Preparation and antibacterial application of dopamine-heme copolymer nanosheet
By self-assembling and synthesizing dopamine-heme copolymer nanosheets in an ammonia/ethanol system, the problems of complex preparation and high toxicity of existing antibacterial materials are solved, and efficient inhibition and sterilization of Gram-positive bacteria are achieved, with good biocompatibility and low toxicity.
Patent Information
- Application Number
- CN202510824234.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-19
- Publication Date
- 2025-09-16
AI Technical Summary
Existing antibacterial materials have problems such as complex preparation process, high cytotoxicity, unassessed environmental impact, and poor antibacterial effect, especially insufficient inhibition and bactericidal effect against Gram-positive bacteria.
Dopamine-heme copolymer nanosheets were self-assembled and synthesized at room temperature in an ammonia/ethanol reaction system using a one-pot method. The preparation process is simple, green and pollution-free, forming regular elliptical nanosheets for the inhibition and sterilization of Gram-positive bacteria.
The prepared dopamine-heme copolymer nanosheets have good biocompatibility and dispersibility, show significant inhibitory and bactericidal effects on Staphylococcus aureus, and have extremely low cytotoxicity, and can achieve efficient sterilization at low concentrations.
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Figure CN120643679A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical polymer materials, and in particular to the preparation of a dopamine-heme copolymer nanosheet and its application in resisting Staphylococcus aureus. Background Art
[0002] Bacteria, tiny, ancient organisms, have a profound impact on human daily life. Aside from some beneficial probiotics, most bacteria cause infectious diseases and pose a threat to human health. Antibiotics were the first drugs used to kill bacteria, but their overuse can lead to bacterial resistance. They can also cause damage to the liver and nervous system, and severely impact hearing and vision.
[0003] Some antibacterial materials that can replace antibiotics, such as quaternary ammonium salts (QAS), metal ions, antimicrobial peptides, graphene oxide (GO) sheets, nitric oxide (NO), etc., have been gradually developed and have shown excellent antibacterial effects. However, they have some shortcomings. The significant cytotoxicity of metal ions / oxides limits the widespread application of these materials; the public safety and environmental impacts of new antibacterial materials such as carbon nanotubes (CNTs) and graphene oxide (GO) have not been fully evaluated. In view of the above reasons, it is still necessary to develop new materials with safe and efficient antibacterial effects. At the same time, the preparation process of the materials needs to be simpler and more convenient, which is more conducive to the popularization and promotion of the materials.
[0004] Polydopamine (PDA) is widely used in the interfacial chemical modification of antibacterial composite materials and functional materials due to its rich chemical properties and unique photothermal, antioxidant, adhesion and biocompatibility. However, the antibacterial ability of PDA is relatively mild and cannot meet higher antibacterial requirements. In order to achieve higher sterilization efficiency, other antibacterial substances still need to be introduced. Therefore, more research is to use PDA as an optimized material in combination with other antibacterial agents to obtain better antibacterial ability. Nano-antibacterial drugs are encapsulated by PDA to obtain better dispersibility and biocompatibility, but the preparation process generally requires multiple steps and the preparation method is cumbersome. Antibacterial substances such as metal ions, antibiotics, QAS can be easily connected to the PDA modified surface through physical adsorption or chemical bonds to achieve controlled release, reduced toxicity and other effects, but they also face the problems of complex preparation process and metal ion cytotoxicity. Summary of the Invention
[0005] The present invention aims to address the aforementioned problems in the prior art by providing a method for preparing dopamine-heme copolymer nanosheets and their antibacterial application. The present invention utilizes a one-pot method to self-assemble dopamine-heme copolymer nanosheets in an ammonia / ethanol reaction system at room temperature. This method is simple, environmentally friendly, and pollution-free, without the need for additional oxidants or surfactants. The resulting elliptical dopamine-heme copolymer nanosheets exhibit regular morphology, good biocompatibility, and excellent antibacterial and bactericidal properties against Gram-positive bacteria (Staphylococcus aureus).
