Nickel-doped histidine carbon nanoszyme and preparation method thereof

Abstract

This invention relates to the field of carbon nanozyme technology, specifically to a nickel-doped histidine carbon nanozyme and its preparation method. Conventional metal-doped carbon nanozymes exhibit POD activity, which is detrimental to the healing of bacterial-infected wounds in the later stages. To address the above problem, this invention provides a nickel-doped histidine carbon nanozyme, which is a product generated by a high-temperature hydrothermal reaction between histidine and a soluble nickel salt. Under high-temperature hydrothermal conditions, nickel ions form coordination bonds with histidine. The carbon nanozyme obtained by this invention has a better photothermal bactericidal effect and does not have POD activity, which is more conducive to the healing of wounds in the later stages.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of carbon nanomzyme, and particularly relates to a nickel-doped histidine carbon nanomzyme and a preparation method thereof. BACKGROUND

[0002] The application of carbon materials in the biomedical field is showing its extraordinary clinical treatment potential, especially as an effective alternative to antibiotics, opening up a new vision for anti-infection treatment. These tiny nanoparticles not only have excellent biocompatibility and low toxicity, but also show high efficiency in targeting and killing pathogens, providing an innovative solution to the increasingly serious problem of drug resistance.

[0003] Although traditional antibiotics have played a key role in treating bacterial infections, the increasing drug resistance caused by long-term and extensive use has become a major challenge to global public health. In contrast, carbon materials, with their unique optical, electrical and chemical properties, can be activated under light or specific conditions to release precise attack power against bacteria, thereby achieving more precise and efficient treatment while reducing the risk of drug resistance.

[0004] However, current metal-doped carbon nanomzymes have been explored for use in the biomedical field and exhibit peroxidase (POD) activity. Although this property has some application value in certain situations, the continuous production of reactive oxygen species (ROS) brings about side effects that cannot be ignored. Excessive generation of ROS not only exacerbates the inflammatory response at the wound site, but also seriously interferes with the normal regeneration process of skin tissue, which poses a major obstacle to the late healing of bacterial infection wounds. Therefore, how to maintain antibacterial efficacy while avoiding or reducing these adverse reactions has become a key problem that needs to be solved.

[0005] It is worth noting that although metal-doped carbon nanomzymes are increasingly widely used in the biomedical field, there are few reports on metal-doped carbon nanomzyme sterilization materials without POD activity. The development of such materials is expected to achieve safer and more efficient control of bacterial infections without triggering excessive inflammatory response, creating a more favorable microenvironment for wound healing and tissue regeneration. In the future, with further research, metal-doped carbon nanomzyme sterilization materials without POD activity are expected to become an important supplement to antibiotic therapy. SUMMARY

[0006] The existing problem in the prior art is that conventional metal-doped carbon nanomzymes have POD activity. To solve the above technical problem, the present application provides a nickel-doped histidine carbon nanomzyme, which is a product generated from histidine and soluble nickel salt through high-temperature hydrothermal reaction. Under high-temperature hydrothermal conditions, nickel ions form a coordination chemical bond with histidine. The particle size range is 100-300 nm.

[0007] Preferably, the soluble nickel salt comprises one or more of nickel chloride, nickel acetate, nickel nitrate, or nickel sulfate.

[0008] Preferably, the temperature of the hydrothermal reaction is 150-220℃.

[0009] Preferably, the preparation method of the nickel-doped histidine carbon nanozyme includes the following steps:

[0010] Histidine was completely dissolved in water, and soluble nickel salt was added. The mixture was stirred until homogeneous. The resulting mixture was then transferred to a hydrothermal reactor for high-pressure hydrothermal reaction. After the reaction was completed, the product was removed from the hydrothermal reactor, ultrasonically mixed until homogeneous, and then freeze-dried to obtain the doped histidine carbon nanozyme.

[0011] Preferably, the ratio of histidine to soluble nickel salt is 1-10:1.

[0012] The present invention has the following beneficial effects:

[0013] (1) The carbon nanozyme obtained by the present invention does not have POD activity and does not generate ROS during use, but can remove ROS. The nanozyme uses photothermal effect, CAT activity and SOD activity for sterilization.

