Monatomic mesoporous carbon coated noble metal alloy antibacterial nano-enzyme and preparation method thereof

Abstract

The invention provides a monatomic mesoporous carbon coated noble metal alloy antibacterial nano-enzyme and a preparation method thereof, a solution containing platinum salt or acid and transition metal copper salt is used as a metal precursor, the metal precursor solution and an organic or aqueous solution dispersed with MZn-SA-C are mixed and stirred uniformly, and the mixture reacts in a constant-temperature oven to obtain PtCuZnM-coated MZn-SA-C. The preparation method comprises the following steps: preparing the PtCuZnM-coated noble metal alloy antibacterial nano-enzyme, and preparing the PtCuZnM-coated noble metal alloy antibacterial nano-enzyme. The PtCuZnM (at) MZn-SA-C which is small in nano particle size, high in dispersion, high in catalase-like activity, high in superoxide dismutase-like activity and high in antibacterial activity is prepared through a solvothermal method, and in the healing treatment process of infected wounds of mice, the wound healing rate is remarkably increased. Belongs to the fields of biological materials, antibiosis and medical wound dressings.

Description

TECHNICAL FIELD

[0001] The present application relates to a single-atom mesoporous carbon-coated hybrid noble metal alloy antibacterial nanoscale enzyme and a preparation method thereof, and belongs to the field of biomaterials, antibacterial and medical wound dressings. BACKGROUND

[0002] Bacterial infection during wound healing exacerbates wound inflammation, greatly restricting skin tissue repair and tissue function integrity. Skin, as the largest organ separating the human body from the external environment, is an important barrier between the human body and the external environment, and therefore, will inevitably be affected by external physical and chemical factors, microorganisms and human factors, etc. to produce a wound. Skin wound healing is a complex biological self-repair process, which will bring a huge economic burden to patients, causing billions of dollars in losses in North America each year; and for diabetic wound healing, the patient's persistent hyperglycemia provides a large amount of nutrients for the infecting bacteria and a powerful environment for bacterial reproduction, and the persistent bacterial infection eventually leads to tissue structure destruction and vascular dysfunction. In addition, wound healing needs to be in a relatively dry and sterile environment, therefore, it is necessary to develop a material that can simultaneously satisfy the requirements of sterilization, liquid absorption, easy gas exchange and promotion of wound healing.

[0003] Antibacterial nanoscale enzymes are a new class of materials that utilize the unique physical and chemical properties of nanomaterials (such as small size effect, high specific surface area, surface plasmon resonance, etc.) to achieve high-efficiency antibacterial function. Compared with antibiotics, antibacterial nanoscale enzymes have good cell membrane permeability and biocompatibility, which makes them less likely to cause bacterial resistance. In addition, antibacterial nanoscale enzymes can exert antibacterial effects by regulating ROS levels, generating HOBr / Cl and scavenging peripheral DNA, which can destroy the cell structure of bacteria, including cell wall, cell membrane, DNA, protein and polysaccharide, thereby killing bacteria or preventing the formation of biofilms.

[0004] At present, commonly used antibacterial nanoscale enzymes include gold nanoparticles (Au NPs), silver nanoparticles (Ag NPs), zinc oxide nanoparticles (ZnO NPs), graphene, etc. However, the above-mentioned nanoscale antibacterial agents still have certain biological toxicity risk because they require a high drug concentration in the treatment of wound healing. SUMMARY

[0005] The present application provides a single-atom mesoporous carbon-coated hybrid noble metal alloy antibacterial nanoscale enzyme and a preparation method thereof, to solve the problem of biological toxicity risk of existing nanoscale antibacterial agents in the treatment of wound healing, which still requires a high drug concentration.

[0006] To solve the above problems, a preparation method of single-atom mesoporous carbon-coated hybrid noble metal alloy antibacterial nanoszyme is adopted, which is as follows: using cobalt, zinc and nitrogen-doped mesoporous carbon MZn-SA-C as a hard template and a carrier, using a solution containing platinum salt or acid and transition metal salt or acid as a metal precursor, mixing and stirring the metal precursor solution with an organic or aqueous solution in which MZn-SA-C is dispersed, and reacting in a constant-temperature oven, PtCuZnM@MZn-SA-C can be obtained.

