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

Abstract

The application relates to a flower-shaped NiCo2O4 nanometer enzyme and a preparation method and application thereof, the preparation method comprises the following steps: urea is added into deionized water and stirred and dissolved, then soluble divalent nickel salt and divalent cobalt salt are added into the urea solution and uniformly dispersed, and the stirring reaction is carried out at room temperature; the prepared mixed solution is placed in a high-pressure closed container, and a Ni-Co precursor is obtained through reaction; then, calcination is carried out, and the NiCo2O4 nanometer enzyme is obtained. The nanometer enzyme has a flower-shaped structure and good photo-thermal antibacterial performance, and has the advantages of good photo-thermal stability, good blood compatibility, low cytotoxicity and the like; by using a large specific surface area and good photo-thermal conversion performance, bacteria infecting a wound surface are killed through a photo-thermal effect, and the nanometer enzyme can be used for a wound infection repair product.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological materials, in particular to a flower-like NiCo2O4 nanoscale enzyme and a preparation method and application thereof. BACKGROUND

[0002] After the wound site is infected by bacteria, it will cause sustained inflammatory response and disturbance of inflammatory microenvironment metabolism, and the wound exudate provides a good natural culture medium for bacteria and promotes their proliferation, sometimes leading to repeated infection, further deterioration of the wound microenvironment, and inhibition of the wound repair process. Repeated infection will prolong or even stop the inflammatory phase of the wound, leading to delayed reepithelialization, impaired angiogenesis, reduced collagen deposition, and ultimately prolonged wound healing time, therefore, adjusting 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 of pathogenic bacteria, in addition, antibiotics not only lack the functions of promoting tissue regeneration or regulating immune microenvironment, but also have toxicity at high doses, therefore, it is urgent to develop new materials with low drug resistance for the repair of infected wounds.

[0004] Nanoscale enzymes have become a new type of nanomaterial, which exhibits similar catalytic activity to natural enzymes in vivo, but has different functional capabilities under specific physiological conditions. Even at extreme temperature and pH, nanoscale enzymes can maintain their biological catalytic activity, and also exhibit anti-degradation. Nanoscale enzymes directly destroy the cell structure of bacteria by producing active substances such as hydroxyl radicals, rather than inhibiting the growth of bacteria through specific targets, making it difficult for bacteria to develop drug resistance. In addition, some nanoscale enzymes also exhibit good photothermal performance, which has 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 almost no drug resistance due to the damage of multiple components such as protein denaturation and cell surface damage to bacteria. SUMMARY

[0005] In view of the deficiencies of the prior art, the first object of the present application is to provide a flower-like antibacterial material NiCo2O4 nanoscale enzyme, which has the advantages of high stability, low biological toxicity and high active oxygen scavenging capacity.

[0006] The second object of the present application is to provide a preparation method of the above-mentioned flower-like NiCo2O4 nanoscale enzyme, which has the advantages of easy availability of raw materials and simple operation.

[0007] The third object of the present application is to provide the application of the above-mentioned flower-like NiCo2O4 nanoscale enzyme, which can be combined with photothermal effect to inhibit the growth of bacteria in infected wounds, and can be used for the repair of infected wounds.

[0008] To achieve the above object, the present application provides the following technical solutions.

[0009] In a first aspect, the present application provides a method for preparing a flower-like NiCo2O4 nanoscale enzyme, comprising the following steps:

[0010] (1) urea is added to deionized water and stirred to dissolve, and then soluble divalent nickel salt and divalent cobalt salt are added to the urea solution and dispersed uniformly, and the reaction is stirred at room temperature;

[0011] (2) the mixed solution prepared in step (1) is placed in a high-pressure sealed container and reacted at 100-150℃ for 8-24h, and after cooling to room temperature, it is washed, vacuum dried, and the Ni-Co precursor is collected;

[0012] (3) the Ni-Co precursor prepared in step (2) is calcined to obtain a NiCo2O4 nanoscale enzyme.

