Flower-like NiCo2O4 nano-enzyme and preparation method and application thereof

Abstract

The invention relates to a flower-like NiCo2O4 nano-enzyme and a preparation method and application thereof.The preparation method comprises the following steps that urea is added into deionized water to be stirred and dissolved, then soluble divalent nickel salt and divalent cobalt salt are added into the urea solution to be evenly dispersed, and stirring reaction is conducted at the room temperature; placing the prepared mixed solution in a high-pressure closed container, and reacting to obtain a Ni-Co precursor; and then calcining to obtain the NiCo2O4 nano enzyme. The nano-enzyme provided by the invention has a flower-like structure and good photo-thermal antibacterial performance, and also has the advantages of good photo-thermal stability, blood compatibility, low cytotoxicity and the like; and by utilizing a relatively large specific surface area and good photothermal conversion performance, bacteria infecting the wound surface are killed through a photothermal effect, and the material can be used for wound surface infection repair products.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a flower-shaped NiCo2O4 nanozyme and a preparation method and application thereof. Background Art

[0002] Bacterial infection of the wound surface will cause persistent inflammatory response and disturbance of inflammatory microenvironment metabolism. Wound exudation sometimes leads to repeated infection by providing a good natural culture medium for bacteria and promoting their proliferation, further deteriorating the wound microenvironment and inhibiting the wound repair process. Repeated infection will prolong or even stagnate the inflammatory phase of the wound, leading to delayed re-epithelialization, impaired angiogenesis, reduced collagen deposition, and ultimately prolonged wound healing time. Therefore, regulating the wound infection microenvironment is the key to promoting the repair of infected wounds.

[0003] The development and application of antibiotics have shortened the wound repair process. However, the long-term and frequent use of antibiotics has led to serious drug resistance in pathogens. In addition, antibiotics not only lack functions such as promoting tissue regeneration or regulating the immune microenvironment, but are also toxic when the dose is too high. Therefore, there is an urgent need to develop new materials with low drug resistance for the repair of infected wounds.

[0004] Nanozymes have emerged as a new type of nanomaterial that exhibits similar catalytic activity to natural enzymes in vivo, but with different functional capabilities under specific physiological conditions. Nanozymes can maintain their biocatalytic activity even at extreme temperatures and pH values, while also exhibiting resistance to degradation. Nanozymes directly destroy the cell structure of bacteria by producing active substances such as hydroxyl radicals, rather than inhibiting bacterial growth through specific targets, making it difficult for bacteria to develop drug resistance. In addition, some nanozymes also exhibit good photothermal properties, with the ability to absorb near-infrared light and convert light into heat, and can be used as an effective material for photothermal sterilization, which has little resistance to drug resistance due to multi-component damage to bacteria such as protein denaturation and cell surface damage. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a flower-shaped antibacterial material NiCo2O4 nanozyme, which has the advantages of high stability, low biological toxicity and high active oxygen scavenging ability.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned flower-shaped NiCo2O4 nanozyme, which has the advantages of easy availability of raw materials and simple operation.

[0007] The third object of the present invention is to provide an application of the above-mentioned flower-shaped NiCo2O4 nanozyme, wherein the flower-shaped NiCo2O4 nanozyme can be combined with the photothermal effect to inhibit the growth of bacteria on the infected wound surface, and can be used for the repair of the infected wound surface.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a method for preparing a flower-shaped NiCo2O4 nanozyme, comprising the following steps:

[0010] (1) adding urea to deionized water and stirring to dissolve, then adding soluble divalent nickel salt and divalent cobalt salt to the urea solution and dispersing them evenly, and stirring to react at room temperature;

[0011] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, reacting at 100-150° C. for 8-24 hours, cooling to room temperature, washing, vacuum drying, and collecting the Ni-Co precursor;

[0012] (3) Calcinate the Ni-Co precursor prepared in step (2) to obtain NiCo2O4 nanozyme.

[0013] Preferably, in step (1), the urea: Ni 2+ :Co 2+ : Deionized water = 0.003-0.009 mol: 0.001-0.003 mol: 0.002-0.006 mol: 20-50 mL.

[0014] Preferably, the soluble divalent nickel salt is one of nickel nitrate, nickel chloride and nickel sulfate.

[0015] Preferably, the soluble divalent cobalt salt is one of cobalt nitrate, cobalt chloride and cobalt sulfate.

