A copper-containing nanosheet nanozyme for sterilization and promoting wound repair, its preparation method and application

Abstract

The present invention provides a copper nanosheet nanozyme for sterilization and promoting wound repair, its preparation method and application. The copper nanosheet is a copper phosphate nanosheet structure chemically coordinated and modified by polyphenolic substances. The preparation method includes the steps of adding octadecylamine to ethanol, stirring until dissolved, adding oleic acid, then adding a copper ion solution, stirring evenly, adding a disodium hydrogen phosphate solution, and subjecting the obtained mixed solution to a hydrothermal reaction; after the reaction is completed, washing and freeze-drying the obtained precipitate to obtain copper phosphate nanosheets; adding the copper phosphate nanosheets to a polyphenol solution, ultrasonicating to disperse them evenly, and stirring for reaction; then centrifuging, washing, and freeze-drying to obtain the product. The preparation method of the copper nanosheets of the present invention is simple and convenient to use, can significantly improve the symptoms of difficult wound healing, increase the wound healing speed, promote wound repair, and at the same time kill bacteria, giving play to the application potential of nanomaterials in antibacterial and accelerating wound healing.

Description

Technical Field

[0001] The present invention relates to a copper-containing nanosheet nanozyme for sterilization and promoting wound repair, its preparation method and application, belonging to the field of medical technology. Background Art

[0002] Wounds can be classified into acute wounds and chronic wounds according to the length of the healing time. Acute wounds can heal rapidly within a short time, while chronic wounds usually refer to intractable wounds with slow healing speed, wounds that have not healed for more than 1 month, or wounds without a tendency to heal. Wound healing is a complex physiological process, which is affected by various factors and occurs frequently in the process of combining multiple chronic diseases and aging. Diabetic (foot) ulcers, traumatic ulcers, pressure ulcers, chronic kidney diseases, tumors, connective tissue diseases, etc. can all lead to difficult wound healing. Due to the prevalence of obesity, diabetes and cardiovascular diseases in the elderly, chronic wounds are becoming an increasingly serious socioeconomic problem in an aging society. And difficult wound healing will increase the risk of bacterial infection, which will further increase the risk of patients suffering from sepsis, and may even lead to amputation or death in severe cases, seriously affecting the quality of life of patients.

[0003] Chronic inflammation is one of the characteristics of chronic wounds and is closely related to immune regulation. The imbalance of the immune system will lead to persistent inflammation and impaired wound healing, and then lead to chronic skin wounds. Studies on chronic inflammation have shown that the accumulation of a large amount of reactive oxygen species increases the level of oxidative stress in the body, leading to the imbalance of the human microenvironment and long-term exposure to an inflammatory microenvironment. Excessive reactive oxygen species can activate four pathogenic downstream pathways: the polyol pathway, the pathway of binding of advanced glycation end products to their receptors, the protein kinase C pathway, and the hexosamine pathway, resulting in lipid peroxidation, protein denaturation or DNA damage, inducing oxidative damage to cells, interfering with the cell cycle process, forming irreversible tissue damage, and reducing the tissue regeneration function. Therefore, regulating the level of reactive oxygen species is very important for the healing of chronic inflammatory wounds.

[0004] At present, it is difficult for existing products to meet the requirements of rapid healing of chronic wounds, reducing infection, moistening wounds, reducing scar formation, etc. In recent years, with the rapid development of nanomaterials, some nanomaterials have shown excellent antibacterial properties, providing new ideas for accelerating wound healing, alleviating and treating ulcers and related complications caused by various reasons.

[0005] As a class of nanomaterials, nanozymes have good effects in scavenging reactive oxygen species and have the following advantages: the action of nanozymes is not restricted by subcellular compartments; their catalytic performance may be higher than that of the natural antioxidant defense system; nanozymes exhibit higher stability and better tolerance to harsh environments. Research shows that copper nanoparticles with intermediate valence states have a variety of mimetic enzymes and broad-spectrum reactive oxygen species scavenging ability, and have a protective effect on reactive oxygen species-mediated cell damage at extremely low doses. They can be used to treat reactive oxygen species-related diseases and significantly improve the treatment outcomes of acute kidney injury, acute liver injury, and wound healing.

