Hypoxia response type gold-copper cluster nano-enzyme microgel as well as preparation method and application of hypoxia response type gold-copper cluster nano-enzyme microgel

Abstract

The invention discloses hypoxia response type gold-copper cluster nano-enzyme microgel and a preparation method and application, and belongs to the technical field of nano biological materials.The preparation process of the gold-copper cluster nano-enzyme microgel comprises the steps that sodium borohydride is used as a reducing agent, D-type penicillamine is used as a surface ligand, and the hypoxia response type gold-copper cluster nano-enzyme microgel is prepared; a mixed solution of chloroauric acid and copper chloride is reduced in a water phase through a one-step method, and the D-type penicillamine gold-copper cluster nano-enzyme is prepared; the D-type penicillamine gold-copper cluster nano enzyme is loaded in the supramolecular microgel which is self-assembled by the beta-cyclodextrin grafted quaternary ammonium salt chitosan and the azobenzene modified chondroitin sulfate, so that the D-type penicillamine gold-copper cluster nano enzyme is obtained. The preparation method of the gold-copper cluster nano-enzyme microgel is simple and easy to implement, the reaction condition is mild, and the gold-copper cluster nano-enzyme microgel obtained through the method has efficient enzyme-like catalysis intelligent antibacterial and oxygen supply performance responding to an anoxic microenvironment and has huge application potential in the biomedical field of bacterial pneumonia infection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-biomaterials and relates to an oxygen-responsive gold-copper cluster nanoenzyme microgel and a preparation method and application thereof. Background Art

[0002] Bacterial pneumonia has become one of the most challenging infectious diseases in the world, with high morbidity and mortality rates in recent years. With the overuse of antibiotics, the prevalence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus has continued to rise, further exacerbating the difficulty of anti-infection treatment. At the same time, bacterial infections are prone to form biofilms, in which extracellular polymers hinder the penetration of antimicrobial drugs and the diffusion of oxygen and nutrients, making bacteria highly resistant to antibiotics. In addition, persistent bacterial infection can induce oxidative stress and persistent inflammation, leading to increased alveolar capillary permeability, the formation of pulmonary edema, obstruction of gas exchange, and local hypoxia. Especially in the site of infection, the proliferation and metabolic activity of bacterial pathogens increase oxygen consumption, further aggravating tissue hypoxia and inducing more severe oxidative stress and inflammatory responses.

[0003] At present, the clinical treatment of bacterial pneumonia mainly relies on oral or intravenous antibiotics (such as penicillin and cephalosporin) to kill pathogenic bacteria. However, this approach has many limitations, including antimicrobial resistance, poor drug targeting, and systemic side effects. The use of broad-spectrum antioxidants such as N-acetylcysteine ​​in the treatment of pneumonia also has problems with unsatisfactory bioavailability and relatively low efficiency. Nanozymes are widely studied as a substitute for natural enzymes due to their low cost, high stability, and easy large-scale preparation. In particular, peroxidase mimetic enzymes can effectively kill bacteria and inhibit infection by catalyzing hydrogen peroxide to generate highly toxic reactive oxygen species without causing bacterial resistance. However, most of the current nanozyme-based catalytic therapies focus on antibacterial effects, and it is difficult to effectively alleviate hypoxia and oxidative stress in non-infected areas. At the same time, most nanozyme preparations lack intelligent controllability in the anti-infection process, making it difficult to achieve the purpose of precision treatment. Therefore, there is an urgent need to develop intelligent and precise pathogen elimination and hypoxia relief strategies to solve the complex pathological problems of bacterial pneumonia and achieve synergistic and efficient treatment of bacterial infection and tissue hypoxia and oxidative stress damage.

[0004] Based on the hypoxic microenvironment of bacterial infection sites and tissue damage sites, designing a hypoxia-responsive drug delivery system has become a new strategy for achieving intelligent precision treatment. At present, some bioreduction molecules such as azobenzene derivatives, nitro groups, and quinone groups can be used as trigger switches to react and release drugs under the stimulation of the hypoxic microenvironment with high expression of endogenous reductases, so as to enhance biofilm penetration, achieve precision treatment, and minimize potential side effects. In addition, since both bacterial infection sites and tissue hypoxia damage sites have unique hypoxic microenvironments, how to achieve the precise antibacterial or hypoxia-relief antioxidant effects of nanozymes in hypoxic microenvironments is crucial for the synergistic treatment of bacterial infection and tissue hypoxia damage. Due to the formation of biofilms at the infection site and the accumulation of acid metabolites caused by anaerobic fermentation in the hypoxic environment, the microenvironment of the bacterial infection site is usually weakly acidic, while the uninfected site is usually at a neutral physiological pH. Based on this feature, it is very critical to develop nanozymes with pH-dependent enzyme-catalyzed antibacterial or oxidative stress relief capabilities. Therefore, constructing a hypoxia-responsive and pH-adaptive nanozyme-catalyzed antibacterial and oxygen supply therapeutic system is expected to achieve efficient and controllable treatment of bacterial pneumonia. Summary of the invention

