Au-Cu-coated MSA nanocluster and preparation method and application thereof
By preparing and embedded in Au-Cu@MSA nanoclusters, the antibacterial defense weakening and tissue necrosis problems at diabetic wounds were solved, and efficient antibacterial and healing effects were achieved.
Patent Information
- Application Number
- CN202510762305.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-09-02
AI Technical Summary
The prior art is difficult to effectively solve the problems of weakening of skin antibacterial defense and tissue necrosis caused by local hypoxia and oxidative stress at diabetic wounds, and traditional nanomaterials have shortcomings in biocompatibility and stability.
The Au-Cu@MSA nanoclusters were prepared by controlling the ratio of gold salts and copper salts to thiomalic acid, and the Au-Cu@MSA nanoclusters with dual enzyme activity were synthesized, and they were embedded in a hydrogel cross-linked chondroitin sulfate and carboxymethyl chitosan to form a multifunctional hydrogel drug.
Au-Cu@MSA nanoclusters show significant antibacterial effects in hydrogels. By generating hydroxyl radicals, they kill drug-resistant strains and consume intracellular glutathione, achieving synergistic antibacterial, anti-inflammatory and regenerative effects of wounds, promoting diabetic wound healing.
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Figure CN120572015A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to an Au-Cu@MSA nanocluster and a preparation method and application thereof, belonging to the technical field of nanomaterial preparation. Background Art
[0002] Diabetes is a chronic metabolic disorder characterized by persistent hyperglycemia. Glucose accumulation, bacterial colonization, and impaired angiogenesis in the patient's wounds can lead to local hypoxia and increased oxidative stress levels, directly weakening the skin's antibacterial defense and causing local ischemia and tissue necrosis, posing a major challenge to wound treatment.
[0003] Given the sensitivity of natural enzymes to pH and oxygen, nanozymes with diverse enzymatic activities have been designed to address the hypoxic microenvironment in diabetic wounds. Gold-based nanomaterials offer advantages such as high biocompatibility, low cytotoxicity, and broad antimicrobial efficacy. Metal nanoclusters (NCs) with quantum sizes <2 nm have become widely used nanomaterials due to their molecular-like properties. Harnessing the enzyme-like activity of metal nanoclusters (NCs) and combining them with other active materials is expected to open new avenues for diabetic wound treatment, significantly advancing the application of metal nanomaterials in biotherapy. Summary of the Invention
[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides an Au-Cu@MSA nanocluster and a preparation method and application thereof.
[0005] In order to achieve the above object, the present invention adopts a method for preparing Au-Cu@MSA nanoclusters, comprising the following steps:
[0006] (1) Use chloroauric acid as the gold source, dissolve it in a solvent, stir, and add thiomalic acid until the color of the solution changes from yellow to light yellow or even colorless;
[0007] (2) Continue to add copper chloride as a copper source, so that the color of the solution changes from colorless to light yellow, and continue to add sodium borohydride aqueous solution, isolating it from air, so that the color of the solution changes to black;
[0008] (3) Add methanol-water solution to wash the black precipitate, which was then dried at room temperature to obtain Au-Cu@MSA nanoclusters.
[0009] As an improvement, the solvent in step (1) is pure water, and the mass volume ratio of the chloroauric acid to pure water is (50-100 mg): (10-15 mL); the mass ratio of the chloroauric acid to thiomalic acid is (1-2): (1-6).
[0010] As an improvement, the mass ratio of the copper chloride in step (2) to the chloroauric acid in step (1) is (0.5-0.7):(1-2).
[0011] As an improvement, the sodium borohydride aqueous solution in step (2) is composed of sodium borohydride and pure water in a mass volume ratio of (40-60 mg): (4-6 mL), and the mass ratio of the sodium borohydride to the chloroauric acid in step (1) is 1: (1-2).
[0012] As an improvement, the methanol-water solution in step (3) is composed of methanol and water in a volume ratio of 5:1.