[0006] Polyphenols are compounds found in plant-based foods with potential health-promoting effects and have antioxidant and anti-inflammatory characteristics. Polyphenols can form metal-polyphenol coordination structures through chemical coordination with many metal ions, including copper ions. Theoretically, polyphenols can transfer electrons to copper ions to change the valence state of copper, thereby enhancing the enzymatic catalytic activity of copper nanosheets and their ability to scavenge ROS. The coordination effect of polyphenols also affects the release of copper ions. Chinese patent document CN114306382A provides a copper-based nanozyme, which is formed by the efficient electrostatic adsorption of metal-organic coordination two-dimensional nanosheets and polydopamine nanoparticles. The metal-organic coordination two-dimensional nanosheets are copper-phenol nanozymes formed by the oxidative coupling self-assembly of copper ion precursors and phenolic compound precursors. The copper ion precursors are one or more water-soluble copper salts, and the phenolic compound precursors are one or more of catechol and its derivatives or polyphenols and their derivatives. However, the in vitro antibacterial effect shown by this copper-based nanozyme material requires the synergistic effect of near-infrared light irradiation.

[0007] Currently, there is no report on using polyphenol-modified copper phosphate nanosheets to sterilize and simultaneously promote wound repair. Therefore, the present invention is proposed. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the present invention provides a copper-containing nanosheet nanozyme for sterilization and promoting wound repair, its preparation method and application. The copper phosphate nanosheets of the present invention have a structure of polyphenol-modified copper phosphate nanosheets. This nanosheet combines the antibacterial properties of copper and the easy surface modification of polyphenols to make them play a synergistic role. The preparation method of the copper nanosheets with both good antibacterial properties and the effect of accelerating wound healing prepared by the present invention is simple and easy to use, can significantly improve the symptoms of difficult wound healing, increase the wound healing speed, promote wound repair, and simultaneously kill bacteria, giving full play to the application potential of nanomaterials in antibacterial and accelerating wound healing.

[0009] The technical solution of the present invention is as follows:

[0010] A copper nanosheet nanozyme for sterilization and promoting wound repair, wherein the copper nanosheet nanozyme is a polyphenol-modified copper phosphate nanosheet.

[0011] Preferably according to the present invention, the polyphenol is quercetin (Quercetin, Que), tannic acid (Tannic acid, TA), catechol, gallic acid or epigallocatechin gallate (EGCG), and more preferably tannic acid (Tannic acid, TA).

[0012] According to the present invention, the preparation method of the above-mentioned copper nanosheet nanozyme for sterilization and promoting wound repair includes the following steps:

[0013] (1) Add octadecylamine to ethanol, stir until dissolved, then add oleic acid, and then add a copper ion solution. After stirring evenly, add a sodium hydrogen phosphate (Na2HPO4) solution, and perform a hydrothermal reaction on the obtained mixed solution; after the reaction is completed, wash the obtained precipitate and freeze-dry it to obtain copper phosphate nanosheets;

[0014] (2) Add the copper phosphate nanosheets prepared in step (1) to a polyphenol solution, disperse them evenly by ultrasonic treatment, and then stir to react; then centrifuge, wash, and freeze-dry to obtain polyphenol-modified copper phosphate nanosheets, which are the copper nanosheet nanozymes for sterilization and promoting wound repair.

[0015] Preferably according to the present invention, the mass ratio of octadecylamine to the volume of ethanol in step (1) is 10 - 50 mg:1 mL, and more preferably 30 - 40 mg:1 mL.

[0016] Preferably according to the present invention, the volume ratio of oleic acid added in step (1) to the mass of octadecylamine is 1 - 100 mL:1 g, and more preferably 1 - 10 mL:1 g.