[0005] In view of the problem that it is difficult to achieve efficient, intelligent and controllable treatment of bacterial pneumonia infection in the prior art, the purpose of the present invention is to provide a hypoxia-responsive gold-copper cluster nanozyme microgel and a preparation method and application. The preparation method prepares a D-type penicillamine-protected gold-copper cluster nanozyme (DPA-AuCuNCs) through the dual effects of ligand design and component regulation, giving it excellent pH-adaptable enzyme-like catalytic activity, and loading it in a hypoxic microenvironment-responsive microgel based on natural polymers. The obtained gold-copper cluster nanozyme microgel has hypoxia-responsive enzyme-like catalytic antibacterial and oxygen supply performance. The preparation method has mild reaction conditions, does not require complex instruments and equipment, and is easy to operate. The gold-copper cluster nanozyme microgel prepared by the present invention not only has hypoxia-responsive release performance, but also has pH-switchable peroxidase-like (weakly acidic) and catalase (neutral) catalytic activity, which helps to achieve intelligent and controllable anti-infection or hypoxia relief under different microenvironments, and has great application potential in the repair of bacterial pneumonia and tissue defects caused by bacterial infection.

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

[0007] A method for preparing hypoxia-responsive gold-copper cluster nanozyme microgel comprises the following steps:

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

[0009] 2) Load the D-penicillamine gold-copper cluster nanozyme into the supramolecular microgel self-assembled from β-cyclodextrin-grafted quaternary ammonium chitosan and azobenzene-modified chondroitin sulfate to obtain the hypoxia-responsive gold-copper cluster nanozyme microgel.

[0010] Further, the specific process of step 1) is as follows: Mix the chloroauric acid solution and the copper chloride solution, add the D-penicillamine solution under stirring, then add the sodium borohydride solution, and react for 2 hours under ice bath to obtain the D-penicillamine gold-copper cluster nanozyme.

[0011] Further, the total amount of chloroauric acid and copper chloride, and the dosage ratio of D-penicillamine to sodium borohydride is 10 μmol: 20 μmol: 100 μmol; among them, 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 through the following process: React chondroitin sulfate with a molecular weight of 30 kDa and p-diaminoazobenzene in an aqueous solution in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide for amidation reaction to obtain the azobenzene-modified chondroitin sulfate.

[0013] Further, the dosage 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: Disperse the D-penicillamine gold-copper cluster nanozyme in the β-cyclodextrin-grafted quaternary ammonium chitosan solution, and then mix and shake it with the azobenzene-modified chondroitin sulfate solution to form the hypoxia-responsive gold-copper cluster nanozyme microgel.

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

[0016] Further, the loading amount of the D-penicillamine gold-copper cluster nanozyme in the hypoxia-responsive gold-copper cluster nanozyme microgel is 40 - 160 μg / mL.

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

[0018] An application of a hypoxia-responsive gold-copper cluster nanozyme microgel in the preparation of antibacterial drugs.

[0019] Further, the antibacterial drug is an anti-bacterial infectious pneumonia drug.

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

[0021] The present invention first selects a thiol small molecule compound D-penicillamine (DPA) with inherent antibacterial effect and excellent metal complexing activity as a surface ligand to synthesize D-penicillamine gold-copper cluster nanozyme (DPA-AuCuNCs). Based on the good biocompatibility and stability of gold elements, the multi-type enzyme activity of the copper coordination structure and the bimetallic synergy, the prepared DPA-AuCuNCs have good stability and excellent pH-adaptive enzyme-like catalytic activity. Under acidic conditions, DPA-AuCuNCs have peroxidase-like catalytic activity, catalyzing hydrogen peroxide to generate hydroxyl radicals with high antibacterial activity, exerting efficient antibacterial efficacy and avoiding the problem of traditional antibiotic resistance; under neutral conditions, based on the catalase-like activity of DPA-AuCuNCs, hydrogen peroxide is decomposed into oxygen and water, which can convert excessively accumulated reactive oxygen into dissolved oxygen at the site of tissue damage, and realize intelligent and controllable anti-infection or hypoxia and oxidative stress relief under different microenvironments. The gold-copper cluster nanozyme microgel of the present invention has excellent biocompatibility and its preparation method is simple and easy, the reaction conditions are mild, and no complex instruments and equipment are required. In the present invention, DPA-AuCuNCs are loaded in a supramolecular microgel based on azobenzene-modified chondroitin sulfate and β-cyclodextrin grafted quaternary ammonium chitosan to construct an hypoxia-responsive nanozyme microgel. The nanozyme microgel can intelligently respond to the release of functional nanozymes at the hypoxic lesion site, improve the delivery efficiency of the drug and the biosafety under normal physiological conditions, and the particle size of the microgel allows it to be administered by aerosol inhalation, further improving the targeting and bioavailability in the treatment of bacterial pneumonia. The quaternary ammonium chitosan in the microgel component has inherent antibacterial activity, which helps to synergistically exert antibacterial efficacy; chondroitin sulfate, as a natural glycosaminoglycan, helps to remodel the extracellular matrix, thereby promoting the lung tissue repair process. At the same time, combined with the pH-adaptive enzyme-like catalytic activity of DPA-AuCuNCs, the nanozyme microgel of the present invention can exert hypoxia-responsive intelligent catalytic antibacterial and hypoxia-alleviating effects, and has great application potential in the repair of tissue defects caused by infections such as bacterial pneumonia and hypoxia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Characterization of the enzyme-like catalytic activities of different Au-Cu ratio Au-Cu cluster nanozymes obtained in Examples 1-5, Comparative Example 1 and Comparative Example 2 of the present invention. Among them, (a) shows the peroxidase-mimicking catalytic ability of DPA-AuCuNCs with different Au / Cu ratios, which is mainly reflected by measuring the absorbance value at 652 nm due to the catalytic oxidation of the substrate 3,3',5,5'-tetramethylbenzidine (TMB) to turn blue in the presence of hydrogen peroxide; (b) shows the catalytic activity of different DPA-AuCuNCs as catalase, which is mainly illustrated by measuring the increased dissolved oxygen after different DPA-AuCuNCs react in a 100 mmol / L hydrogen peroxide solution for 15 minutes.