[0013] In a second aspect, the present invention further provides an Au-Cu@MSA nanocluster prepared by the preparation method; the Au-Cu@MSA nanocluster is uniformly spherical with an average diameter of 1.5-2 nanometers, and the Au, Cu and S elements are uniformly distributed in the cluster; the Au-Cu@MSA nanocluster has dual enzyme activities of peroxidase-like and glutathione peroxidase-like.
[0014] The third aspect of the present invention further provides a use of the Au-Cu@MSA nanoclusters in the preparation of a drug for promoting diabetic wound healing.
[0015] As an improvement, the drug is a hydrogel containing Au-Cu@MSA nanoclusters.
[0016] As an improvement, the hydrogel containing Au-Cu@MSA nanoclusters is formed by embedding Au-Cu@MSA nanoclusters and glucose oxidase into a hydrogel formed by cross-linking oxidized chondroitin sulfate and carboxymethyl chitosan.
[0017] As an improvement, the drug generates hydroxyl radicals, kills methicillin-resistant Staphylococcus aureus and depletes intracellular glutathione levels, thereby achieving an antibacterial effect.
[0018] The reaction mechanism of this invention involves selecting organic thiols (such as thiomalic acid) as ligands based on the distinct coordination properties between different metal atoms (Au, Cu) and ligands. Compared to long-chain organic amines, thiomalic acid offers the following advantages: 1) excellent water solubility and biocompatibility, making it suitable for aqueous synthesis and biomedical applications; 2) the sulfhydryl group (-SH) on thiomalic acid can form stable covalent bonds with Au and Cu atoms. Compared to the coordination bonds formed between long-chain organic amines and Au and Cu atoms, the S-Au / Cu bond energy is stronger, which is crucial for the stability of the nanoparticles; 3) because thiomalic acid forms a -RS-Au / Cu- complex with the metal ions (Au / Cu), it can slowly reduce the metal ions (Au / Cu) under the strong reducing effect of sodium borohydride aqueous solution and effectively prevent their further growth. As a result, the Au-Cu particles obtained with thiomalic acid as a ligand are small, only approximately 2 nm in size, and are well metabolized by the kidney, which is crucial for its future clinical translation. However, when long-chain organic amines are used as ligands, since they only form coordination bonds with the metal ions (Au / Cu), they are rapidly reduced and grow under the strong reducing effect of aqueous sodium borohydride solution. This often results in the formation of relatively large Au-Cu nanoparticles (>50 nm), which are difficult to metabolize through the renal filtration barrier and pose a potential nephrotoxic risk due to long-term accumulation. Therefore, by precisely controlling the ratio of thiomalic acid to metal atoms in the target clusters, as well as the ratios between different metal atoms, the present invention successfully synthesized Au-Cu@MSA NCs with dual enzyme-like activity.
[0019] Compared to existing technologies, this invention successfully prepared stable Au-Cu@MSA NCs using gold and copper salts as metal sources under the protection of an organic thiol ligand. Inductively coupled plasma mass spectrometry (ICP-MS) analysis determined the atomic ratio of gold (Au) to copper (Cu) in the nanoclusters to be 1:2.42. Furthermore, the nanoclusters exhibited dual peroxidase- and glutathione peroxidase-like enzymatic activities, efficiently generating hydroxyl radicals (·OH), effectively killing methicillin-resistant Staphylococcus aureus (MRSA) and depleting intracellular glutathione (GSH), resulting in significant antibacterial effects. This property holds great promise for the application of Au-Cu@MSA NCs in diabetic wound healing. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is the UV-visible absorption spectrum of the AuCu@MSA NCs prepared in Example 1 in pure water;
[0021] Figure 2The high-resolution transmission electron microscopy images of Au-Cu@MSA NCs prepared in Example 1 at 20 nm and 2 nm scales are shown; a is a high-resolution transmission electron microscopy image at a 20 nm scale, and b is a high-resolution transmission electron microscopy image at a 2 nm scale;
