Gold cluster nanozyme ligands and self-assembled hydrogels and their preparation methods

Abstract

This invention provides a ligand for gold cluster nanozymes and a method for preparing self-assembled gold cluster nanozyme hydrogels using the ligand. The method includes the steps of synthesizing gold cluster nanozymes in an aqueous phase using the ligand via a one-step process, adding the gold cluster nanozymes to a completely dissolved gelling agent hot solution, and allowing it to cool and stand at room temperature to obtain the self-assembled gold cluster nanozyme hydrogel. This invention also provides the self-assembled gold cluster nanozyme hydrogel obtained by the described method. The gold cluster nanozyme ligand of this invention possesses both reducing and assembly properties. The method for preparing hydrogels using it is simple and easy to implement, with mild reaction conditions, requiring no complex equipment or other cross-linking agents. It not only improves the uniformity and stability of gold cluster nanozymes in hydrogels but also endows the hydrogels with excellent biocompatibility and peroxidase-like activity, among other biological properties.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterial preparation technology, and in particular to gold cluster nanoenzyme-based hydrogels with self-assembly properties, their ligands, and preparation methods. Background Technology

[0002] Infectious tissue defects are often accompanied by bacterial infection and excessive inflammation, severely hindering tissue repair and regeneration. Among numerous biomaterials, multifunctional hydrogels are ideal for promoting the regeneration of infected tissues due to their good biocompatibility, biodegradability, and extracellular matrix-like three-dimensional structure. However, most multifunctional hydrogels still fall short of clinical needs due to their limited biological functions and complex preparation processes. Therefore, there is an urgent need to develop a novel hydrogel that can simultaneously meet the requirements of simple processing and multifunctionality.

[0003] In recent years, with the development of nanotechnology and nanomaterials, combining nanomaterials with hydrogels has become a new strategy for constructing multifunctional hydrogels. Among them, gold clusters have been extensively studied in the fields of disease diagnosis and treatment and tissue regeneration due to their excellent pharmacokinetic properties, good biocompatibility, and enzyme-like catalytic activity. In particular, gold clusters with peroxidase-like catalytic properties have shown significant efficacy in highly efficient antibacterial and anti-infective effects. Furthermore, gold clusters have abundant and tunable surface ligands, allowing for precise control of their physicochemical properties and biological functions through the design and screening of suitable ligand structures. Therefore, gold clusters possess unique advantages and enormous potential in constructing multifunctional hydrogels.

[0004] Currently, researchers typically encapsulate gold clusters as independent components within the three-dimensional network structure of hydrogels through in-situ encapsulation. While this method is simple, it is prone to problems such as gold cluster aggregation, leakage, and uneven distribution. Alternatively, cross-linking molecules can be used as functional ligands to modify the surface of gold clusters. The physical and chemical cross-linking reactions between functionalized ligands and other polymers can then be used to construct gold cluster composite hydrogels. This approach can prevent gold cluster leakage and improve the mechanical properties of the hydrogel to some extent; however, the complexity of the preparation process and potential biosafety risks limit its application in tissue regeneration.

[0005] Therefore, in order to further improve the stability, uniformity and ease of preparation of gold cluster-based hydrogels, it is urgent to develop more efficient hydrogel preparation strategies. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for synthesizing gold clusters using functionalized dipeptides with assembly capabilities and multiple biological properties as ligands. This method enables the prepared gold clusters to exhibit excellent assembly performance and utilizes non-covalent interactions such as hydrophilic-hydrophobic interactions as the main driving force to promote the self-assembly process of gold cluster structural units, thereby obtaining gold cluster self-assembled hydrogels. The reaction conditions are mild, the operation is simple, and the prepared gold cluster self-assembled hydrogels also exhibit excellent biocompatibility and peroxidase-like properties.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a ligand for gold cluster nanozymes, wherein the ligand for gold cluster nanozymes possesses both reducing and assembly properties, and the ligand structure is a functionalized dipeptide of 9-fluorenylmethoxycarbonyl-phenylalanine-cysteine, with the following molecular structural formula:

[0008]

[0009] The present invention also provides a method for preparing a gold cluster nanozyme self-assembled hydrogel using the ligand, comprising the following steps:

[0010] Step 1: Using the ligands for the gold cluster nanozyme, the gold cluster nanozyme is synthesized in an aqueous phase in one step.