[0023] Figure 2 Characterization of the peroxidase-like and catalase catalytic activities of DPA-AuCuNCs with an Au-Cu ratio of 1.25:1 prepared in Example 1 of the present invention. Among them, (a) shows the peroxidase-like catalytic activity of DPA-AuCuNCs obtained by measuring the absorbance value at 652 nm at different pH values; (b) shows the catalytic activity of DPA-AuCuNCs as catalase under different pH conditions, which is mainly reflected by measuring the increased dissolved oxygen after it reacts in a 100 mmol / L hydrogen peroxide solution for 10 minutes.

[0024] Figure 3 The hydrodynamic diameter of nanozyme microgels prepared with different microgel precursor ratios in the present invention and the elemental distribution of nanozyme microgels obtained in Example 7 by transmission electron microscopy. Among them, (a) shows the hydrodynamic diameter distribution of microgels prepared in Example 1 and Examples 6-9; Figure (b) shows the energy spectrum scanning area of nanozyme microgels, and Figures (c), (d), (e), (f) and (g) are the distribution maps of C, O, S, Au and Cu elements respectively.

[0025] Figure 4 Scanning electron microscopy images and evaluation of the hypoxia-responsive performance of nanozyme microgels of the present invention. Among them, (a) shows the scanning electron microscopy image of nanozyme microgels; (b) shows the scanning electron microscopy image of microgels after incubation with the chemical hypoxia mimetic sodium dithionite (Na 2 S 2 O 4 ).

[0026] Figure 5 Antibacterial effect diagrams of nanozyme microgels of the present invention. Among them, (a) shows the peroxidase-like catalytic antibacterial performance of nanozyme microgels against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAO1), and (b) shows the hypoxia-responsive antibacterial effect of nanozyme microgels against MRSA.

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

[0028] Figure 7 It is the therapeutic effect of the nanozyme microgel of the present invention on mice with bacterial pneumonia, wherein (a), (b), (c) and (d) are hematoxylin-eosin staining (H&E) analysis of lung tissues of healthy mice, MRSA infection group mice, MRSA infection microgel treatment group mice, and MRSA infection nanozyme microgel treatment group mice, respectively. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly and comprehensively understood.

[0030] The present invention selects thiol small molecules with inherent antibacterial activity as ligands and adjusts the metal components to synthesize gold-copper bimetallic cluster nanozymes in the aqueous phase, and loads them in azobenzene microgels based on natural polymers to prepare a hypoxia-responsive gold-copper cluster nanozyme microgel for intelligent and controllable treatment of bacterial pneumonia. DPA, a thiol small molecule compound with inherent antibacterial effect and excellent metal complexing activity, is selected as a surface ligand, and the metal atomic ratio is further adjusted to synthesize D-type penicillamine gold-copper cluster nanozymes, namely DPA-AuCuNCs, in the aqueous phase using a one-step method. In the present invention, the stability of copper nanoclusters can be improved through ligand design and component adjustment, and its pH-adaptive enzyme-like catalytic activity can be adjusted, thereby achieving the catalytic antibacterial effect in acidic environment and the catalytic oxygen supply effect in neutral environment, and realizing the role of intelligent anti-infection or hypoxia relief under different microenvironments. On this basis, DPA-AuCuNCs were in situ encapsulated in supramolecular microgels based on azobenzene-modified chondroitin sulfate and β-cyclodextrin grafted quaternary ammonium chitosan. The resulting nanozyme microgels can not only intelligently respond to the release of functional nanozymes at the hypoxic lesions, improve the drug delivery efficiency and biosafety under normal physiological conditions, but also deliver the drug through aerosol inhalation, further improving the targeting and bioavailability of the treatment, and achieving more efficient and precise treatment effects for bacterial pneumonia.

[0031] The preparation method of the hypoxia-responsive gold-copper cluster nanozyme microgel of the present invention comprises the following steps:

[0032] 1) Preparation of DPA-AuCuNCs: Using sodium borohydride as a reducing agent and D-type penicillamine DPA as a surface ligand, a mixed solution of chloroauric acid and cupric chloride was reduced in an aqueous phase by a one-step method to prepare DPA-AuCuNCs. Specifically, the chloroauric acid solution and the cupric chloride solution were mixed, and then the DPA solution was added under vigorous stirring, and then the sodium borohydride solution was quickly added and reacted in an ice bath for 2 hours.

