3D sea urchin-shaped copper telluride nano-enzyme hydrogel, preparation method thereof and application of hydrogel in preparation of gram-negative bacterium resisting medicine

Abstract

The invention provides 3D sea urchin-shaped copper telluride nano-enzyme hydrogel, a preparation method thereof and application of the 3D sea urchin-shaped copper telluride nano-enzyme hydrogel in preparation of gram-negative bacterium resisting drugs, and belongs to the technical field of inorganic nano-enzymes. The copper telluride nano-enzyme hydrogel provided by the invention can be used for specifically and selectively treating gram-negative bacterium infection due to the fact that 3D sea urchin-shaped copper telluride has double-enzyme activity of oxidase-like and glutathione-like oxidase, and biosynthesis of lipopolysaccharide (LPS) and flagellum can be interfered by consuming glutathione (GSH); the attack ability of reactive oxygen species (ROS) generated by catalysis is enhanced, so that drug-resistant gram negative bacteria and biological membranes thereof can be specifically removed. Besides, the 3D urchin-like copper telluride nano-enzyme hydrogel can significantly inhibit pseudomonas aeruginosa infection and inflammatory response on burn wounds, promotes angiogenesis and cell proliferation at the same time, and has a good treatment effect on the burn wounds infected by pseudomonas aeruginosa.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of inorganic nanoscale enzyme, and particularly relates to a 3D urchin-like cupric telluride nanoscale enzyme hydrogel, a preparation method thereof and application thereof in preparation of anti-Gram-negative bacteria drugs. BACKGROUND

[0002] Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii, have developed resistance to most of the commonly used antibiotics in the clinic. Gram-negative bacteria prevent the effective penetration of antibacterial drugs by adjusting the structure of lipopolysaccharide to reduce the permeability of the outer membrane of bacteria, and use flagella to enhance their ability to colonize the host and escape the attack of antibacterial drugs. In addition, Gram-negative bacteria are prone to form biofilms, which have a dense extracellular polymeric substance barrier and a biofilm microenvironment that can effectively protect bacteria. The above characteristics make Gram-negative bacteria more difficult to kill than other types of bacteria, so it is extremely difficult to develop effective antibacterial drugs against Gram-negative bacteria.

[0003] As a commonly used nanoscale enzyme, copper-based nanoscale enzyme has excellent physical and chemical properties and has attracted widespread attention in antibacterial therapy. Compared with traditional antibiotic drugs, copper-based nanoscale enzyme can release copper ions, catalyze the generation of active oxygen, and physically contact the bacterial cell wall, effectively destroying the structure and metabolic function of bacteria, thereby overcoming bacterial drug resistance. The above multi-modal antibacterial mechanism endows copper-based nanoscale enzyme with high-efficiency and broad-spectrum bactericidal effect, but its indiscriminate bactericidal ability may cause cytotoxicity and microecological imbalance at the lesion site, thereby leading to adverse reactions such as inflammation and immune disorders. Therefore, it is a challenge in the current field to provide a copper-based nanoscale enzyme that selectively targets Gram-negative bacteria. SUMMARY

[0004] In view of this, the present application aims to provide a 3D urchin-like cupric telluride nanoscale enzyme hydrogel, a preparation method thereof and application thereof in preparation of anti-Gram-negative bacteria drugs. The 3D urchin-like cupric telluride nanoscale enzyme hydrogel prepared by the present application can specifically select treatment of Gram-negative bacteria infection.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a 3D urchin-like cupric telluride nanoscale enzyme hydrogel, comprising a hydrogel matrix and 3D urchin-like cupric telluride nanoscale enzyme dispersed in the hydrogel matrix.

[0007] Preferably, the particle size of the 3D urchin-like cupric telluride nanoscale enzyme is 200-300 nm.

[0008] Preferably, the hydrogel matrix comprises one or more of cationic guar gum hydrogel, hyaluronic acid hydrogel, alginate hydrogel and F127 hydrogel.

[0009] The mass ratio of the 3D urchin-like cupric telluride nanoszyme to the hydrogel base is 1:1000-2000.

[0010] The application provides a preparation method of the 3D urchin-like cupric telluride nanoszyme hydrogel.

[0011] The soluble copper salt, telluric acid, dispersant, reducing agent and organic solvent are mixed to perform a coprecipitation reaction to obtain the 3D urchin-like cupric telluride nanoszyme.