[0014] (2) The carbon nanozymes obtained in this invention do not produce ROS while sterilizing, which can significantly reduce the inflammatory response at the wound site, promote the formation of blood vessels at the wound site, and is more conducive to the healing of bacterial infection wounds in the later stage. Attached image description:

[0015] Figure 1 Particle size distribution of NH Nanozyme obtained in Example 1

[0016] Figure 2 Figure 1 shows the test results of the ability of NH Nanozyme to scavenge ABTS free radicals obtained in Example 1.

[0017] Figure 3 Figure 1 shows the test results of the ability of NH Nanozyme to scavenge DPPH free radicals obtained in Example 1.

[0018] Figure 4 : Graph showing the test results of the NH Nanozyme's ability to scavenge ·OH obtained in Example 1.

[0019] Figure 5 SOD activity diagram of NH Nanozyme obtained in Example 1.

[0020] Figure 6 POD activity diagrams of Example 1 and Comparative Examples 1 and 2.

[0021] Figure 7 : Antibacterial effect of NH Nanozyme obtained in Example 1. Detailed implementation method:

[0022] The present invention will be described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely illustrative examples of implementation of the present invention and are not intended to limit the scope of the present invention.

[0023] Example 1

[0024] 0.030 g of histidine was dissolved in 20 mL of distilled water, and then 0.009 g of nickel chloride hexahydrate was added. After stirring until completely dissolved, the mixture was stirred thoroughly for 10 min to obtain a mixture. The mixture was then placed in a hydrothermal reactor and hydrothermally reacted at 180 °C for 12 h. After the reaction was completed, the obtained product was taken out of the hydrothermal reactor, ultrasonically mixed evenly, and freeze-dried to obtain nickel-doped histidine carbon nanozyme, denoted as NH Nanozyme.

[0025] The particle size distribution diagram of the NH Nanozyme obtained in Example 1 is shown in the attached specification. Figure 1 As shown, the particle size of the NH Nanozyme is approximately 139.6 nm.

[0026] The test results of the ability of NH Nanozyme to scavenge ABTS free radicals obtained in Example 1 are shown in the attached instruction manual. Figure 2 As shown.

[0027] The test results of the ability of NH Nanozyme to scavenge DPPH free radicals obtained in Example 1 are shown in the attached instruction manual. Figure 3 As shown.

[0028] The test results of the ability of NH Nanozyme to scavenge hydroxyl radicals (·OH) obtained in Example 1 are shown in the attached instruction manual. Figure 4 As shown.

[0029] The SOD activity diagram of the NH Nanozyme obtained in Example 1 is shown in the attached specification. Figure 5 As shown.

[0030] The POD activity diagram of the NH Nanozyme obtained in Example 1 is shown in the attached specification. Figure 6 As shown.

[0031] The antibacterial effect of NH Nanozyme obtained in Example 1 is shown in the attached diagram of the instruction manual. Figure 7 As shown.

[0032] Example 2 is the same as Example 1, except that the hydrothermal reaction temperature in Example 2 is 200°C.

[0033] Example 3 is the same as Example 1, except that the hydrothermal reaction time in Example 3 is 8 hours.

[0034] Example 4

[0035] 0.030 g of histidine was dissolved in 20 mL of distilled water, and then 0.018 g of nickel nitrate hexahydrate was added. After stirring until completely dissolved, the mixture was stirred thoroughly for 10 min to obtain a mixture. The mixture was then placed in a hydrothermal reactor and hydrothermally reacted at 180 °C for 12 h. After the reaction was completed, the obtained product was taken out of the hydrothermal reactor, ultrasonically mixed evenly, and freeze-dried to obtain nickel-doped histidine carbon nanozyme.

[0036] Example 5

[0037] Dissolve 0.030 g of histidine in 20 mL of distilled water, then add 0.003 g of nickel acetate tetrahydrate. After stirring until completely dissolved, mix thoroughly for 10 min to obtain a mixture. Then, place the mixture in a hydrothermal reactor and hydrothermally react at 180 °C for 12 h. After the reaction is complete, remove the obtained product from the hydrothermal reactor, sonicate to mix evenly, and freeze-dry to obtain nickel-doped histidine carbon nanozyme.