[0011] Step 2: Add the gold cluster nanozyme to the completely dissolved gelling factor hot solution, and after cooling and standing at room temperature, the gold cluster nanozyme self-assembled hydrogel is obtained.

[0012] Furthermore, the one-step method described in step one specifically refers to:

[0013] Sodium hydroxide aqueous solution was added to the aqueous solution of the ligand for the gold cluster nanozyme to obtain a completely dissolved ligand solution; then, chloroauric acid solution was added to the ligand solution, and the mixture was stirred evenly. The mixture was then reacted under the conditions of heating temperature of 40-90℃ and heating time of 10-18 hours to obtain the gold cluster nanozyme modified with ligand for the gold cluster nanozyme with assembly performance.

[0014] In this study, the molar ratio of the ligand to chloroauric acid in the gold cluster nanozyme was 1.5–1.9:1.

[0015] Furthermore, the concentration of the sodium hydroxide aqueous solution is 9.5–15 mmol / L, so that the one-step method is carried out under alkaline conditions with a pH of 9–12.

[0016] Furthermore, after adding chloroauric acid solution to the ligand solution, the specific steps are as follows:

[0017] At room temperature, stir at 500 rpm for at least 10 to 20 minutes, then set the temperature of the reaction system to 40 to 90°C and stir at 500 rpm for 10 to 18 hours to obtain a pale yellow final reaction solution.

[0018] The final reaction solution was centrifuged at 10,000–12,500 rpm for 10–20 minutes to remove the precipitate. The product was then further purified by ultrafiltration centrifugation 3–6 times using ultrafiltration centrifuge tubes with a cutoff molecular weight of 10 kDa. The purified gold cluster nanozyme was concentrated under vacuum to increase its concentration to 10–24 times the original concentration, and then stored at 4°C for later use.

[0019] Furthermore, the gelling factor described in step two has the structure of 9-fluorenylmethoxycarbonyl-phenylalanine.

[0020] Furthermore, step two specifically involves:

[0021] Gel factor powder was added to phosphate buffer solution with a concentration of 50-200 mmol / L, and then heated at a temperature of 50-80℃ for 10-30 min to fully dissolve the gel factor powder, thus obtaining the gel factor hot solution. Then, an aqueous solution of gold cluster nanozyme with a concentration of 8-17 mg / mL was added to the gel factor hot solution, mixed well, and then cooled and allowed to stand at room temperature for 6-24 h to obtain a gold cluster nanozyme self-assembled hydrogel.

[0022] The mass ratio of gelling factor and gold cluster nanozyme in the gold cluster nanozyme self-assembled hydrogel is 3-5 mg: 0.8-1.7 mg.

[0023] The present invention also provides a gold cluster nanozyme self-assembled hydrogel prepared by the method, wherein the gold cluster nanozyme participates in the construction of the hydrogel fiber structure in an assembled manner, and the gold cluster nanozyme is uniformly distributed in the fiber network structure of the hydrogel.

[0024] The beneficial effects of this invention are as follows: This invention designs a functionalized dipeptide with both assembly and reduction properties and multiple biological properties: 9-fluorenylmethoxycarbonyl-phenylalanine-cysteine ​​(Fmoc-FC) as a ligand, and synthesizes a gold cluster nanozyme with assembly properties and peroxidase-like catalytic properties; the aromatic groups on the 9-fluorenylmethoxycarbonyl and phenylalanine side chains in the ligand structure enable the gold cluster nanozyme to participate in the assembly process of hydrogel fiber structure by utilizing π-π stacking and hydrophobic interactions, and to be uniformly distributed on the formed fiber structure, thereby forming a novel gold cluster nanozyme self-assembled hydrogel.

[0025] This preparation method is simple and easy to implement, with mild reaction conditions. It does not require complex instruments or other cross-linking agents. It can not only improve the uniformity and stability of gold cluster nanozymes in hydrogels, but also endow hydrogels with excellent biocompatibility and peroxidase-like activity and other biological properties.

