Hypoxia-responsive gold-copper cluster nanoszyme microgel, preparation method and application thereof

Abstract

The application discloses anoxic response type gold copper cluster nanoszyme microgel, a preparation method and application thereof, and belongs to the technical field of nanobiomaterials, and the preparation process of the gold copper cluster nanoszyme microgel comprises the following steps: taking sodium borohydride as a reducing agent, taking D-penicillamine as a surface ligand, reducing a mixed solution of chloroauric acid and copper chloride in an aqueous phase through a one-step method, and obtaining D-penicillamine gold copper cluster nanoszyme; and the D-penicillamine gold copper cluster nanoszyme is loaded in a self-assembled supramolecular microgel of beta-cyclodextrin grafted quaternary ammonium salt chitosan and azobenzene modified chondroitin sulfate, so that the gold copper cluster nanoszyme microgel is obtained.The preparation method of the gold copper cluster nanoszyme microgel is simple and easy to implement, and the reaction condition is mild; the gold copper cluster nanoszyme microgel obtained by the application has efficient enzyme-like catalytic intelligent antibacterial and oxygen supply performance in response to anoxic microenvironment, and has great application potential in the biomedical field such as bacterial pneumonia infection.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials technology, and relates to an oxygen-deficient gold-copper cluster nanoenzyme microgel, its preparation method, and its application. Background Technology

[0002] Bacterial pneumonia has become one of the most challenging infectious diseases globally, with persistently high morbidity and mortality rates in recent years. The overuse of antibiotics has led to a continuous increase in the prevalence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), further complicating anti-infective treatment. Simultaneously, bacterial infections readily form biofilms, where extracellular polymers hinder the penetration of antimicrobial drugs and the diffusion of oxygen and nutrients, resulting in high antibiotic resistance. Furthermore, persistent bacterial infection triggers oxidative stress and persistent inflammation, leading to increased alveolar capillary permeability, pulmonary edema, impeded gas exchange, and local hypoxia. Particularly at the site of infection, the proliferation and metabolic activity of bacterial pathogens increase oxygen consumption, further exacerbating tissue hypoxia and inducing more severe oxidative stress and inflammatory responses.

[0003] Currently, clinical treatment of bacterial pneumonia mainly relies on oral or intravenous antibiotics (such as penicillin and cephalosporins) to kill pathogens. However, this method has many limitations, including antimicrobial resistance, poor drug targeting, and systemic side effects. The application of broad-spectrum antioxidants such as N-acetylcysteine ​​in pneumonia treatment also suffers from poor bioavailability and relatively low efficiency. Nanozymes, due to their low cost, high stability, and ease of large-scale preparation, are expected to become a substitute for natural enzymes and have been widely studied. In particular, peroxidase mimics can catalyze the generation of highly toxic reactive oxygen species from hydrogen peroxide, effectively killing bacteria and inhibiting infection without inducing bacterial resistance. However, most current nanozyme-based catalytic therapies focus primarily on antibacterial effects, failing to effectively alleviate hypoxia and oxidative stress in non-infected areas. Furthermore, most nanozyme preparations lack intelligent controllability during the anti-infection process, making it difficult to achieve precise treatment. Therefore, there is an urgent need to develop intelligent and precise pathogen elimination and hypoxia relief strategies to address the complex pathological problems of bacterial pneumonia and achieve synergistic and efficient treatment of bacterial infection and tissue hypoxia and oxidative stress damage.

[0004] Designing hypoxia-responsive drug delivery systems based on the hypoxic microenvironment of bacterial infection sites and tissue damage sites has become a new strategy for achieving intelligent and precise treatment. Currently, some bioreducing molecules, such as azobenzene derivatives, nitro groups, and quinone groups, can act as trigger switches, reacting to release drugs in a hypoxic microenvironment with high expression of endogenous reductases. This aims to enhance biomembrane permeability, achieve precise treatment, and minimize potential side effects. Furthermore, since both bacterial infection sites and tissue hypoxia damage sites possess unique hypoxic microenvironments, achieving precise antibacterial or hypoxia-relieving antioxidant effects of nanozymes in hypoxic microenvironments is crucial for the synergistic treatment of bacterial infections and tissue hypoxia damage. Due to biofilm formation at infection sites and the accumulation of acidic metabolites caused by anaerobic fermentation in hypoxic environments, the microenvironment of bacterial infection sites is usually weakly acidic, while uninfected sites are typically at a neutral physiological pH. Based on this characteristic, developing nanozymes with pH-dependent enzyme-catalyzed antibacterial or oxidative stress-relieving capabilities is critical. Therefore, constructing a hypoxia-responsive and pH-adaptive nanozyme-catalyzed antibacterial and oxygen-supplying therapeutic system holds promise for achieving highly efficient and controllable treatment of bacterial pneumonia. Summary of the Invention

[0005] To address the challenge of achieving efficient, intelligent, and controllable treatment of bacterial pneumonia infections in existing technologies, this invention aims to provide a hypoxia-responsive gold-copper cluster nanoenzyme microgel, its preparation method, and its applications. This preparation method utilizes ligand design and component regulation to prepare D-penicillamine-protected gold-copper cluster nanoenzymes (DPA-AuCuNCs), endowing them with excellent pH-adaptive enzyme-like catalytic activity. These nanoenzymes are then loaded into a hypoxia-responsive microenvironment microgel based on natural polymers. The resulting gold-copper cluster nanoenzyme microgel exhibits hypoxia-responsive enzyme-like catalytic antibacterial and oxygen-supplying properties. This preparation method employs mild reaction conditions, requires no complex equipment, and is easy to operate. The gold-copper cluster nanoenzyme microgel prepared by this invention not only possesses hypoxia-responsive release properties but also exhibits pH-switchable peroxidase-like (weakly acidic) and catalase-like (neutral) catalytic activities, facilitating intelligent and controllable anti-infection or hypoxia relief effects under different microenvironments. It holds significant application potential in the repair of bacterial pneumonia and tissue defects caused by bacterial infections.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing hypoxia-responsive gold-copper cluster nanoenzyme microgels includes the following steps:

[0008] 1) Using sodium borohydride as a reducing agent and D-type penicillamine as a surface ligand, a mixed solution of chloroauric acid and copper chloride was reduced in an aqueous phase in one step to prepare D-type penicillamine gold-copper cluster nanozyme.