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A method for preparing dopamine-heme copolymer nanosheets comprises the following steps:
[0008] 1) Prepare a mixed solvent of ultrapure water, anhydrous ethanol and ammonia water, and stir thoroughly in a sealed container to mix evenly;
[0009] 2) dissolving hematin in the mixed solvent prepared in step 1) and stirring in a sealed container to fully dissolve it;
[0010] 3) preparing an aqueous solution of dopamine hydrochloride and adding it dropwise to the solution obtained in step 2), and stirring the mixture in a sealed container for reaction;
[0011] 4) After the reaction is completed, the mixture is washed several times by centrifugation with ultrapure water until the supernatant becomes clear and colorless. The resulting precipitate is the dopamine-heme copolymer nanosheet.
[0012] In step 1), the concentration of ammonia water is 25% to 28%.
[0013] In step 1), the volume ratio of ultrapure water, anhydrous ethanol and ammonia water is (1-14):4:0.2.
[0014] In step 3), the volume ratio of water, ethanol, and aqueous ammonia in the final reaction mixture is (5-15):4:0.2.
[0015] In step 3), the final reaction concentration of hemin is 3-10 mg / mL.
[0016] In step 3), the molar ratio of dopamine hydrochloride to hemin is (0.25-4):1.
[0017] In step 3), the stirring reaction temperature is 20°C to 40°C, and the stirring reaction time is 12 to 24 h.
[0018] A dopamine-hemoglobin copolymer nanosheet is prepared by the above preparation method.
[0019] The dopamine-heme copolymer nanosheet has an elliptical morphology, a major axis length of 6 to 8 μm, a minor axis length of 3 to 4 μm, a single layer thickness of 0.5 to 3 nm, and a final structural stable state thickness of 50 to 200 nm. The nanosheet has good dispersibility in aqueous solution.
[0020] The application of the dopamine-heme copolymer nanosheet is used to kill Staphylococcus aureus.
[0021] The dopamine-heme copolymer nanosheets have extremely low cytotoxicity and good biocompatibility, and have a good sterilization effect on Staphylococcus aureus at a relatively low concentration of 10 μg / mL.
[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:
[0023] (1) The preparation method of the present invention is simple and convenient, the reaction solvent is green and environmentally friendly, and the entire reaction process does not require additional energy supply (such as high temperature, high pressure, microwave, etc.).
[0024] (2) The dopamine-heme copolymer nanosheets prepared by the method of the present invention have good monodispersity and biocompatibility.
[0025] (3) The dopamine-heme copolymer nanosheets prepared by the method of the present invention have significant inhibitory and killing effects on Gram-positive bacteria (Staphylococcus aureus), and a lower dose can achieve a higher bacterial activity inhibition rate. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 (a) and (b) are scanning electron micrographs of the dopamine-heme copolymer nanosheets of Example 1, and (c) and (d) are transmission electron micrographs of the dopamine-heme copolymer nanosheets of Example 1.
[0027] Figure 2 (a) shows the elements and their proportions contained in the dopamine-hemoglobin copolymer nanosheets of Example 1 (the peak area represents the element content), and (b) shows the UV-visible absorption spectra of the dopamine-hemoglobin copolymer nanosheets and hemoglobin of Example 1, wherein DA-hematin represents dopamine-hemoglobin copolymer nanosheets and hematin represents hydroxyhematin.
[0028] Figure 3 3 is a comparison of Fourier transform infrared spectra of hydroxyhematin and the dopamine-heme copolymer nanosheet of Example 1; wherein DA-hematin represents dopamine-heme copolymer nanosheet, and hematin represents hydroxyhematin.
[0029] Figure 4 These are atomic force microscopy images of the dopamine-heme copolymer nanosheets of Example 1; wherein, (a) is a three-dimensional atomic force microscopy image of the dopamine-heme copolymer nanosheet, (b) is a two-dimensional atomic force microscopy image of the dopamine-heme copolymer nanosheet, (c) is a height change diagram in the white dotted box area in Figure (b), (d) is a two-dimensional atomic force microscopy image of the single-layer structure, and (e) is the height change of the nanosheet in the area where the white line segment in Figure (d) is located.