[0022] Figure 3 Elemental mapping of Au-Cu@MSA NCs prepared in Example 1;
[0023] Figure 4 Figure 4 is a graph showing the peroxidase activity test of Au-Cu@MSA NCs; a is a UV-visible spectrum of TMB oxidation catalyzed under different conditions; b is a UV-visible spectrum of OPD oxidation catalyzed under different conditions; c is a UV-visible spectrum of TMB oxidation catalyzed by Au-Cu@MSA NCs at different concentrations in the presence of Glu and GOx; d is a UV-visible spectrum of OPD oxidation catalyzed by Au-Cu@MSANCs at different concentrations in the presence of Glu and GOx;
[0024] Figure 5 Graph showing the glutathione peroxidase activity test of Au-Cu@MSA NCs in Example 5; a shows the relationship between the glutathione peroxidase activity of Au-Cu@MSA NCs and the reaction time; b shows the relationship between the glutathione peroxidase activity of Au-Cu@MSA NCs and the reaction concentration;
[0025] Figure 6 Schematic diagram of the biosafety of the hydrogel of the present invention;
[0026] Figure 7 Figure 1 is an in vitro antibacterial evaluation diagram of the hydrogel of the present invention; a is a photo of surviving MRSA colonies after treatment with different hydrogels, b is the MRSA survival rate measured based on OD600, c is a SEM image of MRSA after treatment with different hydrogels, and d is a live-dead staining image of MRSA after treatment with different hydrogels;
[0027] Figure 8 : is an in vivo therapeutic evaluation diagram of the hydrogel of the present invention; a is a representative wound photo taken at a specified time point, and b is a visual image of the wound healing process during the treatment. DETAILED DESCRIPTION
[0028] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.
[0029] Example 1
[0030] A method for preparing Au-Cu@MSA NCs comprises the following steps:
[0031] (1) Weigh 80 mg of chloroauric acid and dissolve it in 10 mL of pure water. Add 350 mg of thiomalic acid and continue dissolving it under stirring until the color of the mixture changes from yellow to light yellow and finally to colorless.
[0032] (2) Under stirring conditions, 45 mg of copper chloride was directly added to the above reaction mixture. After stirring for 5 min, a sodium borohydride aqueous solution formed by dissolving 50 mg of sodium borohydride in 5 mL of pure water was poured into the above reaction solution. A stopper was added to isolate the air. The color of the solution quickly turned black.
[0033] (3) After overnight reaction, the black precipitate was washed three times with methanol-water solution (v:v = 5:1) and dried at room temperature. The product was Au-Cu@MSA NCs.
[0034] Figure 1 This is the UV-visible absorption spectrum of AuCu@MSA NCs prepared in Example 1 in pure water. It can be seen that Au-Cu@MSA NCs have a weak absorption peak near 380 nm, which is different from the obvious plasmon resonance peak observed in larger-sized (8-13 nm) Au-Cu NPs.
[0035] Figure 2 High-resolution transmission electron microscopy images of the Au-Cu@MSA NCs prepared in Example 1 at 20 nm and 2 nm scales show that the Au–Cu @MSA NCs are uniformly spherical with an average diameter of approximately 1.8 nm, reflecting the stability of the Au−Cu@MSA NCs.
[0036] Figure 3 This is the elemental mapping of Au-Cu@MSA NCs prepared in Example 1. It can be seen that Au, Cu, and S elements are uniformly distributed in Au-Cu@MSA NCs, indicating that Au−Cu@MSA NCs formed a highly uniform structure during the synthesis process.
[0037] Example 2
[0038] A method for preparing Au-Cu@MSA NCs comprises the following steps:
[0039] (1) Weigh 60 mg of chloroauric acid and dissolve it in 10 mL of pure water. Add 300 mg of thiomalic acid and continue dissolving it under stirring until the color of the mixture changes from yellow to light yellow and finally to colorless.
[0040] (2) Under stirring conditions, 40 mg of copper chloride was directly added to the above reaction mixture. After stirring for 5 min, a sodium borohydride aqueous solution formed by dissolving 50 mg of sodium borohydride in 5 mL of pure water was poured into the above reaction solution. A stopper was added to isolate the air. The color of the solution quickly turned black.
