 / 6, 1 / 6 to 1 / 7, 1 / 7 to 1 / 8, 1 / 8 to 1 / 9, or 1 / 9 to 1 / 10).

[0045] In some embodiments, the concentration of the antimicrobial peptide is 0.1M to 1M (e.g., 0.1M to 0.2M, 0.2M to 0.3M, 0.3M to 0.4M, 0.4M to 0.5M, 0.5M to 0.6M, 0.6M to 0.7M, 0.7M to 0.8M, 0.8M to 0.9M, or 0.9M to 1.0M).

[0046] In some embodiments, stirring is performed for 24 to 48 hours.

[0047] In some embodiments, dialysis is performed for 3-4 days.

[0048] This method can be used to prepare targeted nanozymes with AHL-like lipase and peroxidase activities. Furthermore, by adjusting the three-dimensional structure of the antimicrobial peptide and synthesizing the type of metal single atoms, a series of multifunctional nanozymes with different bacterial affinities and different catalytic activity centers can be synthesized.

[0049] The nanozyme of the present invention possesses AHL-like lipase and peroxidase activities. On the one hand, it can catalyze and inhibit the synthesis of AHLs, degrading or hindering their binding to receptors, thereby quenching the QS system of Gram-negative bacteria. On the other hand, it can catalyze the decomposition of H2O2 in the bacterial environment into reactive oxygen species (ROS), producing a highly effective antibacterial effect. The nanozyme of the present invention can inhibit Gram-negative bacteria, inhibit biofilm formation, regulate the dynamic balance of oral flora, and thus be used to prevent or treat oral diseases.

[0050] Therefore, in one aspect, the present application provides a pharmaceutical composition comprising the nanozyme of the present invention, and optionally, further comprising a pharmaceutically acceptable carrier or excipient.

[0051] As used herein, "pharmaceutically acceptable carrier or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, disintegrants, binders, surfactants, glidants, lubricants, pH adjusters, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, antioxidants, colorants, flavorings, preservatives, taste-masking agents, and the like.

[0052] The pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form, in particular a dosage form suitable for oral administration, such as oral tablets (e.g., lozenges, adhesive tablets), injections, gels, aerosols, gargles, films, etc. The pharmaceutical composition can be administered to a patient or subject in need thereof in any suitable manner. In some embodiments, the pharmaceutical composition is administered to a subject in a manner suitable for oral administration (e.g., rubbing, spraying, patching, local injection, etc.).

[0053] In one aspect, the present application provides an antibacterial agent comprising a nanozyme of the present invention. In some embodiments, the antibacterial agent is used to inhibit Gram-negative bacteria, such as Gram-negative bacteria present in the oral cavity, including but not limited to Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythiae, Fusobacterium nucleatum, Prevotella intermedia, and the like.

[0054] In one aspect, the present application provides an oral care product comprising the nanozyme of the present invention. The oral care product includes but is not limited to toothpaste, mouthwash, tooth powder and tooth gel.

[0055] In one aspect, the present application provides a use of the nanozyme of the present invention in the preparation of a drug for preventing or treating a disease caused by oral bacteria in a subject, or regulating the level of oral flora.

[0056] In one aspect, the present application provides a method for preventing or treating a disease caused by oral bacteria in a subject, comprising administering to a subject in need thereof an effective amount of the nanozyme of the present invention.

[0057] In some embodiments, the oral bacteria are Gram-negative bacteria, including but not limited to Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythiae, Fusobacterium nucleatum, Prevotella intermedia, etc. The diseases caused by oral bacteria include but are not limited to dental caries, periodontal disease, bad breath, oral cancer, digestive system diseases, etc.

[0058] As used herein, the term "prevention" refers to the method implemented in order to prevent or delay the occurrence of disease or illness or symptom in a subject. As used herein, the term "treatment" refers to the method implemented in order to obtain beneficial or required clinical results. For the purposes of the present invention, beneficial or required clinical results include but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (that is, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease and alleviating symptoms (no matter in part or in whole), no matter it is detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), extending the survival.

[0059] As used herein, the term "subject" refers to a mammal, eg, bovine, equine, porcine, canine, feline, rodent, primate; eg, a human.

