Multifunctional antibacterial nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional antibacterial nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. The invention provides a multifunctional nano-enzyme which is prepared from polyethylene glycol, polyphenol, manganese chloride tetrahydrate and copper sulfate pentahydrate as raw materials through a reflux one-step method, and the multifunctional antibacterial nano-enzyme composed of carbon, oxygen, copper and manganese elements is prepared. The nano enzyme integrates the physicochemical properties of copper, manganese and polyphenol compounds, and can exert multifunctional enzyme activity superior to that of common copper or manganese metalloenzyme; under the synergistic action of the multifunctional enzyme activity, the nano-enzyme shows excellent antibacterial activity in the presence or absence of hydrogen peroxide, and also shows good biological safety. Therefore, the multifunctional nano enzyme has a great application prospect in the field of bacterial infection treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a multifunctional antibacterial nanozyme and a preparation method and application thereof. Background Art

[0002] With the rapid development of nanotechnology, nanozymes, a functional nanomaterial with simulated enzyme activity, have shown great application prospects in the biomedical field due to their easy synthesis, low cost, modifiable and rich physical and chemical properties. Its modifiable characteristics are to integrate different functions to adapt to inflammation, bacterial infection and tumors. At present, in addition to substances with natural enzyme activity, they are mainly bionic nanozymes composed of metal ions, such as gold, silver, cerium, zinc, copper and other nanozymes. However, the enzyme activity of these reported metal nanozymes often requires a large amount to achieve ideal enzyme activity, and the enzyme activity type is single, which makes it difficult to achieve effective application in complex environments. On the other hand, the reported nanozymes with multi-enzyme activity cannot effectively play the corresponding role due to the competition of their own enzyme activity, thereby greatly reducing their application value. Therefore, it is of great significance to develop nanozymes with synergistic multifunctional enzyme activity.

[0003] Nanozymes offer broad prospects for the treatment of microbial infections due to their unique enzymatic activity. At present, the reported nanozymes are mainly used for antimicrobial applications through external forces such as photothermodynamics, photodynamics, sonodynamics, and chemodynamics, which greatly increases the application cost. The heterogeneity of external forces can easily lead to side effects and increase the cost of treatment. In addition, the reported antibacterial nanozymes are used in large amounts, which can cause potential biological toxicity and further increase the application cost. Therefore, it is urgent to find and develop antibacterial nanozymes with synergistic multifunctional nanozyme activity. Summary of the invention

[0004] In view of the existing technical shortcomings of nanozymes, such as activity competition, single activity, and large antibacterial dosage, the present invention provides a multifunctional antibacterial nanozyme and its preparation method and application. The nanozyme of the present invention is composed of carbon, oxygen, copper and manganese elements, has multifunctional enzyme activity, and the multifunctional nanozyme achieves low-dose high-efficiency antibacterial activity, which has great application prospects in the field of bacterial infection treatment.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for preparing a multifunctional antibacterial nanozyme comprises the following steps: using polyethylene glycol, polyphenol, manganese chloride tetrahydrate and copper sulfate pentahydrate as raw materials, deionized water as solvent, performing a normal pressure reflux reaction for 1-24 hours, and then performing dialysis, centrifugal washing and air drying to obtain the desired multifunctional antibacterial nanozyme.

[0007] Preferably, the relative molecular mass of the polyethylene glycol is 200-2000, the feeding mass ratio of the polyethylene glycol to the polyphenol is 1:5-5:1, the feeding mass ratio of the polyethylene glycol to manganese chloride tetrahydrate is 1:10-10:1, and the feeding mass ratio of the manganese chloride tetrahydrate to copper sulfate pentahydrate is 1:4-10:1.

[0008] Preferably, the mass volume ratio of the polyethylene glycol to the solvent is 0.1:10-1:10, and the reaction temperature is 40-160°C.

[0009] Preferably, the polyphenols include but are not limited to tannic acid and salvianolic acid B.

[0010] The present invention also provides an antibacterial nanozyme with multifunctional enzyme activity prepared by the above preparation method.