[0030] Figure 5 The cytotoxicity results of dopamine-heme copolymer nanosheets of Example 1 at different concentrations on L929 cells (mouse fibroblasts) for 24 h and 48 h are shown.
[0031] Figure 6 The figures show the inhibitory and sterilizing effects of dopamine-heme copolymer nanosheets of Example 1 at different concentrations on Staphylococcus aureus; (a) is a real shot of the inhibitory and sterilizing effects, and (b) is the quantitative result of the inhibition rate. DETAILED DESCRIPTION
[0032] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.
[0033] Example 1
[0034] (1) Preparation of mixed solvent
[0035] Take 9 mL of ultrapure water, 4 mL of anhydrous ethanol, and 0.2 mL of concentrated ammonia (25%-28%), place them in a 25 mL sealed glass container, and stir magnetically for 30 min until the mixture is evenly mixed.
[0036] (2) Dissolving hematin
[0037] To the above mixed solvent was added 83.5 mg (0.132 mmol) of hemin, and the mixture was stirred under sealed conditions for 1 h until it was completely dissolved to obtain a black homogeneous solution.
[0038] (3) Copolymerization
[0039] Prepare dopamine hydrochloride aqueous solution: Dissolve 50 mg (0.264 mmol) of dopamine hydrochloride in 1 mL of ultrapure water. Rapidly add the dopamine hydrochloride aqueous solution dropwise to the solution in step (2) (the total volume ratio of the mixed solution is now water:ethanol:ammonia = 10:4:0.2, and the hematin reaction concentration is 5.88 mg / mL). Stir and react at 25°C under closed conditions for 18 h. The solution gradually turns dark gray.
[0040] (4) Purification
[0041] The reaction solution was centrifuged at 5000 rpm for 15 min, the supernatant was removed, and the precipitate was washed three times with ultrapure water until the supernatant was colorless. Dopamine-heme copolymer nanosheets were obtained after freeze-drying.
[0042] Example 2
[0043] (1) Preparation of mixed solvent
[0044] Take 50 mL of ultrapure water, 40 mL of anhydrous ethanol, and 2 mL of concentrated ammonia (25%-28%), place them in a 250 mL sealed glass container, and stir magnetically for 30 min until the mixture is evenly mixed.
[0045] (2) Dissolving hematin
[0046] To the above mixed solvent was added 835 mg (1.32 mmol) of hemin, and the mixture was stirred under sealed conditions for 1 h until it was completely dissolved to obtain a black homogeneous solution.
[0047] (3) Copolymerization
[0048] Prepare dopamine hydrochloride aqueous solution: dissolve 500 mg (2.64 mmol) of dopamine hydrochloride in 50 mL of ultrapure water.
[0049] The dopamine hydrochloride aqueous solution was rapidly added dropwise to the solution of step (2) (the total volume ratio of the mixed solution at this time was water:ethanol:ammonia water = 10:4:0.2, and the final reaction concentration of hydroxyhematin was 5.88 mg / mL). The reaction was stirred under closed conditions at 25°C for 18 h, and the solution gradually turned dark gray.
[0050] (4) Purification
[0051] The reaction solution was centrifuged at 5000 rpm for 15 min, the supernatant was removed, and the precipitate was washed three times with ultrapure water until the supernatant was colorless. Dopamine-heme copolymer nanosheets were obtained after freeze-drying.
[0052] Example 3
[0053] (1) Preparation of mixed solvent
[0054] Take 9 mL of ultrapure water, 4 mL of anhydrous ethanol, and 0.2 mL of concentrated ammonia (25%-28%), place them in a 25 mL sealed glass container, and stir magnetically for 30 min until the mixture is evenly mixed.
[0055] (2) Dissolving hematin
[0056] To the above mixed solvent was added 48.6 mg (0.0767 mmol) of hemin, and the mixture was stirred under sealed conditions for 1 h until it was completely dissolved to obtain a black homogeneous solution.