[0009] 2) D-type penicillamine gold-copper cluster nanozymes were loaded into supramolecular microgels self-assembled with β-cyclodextrin-grafted quaternary ammonium chitosan and azobenzene-modified chondroitin sulfate to obtain hypoxia-responsive gold-copper cluster nanozyme microgels.

[0010] Further, step 1) specifically involves mixing chloroauric acid solution and copper chloride solution, then adding D-type penicillamine solution under stirring, followed by sodium borohydride solution, and reacting in an ice bath for 2 hours to obtain D-type penicillamine gold-copper cluster nanozyme.

[0011] Furthermore, the total amount of chloroauric acid and copper chloride, and the ratio of D-type penicillamine to sodium borohydride, are 10 μmol: 20 μmol: 100 μmol; wherein the molar ratio of chloroauric acid to copper chloride is 1.5:1-0.25:1.

[0012] Further, in step 2), the azobenzene-modified chondroitin sulfate is prepared by the following process: chondroitin sulfate with a molecular weight of 30 kDa is subjected to an amidation reaction with p-diaminoazobenzene in an aqueous solution in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain azobenzene-modified chondroitin sulfate.

[0013] Furthermore, the ratio of chondroitin sulfate, p-diaminoazobenzene, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 62.5 mg: 0.85 mg: 38 mg: 34.5 mg.

[0014] Further, the specific process of step 2) is as follows: the D-type penicillamine gold-copper cluster nanozyme is dispersed in a β-cyclodextrin-grafted quaternary ammonium salt chitosan solution, and then mixed and shaken with an azobenzene-modified chondroitin sulfate solution to form an oxygen-deficient responsive gold-copper cluster nanozyme microgel.

[0015] Furthermore, the mass ratio of β-cyclodextrin-grafted quaternary ammonium chitosan to azobenzene-modified chondroitin sulfate is 1:6 to 1:10.8.

[0016] Furthermore, the loading capacity of D-type penicillamine gold-copper cluster nanozymes in hypoxia-responsive gold-copper cluster nanozyme microgels was 40-160 μg / mL.

[0017] A hypoxia-responsive gold-copper cluster nanoenzyme microgel.

[0018] Application of a hypoxia-responsive gold-copper cluster nanoenzyme microgel in the preparation of antibacterial drugs.

[0019] Furthermore, the antibacterial drugs are for treating bacterial infections of pneumonia.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] This invention first selects D-type penicillamine (DPA), a thiol-based small molecule compound with inherent antibacterial activity and excellent metal complexing activity, as a surface ligand to synthesize D-type penicillamine gold-copper cluster nanozymes (DPA-AuCuNCs). Based on the good biocompatibility and stability of gold, the multi-enzyme activity of copper coordination structure, and the synergistic effect of bimetals, the prepared DPA-AuCuNCs exhibit good stability and excellent pH-adaptive enzyme-like catalytic activity. Under acidic conditions, DPA-AuCuNCs exhibit peroxidase-like catalytic activity, catalyzing the generation of highly antibacterial hydroxyl radicals from hydrogen peroxide, thus exerting a highly efficient antibacterial effect and avoiding the problem of traditional antibiotic resistance. Under neutral conditions, the catalase-like activity of DPA-AuCuNCs decomposes hydrogen peroxide into oxygen and water, converting excessively accumulated reactive oxygen species into dissolved oxygen at tissue damage sites, achieving intelligent and controllable anti-infection or hypoxia and oxidative stress relief under different microenvironments. The gold-copper cluster nanoenzyme microgel of this invention exhibits excellent biocompatibility and its preparation method is simple and easy to perform, with mild reaction conditions and no need for complex equipment. In this invention, DPA-AuCuNCs are loaded into a supramolecular microgel based on azobenzene-modified chondroitin sulfate and β-cyclodextrin-grafted quaternary ammonium chitosan to construct a hypoxia-responsive nanoenzyme microgel. This nanoenzyme microgel can intelligently release functional nanoenzymes in response to hypoxic lesions, improving drug delivery efficiency and biosafety under normal physiological conditions. Simultaneously, the particle size of the microgel allows for nebulized inhalation administration, further improving targeting and bioavailability in the treatment of bacterial pneumonia. The quaternary ammonium chitosan in the microgel components possesses inherent antibacterial activity, contributing to synergistic antibacterial efficacy; chondroitin sulfate, as a natural glycosaminoglycan, aids in extracellular matrix remodeling, thereby promoting lung tissue repair. Simultaneously, by combining the pH-adaptive enzyme-like catalytic activity of DPA-AuCuNCs, the nanoenzyme microgel of this invention can exert hypoxia-responsive intelligent catalytic antibacterial and hypoxia-relieving effects, and has great application potential in the repair of tissue defects caused by bacterial pneumonia and other infections and hypoxia. Attached Figure Description

[0022] Figure 1This section describes the enzyme-like catalytic activity of gold-copper cluster nanozymes with different gold-copper ratios obtained in Examples 1-5, Comparative Example 1, and Comparative Example 2 of this invention. (a) represents the peroxide-mimicking enzyme catalytic ability of DPA-AuCuNCs with different AuCu ratios, mainly reflected by the absorbance value measured at 652 nm as a result of their catalytic oxidation of the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to blue in the presence of hydrogen peroxide; (b) represents the catalase-like catalytic activity of different DPA-AuCuNCs, mainly illustrated by measuring the increase in dissolved oxygen after reacting with different DPA-AuCuNCs in a 100 mmol / L hydrogen peroxide solution for 15 minutes.