[0011] Preferably, the nanozyme can exhibit excellent multifunctional enzyme activities such as peroxidase, superoxide dismutase and nicotinamide adenine dinucleotide phosphate oxidase within a working concentration range of 1-100 μg / mL.

[0012] Furthermore, the present invention also provides the application of the above-mentioned multifunctional antibacterial nanozyme in the antimicrobial field.

[0013] Preferably, the microorganisms to be killed are pathogenic bacteria methicillin-resistant Staphylococcus aureus.

[0014] Preferably, the nanozyme is used in a lower working concentration range of 10-60 μg / mL, which can effectively kill bacteria at a concentration of 1×10 9 CFU / mL of pathogenic methicillin-resistant Staphylococcus aureus.

[0015] Furthermore, the nanozyme has excellent biosafety, which can ensure its potential application in the prevention and treatment of pathogenic bacterial infections.

[0016] Furthermore, the present invention also provides the application of the above-mentioned multifunctional antibacterial nanozyme in the field of anti-oxidation.

[0017] Preferably, the anti-oxidation comprises scavenging hydrogen peroxide and superoxide anions.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The multifunctional antibacterial nanozyme of the present invention has the characteristics of simple and easy preparation process and good reproducibility; its excellent multifunctional enzyme activity and good biosafety provide guarantee and technical support for its high efficiency in antibacterial and related infection prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the infrared spectrum of the nanozyme of Example 1 of the present invention.

[0021] Figure 2 This is a transmission electron microscopy image of the nanozyme of Example 1 of the present invention.

[0022] Figure 3 Characterization diagram of the peroxidase (left), superoxide dismutase (middle) and nicotinamide adenine dinucleotide phosphate oxidase activity (right) of the nanozyme of Example 1 of the present invention.

[0023] Figure 4 This is the antibacterial activity of the nanozyme in Example 1 of the present invention (colony plate image after co-culture of nanozyme and methicillin-resistant Staphylococcus aureus).

[0024] Figure 5 This is the biosafety of the nanozyme in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1

[0027] Weigh 1g polyethylene glycol (M w =200) and 5g tannic acid were dissolved in 100mL deionized water, and 10g manganese chloride tetrahydrate and 1g copper sulfate pentahydrate were slowly added. After all the substances were dissolved and mixed, they were transferred to a 250mL flask, placed in a 40℃ oil bath, and refluxed for 24h. The obtained product was centrifuged, washed, and dried to obtain a solid nanozyme.

[0028] The infrared spectrum of the nanozyme prepared in this example is shown in Figure 1 , transmission electron microscopy images are shown in Figure 2 .

[0029] Example 2

[0030] Weigh 5 g of polyethylene glycol (M w =2000) and 1g of salvianolic acid B were dissolved in 50mL of deionized water, and 0.5g of manganese chloride tetrahydrate and 2g of copper sulfate pentahydrate were slowly added. After all the substances were dissolved and mixed, they were transferred to a 100mL flask, placed in an oil bath at 160°C, and refluxed for 1h. The obtained product was centrifuged, washed, and dried to obtain a solid nanozyme.

[0031] Example 3

[0032] Weigh 2 g of polyethylene glycol (M w=1000) and 2g tannic acid were dissolved in 100mL deionized water, and 2g manganese chloride tetrahydrate and 1g copper sulfate pentahydrate were slowly added. After all the substances were dissolved and mixed, they were transferred to a 100mL flask, placed in a 100℃ oil bath, refluxed for 12h, and the obtained product was centrifuged, washed, and dried to obtain a solid nanozyme.

[0033] Example 4

[0034] Weigh 1g polyethylene glycol (M w =800) and 2g of salvianolic acid B were dissolved in 70mL of deionized water, and 2g of manganese chloride tetrahydrate and 2g of copper sulfate pentahydrate were slowly added. After all the substances were dissolved and mixed, they were transferred to a 150mL flask, placed in a 60℃ oil bath, and refluxed for 24h. The obtained product was centrifuged, washed, and dried to obtain a solid nanozyme.