[0012] The aqueous solution of the 3D urchin-like cupric telluride nanoszyme is mixed with a hydrogel base to perform gelation to obtain the 3D urchin-like cupric telluride nanoszyme hydrogel.

[0013] Preferably, the soluble copper salt includes one or more of copper chloride, copper acetate, copper sulfate and copper nitrate.

[0014] The dispersant includes one or more of polyvinylpyrrolidone, polyethylene glycol and cellulose ether.

[0015] The reducing agent includes one or more of ascorbic acid, citric acid, oxalic acid, phosphoric acid and malic acid.

[0016] Preferably, the amount of the soluble copper salt is 5-20 parts by mass, the amount of the telluric acid is 8-30 parts, the amount of the dispersant is 100-400 parts, and the amount of the reducing agent is 30-120 parts.

[0017] Preferably, the coprecipitation reaction is performed at a temperature of 160-200 DEG C for 3-5 h.

[0018] Preferably, the gelation is performed under stirring, and the gelation is performed for 5-10 min.

[0019] The application provides an application of the 3D urchin-like cupric telluride nanoszyme hydrogel in preparation of an antibacterial drug or a wound repair drug.

[0020] Preferably, the antibacterial drug includes an anti-Gram-negative bacterial drug.

[0021] The application provides a 3D urchin-like cupric telluride nanoscale enzyme hydrogel, which comprises a hydrogel base and 3D urchin-like cupric telluride nanoscale enzymes dispersed in the hydrogel base. In terms of structure, on the one hand, the 3D urchin-like cupric telluride nanoscale enzyme can physically destroy gram-negative bacteria by using surface spikes, and on the other hand, the 3D urchin-like shape endows the nanoscale enzyme with a high specific surface area, which can expose more catalytic sites to improve catalytic activity. The application takes the hydrogel as the base of the 3D urchin-like cupric telluride nanoscale enzyme, and the hydrogel is an ideal wound dressing with physical properties such as adhesion, self-healing and moisturizing. After loading the nanoscale enzyme, the hydrogel can also play a role in sustained release, greatly enhancing the enrichment of nanomaterials on the surface of the wound and the antibacterial effect. In addition, the application uses the hydrogel as the base of the 3D urchin-like cupric telluride nanoscale enzyme, which has the advantages of low cost and excellent physical properties, and has good water dispersibility. In use, it can be dispersed in water, physiological saline, PBS buffer, cell culture medium and the like, and can also be directly applied to the skin surface as a dressing.

[0022] The cupric telluride nanoscale enzyme hydrogel provided by the application can specifically select gram-negative bacteria for treatment. This is because the 3D urchin-like cupric telluride has dual enzyme activity of oxyenzyme and glutathione oxidase, can interfere with the biosynthesis of lipopolysaccharide (LPS) and flagellum by consuming glutathione (GSH), and enhance the attack ability of active oxygen (ROS) generated by catalysis, so as to specifically remove drug-resistant gram-negative bacteria and their biofilms. In addition, the 3D urchin-like cupric telluride nanoscale enzyme hydrogel of the application can significantly inhibit the infection of Pseudomonas aeruginosa on a burn wound surface and the inflammatory response, and promote angiogenesis and cell proliferation, and has a good treatment effect on a burn wound surface infected with Pseudomonas aeruginosa.

[0023] The application provides a preparation method of the above-mentioned 3D urchin-like cupric telluride nanoscale enzyme hydrogel. The application adopts a co-precipitation method to synthesize the 3D urchin-like cupric telluride nanoscale enzyme in one step, and then performs gelation with a hydrogel base to obtain the 3D urchin-like cupric telluride nanoscale enzyme hydrogel. This method is simple in operation, low in cost and easy to realize industrialized batch production. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A photo of the 3D urchin-like cupric telluride nanoscale enzyme prepared in Example 1 after freeze-drying;

[0025] Figure 2 A photo of the 3D urchin-like cupric telluride nanoscale enzyme prepared in Example 1 in an aqueous solution;

[0026] Figure 3 A photo of the 3D urchin-like cupric telluride nanoscale enzyme hydrogel prepared in Example 1 (from left to right, water, cationic guar gum hydrogel and cupric telluride nanoscale enzyme hydrogel);

[0027] Figure 4 TEM image of 3D urchin-like cupric telluride nanoszyme prepared in Example 1;

[0028] Figure 5 Element mapping image of 3D urchin-like cupric telluride nanoszyme prepared in Example 1;