[0007] In the foregoing method, the reaction temperature in the constant-temperature reaction oven is 160-200 ℃, and the reaction time is 12-24 h.

[0008] The method further includes the following steps:

[0009] Step one, preparation of single-atom mesoporous derived carbon (MZn-SA-C)

[0010] At room temperature, an appropriate amount of zinc salt and dimethyl imidazole are respectively dissolved in an appropriate amount of anhydrous methanol solution, and after stirring, the two solutions are mixed and reacted for 12-18 h; then an appropriate amount of transition metal salt is added to the above reaction, and after stirring for a certain time, M-ZIF-8 is obtained by centrifugal washing with anhydrous methanol; the obtained M-ZIF-8 is dried in a vacuum drying box at 60-80 ℃, and then transferred to a tube furnace for high-temperature calcination in an inert atmosphere to obtain single-atom mesoporous derived carbon (MZn-SA-C).

[0011] Step two, preparation of PtCuZnM@MZn-SA-C antibacterial nanoszyme

[0012] The antibacterial nanoszyme is composed of MZn-SA-C and PtM nanoscale alloy, which is mainly prepared by a solvothermal method.

[0013] Using MZn-SA-C prepared in step one as a carrier and a hard template, an appropriate amount of MZn-SA-C, Pt salt or acid, transition metal salt or acid, surfactant and solvent are added to a polytetrafluoroethylene reaction kettle, and stirred to form a uniform mixed solution; the mixed solution is ultrasonically treated, and then placed in a constant-temperature oven for heating reaction at a certain temperature after stirring; after the reaction in the constant-temperature oven is completed, the product is centrifugally washed with anhydrous ethanol, and dried in a vacuum oven to obtain single-atom coated noble metal alloy antibacterial nanoszyme.

[0014] In step one, an appropriate amount of zinc salt and dimethyl imidazole are respectively dissolved in an appropriate volume of anhydrous methanol solution, and after stirring for 0.5 h, the two solutions are mixed.

[0015] In step two, a proper amount of MZn-SA-C is added into a polytetrafluoroethylene reactor together with a Pt salt or acid, a copper salt of a transition metal, a surfactant and a solvent, and stirred for 1 h to form a uniform mixed solution; the mixed solution is ultrasonically treated for 1 h, and then stirred for 0.5 h before being placed into a constant-temperature oven at a certain temperature; a heating reaction is carried out; after the reaction in the constant-temperature oven is completed, the product is washed by centrifugation with anhydrous ethanol for three times, and dried in a vacuum oven to obtain the single-atom coated noble metal alloy antibacterial nanoscale enzyme.

[0016] In step one, the molar ratio of the zinc salt to dimethylimidazole is 1:3.5-4.5, and the addition amount of the transition metal salt is 0.01-0.1 mol; the transition metal salt is one of nitrate, acetate, chloride or acetylacetone salt of iron (Fe), cobalt (Co), nickel (Ni) and copper (Cu), such as FeCl3, CoCl2, NiCl2 and CuCl2.

[0017] In step one, the dried M-ZIF-8 is calcined at a high temperature of 850-950 ℃ in an inert atmosphere (N2, Ar or He) in a tube furnace.

[0018] In step two, the Pt salt or acid is one of platinum acetylacetone, potassium chloroplatinite, potassium chloroplatinate and chloroplatinic acid, and the Cu salt is one of nitrate, acetate, chloride or acetylacetone salt.

[0019] In step two, the molar ratio of the Pt salt or acid to the Cu salt is 1:1-4.

[0020] In step two, the surfactant is one, two or more combinations of sodium hexadecyl sulfonate, hexadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium chloride, polyether F127 and polyether F123, and the solvent is water, DMF, ethylene glycol or a mixture of two or more thereof.

[0021] In step two, the reaction temperature in the constant-temperature reaction oven is 160-200 ℃, and the reaction time is 12-24 h.