[0026] The gold cluster nanozymes (Fmoc-FC-AuNCs) prepared by the method of this invention exhibit excellent peroxidase-like activity under acidic conditions. The phenylalanine in the gelling factor (Fmoc-F) of the hydrogel structure can dissociate into H+ in a neutral environment. + Furthermore, an acidic microenvironment is provided for Fmoc-FC-AuNCs to enhance their enzyme-like catalytic performance. This allows the hydrogel to overcome the limitations imposed by environmental factors such as pH on the enzyme-like activity of Fmoc-FC-AuNCs, and still exhibit significant enzyme-like activity under near-neutral and other physiological environments, which is beneficial for its wide application in biomedical fields such as tissue regeneration. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope image of the Fmoc-FC-AuNCs of this invention;

[0028] Figure 2 This is the Fourier transform infrared absorption spectrum of the Fmoc-FC ligand and Fmoc-FC-AuNCs nanozyme of the present invention.

[0029] Figure 3 These are transmission electron microscopy (TEM) images and elemental distribution diagrams of the gold cluster nanoenzyme hydrogel of this invention.

[0030] Figure (a) shows the transmission electron microscope image and energy dispersive spectroscopy scan region of the three-dimensional fiber structure of the hydrogel, while Figures (b), (c), (d), (e) and (f) are the distribution maps of C, N, O, S and Au elements.

[0031] Figure 4 This is a characterization of the peroxidase-like catalytic activity of the self-assembled hydrogel of Fmoc-FC-AuNCs and gold cluster nanozymes of this invention.

[0032] Figure (a) shows the catalytic activity characterization at a reaction environment pH of 6.0, and Figure (b) shows the catalytic activity characterization at a reaction environment pH of 7.4. Detailed Implementation

[0033] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0034] Currently, hydrogels composed of functional nanomaterials have multiple advantages in promoting the repair and regeneration of infected tissues, but their complex preparation process and limited biological functions have affected their further application.

[0035] Therefore, this invention designs a functionalized dipeptide Fmoc-FC with excellent assembly performance and a variety of biological properties. Using it as a ligand, Fmoc-FC-AuNCs nanozymes with assembly characteristics are prepared in one step. Furthermore, a novel gold cluster nanozyme self-assembly hydrogel is constructed under the synergistic effect of the gelling factor Fmoc-F.

[0036] This preparation method, on the one hand, enables gold cluster nanozymes to participate in the construction of hydrogel fiber structures in an assembled manner without relying on other chemical cross-linking agents, simplifying the preparation process of gold cluster-based hydrogels and ensuring the stable and uniform distribution of gold cluster nanozymes in hydrogels; on the other hand, based on the inherent peroxidase-like activity of Fmoc-FC-AuNCs and the unique amphiphilicity of its ligand and gel factor structures, the prepared gold cluster nanozyme self-assembled hydrogels not only have good biocompatibility, but also exhibit excellent enzyme-like catalytic activity and great potential for anti-infection applications.

[0037] To achieve the above objectives, the present invention provides the following specific embodiments:

[0038] Example 1: A ligand for gold cluster nanozymes, wherein the ligand possesses both reducing and assembly properties, and the ligand structure is a functionalized dipeptide of 9-fluorenylmethoxycarbonyl-phenylalanine-cysteine, with the following molecular structural formula:

[0039]

[0040] Example 2: A method for preparing a gold cluster nanozyme self-assembled hydrogel using the ligand provided in Example 1, comprising the following steps:

[0041] Step 1: Using the ligands for the gold cluster nanozyme, a one-step synthesis of the gold cluster nanozyme in the aqueous phase is performed, specifically as follows:

[0042] An aqueous solution of sodium hydroxide (9.5–15 mmol / L) was added to the ligand aqueous solution of the gold cluster nanozyme to allow the reaction to proceed under alkaline conditions with a pH of 9–12, resulting in a completely dissolved ligand solution. Then, a chloroauric acid solution was added to the ligand solution, and the mixture was stirred at 500 rpm for at least 10–20 minutes at room temperature. Subsequently, the temperature of the reaction system was set to 40–90 °C, and the mixture was stirred at 500 rpm for 10–18 hours to obtain a pale yellow final reaction solution.