[0038] Comparative Example 1 is the same as Example 1, except that histidine is replaced with cysteine ​​in Comparative Example 1.

[0039] Comparative Example 2 is the same as Example 1, except that nickel chloride hexahydrate is replaced with ferrous chloride tetrahydrate in Comparative Example 2. The molar amount of ferrous ions in ferrous chloride tetrahydrate is equal to the molar amount of nickel ions in nickel chloride hexahydrate in Example 1.

[0040] Comparative Examples 1 and 2 exhibit POD activity, capable of generating hydroxyl radicals, which turn blue upon the addition of TMB. Example 1, however, lacks POD activity, cannot generate hydroxyl radicals, and does not turn blue upon the addition of TMB.

[0041] The POD activity diagrams of NH Nanozyme obtained in Comparative Examples 1 and 2 are shown in the attached instructions. Figure 6 As shown.

[0042] Performance testing

[0043] The carbon nanozymes obtained in the embodiments and comparative examples of the present invention were subjected to relevant performance tests, and the specific test results are shown in Table 1.

[0044] The test method for photothermal sterilization performance is as follows:

[0045] Take 200 μL of a concentration of 10 8 CFU·mL -1Staphylococcus aureus was mixed with 200 μL of a carbon nanozyme aqueous solution with a concentration of 50-150 μg / mL. After incubation at 37°C for 1 h, the mixture was irradiated with a 1.5 W laser at a wavelength of 808 nm for 10 min. Then, 100 μL of the mixed solution was diluted 20,000 times and plated. The plates were then incubated overnight at 37°C. The number of colonies on the plates was then counted.

[0046] The test method for peroxidase (POD) activity is as follows:

[0047] The nanozymes obtained in the embodiments and comparative examples of the present invention were added in equal amounts to the same volume of 100 mM H2O2 aqueous solution. The same amount of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each test solution, and after standing at room temperature for 20 min, the UV absorption of the solution at 650 nm was measured.

[0048] The testing principle is as follows: If the material possesses POD activity, it will catalyze the generation of a large amount of ·OH from H₂O₂ within a short time. At this time, 3,3',5,5'-tetramethylbenzidine (TMB) in the test solution will combine with hydroxyl radicals and be oxidized to ox-TMB, which will exhibit a characteristic UV absorption peak at 650 nm. Conversely, if the material does not possess POD activity, it cannot catalyze the generation of a large amount of hydroxyl radicals from H₂O₂, and no ox-TMB will be generated in the test solution; therefore, no characteristic UV absorption peak will appear at 650 nm.

[0049] The test method for assessing the ability to scavenge ABTS free radicals is as follows:

[0050] The same mass of the carbon nanozymes obtained in the embodiments of the present invention and the comparative examples were added to the same volume and concentration of ABTS working solution. After standing at room temperature for 20 min, the ultraviolet absorption of the solution at 734 nm was tested.

[0051] The testing principle is as follows: When 2,2-azono-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt reacts with potassium persulfate, it generates the blue-green free radical ABTS· + This free radical exhibits a distinct ultraviolet characteristic absorption at a wavelength of 734 nm. When an antioxidant acts on this free radical, the antioxidant donates electrons to react with ABTS· + Free radicals combine, causing the solution to lighten in color, at which point the absorbance at 734 nm will decrease or disappear.

[0052] The ABTS working solution is prepared by dissolving ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) in deionized water, then adding the same volume of potassium persulfate aqueous solution (dissolved completely by ultrasonication), mixing thoroughly, and then letting it stand at room temperature in the dark for 12 hours.

[0053] The test method for assessing the ability to scavenge DPPH free radicals is as follows:

[0054] The same mass of the carbon nanozymes obtained in the embodiments and comparative examples of the present invention were added to the same volume and concentration of DPPH alcohol solution. After standing at room temperature for 20 min, the presence or absence of UV characteristic absorption at 519 nm was tested. The DPPH alcohol solution is a homogeneous solution formed by dissolving DPPH in anhydrous ethanol.