[0023] Figure 2 This invention describes the peroxidase-like and catalase-like catalytic activities of DPA-AuCuNCs with a gold-copper ratio of 1.25:1 prepared in Example 1 of this invention. (a) shows the peroxidase-like catalytic activity of DPA-AuCuNCs at different pH values ​​obtained by measuring the absorbance at 652 nm; (b) shows the catalase-like catalytic activity of DPA-AuCuNCs under different pH conditions, mainly reflected by measuring the increase in dissolved oxygen after reacting in a 100 mmol / L hydrogen peroxide solution for 10 minutes.

[0024] Figure 3 Figure 1 shows the hydrated particle size of nanoenzyme microgels prepared with different microgel precursor ratios in this invention, and the transmission electron microscopy elemental distribution of nanoenzyme microgels obtained in Example 7. Figure 2 shows the hydrated particle size distribution of microgels prepared in Examples 1 and 6-9. Figure 3 shows the energy dispersive spectroscopy (EDS) scanning region of nanoenzyme microgels. Figures 4, 5, 6, 7, 8 and 9 show the elemental distributions of C, O, S, Au and Cu, respectively.

[0025] Figure 4 This document presents scanning electron microscope (SEM) images of the nanoenzyme microgels of this invention and their hypoxia response performance evaluation. Image (a) is a scanning electron microscope (SEM) image of the nanoenzyme microgels; image (b) is a scanning electron microscope (SEM) image of the microgels after incubation with the chemical hypoxia mimic sodium dithionite (Na₂S₂O₄).

[0026] Figure 5 These are images showing the antibacterial effect of the nanoenzyme microgel of the present invention, wherein (a) shows the antibacterial performance of the nanoenzyme microgel against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1) peroxidase catalysis, and (b) shows the antibacterial effect of the nanoenzyme microgel against MRSA in response to hypoxia.

[0027] Figure 6 This is a characterization of the intracellular catalytic oxygen supply performance of the nanoenzyme microgel of the present invention.

[0028] Figure 7 This invention relates to the therapeutic effect of nanoenzyme microgel on bacterial pneumonia in mice. (a), (b), (c), and (d) show the hematoxylin-eosin (H&E) staining analysis of lung tissue from healthy mice, MRSA-infected mice, MRSA-infected mice treated with nanoenzyme microgel, and MRSA-infected mice treated with nanoenzyme microgel, respectively. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0030] This invention synthesizes gold-copper bimetallic cluster nanozymes in an aqueous phase by selecting thiol small molecules with inherent antibacterial activity as ligands and adjusting the metal components. These nanozymes are then loaded onto azophenyl microgels based on natural polymers to prepare a hypoxia-responsive gold-copper cluster nanozyme microgel for intelligent and controllable treatment of bacterial pneumonia. The invention utilizes DPA, a thiol small molecule compound with inherent antibacterial activity and excellent metal complexing activity, as a surface ligand. The metal atom ratio is further controlled, and a one-step synthesis of D-type penicillamine gold-copper cluster nanozymes, namely DPA-AuCuNCs, is performed in an aqueous phase. Through ligand design and component adjustment, the stability of copper nanoclusters can be enhanced, and their pH-adaptive enzyme-like catalytic activity can be regulated, thereby achieving antibacterial catalysis in acidic environments and oxygen supply catalysis in neutral environments. This enables intelligent anti-infection or hypoxia relief in different microenvironments. Based on this, DPA-AuCuNCs were in situ encapsulated in a supramolecular microgel based on azophenyl-modified chondroitin sulfate and β-cyclodextrin grafted quaternary ammonium salt chitosan. The resulting nanoenzyme microgel can not only intelligently respond to release functional nanoenzymes at hypoxic lesion sites, improving drug delivery efficiency and biosafety under normal physiological conditions, but also further improve the targeting and bioavailability of treatment through nebulized inhalation, achieving a more efficient and precise therapeutic effect for bacterial pneumonia.

[0031] The present invention discloses a method for preparing hypoxia-responsive gold-copper cluster nanoenzyme microgels, comprising the following steps:

[0032] 1) Preparation of DPA-AuCuNCs: DPA-AuCuNCs were prepared by reducing a mixed solution of chloroauric acid and copper chloride in an aqueous phase in a one-step process using sodium borohydride as a reducing agent and DPA as a surface ligand. Specifically, a DPA solution was added to a mixture of chloroauric acid and copper chloride solutions under vigorous stirring, followed by the rapid addition of sodium borohydride solution and reaction in an ice bath for 2 hours.

[0033] The total amount of chloroauric acid and copper chloride, and the ratio of DPA to sodium borohydride are 10 μmol: 20 μmol: 100 μmol; the molar ratio of chloroauric acid and copper chloride is 1.5:1-0.25:1, preferably 1.5:1, 1.25:1, 1:1, 0.5:1 and 0.25:1.

[0034] 2) Preparation of quaternary ammonium chitosan grafted with β-cyclodextrin, a precursor for microgels: First, chitosan was quaternized, and then β-cyclodextrin was grafted onto the quaternary ammonium chitosan using epichlorohydrin as a crosslinking agent. Specifically, chitosan was pre-dispersed in an aqueous acetic acid solution, and then epichlorohydrin trimethylammonium chloride aqueous solution was added dropwise. The mixture was heated and stirred at 55°C for 18 hours. After the reaction was complete, the supernatant was obtained by centrifugation, dialyzed, and then freeze-dried to obtain quaternized chitosan. Then, β-cyclodextrin was dissolved in an aqueous sodium hydroxide solution and stirred at 25°C for 12 hours. Epichlorohydrin was added dropwise, and the mixture was stirred at 25°C for another 4.5 hours. Finally, the quaternary ammonium chitosan solution was added, and the reaction was stopped after stirring at 25°C for 5 hours. The mixture was then dialyzed and freeze-dried for later use.