[0043] The final reaction solution was centrifuged at 10,000–12,500 rpm for 10–20 minutes to remove the precipitate. The product was then further purified by ultrafiltration centrifugation 3–6 times using an ultrafiltration centrifuge tube with a cutoff molecular weight of 10 kDa. The purified gold cluster nanozyme was concentrated under vacuum to increase its concentration to 10–24 times the original concentration. It was then stored at 4°C for later use, thus obtaining a ligand-modified gold cluster nanozyme with assembly properties.

[0044] In this study, the molar ratio of the ligand to chloroauric acid in the gold cluster nanozyme was 1.5–1.9:1.

[0045] Step 2: Add the gelling factor powder to a phosphate buffer solution with a concentration of 50–200 mmol / L. The structure of the gelling factor is 9-fluorenylmethoxycarbonyl-phenylalanine. Then, heat at a temperature of 50–80°C for 10–30 min to fully dissolve the gelling factor powder and obtain the gelling factor hot solution. Then, add an aqueous solution of gold cluster nanozyme with a concentration of 8–17 mg / mL to the gelling factor hot solution, mix well, and cool and stand at room temperature for 6–24 h to obtain a gold cluster nanozyme self-assembled hydrogel.

[0046] The mass ratio of gelling factor and gold cluster nanozyme in the gold cluster nanozyme self-assembled hydrogel is 3-5 mg: 0.8-1.7 mg.

[0047] Example 3: A gold cluster nanozyme self-assembled hydrogel prepared by the method of Example 2, wherein the gold cluster nanozyme participates in the construction of the hydrogel fiber structure in an assembly manner, and the gold cluster nanozyme is uniformly distributed in the fiber network structure of the hydrogel.

[0048] To further explain the present invention and its effects, the following specific experimental examples are provided:

[0049] Specific experimental example 1:

[0050] 1) Preparation of gold cluster nanozymes: Using a functionalized dipeptide with the structure 9-fluorenylmethoxycarbonyl-phenylalanine-cysteine ​​(Fmoc-FC) as a ligand, a one-step reduction of chloroauric acid solution in aqueous phase yielded Fmoc-FC modified gold cluster nanozymes (Fmoc-FC-AuNCs). Specifically, 16.8 mg of Fmoc-FC was dissolved in 0.34 mL of ultrapure water, followed by the addition of 12.6 mL of sodium hydroxide aqueous solution (15 mmol / L). After sonication to ensure complete clarification, 1 mL of chloroauric acid solution (20 mmol / L) was added, and the mixture was stirred at 500 rpm for 10 minutes at room temperature. Subsequently, the reaction system was heated to 50 °C and stirred at 500 rpm for 10 hours to obtain a pale yellow final solution. Then, the product was centrifuged at 12,500 rpm for 15 minutes to remove the precipitate in the solution. The product was further purified by ultrafiltration centrifugation six times using an ultrafiltration centrifuge tube with a cutoff molecular weight of 10 kDa. The purified gold cluster nanozyme was concentrated under vacuum to increase its concentration to 24 times the original concentration, and then stored at 4°C for later use.

[0051] 2) Preparation of gold cluster nanozyme self-assembled hydrogel: 4 mg of 9-fluorenylmethoxycarbonyl-phenylalanine (Fmoc-F) powder was added to 0.9 mL of phosphate buffer (50 mmol / L, pH 7.4), and then heated at 80 °C for 20 minutes to fully dissolve Fmoc-F. Then, 0.1 mL of Fmoc-FC-AuNCs aqueous solution (17 mg / mL) was added to the Fmoc-F hot solution, mixed well, and cooled and allowed to stand at room temperature for 6 hours to obtain gold cluster nanozyme self-assembled hydrogel.

[0052] Specific experimental example 2:

[0053] Similar to specific experimental example 1, except that in step 1), 16.8 mg of Fmoc-FC was dissolved in 0.34 mL of ultrapure water, and then 12.6 mL of sodium hydroxide aqueous solution (9.5 mmol / L) was added to the Fmoc-FC aqueous solution. After sonicating to make the solution completely clear, chloroauric acid solution (1 mL, 20 mmol / L) was added and stirred at 500 rpm for 10 minutes at room temperature.