[0022] The application also provides a single-atom mesoporous carbon coated hybrid noble metal alloy antibacterial nanoscale enzyme prepared by the above method, which has multiple enzyme active sites, and the Pt-based nanoparticles have small particle size and are uniformly distributed on the surface and inside the mesopores of the MZn-SA-C.

[0023] Compared with the prior art, the application has the following advantages:

[0024] 1、The nanoenzyme of the application is composed of single-atom mesoporous carbon (MZn-SA-C) coated with a noble metal alloy (such as PtCuZnM), forming a multi-enzyme active site structure, and endowing the material with excellent antibacterial and catalytic properties; it has CAT-like (catalase-like) and SOD-like (superoxide dismutase-like) activities, can efficiently decompose hydrogen peroxide and scavenge superoxide anions, regulate reactive oxygen species (ROS) levels, and thus achieve efficient killing of bacteria; it has a significant inhibitory effect on common drug-resistant bacteria such as drug-resistant Staphylococcus aureus; the synergistic effect of noble metals (Pt, Cu, Co, Zn) improves electron transfer efficiency and catalytic activity, significantly enhancing the antibacterial effect.

[0025] 2、The biological safety is better than that of traditional antibacterial agents: through single-atom limited dispersion and carbon layer coating structure, the release of metal ions is effectively reduced, and the cytotoxicity is reduced; compared with ordinary nano-silver, zinc oxide and other antibacterial materials which require high doses, the material of the application still maintains excellent antibacterial performance at low doses, significantly reducing adverse reactions on the body tissue; the material structure is stable and can coexist with the biological tissue environment, and is suitable for biological application scenarios such as medical dressings.

[0026] 3、Promote wound repair and tissue regeneration: as shown in the animal experiment (such as Figure 6 ), the wound of the treatment group is almost completely healed after 12 days, which is significantly better than that of the control group; the material has liquid absorption and gas exchange performance, can keep the wound dry, inhibit infection, promote the formation of new blood vessels and tissue repair; by regulating the ROS level, it helps to reduce the inflammatory response and accelerate the healing process.

[0027] 4、The application realizes high dispersion of metal atoms in mesoporous carbon, exposes more active sites, and improves catalytic efficiency; the carbon layer coating enhances the structural stability, prevents metal particle aggregation or dissolution, and improves the service life; through the regulation of temperature, solvent, surfactant and other parameters, the alloy particle size, distribution and carrier structure are accurately controlled, and the process is simple and can be produced on a large scale.

[0028] The application realizes high antibacterial efficiency, low biological toxicity, excellent catalytic performance and significant wound healing promotion effect by constructing a single-atom mesoporous carbon coated noble metal alloy structure, and has a wide application prospect in medical antibacterial materials and wound repair compared with traditional nano-antibacterial agents in safety and efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 XRD pattern of the PtCuZnCo@CoZn-SA-C antibacterial nanoenzyme prepared in the example;

[0030] Figure 2 TEM image of the PtCuZnCo@CoZn-SA-C antibacterial nanoenzyme prepared in the example;

[0031] Figure 3 CAT-like graph of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme prepared for the example;

[0032] Figure 4 SOD-like graph of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme prepared for the example;

[0033] Figure 5 In vitro antibacterial performance graph of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme prepared for the example;

[0034] Figure 6 Animal wound healing treatment graph of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme prepared for the example. DETAILED DESCRIPTION

[0035] Exemplary embodiments of the present disclosure will be described in greater detail below. It should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure can be more thoroughly understood, and so that the scope of the present disclosure can be conveyed to those skilled in the art.

[0036] In the following description, certain specific details are set forth in order to provide a thorough understanding of various disclosed embodiments. However, one skilled in the relevant arts will recognize that embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, structures, and processes associated with the present disclosure can not be shown or described in order to avoid obscuring embodiments.

[0037] Reference throughout this specification to "an embodiment" or "the embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0038] In the following description, for the purposes of clarity, directional terms are used to describe the structural and working relationship of the present application. However, the terms "front", "back", "left", "right", "outer", "inner", "outward", "inward", "up", "down", etc. should be understood as convenient language and should not be interpreted as limiting terms.