[0017] Preferably according to the present invention, the copper ion solution in step (1) is obtained by dissolving a copper salt in water, and the concentration of the copper ion solution is 1 - 10 mmol / L; the copper salt is a nitrate, sulfate or chloride of copper ions; the volume ratio of the copper ion solution added to the mass of octadecylamine is 1 - 50 mL:1 g, and more preferably 10 - 20 mL:1 g.

[0018] Preferably according to the present invention, the concentration of the sodium hydrogen phosphate (Na2HPO4) solution in step (1) is 0.5 - 50 mmol / L, and more preferably 1 - 10 mmol / L; the molar ratio of sodium hydrogen phosphate to copper ions in the copper ion solution is 0.1 - 1:1, and more preferably 0.3 - 0.6:1.

[0019] Preferably according to the present invention, in step (1), the temperature of the hydrothermal reaction is 160 - 200 °C, and the time of the hydrothermal reaction is 6 - 12 h.

[0020] Preferably according to the present invention, in step (1), the washing is first washing with n-hexane 3 - 5 times and then washing with ethanol 3 - 5 times.

[0021] Preferably according to the present invention, in step (2), the concentration of the polyphenol solution is 1 - 10 mg / mL; the mass ratio of the copper phosphate nanosheets to the polyphenol in the polyphenol solution is 1:1 - 5, and further preferably 1:1.

[0022] Preferably according to the present invention, in step (2), the stirring speed is 1000 - 1500 rpm; the reaction time is 1 - 3 h.

[0023] Preferably according to the present invention, in step (2), the centrifugation speed is 5000 - 15000 rpm, and the centrifugation time is 5 - 10 minutes; the washing is washing with deionized water 3 - 5 times.

[0024] According to the present invention, the application of the copper nanosheets with bactericidal and wound repair promoting effects in the preparation of preparations with bactericidal and wound repair promoting effects.

[0025] The technical features and beneficial effects of the present invention are as follows:

[0026] 1. The present invention first prepares copper phosphate nanosheets with a specific morphology by an optimized preparation process, and then reacts with a specific amount of polyphenols to obtain polyphenol-modified copper phosphate nanosheet nanoenzymes. The copper nanosheets prepared by the present invention have a structure of polyphenol-modified copper phosphate nanosheets. The nanosheets utilize the antibacterial properties of copper and have a killing effect on representative Gram-negative bacteria and Gram-positive bacteria such as Escherichia coli and Staphylococcus aureus.

[0027] 2. The copper nanosheets prepared by the present invention have the effect of accelerating wound healing. Polyphenol modification can significantly improve the ability of the nanosheets to scavenge reactive oxygen species and reduce the wound inflammation level, can significantly improve the symptoms of difficult wound healing, increase the wound healing speed and thus promote wound repair. Description of the Drawings

[0028] Figure 1 SEM images of the copper phosphate nanosheets (a) and polyphenol-modified copper phosphate nanosheets (b) prepared in Example 1.

[0029] Figure 2 Infrared spectra of tannic acid, the copper phosphate nanosheets prepared in Example 1, and the polyphenol-modified copper phosphate nanosheets prepared in Example 1 - 2.

[0030] Figure 3 Particle size distribution diagrams of copper phosphate nanosheets and copper phosphate nanosheets modified with polyphenols in different ratios.

[0031] Figure 4 Potential distribution diagrams of copper phosphate nanosheets and copper phosphate nanosheets modified with polyphenols in different ratios.

[0032] Figure 5 Effect diagrams of copper phosphate nanosheets and copper phosphate nanosheets modified with polyphenols in killing Staphylococcus aureus in Test Example 1.

[0033] Figure 6 Effect diagrams of copper phosphate nanosheets and copper phosphate nanosheets modified with polyphenols in killing Escherichia coli in Test Example 1.

[0034] Figure 7 Diagrams of the promotion of wound healing by copper phosphate nanosheets and copper phosphate nanosheets modified with polyphenols in Test Example 2.