[0055] The testing principle is as follows: DPPH alcoholic solution is purple, and DPPH has characteristic ultraviolet absorption at 519 nm. It is known that in the presence of antioxidants, DPPH free radicals are eliminated, resulting in a lighter solution color and a decrease or disappearance of ultraviolet absorption at 519 nm.

[0056] The test method for the ability to scavenge hydroxyl radicals (·OH) is as follows:

[0057] FeCl2 was added to an aqueous H2O2 solution and allowed to react for 5-10 minutes. The carbon nanozymes obtained in the examples and comparative examples of this invention were then added to the reaction solution and allowed to stand for 5-10 minutes. Subsequently, 3,3',5,5'-tetramethylbenzidine (TMB) was added, and after standing at room temperature for 20 minutes, the UV absorption of the solution at 650 nm was measured.

[0058] The testing principle is: using the Fenton reaction to generate OH: H₂O₂ + Fe 2+ =OH + H₂O + Fe 3+ If ·OH is present in the test solution, 3,3',5,5'-tetramethylbenzidine (TMB) will combine with ·OH and be oxidized to ox-TMB, which will exhibit a characteristic UV absorption peak at 650 nm. Conversely, if ·OH is absent in the test solution, H₂O₂ cannot be catalyzed to generate a large number of hydroxyl radicals, and therefore ox-TMB will not be produced in the test solution, and thus no characteristic UV absorption peak will appear at 650 nm.

[0059] The method for testing superoxide dismutase (SOD) activity is as follows:

[0060] Methionin and riboflavin were dissolved in PBS, and carbon nanozymes obtained in the examples and comparative examples of this invention were added. After 5 min, NBT was added, and the solution was allowed to stand under ultraviolet light for 30 min. The ultraviolet absorption of the solution at 560 nm was then tested.

[0061] The testing principle is as follows: In the presence of oxidizing substances, riboflavin can be photoreduced. The reduced riboflavin is then readily re-oxidized under aerobic conditions to produce superoxide anion free radicals (O2). - ), superoxide anion radical (·O2) - The enzyme can reduce nitroblue tetrazolium (NBT) to blue formaldehyde hydrazone, which has maximum absorption at 560 nm. Superoxide dismutase (SOD) scavenge superoxide anion radicals, thereby inhibiting formaldehyde hydrazone formation and resulting in reduced UV absorption at 560 nm. Therefore, after the photoreduction reaction, the deeper the blue color of the reaction solution, the lower the enzyme activity, and vice versa.

[0062] Table 1

[0063]

[0064] As shown in Table 1, the carbon nanozymes obtained in the embodiments of the present invention do not possess POD activity, but they do have good bactericidal effects. In contrast, the carbon nanozymes obtained in the comparative examples all possess POD activity.

[0065] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A nickel-doped histidine carbon nanozyme, characterized in that, It is a product generated by the high-temperature hydrothermal reaction of histidine and soluble nickel salt. Under high-temperature hydrothermal conditions, nickel ions form coordination chemical bonds with histidine. The particle size range of histidine carbon nanozymes is 100-300 nm; The temperature for hydrothermal reactions is 150-220℃.

2. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that, The soluble nickel salt includes one or more of nickel chloride, nickel acetate, nickel nitrate, or nickel sulfate.

3. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that, The hydrothermal reaction temperature is 200℃.

4. The nickel-doped histidine carbon nanozyme according to claim 1, characterized in that, The preparation method includes the following steps: Histidine was completely dissolved in water, and soluble nickel salt was added. The mixture was stirred until homogeneous. The resulting mixture was then transferred to a hydrothermal reactor for high-pressure hydrothermal reaction. After the reaction was completed, the product was removed from the hydrothermal reactor, ultrasonically mixed until homogeneous, and then freeze-dried to obtain the doped histidine carbon nanozyme.

5. The nickel-doped histidine carbon nanozyme according to claim 4, characterized in that, The mass ratio of histidine to soluble nickel salt is 1-10:1.

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