[0039] EMBODIMENTS

[0040] Preparation of CoZn-SA-C: 3.211 g of zinc nitrate hexahydrate and 3.9941 dimethylimidazole were dissolved in 60 mL of anhydrous methanol solution at room temperature, and after stirring for 0.5 h, the two solutions were mixed and reacted for 18 h; then 0.2095 g of cobalt nitrate hexahydrate was added to the above reaction, and after stirring for a certain period of time, Co-ZIF-8 was obtained by centrifugal washing with anhydrous methanol; the obtained Co-ZIF-8 was dried in a vacuum drying oven at 60-80 ℃, then transferred to a tube furnace, and pyrolyzed at 950 ℃ for 3 h in an Ar environment to obtain single-atom mesoporous derived carbon CoZn-SA-C;

[0041] 50 mg of CoZn-SA-C, 12 mg of platinum acetylacetonate, 15.8 mg of copper acetylacetonate, 80 mg of PVP, and 8 mL of N,N-dimethylformamide (DMF) were added to a polytetrafluoroethylene reaction kettle and stirred for 1 h to mix uniformly; the above reaction kettle was placed in an ultrasonic device and ultrasonicated for 1 h, then stirred for 0.5 h after the ultrasonication was completed, and then placed in a constant-temperature oven and reacted at 180 ℃ for 18 h; after the reaction was completed, it was naturally cooled, collected by centrifugation, and washed 3 times with ethanol to obtain PtCuZnCo@CoZn-SA-C antibacterial nanoszyme.

[0042] 5 mg of the above-prepared antibacterial nanoszyme was weighed, mixed with 5 mL of a PBS solution, and ultrasonicated until the solution was uniformly mixed to obtain an antibacterial nanoszyme solution. The antibacterial activity of the above-prepared antibacterial nanoszyme is shown in Figure 5 , which shows that the antibacterial rate of the drug-resistant Staphylococcus aureus can reach 100% at a working concentration of 25 ug / mL.

[0043] Figure 1 The XRD pattern of the above-prepared PtCuZnCo@CoZn-SA-C antibacterial nanoszyme, and no obvious Pt, Cu, Co, and Zn elemental diffraction peaks were found in the X-ray diffraction results of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme, proving that the PtCuZnCo alloy nanoparticles were successfully loaded on the CoZn-SA-C;

[0044] The TEM pattern of the above-prepared PtCuZnCo@CoZn-SA-C antibacterial nanoszyme is shown in Figure 2 , and the PtCuZnCo alloy particles on the CoZn-SA-C are clearly visible.

[0045] The CAT-like enzyme activity of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme at different working concentrations was tested on an ultraviolet spectrophotometer instrument, and the results are shown in Figure 3 . As the working concentration increased, the absorption peak of hydrogen peroxide gradually decreased, and when the working concentration was 200 ug / mL, the absorption peak of hydrogen peroxide was 0.

[0046] The results of testing the SOD-like enzyme activity of different working concentrations of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme on a UV spectrophotometer instrument are shown in Figure 4 As the working concentration increases, the absorption peak of the superoxide anion gradually decreases, and when the working concentration is 200 ug / mL, the absorption peak of the superoxide anion is 0.

[0047] The animal wound healing treatment chart of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme is shown in Figure 6 Compared with the wound healing results of the Control group, the wounds of the treatment group are almost completely healed after 12 days, indicating that the animal infected wound healing of the PtCuZnCo@CoZn-SA-C antibacterial nanoszyme is significantly improved.

[0048] The above is the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A preparation method of single-atom mesoporous carbon-coated hybrid noble metal alloy antibacterial nano-enzyme, characterized in that, The specific method is as follows: using the mesoporous carbon MZn-SA-C doped with cobalt, zinc and nitrogen as a hard template and a carrier, using a solution containing a platinum salt or acid and a transition metal salt copper as a metal precursor, mixing the metal precursor solution with an organic or aqueous solution in which the MZn-SA-C is dispersed, stirring uniformly, and reacting in a constant-temperature oven, so that the PtCuZnM@MZn-SA-C is obtained.