[0060] Advantageous Effects of the Invention

[0061] The present invention designs a multifunctional nanozyme antibacterial agent functionalized with antibacterial peptides, which targets and colonizes bacteria through the selective affinity of antibacterial peptides to the bacterial outer membrane, inhibits bacterial biofilm formation and reduces bacterial drug resistance through lipase-like functions, and kills bacteria by generating reactive oxygen through peroxidase-like functions. Different enzyme-like activities can be adaptively adjusted according to the level of oral flora through structural design, thereby providing protection for a healthy microenvironment in the oral system. DETAILED DESCRIPTION

[0062] Below in conjunction with embodiment, embodiment of the present invention is described in detail, but it will be understood by those skilled in the art that the following examples are only used to illustrate the present invention, rather than to limit the scope of the invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

[0063] Preparation Example 1 Synthesis of Single Metal Single Atom Nanozyme

[0064] Zn(NO₃)₂·6H₂O (0.558 g) was dispersed in 15 mL of methanol; after stirring for half an hour, 2-methylimidazole (0.616 g) dispersed in 15 mL of methanol was injected into the solution and vigorously stirred at room temperature for 6 hours. The resulting precipitate was centrifuged and washed three times with methanol and dried under vacuum at 120°C for 12 hours. The resulting powder was placed in a tube furnace and then heated to different temperatures (600°C to 1200°C) at a heating rate of 5°C / min under flowing argon for 2 hours. The powder was allowed to cool naturally to room temperature, treated with 1 M hydrochloric acid for 12 hours, and then washed and dried to obtain the Zn-SA-C sample.

[0065] By replacing Zn(NO3)2·6H2O with equimolar amounts of Fe(NO3)2·9H2O and Co(NO3)2·6H2O, the corresponding single-atom nanozymes Fe-SA-C and Co-SA-C can be obtained.

[0066] Example 1 Preparation of antimicrobial peptide@diatom nanozyme

[0067] 1. Synthesis of bimetallic single-atom nanozymes

[0068] Fe(NO₃)₂·9H₂O (0.758 g) and Zn(NO₃)₂·6H₂O (0.558 g) were weighed and dispersed in 15 mL of methanol. After stirring for half an hour, 2-methylimidazole (0.616 g) dispersed in 15 mL of methanol was injected into the solution, with the ratio of Fe / Zn / MeIM = 1 / 1 / 4. The solution was then vigorously stirred at room temperature for 6 hours. The resulting precipitate was centrifuged and washed three times with methanol and dried under vacuum at 120°C for 12 hours. The resulting powder was placed in a tube furnace and heated at a heating rate of 5°C / min to various temperatures (600°C to 1200°C) under flowing argon for 2 hours. The mixture was allowed to cool naturally to room temperature and treated with 1 M hydrochloric acid for 12 hours. The resulting powder was then washed and dried to obtain the ZnFe-DAs-C sample.

[0069] By replacing Fe(NO3)2·9H2O with an equimolar amount of Co(NO3)2·6H2O, the corresponding double single-atom nanozyme ZnCo-DAs-C can be obtained.

[0070] 2. Synthesis of antimicrobial peptide-anchored ZnCo-DAs-C: 0.15 M antimicrobial peptide (α-antimicrobial peptide, β-antimicrobial peptide or random coil antimicrobial peptide) was mixed with a ZnCo-DAs-C aqueous solution, stirred in the dark at room temperature for 24 h, and then dialyzed in an aqueous solution for 3 days to obtain Pep-ZnCo-DAs-C, which was then placed in a refrigerator at 4°C for use.

[0071] Example 2: Characterization of the morphology and structure of nanozymes

[0072] 1. Transmission electron microscopy

[0073] Figure 1 is a TEM image of the synthetic nanozyme Pep-ZnCo-DAs-C. It can be seen from the figure that Zn and Co do not appear in the form of metal or its oxide nanoparticles, and are evenly dispersed in the carbon skeleton, which preliminarily indicates their atomic state distribution.

[0074] 2. X-ray diffraction (XRD)

[0075] Figure 2 is the XRD image of the synthetic nanozyme Pep-ZnCo-DAs-C. The diffraction pattern does not show the diffraction peaks of metal or oxide crystals of Zn and Co, which further indicates the atomic state distribution of the two metal elements.