[0016] Preferably, in step (1), the dispersion is ultrasonic dispersion, and the ultrasonic time is 10-30 min.

[0017] Preferably, in step (1), the reaction is stirred at room temperature, the stirring speed is 200-800 rpm, and the reaction time is 10-20 h.

[0018] Preferably, in step (3), the calcination temperature is 180-350°C, and the calcination time is 1-4 hours. More preferably, the calcination temperature is 300-350°C, and the calcination time is 2-4 hours.

[0019] The second aspect of the present invention also provides the NiCo2O4 nanozyme prepared by the preparation method.

[0020] Preferably, the NiCo2O4 nanozyme has a particle size of 2-5 μm.

[0021] The third aspect of the present invention further provides an infected wound repair product, comprising the NiCo2O4 nanozyme. The infected wound repair product can be in the form of ointment, dressing, gel, etc.

[0022] Compared with the prior art, the present invention has the following beneficial and unique effects:

[0023] The preparation method of the NiCo2O4 nanozyme of the present invention has the advantages of easy availability of raw materials and simple operation. The prepared NiCo2O4 nanozyme has a flower-like structure and good photothermal antibacterial performance, as well as good photothermal stability, blood compatibility and low cytotoxicity. It utilizes a large specific surface area and good photothermal conversion performance to kill bacteria in infected wounds through the photothermal effect, and can be used in wound infection repair products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the SEM image of NiCo2O4 nanozyme;

[0025] Figure 2 This is the photothermal heating curve of NiCo2O4 nanozyme;

[0026] Figure 3 This is the photothermal cycle curve of NiCo2O4 nanozyme;

[0027] Figure 4 is the toxicity of different concentrations of NiCo2O4 nanozymes to L929 cells;

[0028] Figure 5 The antibacterial effect of NiCo2O4 nanozyme;

[0029] Figure 6 This is a diagram showing the repair effect of NiCo2O4 nanozyme on infected wounds. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1

[0033] (1) 0.36 g of urea was added to 30 mL of deionized water and stirred to dissolve, and then 0.582 g of nickel nitrate hexahydrate and 0.582 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonically dispersed for 20 min, and stirred at 400 rpm at room temperature overnight;

[0034] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, placing it in a muffle furnace and reacting it at 120° C. for 12 h, cooling it to room temperature, washing it with deionized water, and vacuum drying it at 60° C. to collect the Ni-Co precursor;

[0035] (3) The Ni-Co precursor prepared in step (2) is placed in a crucible and calcined in a muffle furnace at 300° C. for 2 h to obtain NiCo2O4 nanozyme.

[0036] Example 2

[0037] (1) 0.36 g of urea was added to 30 mL of deionized water and stirred to dissolve, then 0.582 g of nickel nitrate hexahydrate and 1.164 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonically dispersed for 20 min, and stirred at 400 rpm at room temperature overnight;

[0038] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, placing it in a muffle furnace and reacting it at 120° C. for 12 h, cooling it to room temperature, washing it with deionized water, and vacuum drying it at 60° C. to collect the Ni-Co precursor;

[0039] (3) The Ni-Co precursor prepared in step (2) is placed in a crucible and calcined in a muffle furnace at 300° C. for 2 h to obtain NiCo2O4 nanozyme.

[0040] Example 3

[0041] (1) 0.36 g of urea was added to 30 mL of deionized water and stirred to dissolve, then 0.582 g of nickel nitrate hexahydrate and 1.746 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonically dispersed for 20 min, and stirred at 400 rpm at room temperature overnight;

[0042] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, placing it in a muffle furnace and reacting it at 120° C. for 12 h, cooling it to room temperature, washing it with deionized water, and vacuum drying it at 60° C. to collect the Ni-Co precursor;

[0043] (3) The Ni-Co precursor prepared in step (2) is placed in a crucible and calcined in a muffle furnace at 300° C. for 2 h to obtain NiCo2O4 nanozyme.