[0041] (3) After overnight reaction, the black precipitate was washed three times with methanol-water solution (v:v = 5:1) and dried at room temperature. The product was Au-Cu@MSA NCs.
[0042] Example 3
[0043] A method for preparing Au-Cu@MSA NCs comprises the following steps:
[0044] (1) Weigh 100 mg of chloroauric acid and dissolve it in 10 mL of pure water. Add 400 mg of thiomalic acid and continue dissolving it under stirring until the color of the mixture changes from yellow to light yellow and finally to colorless.
[0045] (2) Under stirring conditions, 50 mg of copper chloride was directly added to the above reaction mixture. After stirring for 5 min, a sodium borohydride aqueous solution formed by dissolving 50 mg of sodium borohydride in 5 mL of pure water was poured into the above reaction solution. A stopper was added to isolate the air. The color of the solution quickly turned black.
[0046] (3) After overnight reaction, the black precipitate was washed three times with methanol-water solution (v:v = 5:1) and dried at room temperature. The product was Au-Cu@MSA NCs.
[0047] Example 4
[0048] The peroxidase activity test of the Au-Cu@MSA NCs prepared in Example 1 was carried out as follows:
[0049] TMB (3,3',5,5'-tetramethylbenzidine) and OPD (o-phenylenediamine) were used as chromogenic substrates to evaluate the peroxidase-like activity of the synthesized Au-Cu@MSA NCs.
[0050] The reactions were performed in phosphate-buffered saline (PBS, pH = 4), and the absorbance measurements of ox-TMB and ox-OPD were recorded at 652 nm and 420 nm, respectively, using a UV-visible spectrophotometer.
[0051] Figure 4 The graph of the peroxidase activity test of Au-Cu@MSA NCs is shown in Figure 2. Figure 4 a and Figure 4b It can be seen that in the presence of Glu (glucose) and GOx (glucose oxidase), the absorbance of Au-Cu@MSA NCs is strongest at 652 nm and 420 nm. Figure 4 c and Figure 4 d shows that the X In the presence of , the peroxidase-like activity of Au-Cu@MSA NCs gradually increased as its concentration increased from 0 to 100 μg / mL, indicating that the peroxidase-like activity of Au-Cu@MSA NCs was concentration-dependent.
[0052] Example 5
[0053] The glutathione peroxide activity test of the Au-Cu@MSA NCs prepared in Example 1 was carried out as follows:
[0054] The glutathione peroxidase-like activity of Au-Cu@MSA NCs was evaluated using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH indicator, and its absorbance at 400−412 nm was recorded using a UV-visible spectrophotometer.
[0055] Figure 5 This is the glutathione peroxidase activity test diagram of Au-Cu@MSA NCs. Figure 5 a and Figure 5 b It can be seen that the glutathione peroxidase-like activity of Au-Cu@MSA NCs depends on the reaction time and NCs concentration. Glutathione (GSH) is almost completely depleted at 90 min, and the minimum effective concentration threshold is 75 μg / mL, which shows that Au-Cu@MSA NCs have excellent glutathione peroxidase-like activity.
[0056] Example 6
[0057] The biosafety of Au-Cu@MSA NCs prepared in Example 1 after loading into different hydrogels is as follows:
[0058] CMC-OCS (carboxymethyl chitosan-oxidized chondroitin sulfate) hydrogels were prepared by mixing 30 mg / mL CMC solution with 45 mg / mL OCS solution at a volume ratio of 1:1. Other hydrogels were prepared by the same method except that different NCs (100 μg / mL in the hydrogel) and / or GOx (20 μg / mL in the hydrogel) were added. L929 cells and HaCat cells (5×10 3) were seeded in a 96-well plate and incubated for 24 h. Subsequently, the culture medium was replaced with 100 μL of different hydrogel extracts and the cells were co-cultured for 24 h. 10 μL of MTT (1 mg / mL) was added to each well and cultured for 4 h. The culture medium in the well was placed in 100 μL of dimethyl sulfoxide (DMSO) and shaken for a few minutes. The 96-well plate was placed in a microplate reader (wavelength of 570 nm) to measure the absorbance value.