[0035] 3) Preparation of chondroitin sulfate modified with azobenzene as a microgel precursor: Chondroitin sulfate was modified with p-diaminoazobenzene via an amidation reaction. Specifically, chondroitin sulfate with a molecular weight of 30 kDa was dissolved in a buffer solution at pH 5.5. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was added and stirred for 10 minutes, followed by the addition of N-hydroxysuccinimide and stirring for 5 minutes. Finally, p-diaminoazobenzene dissolved in dimethyl sulfoxide was added and stirred at 25°C in the dark for 4 hours. After the reaction was complete, the mixture was dialyzed and freeze-dried to obtain a pale yellow sponge-like solid, which was then dried and stored at room temperature for later use.

[0036] The ratio of chondroitin sulfate, p-diaminoazobenzene, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide was 62.5 mg: 0.85 mg: 38 mg: 34.5 mg.

[0037] 4) Preparation of hypoxia-responsive gold-copper cluster nanoenzyme microgels: β-cyclodextrin in β-cyclodextrin-grafted quaternary ammonium chitosan and azobenzene in azobenzene-modified chondroitin sulfate form supramolecular self-assembled microgels through host-guest interactions. Specifically, DPA-AuCuNCs are dispersed in a β-cyclodextrin-grafted quaternary ammonium chitosan solution, and then mixed and shaken with an azobenzene-modified chondroitin sulfate solution for 30 seconds to form hypoxia-responsive gold-copper cluster nanoenzyme microgels.

[0038] The mass ratio of β-cyclodextrin-grafted quaternary ammonium salt chitosan to azobenzene-modified chondroitin sulfate is 1:6-1:10.8.

[0039] The loading capacity of DPA-AuCuNCs in hypoxia-responsive gold-copper cluster nanoenzyme microgels ranged from 40 to 160 μg / mL.

[0040] 5) Biomedical Applications: Nanoenzyme microgels and methicillin-resistant Staphylococcus aureus (MRSA) were co-cultured under hypoxic conditions to investigate the antibacterial properties of the microgels. Furthermore, the nanoenzyme microgels were used as an antibacterial drug for the treatment of mice with MRSA-infected bacterial pneumonia via nebulized inhalation to explore its pro-repair effect on bacterial pneumonia.

[0041] Example 1

[0042] 1) Preparation of DPA-AuCuNCs: The molar ratio of chloroauric acid to copper chloride was 1.25:1. 112 μL (50 mmol / L) of chloroauric acid aqueous solution and 88 μL (50 mmol / L) of copper chloride aqueous solution were added to 1.4 mL of ultrapure water. The mixture was placed in an ice bath, and DPA aqueous solution (0.4 mL, 50 mmol / L) was added with vigorous stirring (1500 rpm). Then, freshly prepared sodium borohydride solution (0.5 mL, 0.2 mol / L) was rapidly added, and the reaction was carried out in an ice bath for 2 hours. Afterward, the mixture was placed in a 4°C refrigerator and allowed to stand overnight to allow for sufficient nucleation and growth. The synthesized DPA-AuCuNCs were purified by ethanol precipitation to remove unreacted molecules and ions. Add three times the volume of ethanol to a certain volume of DPA-AuCuNCs, centrifuge (10000 rpm, 10 min) to produce a brownish-black coarse precipitate, then wash the precipitate thoroughly three times with ethanol, vacuum dry it and add ultrapure water to dissolve it to obtain a DPA-AuCuNCs solution, which is stored in a refrigerator at 4℃ for later use.

[0043] 2) Preparation of β-cyclodextrin-grafted quaternary ammonium chitosan: For specific preparation steps, please refer to: Liu, X.; Zhang, Y.; Liu, Y.; Hua, S.; Meng, F.; Ma, Q.; Kong, L.; Pan, S.; Che, Y., Int. J. Biol. Macromol. 2023, 240, 124365. The experimental steps are as follows: 0.5 g chitosan (viscosity 100-200 mPa·s) was dispersed in 18 mL (0.5% v / v) of acetic acid aqueous solution. Then, 0.47 g glycidyltrimethylammonium chloride was dissolved in 1.5 mL of ultrapure water and added dropwise to the above solution. The mixture was stirred at 55 °C for 18 h. After the reaction was complete, the solution was centrifuged at 6500 rpm for 8 minutes. The supernatant was dialyzed for 3 days and then freeze-dried to obtain quaternary ammonium chitosan. β-Cyclodextrin (1.13 g) was dissolved in an aqueous sodium hydroxide solution (8 mL, 1 mol / L) in a flask and stirred at 25 °C for 12 hours. Then, 90 μL of epichlorohydrin was added dropwise, and the mixture was stirred at 25 °C for 4.5 hours. Next, a pre-dispersed aqueous solution of quaternary ammonium chitosan (10 mL, 20 mg / mL) was added, and the reaction was stopped after stirring at 25 °C for 5 hours. The resulting solution was dialyzed against a dialysis bag with a molecular weight cutoff of 12–14 kDa for 5 days, and then freeze-dried to obtain a white, spongy solid, namely β-cyclodextrin-grafted quaternary ammonium chitosan, which was dried at room temperature and stored for later use.