[0054] When the concentration of sodium hydroxide aqueous solution is not within the range of 9.5–15 mmol / L, Fmoc-FC cannot be completely dissolved after sonication, thus making it impossible to prepare Fmoc-FC-AuNCs. When the concentration of sodium hydroxide aqueous solution is 15 mmol / L, more stable Fmoc-FC-AuNCs with higher overall performance can be obtained.

[0055] Specific Experiment Example 3:

[0056] Similar to specific experimental example 1, except that in step 1), 14.7 mg of Fmoc-FC was dissolved in 0.34 mL of ultrapure water, and then 12.6 mL of sodium hydroxide aqueous solution (15 mmol / L) was added to the Fmoc-FC aqueous solution.

[0057] Specific Experiment Example 4:

[0058] Similar to specific experimental example 3, except that in step 1), 18.6 mg of Fmoc-FC was dissolved in 0.34 mL of ultrapure water, and then 12.6 mL of sodium hydroxide aqueous solution (15 mmol / L) was added to the Fmoc-FC aqueous solution.

[0059] The synthesis conditions of Fmoc-FC-AuNCs were optimized by adjusting the molar ratio of Fmoc-FC to chloroauric acid. When the molar ratio of Fmoc-FC to chloroauric acid was 1.7:1, Fmoc-FC-AuNCs with higher preparation efficiency and higher performance could be obtained.

[0060] Specific experimental example 5:

[0061] Similar to specific experimental example 1, except that in step 1), the temperature of the reaction system was subsequently set to 40°C, and the mixture was stirred at 500 rpm for 10 hours to obtain a pale yellow final reaction solution.

[0062] Specific Experiment Example 6:

[0063] Similar to specific experimental example 5, except that in step 1), the temperature of the reaction system was subsequently set to 90°C, and the mixture was stirred at 500 rpm for 10 hours to obtain a pale yellow final reaction solution.

[0064] Stable Fmoc-FC-AuNCs cannot be synthesized when the temperature of the reaction system is not within the range of 40–90 °C.

[0065] Specific experimental example 7:

[0066] Similar to specific experimental example 1, except that in step 1), the temperature of the reaction system was subsequently set to 50°C, and the mixture was stirred at 500 rpm for 15 hours to obtain a pale yellow final reaction solution.

[0067] Specific experimental example 8:

[0068] Similar to specific experimental example 7, except that in step 1), the temperature of the reaction system was subsequently set to 50°C, and the mixture was stirred at 500 rpm for 18 hours to obtain a pale yellow final reaction solution.

[0069] The stability and fluorescence intensity of Fmoc-FC-AuNCs show a trend of first increasing and then decreasing with the increase of synthesis time. When the synthesis time is 10 hours, the preparation process can be further simplified and the efficiency can be improved while making the gold clusters have higher performance and stability.

[0070] Specific Experiment Example 9:

[0071] Similar to specific experimental example 1, except that in step 2), 3 mg of Fmoc-F powder was added to 0.9 mL of phosphate buffer (50 mmol / L, pH 7.4), and then heated at 80 °C for 20 minutes to fully dissolve Fmoc-F.

[0072] Specific experimental example 10:

[0073] Similar to specific experimental example 9, except that in step 2), 5 mg of Fmoc-F powder was added to 0.9 mL of phosphate buffer (50 mmol / L, pH 7.4), and then heated at 80 °C for 20 minutes to fully dissolve Fmoc-F.

[0074] Specific experimental example 11:

[0075] Similar to specific experimental example 1, except that in step 2), Fmoc-FC-AuNCs aqueous solution (0.1 mL, 8 mg / mL) was added to the Fmoc-F hot solution, mixed well, and then cooled and allowed to stand at room temperature for 6 hours to obtain a gold cluster nanozyme self-assembled hydrogel.

[0076] Specific experimental example 12:

[0077] Similar to specific experimental example 11, except that in step 2), Fmoc-FC-AuNCs aqueous solution (0.1 mL, 11 mg / mL) is added to the Fmoc-F hot solution, mixed well, and then cooled and allowed to stand at room temperature for 6 hours to obtain a gold cluster nanozyme self-assembled hydrogel.