[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 10-20h.

[0018] Preferably, in step (3), the calcination temperature is 180-350℃, and the calcination time is 1-4h. Further preferably, the calcination temperature is 300-350℃, and the calcination time is 2-4h.

[0019] In a second aspect, the present application also provides a NiCo2O4 nanoscale enzyme prepared by the preparation method.

[0020] Preferably, the particle size of the NiCo2O4 nanoscale enzyme is 2-5μm.

[0021] The third aspect of the present application also provides an infected wound repair product comprising the NiCo2O4 nanoscale enzyme. The infected wound repair product can be in the form of a medicinal plaster, a dressing, a gel, etc.

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

[0023] The preparation method of the NiCo2O4 nanoscale enzyme has the advantages of easy availability of raw materials and simple operation. The prepared NiCo2O4 nanoscale enzyme has a flower-like structure and good photo-thermal antibacterial performance, and also has the advantages of good photo-thermal stability, blood compatibility, and low cytotoxicity. By utilizing the large specific surface area and good photo-thermal conversion performance, the bacteria infecting the wound surface can be killed through photo-thermal effect, and the NiCo2O4 nanoscale enzyme can be used for wound infection repair products. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 SEM image of the NiCo2O4 nanoscale enzyme;

[0025] Figure 2 Photo-thermal heating curve of the NiCo2O4 nanoscale enzyme;

[0026] Figure 3 Photo-thermal cycle curve of the NiCo2O4 nanoscale enzyme;

[0027] Figure 4 Toxicity of NiCo2O4 nanoscale enzyme of different concentrations to L929 cells;

[0028] Figure 5 Antibacterial effect of the NiCo2O4 nanoscale enzyme;

[0029] Figure 6 Repair effect of the NiCo2O4 nanoscale enzyme on infected wounds. DETAILED DESCRIPTION

[0030] The present application will be further described below in combination with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[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, then 0.582 g of nickel nitrate hexahydrate and 0.582 g of cobalt nitrate hexahydrate were added to the urea solution, ultrasonic dispersion was performed for 20 min, and stirring was performed at 400 rpm at room temperature overnight;

[0034] (2) The mixed solution prepared in step (1) is placed in a high-pressure sealed container and placed in a muffle furnace at 120°C for 12h. After cooling to room temperature, it is washed with deionized water and dried under vacuum 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 a temperature of 300°C for 2h to obtain a NiCo2O4 nanoscale enzyme.

[0036] Example 2

[0037] (1) 0.36g urea is added to 30mL deionized water and stirred to dissolve, then 0.582g nickel nitrate hexahydrate, 1.164g cobalt nitrate hexahydrate are added to the urea solution, ultrasonic dispersion for 20min, stirring at 400rpm at room temperature overnight;

[0038] (2) The mixed solution prepared in step (1) is placed in a high-pressure sealed container and placed in a muffle furnace at 120°C for 12h. After cooling to room temperature, it is washed with deionized water and dried under vacuum 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 a temperature of 300°C for 2h to obtain a NiCo2O4 nanoscale enzyme.

[0040] Example 3

[0041] (1) 0.36g urea is added to 30mL deionized water and stirred to dissolve, then 0.582g nickel nitrate hexahydrate, 1.746g cobalt nitrate hexahydrate are added to the urea solution, ultrasonic dispersion for 20min, stirring at 400rpm at room temperature overnight;

[0042] (2) The mixed solution prepared in step (1) is placed in a high-pressure sealed container and placed in a muffle furnace at 120°C for 12h. After cooling to room temperature, it is washed with deionized water and dried under vacuum 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 a temperature of 300°C for 2h to obtain a NiCo2O4 nanoscale enzyme.