[0033] The total amount of chloroauric acid and cupric chloride, and the dosage ratio of DPA to sodium borohydride are 10 μmol: 20 μmol: 100 μmol; the molar ratio of chloroauric acid and cupric 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 as microgel precursor: First, chitosan is quaternized, and then β-cyclodextrin is grafted onto quaternary ammonium chitosan using epichlorohydrin as a crosslinking agent. The specific process is as follows: chitosan is pre-dispersed in an acetic acid aqueous solution, and then an aqueous solution of epoxypropyltrimethylammonium chloride is added dropwise, and heated and stirred at 55°C for 18 hours. After the reaction is completed, the supernatant is obtained by centrifugation, and then freeze-dried after dialysis to obtain quaternary ammonium chitosan. Then β-cyclodextrin is dissolved in an aqueous sodium hydroxide solution and stirred at 25°C for 12 hours. Epichlorohydrin is added dropwise and stirred at 25°C for another 4.5 hours. After that, the quaternary ammonium chitosan solution is added and stirred at 25°C for 5 hours to terminate the reaction. After dialysis, freeze-dry for use.

[0035] 3) Preparation of azobenzene-modified chondroitin sulfate as a microgel precursor: p-diaminoazobenzene was modified onto chondroitin sulfate by amidation reaction. The specific process was as follows: 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, and then N-hydroxysuccinimide was added and stirred 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 completed, dialyzed and freeze-dried to obtain a light yellow sponge-like solid, which was dried at room temperature and stored for later use.

[0036] The usage 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.

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

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

[0039] The loading amount of DPA-AuCuNCs in hypoxia-responsive gold-copper cluster nanozyme microgels was 40-160 μg / mL.

[0040] 5) Biomedical applications: Nanozyme microgels and methicillin-resistant Staphylococcus aureus were co-cultured under hypoxic conditions to examine the antibacterial properties of the microgels. Furthermore, nanozyme microgels were used as antibacterial drugs to treat bacterial pneumonia mice infected with methicillin-resistant Staphylococcus aureus by aerosol inhalation to explore their repair-promoting effects on bacterial pneumonia.

[0041] Example 1

[0042] 1) Preparation of DPA-AuCuNCs: The molar ratio of chloroauric acid and copper chloride was 1.25:1. Aqueous chloroauric acid solution (112 μL, 50 mmol / L) and aqueous copper chloride solution (88 μL, 50 mmol / L) were added to 1.4 mL of ultrapure water. DPA solution (0.4 mL, 50 mmol / L) was added under ice bath and vigorous stirring (1500 rpm). Freshly prepared sodium borohydride solution (0.5 mL, 0.2 mol / L) was then quickly added and reacted for 2 hours under ice bath conditions. After that, it was placed in a refrigerator at 4 °C overnight to allow it to fully nucleate and grow. The synthesized DPA-AuCuNCs were purified by ethanol precipitation to remove unreacted molecules and ions. Three times the volume of ethanol was added to a certain volume of DPA-AuCuNCs, and a brown-black crude precipitate was produced after centrifugation (10000 rpm, 10 min). The precipitate was then thoroughly washed three times with ethanol, vacuum dried, and then dissolved in ultrapure water to obtain a DPA-AuCuNCs solution, which was stored in a 4°C refrigerator for later use.

[0043] 2) Preparation of quaternary ammonium salt chitosan grafted with β-cyclodextrin: The specific preparation steps are as follows: 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: 0.5g chitosan (viscosity 100-200mPa.s) was dispersed in 18mL (0.5% v / v) of acetic acid aqueous solution, and then 0.47g of glycidyl trimethylammonium chloride was dissolved in 1.5mL ultrapure water and added dropwise to the above solution, and stirred at 55°C for 18h. After the reaction was completed, the solution was centrifuged at 6500rpm for 8 minutes, and the supernatant was dialyzed for 3 days and freeze-dried to obtain quaternary ammonium salt chitosan. Add β-cyclodextrin (1.13 g) to the flask and dissolve it in sodium hydroxide aqueous solution (8 mL, 1 mol / L), and stir at 25°C for 12 hours. Then add 90 μL of epichlorohydrin dropwise and stir at 25°C for 4.5 hours. Then add the pre-dispersed quaternary ammonium chitosan aqueous solution (10 mL, 20 mg / mL), and stir at 25°C for 5 hours to end the reaction. The obtained solution was dialyzed for 5 days using a dialysis bag with a cutoff molecular weight of 12 to 14 kDa, and then freeze-dried to obtain a white spongy solid, i.e., quaternary ammonium chitosan grafted with β-cyclodextrin, which was dried at room temperature and stored for later use.

[0044] 3) Preparation of azobenzene-modified chondroitin sulfate: Dissolve chondroitin sulfate (62.5 mg) with a molecular weight of 30 kDa in 2-morpholineethanesulfonic acid buffer (5 mL, 10 mM, pH = 5.5), add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (38 mg) and stir for 10 minutes, then add N-hydroxysuccinimide (34.5 mg) and stir for 5 minutes. Finally, add p-diaminoazobenzene (0.85 mg) dissolved in dimethyl sulfoxide and stir at 25°C in the dark for 4 hours. After the reaction is completed, dialyze for 3 days using a dialysis bag with a cutoff molecular weight of 12 to 14 kDa, then freeze-dry to obtain a light yellow sponge-like solid, dry at room temperature and store for later use.