[0044] 3) Preparation of azobenzene-modified chondroitin sulfate: Chondroitin sulfate (62.5 mg) with a molecular weight of 30 kDa was dissolved in 2-morpholine ethanesulfonic acid buffer (5 mL, 10 mM, pH = 5.5), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg) was added and stirred for 10 minutes. Then, N-hydroxysuccinimide (34.5 mg) was added and stirred for 5 minutes. Finally, p-diaminoazobenzene (0.85 mg) dissolved in dimethyl sulfoxide was added, and the mixture was stirred at 25°C in the dark for 4 hours. After the reaction was complete, the mixture was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 12–14 kDa, and then freeze-dried to obtain a pale yellow sponge-like solid, which was then dried at room temperature and stored for later use.

[0045] 4) Preparation of hypoxia-responsive gold-copper cluster nanoenzyme microgels: 48 μg of DPA-AuCuNCs were pre-dispersed in 200 μL of a 0.25% wt β-cyclodextrin-grafted quaternary ammonium chitosan aqueous solution and mixed thoroughly. Then, an equal volume of azobenzene-modified chondroitin sulfate aqueous solution (1.5% wt) was added, and the mixture was immediately vortexed for 30 seconds to form a microgel. The microgel was centrifuged (3000 rpm) for 3 minutes, the supernatant was discarded, and the precipitate was resuspended in ultrapure water. The precipitate was washed three times to obtain the purified hypoxia-responsive gold-copper cluster nanoenzyme microgel. The mass ratio of β-cyclodextrin-grafted quaternary ammonium chitosan to azobenzene-modified chondroitin sulfate was 1:6. The loading capacity of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanoenzyme microgel was 120 μg / mL.

[0046] 5) Biomedical Applications: Gold-copper cluster nanoenzyme microgels and methicillin-resistant Staphylococcus aureus (MRSA) were co-cultured under hypoxic conditions to investigate the antibacterial properties of the microgels. Furthermore, the gold-copper cluster nanoenzyme microgels were used as an antibacterial drug for the treatment of methicillin-resistant Staphylococcus aureus-infected bacterial pneumonia mice via nebulized inhalation to explore its pro-repair efficacy against bacterial pneumonia.

[0047] Example 2

[0048] Same as Example 1, except that in step 1), the total number of moles of metal atoms remains unchanged, and the molar ratio of chloroauric acid to copper chloride is set to 1.5:1, that is, chloroauric acid aqueous solution (120 μL, 50 mmol / L) and copper chloride aqueous solution (80 μL, 50 mmol / L) are added to 1.4 mL of ultrapure water.

[0049] Example 3

[0050] Same as Example 1, except that in step 1), the total number of moles of metal atoms remains unchanged, and the molar ratio of chloroauric acid and copper chloride is set to 1:1, that is, chloroauric acid aqueous solution (100μL, 50mmol / L) and copper chloride aqueous solution (100μL, 50mmol / L) are added to 1.4mL of ultrapure water.

[0051] Example 4

[0052] Similar to Example 1, except that in step 1), the total number of molar metal atoms remains unchanged, and the molar ratio of chloroauric acid to copper chloride is set to 0.5:1, that is, chloroauric acid aqueous solution (67 μL, 50 mmol / L) and copper chloride aqueous solution (133 μL, 50 mmol / L) are added to 1.4 mL of ultrapure water.

[0053] Example 5

[0054] Same as Example 1, except that in step 1), the total number of molar metal atoms remains unchanged, and the molar ratio of chloroauric acid to copper chloride is set to 0.25:1, that is, chloroauric acid aqueous solution (40 μL, 50 mmol / L) and copper chloride aqueous solution (160 μL, 50 mmol / L) are added to 1.4 mL of ultrapure water.

[0055] Example 6

[0056] Same as Example 1, except that in step 4), the mass ratio of β-cyclodextrin-grafted quaternary ammonium salt chitosan to azobenzene-modified chondroitin sulfate is 1:7.2, that is, the concentration of the aqueous solution of β-cyclodextrin-grafted quaternary ammonium salt chitosan is 0.25%wt, and the concentration of the aqueous solution of azobenzene-modified chondroitin sulfate is 1.8%wt.

[0057] Example 7

[0058] Similar to Example 1, except that in step 4), the mass ratio of β-cyclodextrin-grafted quaternary ammonium salt chitosan to azobenzene-modified chondroitin sulfate is 1:8.4, that is, the concentration of the aqueous solution of β-cyclodextrin-grafted quaternary ammonium salt chitosan is 0.25%wt, and the concentration of the aqueous solution of azobenzene-modified chondroitin sulfate is 2.1%wt.

[0059] Example 8

[0060] Same as Example 1, except that in step 4), the mass ratio of β-cyclodextrin-grafted quaternary ammonium salt chitosan to azobenzene-modified chondroitin sulfate is 1:9.6, that is, the concentration of the aqueous solution of β-cyclodextrin-grafted quaternary ammonium salt chitosan is 0.25%wt, and the concentration of the aqueous solution of azobenzene-modified chondroitin sulfate is 2.4%wt.

[0061] Example 9

[0062] Similar to Example 1, except that in step 4), the mass ratio of β-cyclodextrin-grafted quaternary ammonium salt chitosan to azobenzene-modified chondroitin sulfate is 1:10.8, that is, the concentration of the aqueous solution of β-cyclodextrin-grafted quaternary ammonium salt chitosan is 0.25%wt, and the concentration of the aqueous solution of azobenzene-modified chondroitin sulfate is 2.7%wt.

[0063] Example 10

[0064] Same as Example 7, except that in step 4), the loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanoenzyme microgel is 40 μg / mL, that is, the amount of DPA-AuCuNCs in the aqueous solution of β-cyclodextrin-grafted quaternary ammonium chitosan is 16 μg.