[0044] Example 4

[0045] (1) 0.36g urea is added to 30mL deionized water and stirred to dissolve, then 0.582g nickel nitrate hexahydrate, 1.164g cobalt nitrate hexahydrate are added to the urea solution, ultrasonic dispersion for 20min, stirring at 400rpm at room temperature overnight;

[0046] (2) The mixed solution prepared in step (1) is placed in a high-pressure sealed container and placed in a muffle furnace at 150°C for 12h. After cooling to room temperature, it is washed with deionized water and vacuum dried 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 a temperature of 300°C for 2h to obtain a NiCo2O4 nanoscale enzyme.

[0048] Example 5

[0049] (1) 0.36g urea is added to 30mL deionized water and stirred to dissolve, then 0.582g nickel nitrate hexahydrate, 1.164g cobalt nitrate hexahydrate are added to the urea solution, ultrasonic dispersion for 20min, stirring at 400rpm at room temperature overnight;

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

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

[0052] Example 6

[0053] (1) 0.36g urea is added to 30mL deionized water and stirred to dissolve, then 0.582g nickel nitrate hexahydrate, 1.164g cobalt nitrate hexahydrate are added to the urea solution, ultrasonic dispersion for 30min, stirring at 200rpm at room temperature overnight;

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

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

[0056] Test Example

[0057] 1. Morphology characterization

[0058] Test method: The NiCo2O4 nanoscale enzyme is laid on a sample table with conductive glue, the sample not stuck is blown away gently, gold is sprayed on the surface of the sample, and then the morphology is observed by SEM.

[0059] Figure 1 The SEM morphology of the NiCo2O4 nanoszyme synthesized in Examples 1-6 can be seen to have a clear flower-like structure, and thus has a good specific surface area.

[0060] The NiCo2O4 nanoszyme obtained in Example 2 was used to perform the following tests.

[0061] 2. Photothermal performance

[0062] Test method: A solution of NiCo2O4 nanoszyme with different concentrations (0.25, 0.50, 0.75 and 1.00 mg / mL) was prepared, and irradiated with an 808 nm NIR laser for 10 minutes at a power of 1.0 W / cm 2 A thermocouple thermometer was used to record the real-time temperature, and the real-time temperature was recorded every 20 s. The obtained data were plotted into a temperature rise curve with time as the abscissa and temperature as the ordinate.

[0063] The test results are shown in Figure 2 and Figure 3 It can be seen that the photothermal effect becomes more significant as the concentration increases, but tends to be stable after a certain concentration, and the difference between 0.75 mg / mL and 1 mg / mL is not very large. Moreover, the NiCo2O4 nanoszyme synthesized in the examples of the present application has good photothermal cycling performance.

[0064] 3. Cytotoxicity

[0065] Test method: The cultured L929 cells were digested and suspended with trypsin, inoculated into a 48-well plate, and after 12 hours of culture, the original culture solution was removed, and a cell culture solution containing NiCo2O4 nanoszyme with different concentrations (0.10, 0.25, 0.50, 1.00, 2.50 and 5.00 mg / mL) was added for continued culture for 24 hours. Subsequently, CCK-8 solution was added to each well, and incubated in an incubator for 1-2 hours, and then the absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader to detect the cell survival rate.

[0066] The test results are shown in Figure 4 It can be seen that the NiCo2O4 nanoszyme synthesized in the examples of the present application has good biocompatibility.

[0067] 4. Antibacterial effect

[0068] Test method: After recovering, E. coli and S. aureus were 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. The NiCo2O4 nanoszyme corresponding to a certain concentration was added in the 24-well plate. Then 100 μL of diluted bacterial suspension and 1 mL of sterile normal saline were added in each well, and incubated at 37 °C for 24 hours, gradient dilution and then coated on LB agar plate for counting. The control group was the normal saline group.