[0035] Figure 8 Results of the DPPH scavenging experiment of copper phosphate nanosheets (a) and copper phosphate nanosheets modified with polyphenols (b) in Test Example 3.

[0036] Figure 9 Results of the material toxicity of tannic acid, copper phosphate nanosheets and copper phosphate nanosheets modified with polyphenols in Test Example 4.

[0037] Figure 10 Results of the live / dead staining of copper phosphate nanosheets in Test Example 4.

[0038] Figure 11 Results of the live / dead staining of copper phosphate nanosheets modified with polyphenols in Test Example 4. Specific implementation manners

[0039] The present invention will be further described below through specific embodiments. The embodiments of the present invention are to enable those skilled in the art to better understand the present invention and do not impose any limitations on the present invention.

[0040] Meanwhile, in the following embodiments, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0041] Example 1

[0042] A preparation method of a copper-containing nanosheet nanozyme for sterilization and promoting wound repair, comprising the following steps:

[0043] (1) Add 0.5556 g of octadecylamine to 16 mL of absolute ethanol. After stirring until fully dissolved, continue stirring and add 4 mL of oleic acid. Then add 9 mL of 2 mmol / L copper nitrate solution, and the solution turns dark blue. After continuing to stir for 10 min, slowly add 7 mL of 1.2 mmol / L disodium hydrogen phosphate (Na2HPO4) solution drop by drop. During the addition process, the color of the solution gradually fades and finally becomes light blue. After stirring for 5 minutes, transfer it to a reaction kettle and react at 160 °C for 6 h. After the reaction is completed, discard the upper oily substance, take out the obtained blue precipitate, wash it 5 times with n-hexane first and then 5 times with ethanol, and freeze-dry the obtained solid to get a blue substance, that is, copper phosphate nanosheets, denoted as Cu NPs, for standby.

[0044] (2) Add 0.2 g of the copper phosphate nanosheets prepared in step (1) to 100 mL of tannic acid aqueous solution with a concentration of 2 mg / mL. After ultrasonic dispersion to make it uniform, stir and react for 2 h with a magnetic stirrer (rotation speed of 1000 rpm); after the reaction is completed, centrifuge the obtained reaction solution at a rotation speed of 8000 rpm for 10 min, wash the obtained solid 3 times with deionized water, and freeze-dry to obtain polyphenol-modified copper phosphate nanosheets, which are copper-containing nanosheet nanoenzymes with bactericidal and wound repair-promoting functions, denoted as TA-Cu NPs.

[0045] The SEM images of the copper phosphate nanosheets and polyphenol-modified copper phosphate nanosheets prepared in this example are shown in Figure 1 (a)-(b) respectively. It can be seen from Figure 1 that the size of the polyphenol-modified copper phosphate nanosheets is larger than that of the copper phosphate nanosheets without polyphenol modification. Figure 2 The infrared results prove that polyphenol substances have been successfully modified on the copper phosphate nanosheets.

[0046] Example 2

[0047] A method for preparing a copper-containing nanosheet nanoenzyme with bactericidal and wound repair-promoting functions is as described in Example 1, except that the concentration of tannic acid is different, and the concentrations of the tannic acid solutions used are 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL (i.e., the ratio of Cu to TA is 1:2, 1:3, 1:4, 1:5).

[0048] The particle size distribution diagrams and potential distribution diagrams of the copper phosphate nanosheets and polyphenol-modified copper phosphate nanosheets with different ratios are shown in Figure 3 、 Figure 4 respectively. The sizes of the materials synthesized by tannic acid do not differ much, but the dispersion stability is better when the ratio is 1:1.

[0049] The copper phosphate nanosheets and polyphenol-modified copper phosphate nanosheets used in the following test examples are all prepared in Example 1.