[0045] 4) Preparation of hypoxia-responsive gold-copper cluster nanozyme microgel: 48 μg DPA-AuCuNCs were pre-dispersed in 200 μL of 0.25% wt β-cyclodextrin grafted quaternary ammonium chitosan aqueous solution, mixed evenly, and then an equal volume of azobenzene-modified chondroitin sulfate aqueous solution (1.5% wt) was added, and the microgel was immediately formed after mixing with a Votex vortex for 30 seconds. Centrifuge (3000 rpm) for 3 minutes, discard the supernatant and add ultrapure water to resuspend, and wash the precipitate three times to obtain the purified hypoxia-responsive gold-copper cluster nanozyme microgel. Among them, the mass ratio of β-cyclodextrin grafted quaternary ammonium chitosan and azobenzene-modified chondroitin sulfate is 1:6. The loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanozyme microgel is 120 μg / mL.

[0046] 5) Biomedical applications: Co-culture the gold-copper cluster nanozyme microgels and methicillin-resistant Staphylococcus aureus under hypoxic conditions to examine the antibacterial properties of the microgels. Furthermore, the gold-copper cluster nanozyme microgels were used as antibacterial drugs to treat bacterial pneumonia mice infected with methicillin-resistant Staphylococcus aureus by aerosol inhalation to explore their repair-promoting effects on bacterial pneumonia.

[0047] Example 2

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

[0049] Example 3

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

[0051] Example 4

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

[0053] Example 5

[0054] The same as Example 1, but different from Example 1, in step 1), the total molar number of metal atoms is ensured to remain unchanged, and the molar ratio of chloroauric acid and cupric chloride is set to 0.25:1, that is, chloroauric acid aqueous solution (40 μL, 50 mmol / L) and cupric chloride aqueous solution (160 μL, 50 mmol / L) are added to 1.4 mL of ultrapure water.

[0055] Example 6

[0056] The same as Example 1, but different from Example 1, in step 4), the mass ratio of quaternary ammonium chitosan grafted with β-cyclodextrin and chondroitin sulfate modified with azobenzene is 1:7.2, that is, the concentration of the aqueous solution of quaternary ammonium chitosan grafted with β-cyclodextrin is 0.25%wt, and the concentration of the aqueous solution of chondroitin sulfate modified with azobenzene is 1.8%wt.

[0057] Example 7

[0058] The same as Example 1, but different from Example 1, in step 4), the mass ratio of quaternary ammonium chitosan grafted with β-cyclodextrin and chondroitin sulfate modified with azobenzene is 1:8.4, that is, the concentration of the aqueous solution of quaternary ammonium chitosan grafted with β-cyclodextrin is 0.25%wt, and the concentration of the aqueous solution of chondroitin sulfate modified with azobenzene is 2.1%wt.

[0059] Example 8

[0060] The same as Example 1, but different from Example 1, in step 4), the mass ratio of quaternary ammonium chitosan grafted with β-cyclodextrin and chondroitin sulfate modified with azobenzene is 1:9.6, that is, the concentration of the aqueous solution of quaternary ammonium chitosan grafted with β-cyclodextrin is 0.25%wt, and the concentration of the aqueous solution of chondroitin sulfate modified with azobenzene is 2.4%wt.

[0061] Example 9

[0062] The same as Example 1, but different from Example 1, in step 4), the mass ratio of quaternary ammonium chitosan grafted with β-cyclodextrin and chondroitin sulfate modified with azobenzene is 1:10.8, that is, the concentration of the aqueous solution of quaternary ammonium chitosan grafted with β-cyclodextrin is 0.25%wt, and the concentration of the aqueous solution of chondroitin sulfate modified with azobenzene is 2.7%wt.

[0063] Example 10

[0064] The same as Example 7, but different from Example 7, in step 4), the loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanozyme microgel is 40 μg / mL, that is, 16 μg of DPA-AuCuNCs is added to the β-cyclodextrin grafted quaternary ammonium chitosan aqueous solution.

[0065] Embodiment 11

[0066] The same as Example 7, but different from Example 7, in step 4), the loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanozyme microgel is 80 μg / mL, that is, 32 μg of DPA-AuCuNCs are added to the β-cyclodextrin grafted quaternary ammonium chitosan aqueous solution.

[0067] Example 12

[0068] The same as Example 7, but different from Example 7, in step 4), the loading amount of DPA-AuCuNCs in the hypoxia-responsive gold-copper cluster nanozyme microgel is 160 μg / mL, that is, the amount of DPA-AuCuNCs added to the β-cyclodextrin grafted quaternary ammonium chitosan aqueous solution is 64 μg.

[0069] Comparative Example 1

[0070] The same as Example 1, but different from Example 1, in step 1), the total molar number of metal atoms is ensured to remain 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] The same as Example 1, but different from Example 1, in step 1), the total molar number of metal atoms is ensured to remain 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] The same as Example 7, except that in step 4), no DPA-AuCuNCs were added to prepare hypoxia-responsive microgels.