[0035] Example 5

[0036] Peroxidase activity assay

[0037] This example uses the nanozyme prepared in Example 1 of the present invention to investigate and characterize its peroxidase activity: 3,3',5,5'-tetramethylbenzidine (TMB) is used as an indicator to prepare a nanozyme solution with a concentration of 1 mg / mL and a TMB solution with a concentration of 10 mmol / L. Different volumes of nanozyme solution (final concentrations of 0, 10, 20, 40, 60, 80 and 100 μg / mL) were measured and mixed with 20 μL of TMB solution, and the Tris-HCl solution was supplemented to 100 μL, followed by adding 100 μL of 10 mmol / L H2O2 solution. The absorbance values ​​of different solutions at 652 nm were immediately measured using an ELISA instrument, and the measurement was performed once every 1 minute for a total of 30 minutes to evaluate the peroxidase activity of the nanozyme. The corresponding results are shown in Figure 3 .

[0038] Superoxide dismutase activity assay

[0039] In this example, the nanozyme prepared in Example 1 of the present invention was used to investigate and characterize its superoxide dismutase activity: a total superoxide dismutase activity detection kit (WST-8 method) was used for determination. According to the instructions of the kit, 20 μL of nanozyme solutions of different concentrations (final concentrations of 0, 10, 20, 40, 60, 80 and 100 μg / mL) were measured and mixed with 160 μL of WST-8 / enzyme working solution, and then 20 μL of starting solution was added. The mixture was incubated at room temperature in the dark for 30 minutes, and the absorbance value of the solution at 450 nm was determined using an ELISA instrument. The superoxide dismutase activity of the nanozyme was evaluated by using a mixture of superoxide dismutase detection buffer, WST-8 / enzyme working solution and reaction starting solution as blank control 1, a mixture of superoxide dismutase detection buffer and WST-8 / enzyme working solution as blank control 2, and a mixture of nanozyme, superoxide dismutase detection buffer and WST-8 / enzyme working solution as blank control 3 (superoxide dismutase activity % = [(A 空白对照1 -A 空白对照2 )-(A 样品 -A 空白对照3 )] / (A 空白对照1 -A 空白对照2 )×100%), the corresponding results are shown in Figure 3 .

[0040] Nicotinamide adenine dinucleotide phosphate oxidase activity assay

[0041] In this example, the nanozyme prepared in Example 2 of the present invention was used to investigate and characterize its nicotinamide adenine dinucleotide phosphate oxidase activity: a nicotinamide adenine dinucleotide phosphate solution with a concentration of 600 μmol / L and a 120 μg / mL nanozyme solution were prepared respectively, and then equal volumes of the solutions were measured and mixed, and the absorption spectrum of the sample solution in the wavelength range of 250-450 nm was immediately detected by a UV-visible spectrophotometer, and the measurement was performed every 5 minutes for a total of 60 minutes, and then the nicotinamide adenine dinucleotide phosphate oxidase activity of the nanozyme was analyzed and evaluated. The corresponding results are shown in Figure 3 .

[0042] Example 6

[0043] Evaluation of antimicrobial activity:

[0044] This example uses the nanozyme prepared in Example 1 of the present invention as an example to evaluate its ability to kill pathogenic bacteria, methicillin-resistant Staphylococcus aureus: fresh methicillin-resistant Staphylococcus aureus bacterial solution in the logarithmic growth period was centrifuged and washed with PBS, the cells were collected and resuspended with PBS, and the concentration of the bacterial solution was adjusted to make its OD 600 ≈2.0(2.0×10 9CFU / mL). Subsequently, part of the bacterial solution was mixed with equal volumes of nanozyme solutions at concentrations of 40 and 80 μg / mL, and the other part of the bacterial solution was mixed with equal volumes of nanozyme solutions at concentrations of 40 and 80 μg / mL containing 200 μmol / L hydrogen peroxide, mixed evenly, and placed in a 37°C incubator for 3 hours. 100 μL of the incubated bacterial solution was evenly applied to LB solid culture medium plates, placed in a 37°C incubator for 24 hours, and the number of colonies was checked and analyzed to evaluate the bactericidal ability of the nanozyme. The corresponding results are shown in Figure 4 .