[0059] Figure 6 As a biosafety result, it can be seen that the viability of L929 and HaCat cells is close to 100% when treated with CMC-OCS (carboxymethyl chitosan-oxidized chondroitin sulfate), Au-Cu / CMC-OCS, and Au-Cu / GOx / CMC-OCS hydrogels.
[0060] Example 7
[0061] In vitro antibacterial evaluation of Au-Cu@MSA NCs prepared in Example 1 after loading into different hydrogels. The specific operation process is as follows:
[0062] PBS, CMC-OCS, Au-Cu / CMC-OCS and Au-Cu / GO X / CMC-OCS hydrogels were incubated with MRSA (containing 10 mM Glu) at 37°C for 12 h and diluted 10 5 The concentration of MRSA samples was increased by 100 μg / mL and the incubation was continued at 37°C for 15 h. The photos were taken and processed using ImageJ. The morphological changes of MRSA were detected by scanning electron microscopy (SEM). MRSA samples from different treatment groups were collected, washed 2 to 3 times with PBS, and fixed with 2.5% glutaraldehyde at 4°C for 4 h. Dehydrated with different concentrations of ethanol gradient, dried at room temperature, and prepared for observation under SEM. In addition, in the presence of 10 mM Glu, MRSA was observed in the presence of PBS, CMC–OCS, Au–Cu / CMC–OCS, and Au–Cu / GO. X The / CMC–OCS hydrogels were cultured at 37°C for 12 h, and SYTO9 and PI were added (in the dark) to culture together. The experimental results were recorded using an inverted fluorescence microscope.
[0063] Figure 7 In vitro antibacterial results were obtained by Figure 7 a It can be seen that the addition of Au-Cu@MSA NCs and GO X AuCu / GO hydrogel X / CMC-OCS showed the most significant antibacterial activity.
[0064] Figure 7b shows that the MRSA survival rate decreased to 8.62% after treatment with Au-Cu / CMC-OCS hydrogel compared with CMC–OCS (56.36%), and the MRSA survival rate further decreased to 3.78% when exposed to Au-Cu / GOx / CMC-OCS hydrogel.
[0065] Figure 7 c shows that the control group (PBS) has intact MRSA morphology, but bacterial cells treated with CMC-OCS and Au-Cu / CMC-OCS hydrogels both show a certain degree of membrane damage. Notably, MRSA treated with Au-Cu / GOx / CMC-OCS hydrogels exhibited severe shrinkage and membrane rupture.
[0066] Bacterial live / dead fluorescence assay ( Figure 7 The results observed in d) are consistent with the SEM results. MRSA treated with Au-Cu / GOx / CMC-OCS hydrogel showed large-scale death, which is in contrast to the high survival rate of bacteria in other treatment groups.
[0067] Example 8
[0068] In Example 1, Au-Cu@MSA NCs were loaded into different hydrogels for in vivo therapeutic evaluation. The specific procedures were as follows: Male BALB / c mice were used to establish a diabetic wound infection model. A diabetic model was established by intraperitoneal injection of 50 mg / kg STZ (dissolved in sodium citrate, pH 4.5) once daily for 5 consecutive days. Blood glucose levels were monitored; mice with blood glucose levels exceeding 16.7 mmol / L were considered diabetic.
[0069] Skin wounds (1 cm × 1 cm) were created in the dorsal region of mice, and 50 μL of methicillin-resistant Staphylococcus aureus (1 × 10 8 CFU / mL) were applied to the wound site, and wound healing was monitored on days 0, 4, 8, and 12.
[0070] Diabetic mice were randomly divided into 4 groups (n = 4):
[0071] (1) PBS (control), (2) CMC-OCS hydrogel, (3) Au-Cu / CMC-OCS hydrogel, and (4) Au-Cu / GOx / CMC-OCS hydrogel. Figure 8 To evaluate the results of hydrogel in vivo treatment, Figure 8 a and Figure 8 b It can be seen that the Au-Cu / GOx / CMC-OCS hydrogel group can promote wound healing.