[0076] 3. Inductively coupled plasma (ICP) testing

[0077] Table 1

[0078] Table 1 shows the ICP test results of the synthesized nanozyme Pep-ZnCo-DAs-C, which shows that the synthesized nanozyme contains Co and Zn elements, and the contents are 1.73 wt% and Zn0.12 wt%, respectively.

[0079] 4. X-ray Photoelectron Spectroscopy (XPS)

[0080] Figure 3 is the XPS spectrum of the synthesized nanozyme Pep-ZnCo-DAs-C, which shows that Co has Co 2+ 2p 1 / 2, 2p / 3 / 2 orbitals and Co-N bonds, Zn has Zn 2+ 2p 1 / 2 and 2p / 3 / 2 orbitals.

[0081] Example 3 Catalytic Activity Test of Nanozyme

[0082] In this example, the peroxidase-like activity of the nanozyme was evaluated by a 3,3',5,5'-tetramethylbenzidine-hydrogen peroxide colorimetric reaction. The intralipase-like activity of the nanozyme was evaluated by a 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) hydrolysis reaction. Peroxidase-like activity: 40 μg / mL of Pep-ZnCo-DAs-C was added to an acetic acid-ammonium acetate (50 mM, pH 5) solution containing 1 mM H2O2 and 0.1 mM 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was carried out in the dark for 5 minutes. The UV-visible absorption spectra of TMB, TMB-H2O2, and TMB-H2O2-Pep-ZnCo-DAs-C solutions were measured in the range of 350 to 700 nm. Lipase activity: 40 μg / mL of Pep-ZnFe-DAs-C was added to a 1 mM DCFH-DA solution. After a period of reaction in the dark, the fluorescence intensity (It) of the reaction series was recorded using a microreader. The excitation and emission wavelengths were 495 nm and 520 nm, respectively.

[0083] Comparative example: The catalytic performance of the single-atom nanozyme obtained in Preparation Example 1 on hydrogen peroxide was tested to obtain the UV-visible absorption spectrum of TMB-H2O2, and the UV-visible absorption spectrum was compared with the UV-visible absorption spectrum of hydrogen peroxide catalyzed by the diatomic nanozyme.

[0084] Comparative example: The lipase-like catalytic performance of the single-atom nanozyme obtained in Preparation Example 1 was tested to obtain an absorbance-time curve, which was compared with the absorbance-time curve obtained by diatomic nanozyme catalysis.

[0085] Figure 4-Left shows the UV absorption spectra of the prepared single-atom nanozymes Co-SA-C and Zn-SA-C, and the diatomic nanozyme ZnCo-DAs-C catalyzing the TMB-H2O2 color development reaction. In the TMB-H2O2 solution without nanozymes (Backgroud curve in the figure), the decomposition rate of H2O2 is slow, and it is difficult to make TMB color in a short time, resulting in UV-visible spectrum absorption; after adding the above-prepared ZnCo-DAs-C diatomic nanozyme to the solution, the UV-visible spectrum absorption of the solution is significantly enhanced, indicating that it has efficient catalase-like activity and quickly catalyzes the decomposition of hydrogen peroxide. The single-atom catalyst in the comparative example has weaker peroxidase activity. Figure 4-Right shows the UV-visible absorption curve of the ZnCo-DAs-C nanozyme catalyzed at different H2O2 concentrations.

[0086] Figure 5 shows the fluorescence absorption spectra of the prepared single-atom nanozymes Co-SA-C and Zn-SA-C, and the diatomic nanozyme ZnCo-DAs-C catalyzing the hydrolysis of the DCFH-DA lactone bond. The DCFH-DA lactone bond shows fluorescence characteristics after being hydrolyzed. After adding the above-prepared ZnCo-DAs-C diatomic nanozyme to the DCFH-DA solution, the fluorescence value of the solution increases with the reaction time, indicating that ZnCo-DAs-C has efficient lipase-like catalytic activity. The single-atom catalyst in the comparative example has a weaker lipase-like activity.

[0087] Example 4 Targeting Test of Nanozymes

[0088] Bacterial infection causes a decrease in the pH of the surrounding tissue fluid, while the pH range of the normal cell surrounding environment is 6.5-7.5. Therefore, this example tests the targeted antibacterial activity of nanozymes by measuring their antibacterial activity under different pH conditions.