[0045] The corresponding bactericidal rates of the nanozymes prepared in Examples 1-4 against methicillin-resistant Staphylococcus aureus are shown in Table 1.

[0046] Table 1 Bactericidal rate of nanozymes prepared in Examples 1-4 against methicillin-resistant Staphylococcus aureus

[0047]

[0048] Example 7

[0049] Biosafety Assessment:

[0050] In this example, the biosafety of the nanozyme prepared in Example 1 was evaluated by blood compatibility: freshly purchased rabbit red blood cells were collected by centrifugation, the red blood cells were washed with phosphate buffer (pH = 7.4) until the supernatant was clear, and the red blood cells were resuspended to a content of 2%. Then, different concentrations of nanozyme solutions (final concentrations of 0, 20, 40, 80, 120, 160 and 200 μmol / L) were mixed with an equal volume of 2% red blood cells, Triton-X was used as a positive control, and red blood cell phosphate buffer without nanozyme was used as a negative control. The mixture was incubated at 37°C for 2 hours, and then the supernatant was collected by centrifugation. The absorbance of the supernatant at 540 nm was measured using an ELISA instrument to calculate the hemolysis rate (hemolysis rate % = (A 样品 -A 阴性对照 ) / (A 阳性对照 -A 阴性对照 )×100%), the corresponding results are shown in Figure 5 .

[0051] In summary, the nanozyme of the present invention has excellent multifunctional nanozyme activity, antibacterial activity and biosafety.

[0052] The above description is only a preferred embodiment of the present invention and is used to limit the present invention. Any modification, equivalent substitution, improvement, etc. made within the principle and content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a multifunctional antibacterial nanozyme, characterized in that: The method comprises the following steps: using polyethylene glycol, polyphenol, manganese chloride tetrahydrate and copper sulfate pentahydrate as raw materials, using deionized water as solvent, subjecting to normal pressure reflux reaction for 1-24 hours, and obtaining the desired multifunctional antibacterial nanozyme through dialysis, centrifugal washing and air drying.

2. The preparation method according to claim 1, characterized in that: The relative molecular mass of the polyethylene glycol is 200-2000, the feeding mass ratio of the polyethylene glycol to the polyphenol is 1:5-5:1, the feeding mass ratio of the polyethylene glycol to the manganese chloride tetrahydrate is 1:10-10:1, and the feeding mass ratio of the manganese chloride tetrahydrate to the copper sulfate pentahydrate is 1:4-10:

1.

3. The preparation method according to claim 1, characterized in that: The mass volume ratio of the polyethylene glycol to the solvent is 0.1:10-1:10, and the reaction temperature is 40-160°C.

4. The multifunctional antibacterial nanozyme prepared according to the preparation method according to any one of claims 1 to 3.

5. The multifunctional antibacterial nanozyme according to claim 4, characterized in that: The nanozyme has peroxidase, superoxide dismutase or nicotinamide adenine dinucleotide phosphate oxidase activity.

6. Application of the multifunctional antibacterial nanozyme according to claim 4 in the antimicrobial field.

7. The use according to claim 6, characterized in that: The microorganisms are targeted for killing pathogenic methicillin-resistant Staphylococcus aureus.

8. The use according to claim 6, characterized in that: The nanozyme has a lower working concentration range of 10-60 μg / mL, which can effectively kill bacteria at a concentration of 1×10 9 CFU / mL of pathogenic methicillin-resistant Staphylococcus aureus.

9. Application of the multifunctional antibacterial nanozyme according to claim 4 in the field of anti-oxidation.

10. The use according to claim 9, characterized in that: The anti-oxidation includes scavenging hydrogen peroxide and superoxide anions.

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