[0050] Experimental Example 1: Antibacterial Property Test

[0051] 100 μL of the physiological saline dispersion of copper phosphate nanosheets and 100 μL of the physiological saline dispersion of polyphenol-modified copper phosphate nanosheets were respectively mixed with 100 μL of a 10 7 CFU / mL Staphylococcus aureus solution and 800 μL of physiological saline to form a 1000 μL system, so that the final concentration of the bacterial solution in the system was 10 6 CFU / mL, and the final concentrations of copper phosphate nanosheets or polyphenol-modified copper phosphate nanosheets in the system were 0 μg / mL (NC group), 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively; then after incubation in a 37 °C incubator for 1 h, the bacterial solution was diluted to an appropriate concentration (10 4 CFU / mL) and spread on an agar plate, and the colony growth was observed after overnight incubation. The results are as Figure 5 shown.

[0052] The antibacterial property against Escherichia coli was tested according to the above method, and the results are as Figure 6 shown.

[0053] It can be seen from Figure 5-6 that both copper nanosheets and polyphenol-modified copper nanosheets have antibacterial effects on Staphylococcus aureus and Escherichia coli.

[0054] Experimental Example 2: Property Test for Promoting Wound Healing

[0055] Six-week-old male Sprague-Dawley (SD) rats were used. After 12 h of feeding, they were intraperitoneally injected with streptozotocin (STZ). After monitoring blood glucose for 3 weeks, rats with successful establishment of diabetes models were selected for the establishment of diabetic skin defect models. The specific modeling steps were as follows: After anesthetizing the SD rats, a puncher was used to punch holes (6 mm in diameter) on their backs. After cutting off the full-thickness skin with scissors, a skin dermal splint model was adopted. After adding drugs (the drugs were 0.01 mg / mL Cu NPs PBS solution of Example 1 and 0.01 mg / mL TA-Cu NPs PBS solution of Example 1, and the drug addition amount was 20 μL), a 3M dressing was placed and fixed with a bandage. The wound dressing was changed and the wound healing was photographed and recorded at 0, 3, 7, 9, 11, and 14 days after surgery. The results are as Figure 7 shown.

[0056] It can be seen from Figure 7 that compared with other groups, the wounds at the skin defect sites of rats in the polyphenol-modified copper nanosheet group (TA-Cu NPs) were basically healed 7 days after surgery.

[0057] Experimental Example 3: DPPH Scavenging Experiment

[0058] (1) DPPH scavenging experiment: (1) Dissolve DPPH reagent in absolute ethanol to prepare a solution with a concentration of 20 mmol / L; (2) Dilute the 20 mmol / L DPPH solution 100 times with 95% ethanol and measure the absorbance at 515 nm. Adjust the DPPH reagent to an absorbance of 0.94 - 0.97; (3) Mix 100 μL of the ethanol solution of Cu NPs or TA-Cu NPs with 900 μL of the DPPH solution respectively. The final concentrations of Cu NPs or TA-Cu NPs in the mixture are 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1 mg / mL. React in the dark at room temperature for 30 minutes; (4) Use a UV spectrophotometer to measure the absorbance of the solution at 515 nm.

[0059] The results are as Figure 8 shown. It can be seen from Figure 8 that compared with copper phosphate nanosheets, polyphenol-modified copper phosphate nanosheets have a more obvious DPPH scavenging effect.

[0060] Test Example 4: Toxicity experiment

[0061] Detect the material toxicity through cell proliferation experiment. The specific steps are as follows:

[0062] When human dermal fibroblasts are cultured to the 5th passage, plate them in a 96-well plate with a cell density of 5×10 4 / mL, add 100 μL of cell suspension to each well. After overnight incubation, add the materials for stimulation. The concentrations of the three materials (TA, Cu NPs, and TA-Cu NPs) are 0.0001, 0.001, 0.01, 0.5, 1 mg / mL respectively. After 24 hours, wash 3 times with PBS, add 100 μL of diluted CCK-8 detection solution to each well. After placing it in a cell incubator for 2 hours, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each well at 450 nm to reflect cell viability. The results are as Figure 9 shown. It can be seen from Figure 9 that TA modification can significantly reduce the toxicity of copper phosphate nanosheets.