[0089] The test process is as follows: pick up a single E. coli colony with a pipette tip, culture it in LB broth medium on a shaker for 24 hours until the logarithmic growth phase (37°C, 200 rpm), and dilute the bacterial solution to 1*10 7 CFU / ml. Take 50 microliters of bacterial solution and add it to 50 microliters of nanozyme solution (0.2mg / ml) and culture for 12 hours. The pH of the nanozyme solution is pH 7.5, pH 6.5, pH 5.5, and pH 4.5, respectively. The buffer solution without nanozyme is the control group. Dilute the bacterial solution after co-culture 100 times, take 100 microliters and spread it on agar medium. After culturing for 12 hours, take a picture and calculate the number of colonies. According to the formula, the inhibition rate = (C对照组 -C 实验组 ) / C 对照组 *100%, calculate the inhibition rate.

[0090] Figure 6 shows the antibacterial effect of the synthetic nanozyme Pep-ZnCo-DAs-C in the normal tissue pH range and the bacterial infection pH range. The results show that the nanozyme has low antibacterial efficiency within the normal tissue pH range. However, in the acidic pH environment caused by bacterial overgrowth, the nanozyme's catalytic activity is significantly enhanced, with an inhibition rate of 99%, demonstrating its ability to target bacterial infection environments and cause minimal damage to normal physiological tissues.

[0091] Example 5 Bacterial biofilm inhibition ability test

[0092] In this example, the effects of the bioenzyme and the constructed nanozyme on the secretion level of AHLs by Gram-negative bacteria were tested using natural AHLs lipase as a control; the relative percentage of bacterial biofilm formation was calculated by crystal violet staining, and the biofilm formation of Gram-negative bacteria under untreated, bioenzyme-treated and nanozyme-treated conditions was compared. The test process is as follows: Michaelis constant determination: 0.2 mg / mL of nanozyme or bioenzyme was added to a DCFH-DA substrate solution containing 0.005 mM, 0.01 mM, 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.8 mM, 1.2 mM, 1.6 mM, 2 mM, and 2.6 mM, and the reaction was carried out in the dark for 15 minutes. The reaction rates of the substrate solutions with different concentrations were tested, and the Michaelis constant was calculated based on the relationship between substrate concentration and reaction rate.

[0093] Biofilm inhibition test: Sterile coverslips (10 × 10 mm, 0.13 mm) were placed in a 24-well plate. E. coli (20 μL) with an OD of 0.5 and 40 μL of nanozyme solution (0.2 mg / mL) or biotin were cultured in the 24-well plate containing the coverslips and incubated at 37°C for 72–108 h.

[0094] The coverslips with biofilm growth were transferred to new 24-well plates and stained with 200 μL crystal violet (1% w / v, Sigma) at 37°C for 15 minutes. The coverslips were then washed three times with 2 mL of NaCl solution (0.9%). Afterwards, the crystal violet bound to the biofilm on the coverslips was eluted with 200 μL of 70% (v / v) ethanol, and the absorbance of the eluate was measured at 595 nm. The relative percentage of biofilm inhibition was calculated by the following formula: Biofilm (%) = (A 对照组 -A 实验组 ) / A 对照组 *100%.

[0095] Table 2

[0096] Table 2 shows the catalytic performance and biofilm inhibition rate of the synthetic nanozyme compared with the biological enzyme, indicating that the nanozyme has a catalytic activity comparable to that of the biological enzyme.

[0097] From the experiments on the catalytic performance of nanozymes on hydrogen peroxide, the lipase-like effect of nanozymes and their ability to inhibit biofilms, and the experiments on the inhibition of nanozymes on Gram-negative bacteria under different pH conditions, it can be proved that the antibacterial efficiency of nanozymes is related to the bacterial proliferation state, rather than being quantitative. When bacteria proliferate in large quantities, biofilms are formed, hydrogen peroxide is secreted, and a local acidic environment is formed, which leads to the enhancement of the catalytic antibacterial activity of nanozymes. Therefore, it can exhibit adaptive antibacterial properties according to the differences in the levels of oral flora.