[0065] Example 11

[0066] Same as Example 7, except that in step 4), the loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanoenzyme microgel is 80 μg / mL, that is, the amount of DPA-AuCuNCs in the aqueous solution of β-cyclodextrin-grafted quaternary ammonium chitosan is 32 μg.

[0067] Example 12

[0068] Same as Example 7, except that in step 4), the loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanoenzyme microgel is 160 μg / mL, that is, the amount of DPA-AuCuNCs in the aqueous solution of β-cyclodextrin-grafted quaternary ammonium chitosan is 64 μg.

[0069] Comparative Example 1

[0070] Same as Example 1, except that in step 1), the total number of molar metal atoms remains unchanged, and only chloroauric acid aqueous solution (200 μL, 50 mmol / L) is added to prepare DPA-modified gold cluster nanozymes (DPA-AuNCs).

[0071] Comparative Example 2

[0072] Same as Example 1, except that in step 1), the total number of molar metal atoms remains unchanged, and only copper chloride aqueous solution (200 μL, 50 mmol / L) is added to prepare DPA-modified copper cluster nanozymes (DPA-CuNCs).

[0073] Comparative Example 3

[0074] Similar to Example 7, except that DPA-AuCuNCs were not added in step 4), and hypoxia-responsive microgels were prepared.

[0075] Figure 1 This invention characterizes the enzyme-like catalytic activity of a series of gold-copper cluster nanozymes with different gold-copper ratios synthesized in this invention. DPA-AuCuNCs with different gold-copper ratios, as well as DPA-AuNCs and DPA-CuNCs, were obtained from Examples 1-5, Comparative Example 1, and Comparative Example 2, respectively. Figure 1 As shown in Figure (a), the prepared bimetallic DPA-AuCuNCs exhibited significantly higher peroxidase-like catalytic activity than the single-metal DPA-AuNCs and DPA-CuNCs, indicating that the synergistic effect of the bimetals significantly enhanced their peroxidase-like catalytic performance. The catalase-like catalytic activity was characterized by measuring the dissolved oxygen production of different DPA-AuCuNCs in hydrogen peroxide (100 mmol / L, pH = 7.4) solution using a portable dissolved oxygen meter. Figure 1 As shown in (b), the catalase-like catalytic activity increases with decreasing AuCu ratio, indicating that copper plays a key role in regulating the catalase-like catalytic activity of DPA-AuCuNCs. These results demonstrate that this invention has prepared a series of gold-copper cluster nanozymes possessing both peroxidase-like and catalase-like catalytic activities.

[0076] Figure 2 This section characterizes the peroxidase-like and catalase-like catalytic activities of DPA-AuCuNCs obtained in Example 1 of this invention under different pH conditions. Given the regulatory role of multivalent metal components, especially multivalent Cu, in the catalytic activity of DPA-AuCuNCs, its pH-dependent dual-enzyme catalytic activity is further investigated. Figure 2 As shown in (a), under acidic conditions (pH 4.6, 6.0), the absorbance at 652 nm in the hydrogen peroxide-TMB system in the presence of DPA-AuCuNCs was significantly higher than under neutral or alkaline conditions (pH 7.4, 8.2), with the highest absorbance value at pH 6. This result indicates that DPA-AuCuNCs can exert its peroxidase-like activity to catalyze antibacterial activity in the weakly acidic microenvironment of bacterial infection, without harmful effects on normal tissues with a physiologically neutral pH. Simultaneously, due to… Figure 2 As shown in (b), DPA-AuCuNCs exhibit superior catalase-like catalytic activity in neutral or slightly alkaline buffer solutions compared to weakly acidic microenvironments. At pH 7.4, dissolved oxygen in the solution increased by 5 mg / L, enabling it to alleviate oxidative stress induced by hydrogen peroxide in uninfected tissues. Therefore, this favorable pH-dependent enzyme-like catalytic activity of DPA-AuCuNCs allows it to achieve intelligent anti-infection or hypoxia-relieving effects under different microenvironments.

[0077] Figure 3 This describes the hydrated particle size of nanoenzyme microgels prepared with different microgel precursor ratios according to the present invention, and the transmission electron microscopy elemental distribution of the nanoenzyme microgels obtained in Example 7. Figure 3 The hydrated particle size distribution of the microgels prepared in Examples 1 and 6-9 (a) shows that when the ratio of β-cyclodextrin-grafted quaternary ammonium chitosan (QC-β-CD) to azobenzene-modified chondroitin sulfate (CS-Azo) is 1:6-1:10.8, microgels with a hydrated particle size range of 0.5-2.3 micrometers and uniform particle size distribution can be obtained. Furthermore, the particle size of the prepared microgel increases with the increase of the ratio of the two components. Elemental analysis was performed on the nanoenzyme microgel obtained in Example 7. Figure 3 (b) shows the energy spectrum scanning region of the nanoenzyme microgel. Figure 3(c), (d), (e), (f), and (g) are the distribution diagrams of C, O, S, Au, and Cu elements, respectively. It can be seen that the nanoenzyme microgel in Example 7 is a spherical shape with a size of micrometer. The distribution of Au and Cu elements indicates that DPA-AuCuNCs are uniformly distributed inside the microgel.

[0078] Figure 4 This document presents scanning electron microscopy (SEM) images and an evaluation of the hypoxia response performance of the nanoenzyme microgels (NC@mGels) prepared in Example 7 of this invention. Currently, the commonly used chemical hypoxia mimicking agent is sodium dithionite (Na2S2O4), which has strong reducing properties and can rapidly react with oxygen in solution to remove dissolved oxygen. It can also reduce the azo group (-N=N-) in azobenzene to the aniline group (-NH2). Therefore, the hypoxia response performance of NC@mGels was investigated using Na2S2O4, primarily by characterizing the particle size change before and after incubation with Na2S2O4. The SEM results show that the NC@mGels prepared in Example 7 are well-dispersed and uniformly shaped spherical particles. Figure 4 (a) but after incubation with Na2S2O4 for 30 minutes, it degrades into smaller nanoparticles (a). Figure 4 (b)). The average particle size of NC@mGels, obtained from the fitting of the scanning electron microscope image, decreased from 662 nm to 90 nm after incubation with Na2S2O4, indicating that the nanoenzyme microgel of the present invention has excellent hypoxia response performance.