[0044] Example 4

[0045] (1) 0.36 g of urea was added to 30 mL of deionized water and stirred to dissolve, then 0.582 g of nickel nitrate hexahydrate and 1.164 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonically dispersed for 10 min, and stirred at 600 rpm at room temperature overnight;

[0046] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, placing it in a muffle furnace and reacting it at 150° C. for 12 h, cooling it to room temperature, washing it with deionized water, and vacuum drying it at 60° C. to collect the Ni-Co precursor;

[0047] (3) The Ni-Co precursor prepared in step (2) is placed in a crucible and calcined in a muffle furnace at 300° C. for 2 h to obtain NiCo2O4 nanozyme.

[0048] Example 5

[0049] (1) 0.36 g of urea was added to 30 mL of deionized water and stirred to dissolve, then 0.582 g of nickel nitrate hexahydrate and 1.164 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonically dispersed for 20 min, and stirred at 400 rpm at room temperature overnight;

[0050] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, placing it in a muffle furnace and reacting it at 120° C. for 24 h, cooling it to room temperature, washing it with deionized water, and vacuum drying it at 60° C. to collect the Ni-Co precursor;

[0051] (3) The Ni-Co precursor prepared in step (2) was placed in a crucible and calcined in a muffle furnace at 350° C. for 2 h to obtain NiCo2O4 nanozyme.

[0052] Example 6

[0053] (1) 0.36 g of urea was added to 30 mL of deionized water and stirred to dissolve, then 0.582 g of nickel nitrate hexahydrate and 1.164 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonically dispersed for 30 min, and stirred at 200 rpm at room temperature overnight;

[0054] (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, placing it in a muffle furnace and reacting it at 120° C. for 12 h, cooling it to room temperature, washing it with deionized water, and vacuum drying it at 60° C. to collect the Ni-Co precursor;

[0055] (3) The Ni-Co precursor prepared in step (2) was placed in a crucible and calcined in a muffle furnace at 300° C. for 4 h to obtain NiCo2O4 nanozyme.

[0056] Test Case

[0057] 1. Morphology characterization

[0058] Test method: Spread the NiCo2O4 nanozyme flat on a sample table covered with conductive glue, gently blow away the unsticky sample, spray gold on the sample surface, and then observe its morphology through SEM.

[0059] Figure 1 The SEM morphology of the NiCo2O4 nanozymes synthesized in Examples 1-6 shows that the nanozymes synthesized in the examples of the present invention have obvious flower-like structures and therefore have better specific surface areas.

[0060] The following test was carried out using the NiCo2O4 nanozyme obtained in Example 2.

[0061] 2. Photothermal performance

[0062] Test method: NiCo2O4 nanozyme solutions of different concentrations (0.25, 0.50, 0.75 and 1.00 mg / mL) were prepared and irradiated with 808 nm NIR laser for 10 min at a power of 1.0 W / cm 2 , use a thermocouple thermometer to record the real-time temperature, record the real-time temperature every 20 seconds, use time as the horizontal axis and temperature as the vertical axis, and plot the obtained data into a heating curve.

[0063] The test results are as follows Figure 2 and Figure 3 As shown, it can be seen that as the concentration increases, the photothermal effect becomes more significant, but after a certain concentration, it tends to be stable, and the difference between 0.75 mg / mL and 1 mg / mL is not very large. In addition, the NiCo2O4 nanozyme synthesized in the embodiment of the present invention has good photothermal cycle performance.

[0064] 3. Cytotoxicity

[0065] Test method: Digest and suspend the cultured L929 cells with trypsin, inoculate them into 48-well plates, remove the original culture medium after 12 hours of culture, add NiCo2O4 nanozyme cell culture medium containing different concentrations (0.10, 0.25, 0.50, 1.00, 2.50 and 5.00 mg / mL) and continue to culture for 24 hours. Then add CCK-8 solution to each well, culture in an incubator for 1-2 hours, and then use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nanometers to detect cell survival rate.

[0066] The test results are as follows Figure 4 As shown, it can be seen that the NiCo2O4 nanozyme synthesized in the embodiment of the present invention has good biocompatibility.

[0067] 4. Antibacterial effect

[0068] Test method: Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) were revived and cultured to the logarithmic growth phase, centrifuged, and the bacteria were collected and resuspended in physiological saline. The final bacterial solution concentration was 1×10 8CFU / mL. A certain concentration of NiCo2O4 nanozyme was added to a 24-well plate. Then 100 μL of diluted bacterial suspension and 1 mL of sterile saline were added to each well, incubated at 37°C for 24 hours, and then spread on LB agar plates after gradient dilution and counted. The control group was the saline group.