[0078] By adjusting the ratio of Fmoc-F and Fmoc-FC-AuNCs in the hydrogel system, the preparation conditions of the hydrogel were optimized. When the concentrations of Fmoc-F and Fmoc-FC-AuNCs were 4 mg / mL and 1.7 mg / mL, respectively, a gold cluster nanozyme self-assembled hydrogel with a faster gelation rate and higher stability could be obtained.

[0079] The gold cluster nanozyme solution prepared by this invention is a pale yellow liquid. Figure 1 These are transmission electron microscope (TEM) images of Fmoc-FC-AuNCs prepared in Experimental Example 1 of this invention. Figure 1It can be seen that it has uniform size and good dispersibility, with an average particle size of 2.19 nm.

[0080] Figure 2 These are the Fourier transform infrared absorption spectra of the ligand Fmoc-FC selected in this invention and the Fmoc-FC-AuNCs nanozyme prepared in specific experimental example 1. Figure 2 As shown, compared to pure Fmoc-FC ligands, Fmoc-FC-AuNCs located at 2550 cm⁻¹... -1 The disappearance of the thiol (-SH) characteristic peak while the characteristic peaks of other functional groups in the ligand remain indicates the successful preparation of Fmoc-FC-AuNCs and that the ligand structure with assembly properties remains unchanged.

[0081] Figure 3 This is a transmission electron microscope (TEM) image of the Fmoc-FC-AuNCs nanozyme hydrogel prepared in Experimental Example 1 of this invention, wherein... Figure 3 (a) shows the energy spectrum scanning region of the sample. Figure 3 (b), (c), (d), (e), and (f) are the distribution diagrams of C, N, O, S, and Au elements, respectively. The figures show that the hydrogel forms a loose, porous three-dimensional fiber network structure, with S and Au elements uniformly distributed on the fiber structure, indicating that Fmoc-FC-AuNCs are uniformly distributed within the fiber network structure of the hydrogel.

[0082] Figure 4 This paper describes the peroxidase-like catalytic activity characterization of the Fmoc-FC-AuNCs nanozyme and Fmoc-FC-AuNCs nanozyme hydrogel prepared in Experimental Example 1 of this invention. Figure (a) shows the catalytic activity characterization at a reaction environment of pH 6.0, and Figure (b) shows the catalytic activity characterization at a reaction environment of pH 7.4. The peroxidase-like properties of different materials were detected using the substrate o-phenylenediamine (OPD). 100 μL of Fmoc-FC-AuNCs solution or Fmoc-FC-AuNCs hydrogel (Fmoc-FC-AuNCs final concentration 560 μg / mL), 25 μL of OPD solution (final concentration 12.5 mmol / L), and 25 μL of H2O2 solution (final concentration 40 mmol / L) were added sequentially to 150 μL of phosphate buffer at pH 6.0 and 7.4, respectively. The mixture was incubated at 37 °C for 30 minutes, and then the absorption spectra of different samples were recorded using UV-Vis absorption spectroscopy. Fmoc-FC-AuNCs can catalyze the decomposition of H₂O₂ to generate hydroxyl radicals, oxidizing OPD into a yellow oxidation product with a distinct characteristic peak at 450 nm. Figure 4 It can be seen that Fmoc-FC-AuNCs exhibit excellent peroxidase-like activity under acidic conditions. Figure 4 In (a)), the Fmoc-FC-AuNCs nanoenzyme hydrogel can provide an acidic microenvironment for Fmoc-FC-AuNCs, significantly enhancing the peroxidase-like activity of the hydrogel in a neutral environment. Figure 4 (b)

[0083] This invention designs and uses Fmoc-FC as a ligand to synthesize gold cluster nanozymes (Fmoc-FC-AuNCs) with assembly properties. Furthermore, it constructs a self-assembling hydrogel of the gold cluster nanozymes through non-covalent interactions such as hydrophilic-hydrophobic interactions and π-π stacking interactions. Utilizing functionalized dipeptides with assembly properties as ligands not only allows for precise regulation of the biological properties of gold clusters but also enables the synthesized gold cluster nanozymes to participate in the efficient construction of multifunctional hydrogel networks as structural units.