[0075] Figure 1 The enzyme-like catalytic activity of a series of Au-Cu cluster nanozymes with different Au-Cu ratios synthesized in the present invention is characterized. DPA-AuCuNCs with different Au-Cu ratios and DPA-AuNCs and DPA-CuNCs are obtained from Examples 1-5, Comparative Example 1 and Comparative Example 2, respectively. Figure 1 As shown in (a), the prepared bimetallic DPA-AuCuNCs have a peroxidase-like catalytic activity significantly higher than that of single metal DPA-AuNCs and DPA-CuNCs, indicating that the synergistic effect of the bimetallics significantly improves its peroxidase-like catalytic performance. A portable dissolved oxygen meter was used to measure the amount of dissolved oxygen produced by different DPA-AuCuNCs in hydrogen peroxide (100 mmol / L, pH = 7.4) solution to characterize its catalase-like catalytic activity. Figure 1 As shown in (b), as the AuCu ratio decreases, its catalase-like catalytic activity increases, indicating that copper plays a core role in regulating the catalase-like catalytic activity of DPA-AuCuNCs. The above results indicate that the present invention has prepared a series of gold-copper cluster nanozymes with both peroxidase-like and catalase catalytic activities.

[0076] Figure 2 The peroxidase-like and catalase-like catalytic activities of DPA-AuCuNCs obtained in Example 1 of the present invention under different pH conditions are characterized. In view of the regulatory performance of multivalent metal components in DPA-AuCuNCs, especially multivalent Cu, on catalytic activity, the pH-dependent bienzyme-like catalytic activity was further studied. Figure 2 As shown in (a), under acidic conditions (pH 4.6, 6.0), the absorbance at 652nm in the hydrogen peroxide-TMB system in the presence of DPA-AuCuNCs was significantly higher than that in neutral or alkaline conditions (pH 7.4, 8.2), and the absorbance value was the highest at pH = 6. This result shows that DPA-AuCuNCs can exert its peroxidase-like activity to catalyze antibacterial activity in the weakly acidic microenvironment of bacterial infection, without any harmful effects on normal tissues with a physiologically neutral pH. Figure 2 As shown in (b), DPA-AuCuNCs has excellent catalase-like catalytic activity in neutral or slightly alkaline buffers compared to weakly acidic microenvironments. At pH = 7.4, the dissolved oxygen in its solution increased by 5 mg / L, which can alleviate the oxidative stress caused by hydrogen peroxide in uninfected tissues. Therefore, the good pH-dependent enzyme-like catalytic activity of DPA-AuCuNCs enables it to achieve intelligent anti-infection or hypoxia relief in different microenvironments.

[0077] Figure 3 The hydrated particle size of the nanozyme microgel prepared by different microgel precursor ratios of the present invention and the transmission electron microscopic element distribution of the nanozyme microgel obtained in Example 7 are shown in FIG. Figure 3 The hydrated particle size distribution of the microgels prepared in Example 1 and Examples 6-9 in (a) can be obtained. When the ratio of β-cyclodextrin grafted quaternary ammonium chitosan (QC-β-CD) and azobenzene-modified chondroitin sulfate (CS-Azo) is 1:6-1:10.8, a microgel with a hydrated particle size range of 0.5 to 2.3 microns and uniform particle size distribution can be obtained, and as the ratio of the two increases, the particle size of the prepared microgel increases. The nanozyme microgel obtained in Example 7 was selected for elemental analysis. Figure 3 (b) is the energy spectrum scanning area of ​​the nanozyme 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 nanozyme microgel in Example 7 is a micron-sized sphere. The distribution of Au and Cu elements shows that DPA-AuCuNCs are evenly distributed inside the microgel.

[0078] Figure 4 The following is a scanning electron microscope image of the nanoenzyme microgel (NC@mGels) prepared in Example 7 of the present invention and an evaluation of the hypoxia response performance. Currently, the commonly used chemical hypoxia mimicking agents are mainly sodium dithionite (Na 2 S 2 O 4 ), which has strong reducing properties and can quickly react chemically with oxygen in the solution to remove dissolved oxygen, while also reducing the azo group (-N=N-) in azobenzene to an aniline group (-NH 2 ). Therefore, using Na 2 S 2 O 4 The hypoxia response performance of NC@mGels was investigated by characterizing its 2 S 2 O 4 The particle size changes before and after incubation. Scanning electron microscopy results show that the NC@mGels prepared in Example 7 are well-dispersed and uniform spherical particles ( Figure 4 (a)), but in the case of Na 2 S 2 O 4 After 30 min of incubation, it degraded into smaller nanoparticles ( Figure 4 (b)). The average particle size of NC@mGels obtained by fitting the SEM images is similar to that of Na 2 S 2 O 4 After incubation, the wavelength dropped from 662 nm to 90 nm, indicating that the nanozyme microgel of the present invention has excellent hypoxia response performance.