2. The method of claim 1, wherein: The reaction temperature in the constant-temperature reaction oven is 160-200 DEG C, and the reaction time is 12-24 h.

3. The method of claim 1, wherein, The specific method is as follows: using the mesoporous carbon MZn-SA-C doped with cobalt, zinc and nitrogen as a hard template and a carrier, using a solution containing a platinum salt or acid and a transition metal salt copper as a metal precursor, mixing the metal precursor solution with an organic or aqueous solution in which the MZn-SA-C is dispersed, stirring uniformly, and reacting in a constant-temperature oven, so that the PtCuZnM@MZn-SA-C is obtained. The specific method is as follows: using the mesoporous carbon MZn-SA-C doped with cobalt, zinc and nitrogen as a hard template and a carrier, using a solution containing a platinum salt or acid and a transition metal salt copper as a metal precursor, mixing the metal precursor solution with an organic or aqueous solution in which the MZn-SA-C is dispersed, stirring uniformly, and reacting in a constant-temperature oven, so that the PtCuZnM@MZn-SA-C is obtained. The specific method is as follows: using the mesoporous carbon MZn-SA-C doped with cobalt, zinc and nitrogen as a hard template and a carrier, using a solution containing a platinum salt or acid and a transition metal salt copper as a metal precursor, mixing the metal precursor solution with an organic or aqueous solution in which the MZn-SA-C is dispersed, stirring uniformly, and reacting in a constant-temperature oven, so that the PtCuZnM@MZn-SA-C is obtained. The specific method is as follows: using the mesoporous carbon MZn-SA-C doped with cobalt, zinc and nitrogen as a hard template and a carrier, using a solution containing a platinum salt or acid and a transition metal salt copper as a metal precursor, mixing the metal precursor solution with an organic or aqueous solution in which the MZn-SA-C is dispersed, stirring uniformly, and reacting in a constant-temperature oven, so that the PtCuZnM@MZn-SA-C is obtained. In step one, the zinc salt and the dimethyl imidazole are respectively dissolved in a proper amount of anhydrous methanol solution, and after stirring for 0.5 h, the two solutions are mixed.

4. The method of claim 3, wherein: In step two, after the MZn-SA-C, the Pt salt or acid, the transition metal copper salt, the surfactant and the solvent are added into the polytetrafluoroethylene reaction kettle, the mixture is stirred for 1 h to form a uniform mixed solution; the mixed solution is ultrasonically treated for 1 h, and then stirred for 0.5 h; the mixture is put into a constant-temperature oven for heating reaction at a certain temperature; after the reaction in the constant-temperature oven is completed, the product is washed by centrifugation with anhydrous ethanol for three times, and then dried in a vacuum oven, so that the single-atom coated noble metal alloy antibacterial nanoszyme is obtained.

5. The method of claim 3, wherein: In step one, the molar ratio of the zinc salt to the dimethyl imidazole is 1:3.5-4.5; preferably, the transition metal salt is one of nitrate, acetate, chloride or acetylacetone salt of iron, cobalt, nickel and copper, and the amount of substance of the added transition metal salt is 0.01-0.1 mol; preferably, the Pt salt or acid is one of platinum acetylacetone, potassium chloroplatous acid, potassium chloroplatinate and chloroplatinic acid, and the amount of substance ratio of the Pt salt or acid to the copper salt is 1:1-4.

6. The method of claim 3, wherein: ​ 7. The method of claim 3, wherein: ​ 8. The method of claim 3, wherein: In step one, the dried M-ZIF-8 is calcined at a high temperature of 850-950 ℃ in a tube furnace under an inert atmosphere.

9. The method of claim 3, wherein: The surfactant is one, two or more combinations of sodium hexadecyl sulfonate, hexadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium chloride, polyether F127, polyether F123, and the solvent is water, DMF, ethylene glycol or a mixture of two or more thereof.

10. A single-atom mesoporous carbon-coated hybrid noble metal alloy antimicrobial nano-enzyme prepared from any one of claims 1 to 9, which has multiple enzyme active sites.

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