[0084] This invention differs from traditional in-situ coating and chemical cross-linking strategies for preparing gold cluster-based hydrogels. By designing and selecting appropriate ligand structures, gold clusters can participate in the construction of hydrogel networks in an assembled form, thereby effectively avoiding adverse factors such as easy leakage and uneven distribution of gold cluster nanozymes and potential biosafety risks.

[0085] The ligands and gelling factors designed in this invention possess excellent biocompatibility and various potential biological properties. Furthermore, the preparation method is simple, with mild reaction conditions, requiring no complex equipment. This not only simplifies the preparation process of gold cluster-based hydrogels and improves their efficiency but also enhances the stability and uniformity of gold clusters within the hydrogel. The resulting gold cluster nanozyme self-assembled hydrogel exhibits good biocompatibility and excellent enzyme-like activity, and holds promise for killing bacteria through in-situ catalytic generation of hydroxyl radicals, demonstrating significant application potential in the repair and regeneration of infected tissues.

[0086] The above description is merely a preferred experimental example of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a self-assembled hydrogel of gold cluster nanozymes, characterized in that, Includes the following steps: Step 1: Using ligands for gold cluster nanozymes, gold cluster nanozymes are synthesized in an aqueous phase in a one-step process, specifically as follows: Sodium hydroxide aqueous solution was added to the aqueous solution of the ligand for the gold cluster nanozyme to obtain a completely dissolved ligand solution; then, chloroauric acid solution was added to the ligand solution, mixed and stirred evenly, and reacted under the conditions of heating temperature of 40-90℃ and heating time of 10-18 hours to obtain the gold cluster nanozyme modified with ligand for the gold cluster nanozyme. In this case, the molar ratio of the ligand to chloroauric acid in the gold cluster nanozyme is 1.5–1.9:1; The ligand for the gold cluster nanozyme possesses both reducing and assembly properties. The ligand structure is a functionalized dipeptide of 9-fluorenylmethoxycarbonyl-phenylalanine-cysteine, with the following molecular structure: ； Step 2: Add the gold cluster nanozyme to the completely dissolved gelling factor hot solution, and after cooling and standing at room temperature, obtain the gold cluster nanozyme self-assembled hydrogel. Specifically: Gel factor powder was added to phosphate buffer solution with a concentration of 50-200 mmol / L, and then heated at a temperature of 50-80℃ for 10-30 min to fully dissolve the gel factor powder, thus obtaining the gel factor hot solution. Then, an aqueous solution of gold cluster nanozyme with a concentration of 8-17 mg / mL was added to the gel factor hot solution, mixed well, and then cooled and allowed to stand at room temperature for 6-24 h to obtain a gold cluster nanozyme self-assembled hydrogel. The mass ratio of gelling factor and gold cluster nanozyme in the gold cluster nanozyme self-assembled hydrogel is 3-5 mg: 0.8-1.7 mg. The structure of the gelling factor is 9-fluorenylmethoxycarbonyl-phenylalanine.

2. The method for preparing the gold cluster nanozyme self-assembled hydrogel as described in claim 1, characterized in that, The concentration of the sodium hydroxide aqueous solution is 9.5–15 mmol / L, so that the one-step method is carried out under alkaline conditions with a pH of 9–12.

3. The method for preparing the gold cluster nanozyme self-assembled hydrogel as described in claim 1, characterized in that, After adding chloroauric acid solution to the ligand solution, specifically: At room temperature, stir at 500 rpm for at least 10 to 20 minutes, then set the temperature of the reaction system to 40 to 90°C and stir at 500 rpm for 10 to 18 hours to obtain a pale yellow final reaction solution. The final reaction solution was centrifuged at 10,000–12,500 rpm for 10–20 minutes to remove the precipitate. The product was then further purified by ultrafiltration centrifugation 3–6 times using ultrafiltration centrifuge tubes with a cutoff molecular weight of 10 kDa. The purified gold cluster nanozyme was concentrated under vacuum to increase its concentration to 10–24 times the original concentration, and then stored at 4°C for later use.

4. A gold cluster nanozyme self-assembled hydrogel prepared by the method according to any one of claims 1-3, characterized in that, The gold cluster nanozymes participate in the construction of the hydrogel fiber structure in an assembled manner, and the gold cluster nanozymes are uniformly distributed in the fiber network structure of the hydrogel.

Images