[0079] Figure 5 Graph showing the antibacterial effect of the nanoenzyme microgel of the present invention. 2 O 2 ) was used to evaluate the peroxidase-like synergistic catalytic antibacterial properties of NC@mGels. 2 O 2 (10 μmol / L) and mGels (microgel prepared in Comparative Example 3) and NC@mGels (nanozyme microgel prepared in Example 7) and bacterial suspension (10 6CFU / mL) for 4 hours. Then, 10 μL of the diluted co-incubation solution was evenly dropped on the agar plate, which was then placed upside down in a 37°C incubator for 18 hours. The number of colonies on the agar plate was recorded and statistically analyzed. Figure 5 The results in (a) show that both mGels and NC@mGels showed good antibacterial effects against methicillin-resistant Staphylococcus aureus (MRSA), with the survival rates reduced to 11.8% and 6.7%, respectively, which was mainly attributed to the grafted quaternary ammonium groups and the loaded DPA-AuCuNCs in the microgels. Furthermore, DPA-AuCuNCs have excellent peroxidase-like activity and can be used in low concentrations of H 2 O 2 Under these conditions, the active oxygen produced by the oxidative stress can produce good synergistic antibacterial properties. 2 O 2 After reacting with NC@mGels, the effective killing rate of the material against MRSA was 100% (see Figure 5 (a)). This enzyme-like catalytic synergistic bactericidal effect is more obvious against Pseudomonas aeruginosa (PAO1). NC@mGels+H 2 O 2 The survival rate of PAO1 in the treatment group dropped to 16.2% (see Figure 5 (a)), which is much lower than the survival rate of NC@mGels alone after interaction with PAO1 (76.1%). The plate count method was used to study the hypoxic response antibacterial effect of NC@mGels using MRSA as a model bacterium. Figure 5 As shown in (b), the survival rate of MRSA in the NC@mGels co-culture group under hypoxia was only 0.3%, which was 3.5% lower than that in the normoxic group, indicating that the release of DPA-AuCuNCs by NC@mGels in hypoxia enhanced its antibacterial ability. These results indicate that NC@mGels has excellent enzyme-like catalysis and hypoxia-responsive antibacterial properties, and has great potential for in vivo anti-infection treatment.

[0080] Figure 6 The intracellular oxygen supply performance of the nanozyme microgel of the present invention is characterized, wherein DPA-AuCuNCs and NC@mGels are the gold-copper cluster nanozyme and nanozyme microgel obtained in implementation 7, respectively. Using the oxygen-sensitive fluorescent probe Ru(dpp) 3 Cl 2 The catalytic oxygen supply capacity of the materials for fibroblasts (L929) was determined. L929 was seeded in 8-well chamber coverslips at a density of 2×10 4cells / well and incubated under hypoxic conditions for 18 hours. A hypoxic chamber was used to simulate the hypoxic environment of cell culture, which provided 1% oxygen, 5% carbon dioxide, and 95% nitrogen. DPA-AuCuNCs and NC@mGels were incubated with H 2 O 2 (100 μmol / L) was incubated with L929 for 12 h, and then with Ru(dpp) 3 Cl 2 The oxygen-sensitive fluorescent probe was cultured for another 4 hours under hypoxic conditions. The relative fluorescence intensity (λex = 488 nm) of the fluorescent probe was measured using a fluorescence confocal microscope to reflect the intracellular oxygen level. Figure 6 Available, control group and H alone 2 O 2 The treatment group showed obvious fluorescence signals, indicating that the oxygen content was low. In comparison, the fluorescence intensity of the DPA-AuCuNCs group decreased, and the NC@mGels treatment group showed the lowest fluorescence signal, indicating that the nanozyme microgels in the present invention have good intracellular catalytic oxygen supply performance.

[0081] Figure 7 This is the therapeutic effect of the nanozyme microgel of the present invention on mice with bacterial pneumonia. Figure 7 (a), (b), (c) and (d) are lung tissue hematoxylin-eosin staining (H&E) analysis of healthy mice, MRSA infection group mice, MRSA infection microgel treatment group mice (microgels prepared in comparative example 3, i.e. mGels), and MRSA infection nanozyme microgel treatment group mice (nanozyme microgels prepared in example 7, i.e. NC@mGels). 8 CFU / mL) induced BALB / c mice to construct a mouse model of bacterial pneumonia infected with MRSA. Mice were treated with nebulization inhalation 24 hours after MRSA infection. Nebulization inhalation treatment was performed again 24 hours later. Lung tissues were collected 72 hours after treatment, fixed with 4% paraformaldehyde, and embedded in sections for H&E tissue staining. The results showed that compared with the healthy group mice ( Figure 7 Compared with (a), MRSA infection in untreated mice ( Figure 7 (b) A large number of inflammatory cells infiltrated the lungs, the alveolar wall was edematous, and the alveolar structure was severely damaged. After 72 hours of aerosol treatment, the MRSA infected mGels treated mice ( Figure 7 (c) The infiltration of inflammatory cells in lung tissue was moderate, while that in the NC@mGels treated mice ( Figure 7(d) The alveolar histology of the lungs is clear and complete, the edema of the alveolar wall and the infiltration of inflammatory cells are significantly reduced, and the lung tissue has returned to a healthy state. In summary, the hypoxia-responsive gold-copper cluster nanozyme microgel prepared in Example 7 has a good effect in treating bacterial pneumonia.