[0079] Figure 5 This is a diagram illustrating the antibacterial effect of the nanoenzyme microgel of the present invention. The peroxidase-like synergistic antibacterial performance of NC@mGels was evaluated in the presence of hydrogen peroxide (H2O2). H2O2 (10 μmol / L) was mixed with mGels (the microgel prepared in Comparative Example 3), NC@mGels (the nanoenzyme microgel prepared in Example 7), and a bacterial suspension (10 μmol / L). 6 Co-incubate with CFU / mL for 4 hours. Then, take 10 μL of the diluted co-incubated bacterial solution and evenly drop it onto an agar plate. Invert the plate and incubate at 37°C for 18 hours. Afterward, photograph and record the number of colonies on the agar plate and perform statistical analysis. Figure 5As shown in (a), both mGels and NC@mGels exhibited good antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA), reducing its survival rate to 11.8% and 6.7%, respectively. This is mainly attributed to the quaternary ammonium salt groups grafted into the microgels and the DPA-AuCuNCs loaded thereon. Furthermore, due to the excellent peroxidase-like activity of DPA-AuCuNCs, they can exert good synergistic antibacterial performance by generating reactive oxygen species under low concentration H2O2 conditions. Therefore, after treatment with 10 μmol / L H2O2 and NC@mGels, the effective killing rate of the material against MRSA was 100% (see [reference needed]). Figure 5 (a)). This enzyme-catalyzed synergistic bactericidal effect was more pronounced against Pseudomonas aeruginosa (PAO1). The survival rate of PAO1 in the NC@mGels+H2O2 treatment group decreased to 16.2% (see [reference needed]). Figure 5 The survival rate of NC@mGels in the middle (a) was significantly lower than that of NC@mGels alone after treatment with PAO1 (76.1%). The antibacterial effect of NC@mGels in response to hypoxia was studied using the plate count method with MRSA as a model bacterium. Figure 5 As shown in (b), the survival rate of MRSA in the NC@mGels co-culture group was only 0.3% under hypoxic conditions, a decrease of 3.5% compared to the normoxic group. This indicates that the release of DPA-AuCuNCs by NC@mGels under hypoxic conditions enhances their antibacterial activity. These results suggest that NC@mGels possess excellent enzyme-like catalytic and hypoxia-responsive antibacterial properties, demonstrating great potential for in vivo anti-infective therapy.

[0080] Figure 6 This invention characterizes the intracellular oxygen supply performance of the nanozyme microgel, wherein DPA-AuCuNCs and NC@mGels are the gold-copper cluster nanozyme and nanozyme microgel obtained in Experiment 7, respectively. The catalytic oxygen supply capacity of the materials for fibroblasts (L929) was determined using the oxygen-sensitive fluorescent probe Ru(dpp)3Cl2. L929 cells were seeded in 8-well coverslips at a density of 2 × 10⁻⁶. 4 Cells / wells were incubated under hypoxic conditions for 18 hours. A hypoxic chamber was used to simulate the hypoxic environment of cell culture, providing 1% oxygen, 5% carbon dioxide, and 95% nitrogen. DPA-AuCuNCs and NC@mGels were co-incubated with H2O2 (100 μmol / L) with L929 cells for 12 hours, followed by further incubation under hypoxic conditions for 4 hours with a Ru(dpp)3Cl2 oxygen-sensitive fluorescent probe. The relative fluorescence intensity of the fluorescent probe (λex = 488 nm) was measured using fluorescence confocal microscopy to reflect the intracellular oxygen level. Figure 6It was found that the control group and the H2O2-only treatment group showed obvious fluorescence signals, indicating low oxygen content. In contrast, the fluorescence intensity of the DPA-AuCuNCs group was reduced, and the NC@mGels treatment group showed the lowest fluorescence signal, indicating that the nanoenzyme microgel in this invention has good intracellular catalytic oxygen supply performance.

[0081] Figure 7 This invention relates to the therapeutic effect of nanoenzyme microgels on bacterial pneumonia in mice. Figure 7 (a), (b), (c), and (d) show the hematoxylin and eosin (H&E) staining analysis of lung tissue from healthy mice, MRSA-infected mice, MRSA-infected mice treated with microgels (mGels prepared in Comparative Example 3), and MRSA-infected mice treated with nanozyme microgels (NC@mGels prepared in Example 7), respectively. MRSA bacterial suspension (20 μL, 10 μL) was inoculated intranasally. 8 A bacterial pneumonia mouse model of MRSA infection was established in BALB / c mice induced with CFU / mL. Mice were treated with nebulized inhalation 24 hours after MRSA infection. Nebulized inhalation was repeated 24 hours later. Lung tissue was collected 72 hours after treatment, fixed with 4% paraformaldehyde, embedded, and sectioned for H&E staining. Results showed that compared with healthy mice (… Figure 7 Compared to (a)), MRSA-infected untreated mice ( Figure 7 In the middle (b) group, numerous inflammatory cells infiltrated the lungs, alveolar walls were edematous, and alveolar structure was severely damaged. 72 hours after nebulization treatment, mice in the MRSA-infected mGels treatment group ( Figure 7 In the middle (c) group of mice, the degree of inflammatory cell infiltration in the lung tissue was moderate, while in the NC@mGels treatment group ( Figure 7 In the middle (d) lung, the alveolar histology was clear and intact, with significantly reduced alveolar wall edema and inflammatory cell infiltration, indicating that the lung tissue had returned to a healthy state. In summary, the hypoxia-responsive gold-copper cluster nanoenzyme microgel prepared in Example 7 exhibits good therapeutic effects on bacterial pneumonia.