[0069] The test results are shown in Table 1. Figure 5 As can be seen from Table 1, the NiCo2O4 nanoszyme synthesized in the embodiment of the application has good antibacterial effect. The NiCo2O4 nanoszyme realizes good antibacterial effect by decomposing H2O2 into ·OH. Further under NIR irradiation, the nanoszyme of the application can also absorb photons, convert light energy into heat energy, and increase the temperature of the infected wound site, resulting in the death of bacteria. Since the human body tissue is not easy to absorb near-infrared radiation, and the photosensitive material has excellent optical properties and can effectively absorb near-infrared radiation, the use of photo-thermal nanoszyme can effectively induce local temperature and produce reactive oxygen, thereby effectively killing bacteria. Moreover, compared with the bulk structure, the flower-like structure NiCo2O4 nanoszyme of the application can significantly increase the photo-thermal conversion efficiency due to the capture of photo-generated electrons by oxygen vacancies and the enhancement of photo-thermal conversion. The photo-thermal flower-like antibacterial material NiCo2O4 nanoszyme prepared in the application can kill bacteria on the infected wound surface and promote the repair of the infected wound surface by using the large specific surface area and good photo-thermal conversion performance through photo-thermal effect.

[0070] 5. Wound repair performance

[0071] Test method: After the rats were anesthetized, the hair on the back skin of the rats was shaved, and the exposed skin was disinfected. A full-thickness circular skin defect with a diameter of 12 mm was caused by surgical scissors. Then, a mixture of E. coli and S. aureus (1 x 10 8 CFU / mL) was injected into the skin defect site, and 3M Tegaderm waterproof agent was used to fix for 24 hours to cause infection. The infected rats were randomly divided into groups, and the nanoszyme was injected at the wound site as the experimental group. The wound healing was observed at 3, 7, 10, and 14 days after the operation. For the near-infrared laser irradiation group, the wound site was irradiated with an 808 nm near-infrared laser with a power of 1.0 W / cm 2 .

[0072] Test results: As shown in Table 2, the wounds of rats in different groups were photographed and recorded, and the healing traces of each wound were simulated and statistically analyzed. As can be seen from the results, after 3 days of NiCo2O4 nanoszyme treatment, the wound had obviously scabbed and healed, and after 10 days of treatment, it was almost completely closed. Figure 6

[0073] ​Any technical features in the above-described embodiments can be combined in any manner, and for the sake of brevity, not all possible combinations are described, however, it is to be understood that the scope of the disclosure encompasses all possible combinations of the technical features. It should be noted that, for those of ordinary skill in the art, some modifications and improvements can be made to the present application without departing from the concept of the present application, and these should all fall within the scope of the present application. Therefore, the scope of protection of the present application should be subject to the appended claims.

Claims

1. The application of a flower-shaped NiCo2O4 nanozyme in the preparation of products for repairing infected wounds, characterized in that, The preparation method of the flower-shaped NiCo2O4 nanozyme includes the following steps: (1) Urea was added to deionized water and stirred to dissolve. Then, nickel nitrate and cobalt nitrate were added to the urea solution and dispersed evenly. The mixture was stirred and reacted at room temperature. 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; (2) Place the mixed solution prepared in step (1) in a high-pressure sealed container and react at 100-150℃ for 8-24h. After cooling to room temperature, wash, vacuum dry, and collect the Ni-Co precursor. (3) The Ni-Co precursor prepared in step (2) is calcined to obtain flower-shaped NiCo2O4 nanozyme.

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

3. The application according to claim 1, characterized in that, In step (1) of the preparation method, the reaction is carried out at room temperature with stirring at a speed of 200-800 rpm for 10-20 h.

4. The application according to claim 1, characterized in that, In step (3) of the preparation method, the calcination temperature is 180-350℃ and the calcination time is 1-4h.

5. The application according to claim 1, characterized in that, The flower-shaped NiCo2O4 nanozyme has a particle size of 2-5 μm.

6. A product for repairing infected wounds, characterized in that, It contains the flower-shaped NiCo2O4 nanozyme as described in any one of claims 1-5.

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