[0098] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A multifunctional nanozyme functionalized with an antimicrobial peptide, wherein the nanozyme is a bimetallic single-atom nanozyme with a nitrogen-doped zeolite imidazolate framework material as a carrier, wherein the metal is selected from two of zinc, iron, and cobalt, and an antimicrobial peptide is anchored on the surface of the nanozyme; Preferably, the nitrogen-doped zeolite imidazolate framework material as a carrier is ZIF-8; Preferably, the metal contained in the bimetallic single-atom nanozyme is selected from Fe(II), Zn(II), and Co(II); Preferably, the metals contained in the bimetallic single-atom nanozyme are Fe(II) and Zn(II), or Co(II) and Zn(II); Preferably, the antimicrobial peptide is selected from α-antimicrobial peptide, β-antimicrobial peptide and random coil antimicrobial peptide.

2. A method for preparing a nanozyme, the method comprising: Using nitrogen-doped zeolite imidazolate framework materials as carriers, bimetallic single-atom nanozymes were prepared by thermal decomposition. as well as The antimicrobial peptide is used as a surface anchor and modified on the surface of the bimetallic single-atom nanozyme.

3. The method of claim 2, wherein the nitrogen-doped zeolite imidazolate framework material is obtained by a stirring method; Preferably, the stirring method comprises: Dispersing or dissolving the hydrated nitrate of the metal and 2-methylimidazole (MeIM) in a solvent respectively, mixing the two and stirring at room temperature to obtain a solid and separate it; Preferably, the hydrated nitrate of the metal is selected from Fe(NO3)2·9H2O, Zn(NO3)2·6H2O, Co(NO3)2·6H2O; Preferably, the hydrated nitrates of the metals are Fe(NO3)2·9H2O and Zn(NO3)2·6H2O; preferably, the two salts are fed in equimolar amounts; Preferably, the hydrated nitrates of the metals are Co(NO3)2·6H2O and Zn(NO3)2·6H2O; Preferably, the two salts are fed in equimolar amounts; Preferably, the solvents are all alcohols, such as methanol; Preferably, the stirring method comprises the following steps: (1) dissolving or dispersing two of Fe(NO3)2·9H2O, Zn(NO3)2·6H2O, and Co(NO3)2·6H2O in a solvent, stirring to form a salt solution or salt dispersion, injecting a solution or dispersion of 2-methylimidazole into the salt solution or salt dispersion, and vigorously stirring at room temperature for a certain period of time; (2) Separate and wash the precipitate with a solvent, and heat and vacuum the precipitate. After drying, a nitrogen-doped zeolite imidazolate framework material is obtained.

4. The method of claim 2 or 3, wherein the pyrolysis process comprises: The material is heated in an inert atmosphere (e.g., flowing argon) at a high temperature (e.g., 600° C. to 1200° C.), and naturally cooled to room temperature; Preferably, the pyrolysis is carried out in a tube furnace; Preferably, the method further comprises: treating the product obtained by pyrolysis with acid, washing and drying.

5. The method of any one of claims 2 to 4, wherein the modification process comprises: The antimicrobial peptide was mixed with the aqueous solution of the bimetallic single-atom nanozyme, stirred at room temperature in the dark, and then dialyzed.

6. A pharmaceutical composition comprising the nanozyme of claim 1 or the nanozyme prepared by any one of claims 2 to 5, and optionally, further comprising a pharmaceutically acceptable carrier or excipient; Preferably, the pharmaceutical composition is prepared into a dosage form suitable for oral administration, such as oral tablets (such as lozenges, adhesive tablets), injections, gels, aerosols, gargles, films, etc.

7. An antibacterial agent comprising the nanozyme of claim 1 or the nanozyme prepared by any one of the methods of claims 2 to 5; Preferably, the antimicrobial agent is used to inhibit Gram-negative bacteria.

8. An oral care product comprising the nanozyme of claim 1 or the nanozyme prepared by any one of the methods of claims 2 to 5; Preferably, the oral care product is selected from toothpaste, mouthwash, tooth powder and tooth gel.

9. Use of the nanozyme of claim 1 or the nanozyme prepared by any one of the methods of claims 2 to 5 in the preparation of a drug for preventing or treating a disease caused by oral bacteria in a subject, or for regulating the level of oral flora.

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