[0069] The test results are as follows Figure 5 As shown, it can be seen that the NiCo2O4 nanozyme synthesized in the embodiment of the present invention has a good antibacterial effect. NiCo2O4 nanozyme achieves a good antibacterial effect by decomposing H2O2 into OH. Further under NIR irradiation, the nanozyme of the present application can also absorb photons, convert light energy into heat energy, increase the temperature of the infected wound, and cause bacterial death. Since human tissue is not easy to absorb near-infrared radiation, and photosensitive materials have excellent optical properties and can effectively absorb near-infrared radiation, the use of photothermal nanozymes can effectively induce local temperature and generate reactive oxygen, thereby effectively killing bacteria. Moreover, compared with the block structure, the flower-like structure NiCo2O4 nanozyme of the present invention can significantly increase the photothermal conversion efficiency due to oxygen vacancies capturing photogenerated electrons and enhancing photothermal conversion. The photothermal flower-shaped antibacterial material NiCo2O4 nanozyme prepared by the present invention utilizes a large specific surface area and good photothermal conversion performance, and can kill bacteria in the infected wound through the photothermal effect, and promote the repair of the infected wound.

[0070] 5. Wound repair performance

[0071] Test method: After the rats were anesthetized, the hair on the back of the rats was shaved, and the exposed skin was disinfected. A full-thickness circular skin defect with a diameter of 12 mm was created with surgical scissors. Subsequently, a mixture of Escherichia coli and Staphylococcus aureus (1×10 8 CFU / mL) and fixed with 3M Tegaderm waterproofing agent for 24 hours to induce infection. The infected rats were randomly divided into experimental groups and nanozymes were injected into the wounds. The wound healing was observed at 3, 7, 10 and 14 days after surgery. For the near-infrared laser irradiation group, the power was 1.0W / cm 2 The wound was irradiated with 808nm near-infrared laser for 10 minutes. Among them, the control group: the normal saline group.

[0072] Test results: Figure 6 As shown in the figure, the wounds of rats in different groups and given different treatments were photographed and recorded, and the healing traces of each wound were simulated and statistically analyzed. From the results, it can be seen that after 3 days of treatment with NiCo2O4 nanozymes, the wounds have obviously scabbed and healed, and after 10 days of treatment, they are almost completely closed.

[0073] The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for preparing a flower-shaped NiCo2O4 nanozyme, characterized in that: The following steps are involved: (1) adding urea to deionized water and stirring to dissolve, then adding soluble divalent nickel salt and divalent cobalt salt to the urea solution and dispersing them evenly, and stirring to react at room temperature; (2) placing the mixed solution prepared in step (1) in a high-pressure sealed container, reacting at 100-150° C. for 8-24 hours, cooling to room temperature, washing, vacuum drying, and collecting the Ni-Co precursor; (3) Calcinate the Ni-Co precursor prepared in step (2) to obtain NiCo2O4 nanozyme.

2. The preparation method according to claim 1, characterized in that: In step (1), the urea: Ni 2+ :Co 2+ : Deionized water = 0.003-0.009 mol: 0.001-0.003 mol: 0.002-0.006 mol: 20-50 mL.

3. The preparation method according to claim 1, characterized in that: The soluble divalent nickel salt is one of nickel nitrate, nickel chloride and nickel sulfate.

4. The preparation method according to claim 1, characterized in that: The soluble divalent cobalt salt is one of cobalt nitrate, cobalt chloride and cobalt sulfate.

5. The preparation method according to claim 1, characterized in that: In step (1), the dispersion is ultrasonic dispersion, and the ultrasonic time is 10-30 min.

6. The preparation method according to claim 1, characterized in that: In step (1), the reaction is stirred at room temperature, the stirring speed is 200-800 rpm, and the reaction time is 10-20 hours.

7. The preparation method according to claim 1, characterized in that: In step (3), the calcination temperature is 180-350° C. and the calcination time is 1-4 hours.

8. The NiCo2O4 nanozyme prepared by the preparation method according to any one of claims 1 to 7.

9. The NiCo2O4 nanozyme according to claim 8, characterized in that The particle size of the NiCo2O4 nanozyme is 2-5 μm.

10. An infected wound repair product, characterized in that: Comprising the NiCo2O4 nanozyme as described in claim 8 or 9.

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