[0057] (3) Copolymerization
[0058] Prepare dopamine hydrochloride aqueous solution: dissolve 5 mg (0.026 mmol) of dopamine hydrochloride in 3 mL of ultrapure water.
[0059] The dopamine hydrochloride aqueous solution was rapidly added dropwise to the solution of step (2) (the total volume ratio of the mixed solution at this time was water:ethanol:ammonia water = 12:4:0.2, and the hematin reaction concentration was 3 mg / mL). The reaction was stirred under closed conditions at 25°C for 18 h, and the solution gradually turned dark gray.
[0060] (4) Purification
[0061] The reaction solution was centrifuged at 5000 rpm for 15 min, the supernatant was removed, and the precipitate was washed three times with ultrapure water until the supernatant was colorless. Dopamine-heme copolymer nanosheets were obtained after freeze-drying.
[0062] Example 4
[0063] (1) Preparation of mixed solvent
[0064] Take 5 mL of ultrapure water, 4 mL of anhydrous ethanol, and 0.2 mL of concentrated ammonia (25%-28%), place them in a 25 mL sealed glass container, and stir magnetically for 30 min until the mixture is evenly mixed.
[0065] (2) Dissolving hematin
[0066] To the above mixed solvent was added 142 mg (0.224 mmol) of hemin, and the mixture was stirred under sealed conditions for 1 h until it was completely dissolved to obtain a black homogeneous solution.
[0067] (3) Copolymerization
[0068] Prepare dopamine hydrochloride aqueous solution: dissolve 85 mg (0.448 mmol) of dopamine hydrochloride in 5 mL of ultrapure water.
[0069] The dopamine hydrochloride aqueous solution was rapidly added dropwise to the solution of step (2) (the total volume ratio of the mixed solution at this time was water:ethanol:ammonia water = 10:4:0.2, and the final reaction concentration of hydroxyhematin was 10 mg / mL). The reaction was stirred at 30°C under closed conditions for 24 h, and the solution gradually turned dark gray.
[0070] (4) Purification
[0071] The reaction solution was centrifuged at 5000 rpm for 15 min, the supernatant was removed, and the precipitate was washed three times with ultrapure water until the supernatant was colorless. Dopamine-heme copolymer nanosheets were obtained after freeze-drying.
[0072] Example 5
[0073] (1) Preparation of mixed solvent
[0074] Take 5 mL of ultrapure water, 4 mL of anhydrous ethanol, and 0.2 mL of concentrated ammonia (25%-28%), place them in a 25 mL sealed glass container, and stir magnetically for 30 min until the mixture is evenly mixed.
[0075] (2) Dissolving hematin
[0076] To the above mixed solvent was added 113.6 mg (0.179 mmol) of hemin, and the mixture was stirred under sealed conditions for 1 h until it was completely dissolved to obtain a black homogeneous solution.
[0077] (3) Copolymerization
[0078] Prepare dopamine hydrochloride aqueous solution: dissolve 136 mg (0.716 mmol) of dopamine hydrochloride in 5 mL of ultrapure water.
[0079] The dopamine hydrochloride aqueous solution was rapidly added dropwise to the solution of step (2) (the total volume ratio of the mixed solution at this time was water:ethanol:ammonia water = 10:4:0.2, and the final reaction concentration of hydroxyhematin was 8 mg / mL). The reaction was stirred at 30°C under closed conditions for 24 h, and the solution gradually turned dark gray.
[0080] (4) Purification
[0081] The reaction solution was centrifuged at 5000 rpm for 15 min, the supernatant was removed, and the precipitate was washed three times with ultrapure water until the supernatant was colorless. Dopamine-heme copolymer nanosheets were obtained after freeze-drying.
[0082] Figure 1 (a) and (b) are scanning electron micrographs of dopamine-heme copolymer nanosheets, and (c) and (d) are transmission electron micrographs of dopamine-heme copolymer nanosheets. Figure 1 As shown in the figure, the prepared dopamine-heme copolymer nanosheets are elliptical sheet structures with good monodispersity and regular morphology, with a major axis length of 6~8 μm and a minor axis length of 3~4 μm.