[0082] The present invention prepares DPA-AuCuNCs in an aqueous phase in one step through the dual regulation of ligands and components, and further loads DPA-AuCuNCs in azobenzene natural polymer microgel networks to prepare hypoxia-responsive gold-copper cluster nanozyme microgels. On the one hand, the intrinsic antibacterial activity of the DPA ligand improves the antibacterial performance of the gold-copper cluster nanozyme. On the other hand, the synergistic effect of the gold-copper bimetallic components gives the gold-copper cluster nanozyme controllable pH-adaptive enzyme-like catalytic activity, which is the key to its intelligent and controllable anti-infection or hypoxia relief under different pH microenvironments. The DPA-AuCuNCs of the present invention exert peroxidase-like catalytic activity in an acidic infection microenvironment, catalyzing hydrogen peroxide to generate hydroxyl radicals with high antibacterial activity, achieving efficient antibacterial and avoiding the problem of traditional antibiotic resistance; at the site of neutral tissue damage, its catalase-like activity decomposes hydrogen peroxide into oxygen and water, converts excessively accumulated reactive oxygen into dissolved oxygen, and achieves the role of in-situ oxygen supply and relief of oxidative stress.

[0083] The main components of the nanozyme microgel of the present invention are derived from natural active substances, have excellent biosafety, and the preparation method is simple and easy, the reaction conditions are mild, and no complex instruments and equipment are required. The supramolecular microgel loaded with DPA-AuCuNCs constructed by azobenzene-modified chondroitin sulfate and beta-cyclodextrin grafted quaternary ammonium salt chitosan can achieve controlled response release at the hypoxic lesion site, further exert pH-adaptive enzyme-like catalytic activity, improve the delivery efficiency of the drug and the biosafety under normal physiological conditions. In addition, the particle size of the microgel allows it to be administered by atomized inhalation, further improving the targeting and bioavailability of bacterial pneumonia treatment. The nanozyme microgel of the present invention has good biocompatibility, excellent hypoxia-responsive intelligent enzyme-like catalytic antibacterial and oxygen supply activity, and has great application potential in the repair of tissue damage related to bacterial infections such as bacterial pneumonia.

[0084] The above description is only for the best embodiment of the present invention, but it should not be understood as limiting the claims. The present invention is not limited to the above embodiments, and its specific structure is allowed to be changed. However, all changes made within the protection scope of the independent claims of the present invention are within the protection scope of the present invention.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

Claims

1. A method for preparing hypoxia-responsive gold-copper cluster nanozyme microgel, characterized in that: The following steps are involved: 1) Using sodium borohydride as a reducing agent and D-penicillamine as a surface ligand, a mixed solution of chloroauric acid and copper chloride was reduced in an aqueous phase by a one-step method to prepare D-penicillamine gold-copper cluster nanozyme; 2) The D-penicillamine gold-copper cluster nanozyme was loaded into the self-assembled supramolecular microgel of quaternary ammonium chitosan grafted with β-cyclodextrin and chondroitin sulfate modified by azobenzene to obtain the hypoxia-responsive gold-copper cluster nanozyme microgel.

2. The method for preparing the hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 1, characterized in that: The specific process of step 1) is as follows: after mixing the chloroauric acid solution and the cupric chloride solution, the D-type penicillamine solution is added under stirring, followed by the addition of the sodium borohydride solution, and the mixture is reacted under ice bath for 2 hours to obtain the D-type penicillamine gold-copper cluster nanozyme.

3. The method for preparing the hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 2, characterized in that: The total amount of chloroauric acid and cupric chloride, and the dosage ratio of D-type penicillamine to sodium borohydride are 10 μmol: 20 μmol: 100 μmol; wherein the molar ratio of chloroauric acid to cupric chloride is 1.5:1-0.25:

1.

4. The method for preparing the hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 1, characterized in that: In step 2), 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.

5. The method for preparing the hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 4, characterized in that: The usage 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.

6. The method for preparing hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 1, characterized in that: The specific process of step 2) is: dispersing the D-type penicillamine gold-copper cluster nanozyme in the quaternary ammonium salt chitosan solution grafted with β-cyclodextrin, and then mixing and shaking with the azobenzene-modified chondroitin sulfate solution to form an oxygen-responsive gold-copper cluster nanozyme microgel.

7. The method for preparing hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 1, characterized in that: The mass ratio of quaternary ammonium salt chitosan grafted with beta-cyclodextrin to azobenzene-modified chondroitin sulfate is 1:6-1:10.

8.

8. The method for preparing hypoxia-responsive gold-copper cluster nanozyme microgel according to claim 1, characterized in that: The loading amount of D-type penicillamine gold-copper cluster nanozyme in the hypoxia-responsive gold-copper cluster nanozyme microgel was 40-160 μg / mL.

9. A hypoxia-responsive gold-copper cluster nanozyme microgel prepared according to the method according to any one of claims 1 to 8.

10. Use of the hypoxia-responsive gold-copper cluster nanozyme microgel prepared according to the method according to any one of claims 1 to 8 in the preparation of antibacterial drugs.

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