[0072] The Au-Cu@MSA NCs of the present invention exhibit excellent peroxidase-like and glutathione catalase-like activities, achieving synergistic therapeutic effects by cascade catalytic therapy and promoting wound healing. X Embedded in a hydrogel formed by crosslinking OCS and CMC, a multifunctional Au-Cu / GOx / CMC-OCS hydrogel was successfully constructed. The Schiff bonds in the Au-Cu / GOx / CMC-OCS hydrogel can be destroyed in response to the low pH of the wound environment. This destruction allows the release of chondroitin sulfate (CS), effectively reducing inflammatory factors at the wound site, achieving synergistic antibacterial, anti-inflammatory, and regenerative effects, and providing a powerful platform for effective diabetic wound treatment.
[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A method for preparing Au-Cu@MSA nanoclusters, characterized in that: The following steps are involved: (1) Use chloroauric acid as the gold source, dissolve it in a solvent, stir, and add thiomalic acid until the color of the solution changes from yellow to light yellow or even colorless; (2) Continue to add copper chloride as a copper source, so that the color of the solution changes from colorless to light yellow, and continue to add sodium borohydride aqueous solution, isolating it from air, so that the color of the solution changes to black; (3) A methanol-water solution was added to wash the black precipitate, which was then dried at room temperature to obtain Au−Cu@MSA nanoclusters.
2. The method for preparing Au-Cu@MSA nanoclusters according to claim 1, wherein: The solvent in step (1) is pure water, and the mass volume ratio of the chloroauric acid to pure water is (50-100 mg): (10-15 mL); the mass ratio of the chloroauric acid to thiomalic acid is (1-2): (1-6).
3. The method for preparing Au-Cu@MSA nanoclusters according to claim 1, wherein: The mass ratio of the copper chloride in step (2) to the chloroauric acid in step (1) is (0.5-0.7):(1-2).
4. The method for preparing Au-Cu@MSA nanoclusters according to claim 1, wherein: The sodium borohydride aqueous solution in step (2) is composed of sodium borohydride and purified water in a mass volume ratio of (40-60 mg): (4-6 mL), and the mass ratio of the sodium borohydride to the chloroauric acid in step (1) is 1: (1-2).
5. The method for preparing Au-Cu@MSA nanoclusters according to claim 1, wherein: The methanol-water solution in step (3) is composed of methanol and water in a volume ratio of 5:
1.
6. An Au-Cu@MSA nanocluster, characterized in that: The Au-Cu@MSA nanoclusters are prepared by the preparation method according to any one of claims 1 to 5; the Au-Cu@MSA nanoclusters are uniformly spherical with an average diameter of 1.5-2 nanometers, and the Au, Cu and S elements are uniformly distributed in the clusters; the Au-Cu@MSA nanoclusters have dual enzyme activities of peroxidase-like and glutathione peroxidase-like.
7. Use of the Au-Cu@MSA nanoclusters according to claim 6 in preparing a drug for promoting diabetic wound healing.
8. The use of the Au-Cu@MSA nanoclusters in the preparation of a drug for promoting diabetic wound healing according to claim 7, characterized in that: The drug is a hydrogel containing Au-Cu@MSA nanoclusters.
9. The use of the Au-Cu@MSA nanoclusters in the preparation of a drug for promoting diabetic wound healing according to claim 8, characterized in that: The hydrogel containing Au-Cu@MSA nanoclusters is formed by embedding Au-Cu@MSA nanoclusters and glucose oxidase into a hydrogel formed by cross-linking oxidized chondroitin sulfate and carboxymethyl chitosan.
10. The use of the Au-Cu@MSA nanoclusters in the preparation of a drug for promoting diabetic wound healing according to claim 8, characterized in that: The drug generates hydroxyl radicals, kills methicillin-resistant Staphylococcus aureus and depletes intracellular glutathione levels, thereby achieving an antibacterial effect.