[0083] Figure 2 (a) shows the elements and their proportions contained in the dopamine-heme copolymer nanosheets (the peak area represents the element content), and (b) shows the UV-visible absorption spectrum of the dopamine-heme copolymer nanosheets and heme. Figure 2Panel (a) shows that the dopamine-heme copolymer nanosheets contain four major elements: C, N, O, and Fe. Table 1 calculates the content of each element based on the peak area at the element's location. Reference to the elements and their contents in heme and dopamine indicates that the elemental composition of the resulting dopamine-heme copolymer nanosheets is consistent with that of dopamine and heme. Significant changes in the content of some elements (particularly N and Fe) compared to the monomeric raw materials (see Table 1), combined with changes in the infrared and ultraviolet spectra, indicate a chemical reaction between dopamine and heme.
[0084] Table 1 shows the elemental composition and content of heme, dopamine and dopamine-heme copolymer nanosheets (according to Figure 2 (Calculated from the element peak area in (a)).
[0085] Table 1
[0086]
[0087] Figure 2 In the heme UV-visible absorption spectrum in (b), the absorption peaks at 400 nm, between 500 nm and 600 nm, and at 650 nm represent the characteristic absorption peaks of the heme porphyrin ring. The dopamine-heme copolymer nanosheets retain these characteristic peaks, albeit with a slight red shift. This indicates that the synthesized dopamine-heme copolymer nanosheets retain the heme porphyrin ring structure, but the atoms on the porphyrin ring undergo vibration or rotation due to the reaction with dopamine, resulting in an increase in energy. Furthermore, the red shift in the characteristic peaks in the UV-visible absorption spectrum indicates that π-π stacking between the heme porphyrin rings and between the porphyrin rings and the benzene rings of dopamine increases the atomic energy. These results demonstrate the successful preparation of the dopamine-heme copolymer nanosheets.
[0088] Figure 3 Comparison of Fourier transform infrared spectra of hemin and dopamine-heme copolymer nanosheets. The dopamine-heme copolymer nanosheets have a wavelength of 2830 cm -1 and 2715 cm -1 The peak at 1601 cm represents the stretching vibration of the C-H bonds on the porphyrin ring and the pyrrole ring. -1 The peak at 1362 cm represents the stretching vibration of C=C on the porphyrin ring. -1 The peak at 1130 cm represents the asymmetric stretching vibration of the carboxyl group. -1 The peak at 776 cm represents the coordination between Fe and the porphyrin ring, namely the Fe-N coordination bond. -1The peak at represents the out-of-plane bending vibration of the C-H bond on the porphyrin ring. The dopamine-heme copolymer nanosheets retain the characteristic peaks of the heme porphyrin ring itself and the Fe coordinated to it. Compared with the characteristic peaks of hemin, the peak positions shift slightly due to a shift in the frequency of the chemical bond stretching vibrations caused by the reaction between the atoms on the porphyrin ring and dopamine. These results indicate that the newly synthesized dopamine-heme copolymer nanosheets retain the porphyrin ring structure and the Fe coordination.
[0089] Figure 4 This is an atomic force microscopy image of dopamine-heme copolymer nanosheets. Figure 4 (a) is a three-dimensional atomic force microscopy image of dopamine-heme copolymer nanosheets, (b) is a two-dimensional atomic force microscopy image of dopamine-heme copolymer nanosheets, (c) is the height change diagram in the white dotted box area in Figure (b), (d) is a two-dimensional atomic force microscopy image of a single-layer structure, and (e) is the height change of the nanosheet in the area where the white line segment in Figure (d) is located. Figure 4 (a), (b) and (c) show that the thickness of the finally synthesized dopamine-hemoglobin copolymer nanosheet is about 60 nm, the surface of the nanosheet has a certain degree of roughness, and is formed by a multilayer structure stacking. Figure 4 Images (d) and (e) show a monolayer structure approximately 2 nm thick, believed to be an intermediate in the formation of dopamine-heme copolymer nanosheets. Overall, the final, stable, elliptical dopamine-heme copolymer nanosheets are formed by the self-assembly of these monolayers through π-π stacking.