[0082] This invention enables the one-step preparation of DPA-AuCuNCs in an aqueous phase through dual regulation of ligands and components. Furthermore, DPA-AuCuNCs are loaded onto an azophenyl natural polymer microgel network to prepare an oxygen-responsive gold-copper cluster nanoenzyme microgel. On one hand, the inherent antibacterial activity of the DPA ligand enhances the antibacterial performance of the gold-copper cluster nanoenzyme. On the other hand, the synergistic effect of the gold-copper bimetallic components endows the gold-copper cluster nanoenzyme with controllable pH-adaptive enzyme-like catalytic activity, which is key to its intelligent and controllable anti-infection or hypoxia relief under different pH microenvironments. In acidic infection microenvironments, the DPA-AuCuNCs of this invention exhibit peroxidase-like catalytic activity, catalyzing hydrogen peroxide to generate highly antibacterial hydroxyl radicals, achieving efficient antibacterial activity and avoiding the problem of traditional antibiotic resistance. In neutral tissue injury sites, its catalase-like activity decomposes hydrogen peroxide into oxygen and water, converting excessively accumulated reactive oxygen species into dissolved oxygen, achieving in-situ oxygen supply and alleviating oxidative stress.

[0083] The main components of the nanoenzyme microgel of this invention are derived from natural active substances, exhibiting excellent biocompatibility. Its preparation method is simple and easy to perform, with mild reaction conditions and no need for complex equipment. The supramolecular microgel constructed from azophenyl-modified chondroitin sulfate and β-cyclodextrin grafted with quaternary ammonium chitosan, loaded with DPA-AuCuNCs, can achieve controlled release at hypoxic lesion sites, further exerting pH-adaptive enzyme-like catalytic activity, improving drug delivery efficiency and biocompatibility under normal physiological conditions. Furthermore, the microgel's particle size allows for nebulized inhalation, further enhancing the targeting and bioavailability for treating bacterial pneumonia. The nanoenzyme microgel of this invention possesses good biocompatibility and excellent hypoxia-responsive intelligent enzyme-like catalytic antibacterial and oxygen-supplying activity, showing great application potential in the repair of tissue damage associated with bacterial infections such as bacterial pneumonia.

[0084] The above description is only of the preferred embodiment of the present invention and should not be construed as limiting the scope of the claims. The present invention is not limited to the above embodiments, and variations in its specific structure are permitted. All variations made within the scope of the independent claims of the present invention are also within the scope of protection of the present invention.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

Claims

1. A method for preparing an oxygen-insensitive gold-copper cluster nanoenzyme microgel, characterized in that, Includes the following steps: 1) Using sodium borohydride as a reducing agent and D-type penicillamine as a surface ligand, a mixed solution of chloroauric acid and copper chloride was reduced in an aqueous phase in one step to prepare a gold-copper cluster nanozyme with pH-adaptive catalytic activity. The total amount of chloroauric acid and copper chloride, and the ratio of D-type penicillamine to sodium borohydride were 10 μmol: 20 μmol: 100 μmol; the molar ratio of chloroauric acid and copper chloride was 1.5:1-0.25:

1. 2) The gold-copper cluster nanozyme was loaded into a supramolecular microgel that was self-assembled with β-cyclodextrin-grafted quaternary ammonium salt chitosan and azobenzene-modified chondroitin sulfate to obtain an oxygen-insensitive gold-copper cluster nanozyme microgel. The azobenzene-modified chondroitin sulfate is prepared by the following process: chondroitin sulfate with a molecular weight of 30 kDa is subjected to an amidation reaction with p-diaminoazobenzene in an aqueous solution in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to obtain azobenzene-modified chondroitin sulfate; the ratio of chondroitin sulfate, p-diaminoazobenzene, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide is 62.5 mg: 0.85 mg: 38 mg: 34.5 mg.

2. The method for preparing hypoxia-responsive gold-copper cluster nanoenzyme microgels according to claim 1, characterized in that, Step 1) The specific process is as follows: After mixing chloroauric acid solution and copper chloride solution, add D-type penicillamine solution while stirring, then add sodium borohydride solution, and react in an ice bath for 2 hours to obtain a pH-adaptive catalytically active gold-copper cluster nanozyme.

3. The method for preparing hypoxia-responsive gold-copper cluster nanoenzyme microgels according to claim 1, characterized in that, Step 2) involves dispersing gold-copper cluster nanozymes in a β-cyclodextrin-grafted quaternary ammonium salt chitosan solution, then mixing and shaking it with an azobenzene-modified chondroitin sulfate solution to form an oxygen-deficient responsive gold-copper cluster nanozyme microgel.

4. The method for preparing hypoxia-responsive gold-copper cluster nanoenzyme microgels according to claim 1, characterized in that, The mass ratio of β-cyclodextrin-grafted quaternary ammonium chitosan to azobenzene-modified chondroitin sulfate was 1:6 to 1:10.

8.

5. The method for preparing hypoxia-responsive gold-copper cluster nanoenzyme microgels according to claim 1, characterized in that, The loading capacity of gold-copper cluster nanozymes in hypoxia-responsive gold-copper cluster nanozyme microgels ranged from 40 to 160 μg / mL.

6. An oxygen-responsive gold-copper cluster nanoenzyme microgel prepared according to any one of claims 1-5.

7. The application of a hypoxia-responsive gold-copper cluster nanoenzyme microgel prepared according to any one of claims 1-5 in the preparation of a smart and controllable drug for treating bacterial pneumonia infection.

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