[0063] Verify the material toxicity by cell live / dead staining simultaneously. The specific steps are as follows:

[0064] When human dermal fibroblasts are cultured to the 5th passage, plate them in a 24-well plate with a cell density of 5×10 4 / mL, 500 μL of cell suspension per well. After overnight incubation, materials were added for stimulation. The concentrations of the two materials (Cu NPs and TA-Cu NPs) were 0.0001, 0.001, 0.01, 0.5, and 1 mg / mL respectively. After 24 h, the cells were washed three times with PBS. 250 μL of cell viability / cytotoxicity assay reagent was added to each well. After incubation in the cell culture incubator for 0.5 h, the cells were washed three times with PBS and photographed using a fluorescence microscope. The results are shown as Figure 10-11 shown. It can be seen from Figure 10-11 that the biocompatibility of copper phosphate nanosheets is inferior to that of the TA-modified materials. It can be seen that when the material concentration is 0.01 mg / mL, the number of dead cells of the TA-modified materials is significantly reduced.

Claims

1. A copper-containing nanosheet nanozyme for sterilization and promoting wound repair, characterized in that, The copper-containing nanosheet nanozyme is a tannic acid-modified copper phosphate nanosheet; the polyphenol is tannic acid; Its preparation method includes the following steps: (1) Add octadecylamine to ethanol, stir until dissolved, then add oleic acid, and then add a copper ion solution. After stirring evenly, add a disodium hydrogen phosphate solution, and perform a hydrothermal reaction on the obtained mixed solution; after the reaction is completed, wash the obtained precipitate and freeze-dry it to obtain copper phosphate nanosheets; the molar ratio of disodium hydrogen phosphate to copper ions in the copper ion solution is 0.1-1:1; (2) Add the copper phosphate nanosheets prepared in step (1) to a polyphenol solution, disperse them evenly by ultrasonic treatment, and then stir to react; then centrifuge, wash, and freeze-dry to obtain tannic acid-modified copper phosphate nanosheets, which are the copper-containing nanosheet nanozymes for sterilization and promoting wound repair; the mass ratio of copper phosphate nanosheets to polyphenols in the polyphenol solution is 1:1-5.

2. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (1), the mass ratio of octadecylamine to the volume of ethanol is 10-50 mg:1 mL; the added volume of oleic acid to the mass of octadecylamine is 1-100 mL:1 g.

3. The copper nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (1), the mass ratio of octadecylamine to the volume of ethanol is 30-40 mg:1 mL; the added volume of oleic acid to the mass of octadecylamine is 1-10 mL:1 g.

4. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (1), the copper ion solution is obtained by dissolving a copper salt in water, and the copper salt is a nitrate, sulfate or chloride of copper ions; the added volume of the copper ion solution to the mass of octadecylamine is 1-50 mL:1 g.

5. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (1), the added volume of the copper ion solution to the mass of octadecylamine is 10-20 mL:1 g.

6. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (1), the molar ratio of disodium hydrogen phosphate to copper ions in the copper ion solution is 0.3-0.6:

1.

7. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (1), the temperature of the hydrothermal reaction is 160-200 °C, and the time of the hydrothermal reaction is 6-12 h; the washing is to wash with n-hexane 3-5 times and then wash with ethanol 3-5 times.

8. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (2), the concentration of the polyphenol solution is 1-10 mg / mL; the mass ratio of copper phosphate nanosheets to polyphenols in the polyphenol solution is 1:

1.

9. The copper-containing nanosheet nanozyme for sterilization and promoting wound repair according to claim 1, wherein In step (2), the stirring speed is 1000-1500 rpm; the reaction time is 1-3 h; the centrifugation speed is 5000-15000 rpm, and the centrifugation time is 5-10 minutes; the washing is to wash with deionized water 3-5 times.

10. Use of the copper-containing nanosheets for sterilization and promoting wound repair according to claim 1 in the preparation of a drug for sterilization and promoting wound repair.

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