[0090] Figure 5 The following table shows the cytotoxicity of L929 cells (mouse fibroblasts) treated with varying concentrations of dopamine-heme copolymer nanosheets (solvent: PBS, pH 7.4) for 24 and 48 hours. After incubation of mouse fibroblasts with varying concentrations of nanosheets in complete culture medium supplemented with 10% serum for 24 and 48 hours, cell survival rates remained above 90%, regardless of nanosheet concentration. This demonstrates the material's extremely low cytotoxicity and excellent biocompatibility.
[0091] Figure 6 The inhibition and sterilization effects of different concentrations of dopamine-heme copolymer nanosheets (solvent is PBS at pH 7.4) on Staphylococcus aureus were shown in Figure 2. 1×10 6CFU / mL Staphylococcus aureus, and can inhibit 99.12% of bacteria at a lower concentration of 10 μg / mL. When the concentration of dopamine-heme copolymer nanosheets is 75 μg / mL, it can completely inhibit the growth of Staphylococcus aureus and kill all bacteria, indicating that the synthesized dopamine-heme copolymer nanosheets have a good sterilization effect on Staphylococcus aureus.
[0092] The above embodiments are only preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any equivalent changes, substitutions, and modifications made in accordance with the scope of the present invention and the contents of the specification are included in the scope of protection of the present invention.
Claims
1. A method for preparing dopamine-heme copolymer nanosheets, characterized in that: The following steps are involved: 1) Prepare a mixed solvent of ultrapure water, anhydrous ethanol and ammonia water, and stir thoroughly in a sealed container to mix evenly; 2) dissolving hematin in the mixed solvent prepared in step 1) and stirring in a sealed container to fully dissolve it; 3) preparing an aqueous solution of dopamine hydrochloride and adding it dropwise to the solution obtained in step 2), and stirring the mixture in a sealed container for reaction; 4) After the reaction is completed, the mixture is washed several times by centrifugation with ultrapure water until the supernatant becomes clear and colorless. The resulting precipitate is the dopamine-heme copolymer nanosheet.
2. The method for preparing a dopamine-heme copolymer nanosheet according to claim 1, wherein: In step 1), the concentration of ammonia water is 25% to 28%.
3. The method for preparing a dopamine-heme copolymer nanosheet according to claim 1, wherein: In step 1), the volume ratio of ultrapure water, anhydrous ethanol and ammonia water is (1-14):4:0.
2.
4. The method for preparing a dopamine-heme copolymer nanosheet according to claim 1, wherein: In step 3), the volume ratio of water, ethanol, and aqueous ammonia in the final reaction mixture is (5-15):4:0.
2.
5. The method for preparing a dopamine-heme copolymer nanosheet according to claim 1, wherein: In step 3), the final reaction concentration of hemin is 3-10 mg / mL.
6. The method for preparing a dopamine-heme copolymer nanosheet according to claim 1, wherein: In step 3), the molar ratio of dopamine hydrochloride to hemin is (0.25-4):
1.
7. The method for preparing a dopamine-heme copolymer nanosheet according to claim 1, wherein: In step 3), the stirring reaction temperature is 20°C to 40°C, and the stirring reaction time is 12 to 24 h.
8. A dopamine-heme copolymer nanosheet, characterized in that: Prepared by the preparation method according to any one of claims 1 to 7.
9. The dopamine-heme copolymer nanosheet according to claim 8, wherein: Its morphology is an elliptical nanosheet with a major axis length of 6~8 μm, a minor axis length of 3~4 μm, a single layer thickness of 0.5~3 nm, and a final structural stable state thickness of 50~200 nm. The nanosheet has good dispersibility in aqueous solution.
10. The use of the dopamine-heme copolymer nanosheet according to claim 8, characterized in that: Used to kill Staphylococcus aureus.