[0029] Figure 6 XRD image of 3D urchin-like cupric telluride nanoszyme prepared in Example 1;

[0030] Figure 7 XPS image of 3D urchin-like cupric telluride nanoszyme prepared in Example 1, wherein: A is the full spectrum of cupric telluride, B is the fine spectrum of tellurium element, and C is the fine spectrum of copper element;

[0031] Figure 8 Enzyme-like catalytic activity image of 3D urchin-like cupric telluride nanoszyme prepared in Example 1, wherein: A is the image of detecting the oxidation performance of cupric telluride nanoszyme with OPD probe, B is the image of detecting the performance of cupric telluride nanoszyme in generating superoxide anion by electron spin resonance, and C is the image of detecting the ability of cupric telluride nanoszyme in consuming glutathione;

[0032] Figure 9 Digital photo of 3D urchin-like cupric telluride nanoszyme prepared in Example 1;

[0033] Figure 10 Image for proving that 3D urchin-like cupric telluride nanoszyme prepared in Example 1 specifically kills gram-negative bacteria, wherein A is the bacterial growth curve of methicillin-resistant Staphylococcus aureus (MRSA), B is the bacterial growth curve of S. aureus, C is the bacterial growth curve of Enterococcus faecalis, D is the bacterial growth curve of Escherichia coli, E is the bacterial growth curve of Pseudomonas aeruginosa, and F is the bacterial growth curve of Klebsiella pneumoniae;

[0034] Figure 11 Scanning electron microscope image of 3D urchin-like cupric telluride nanoszyme prepared in Example 1 killing different bacteria, wherein: A is the scanning electron microscope image of MRSA bacteria treated by 3D urchin-like cupric telluride nanoszyme; B is the scanning electron microscope image of E. coli bacteria treated by 3D urchin-like cupric telluride nanoszyme; and C is the scanning electron microscope image of P. aeruginosa bacteria treated by 3D urchin-like cupric telluride nanoszyme;

[0035] Figure 12 Microscopic scanning electron microscope image of 3D urchin-like cupric telluride nanoszyme hydrogel prepared in Example 1;

[0036] Figure 13 Pictures of antibacterial performance of 3D urchin-like cupric telluride nanoszyme hydrogel prepared for example 1;

[0037] Figure 14 Pictures of wound healing and statistics after treatment of mouse burn wound infected with pseudomonas aeruginosa by 3D urchin-like cupric telluride nanoszyme hydrogel prepared for example 1;

[0038] Figure 15 Bacterial plating and statistics in wound after treatment of mouse burn wound infected with pseudomonas aeruginosa in example 1. DETAILED DESCRIPTION

[0039] The application provides a 3D urchin-like cupric telluride nanoszyme hydrogel, which comprises a hydrogel matrix and 3D urchin-like cupric telluride nanoszyme dispersed in the hydrogel matrix.

[0040] In the application, the particle size of the 3D urchin-like cupric telluride nanoszyme is preferably 200-300 nm, and can be 200 nm, 250 nm or 300 nm. x Te, wherein Cu exists in mixed valence states of Cu 2+ and Cu + , and Te exists in mixed valence states of Te 2- and Te 4+ .

[0041] In the application, the hydrogel matrix comprises one or more of cationic guar gum hydrogel, hyaluronic acid hydrogel, alginate hydrogel and F127 hydrogel.

[0042] The application provides a preparation method of the above 3D urchin-like cupric telluride nanoszyme hydrogel, comprising the following steps:

[0043] The soluble copper salt, telluric acid, dispersant, reducing agent and organic solvent are mixed to perform a coprecipitation reaction, so as to obtain 3D urchin-like cupric telluride nanoszyme.

[0044] The aqueous solution of the 3D urchin-like cupric telluride nanoszyme is mixed with the hydrogel matrix to perform gelation, so as to obtain 3D urchin-like cupric telluride nanoszyme hydrogel.

[0045] The present application mixes soluble copper salt, telluric acid, dispersant, reducing agent and organic solvent, carries out coprecipitation reaction, and obtains 3D urchin-like cuprous telluride nanoscale enzyme. In the present application, the soluble copper salt preferably includes one or more of copper chloride, copper acetate, copper sulfate and copper nitrate, and more preferably is copper chloride. In the present application, the copper chloride can be anhydrous copper chloride and / or copper chloride hydrate, the copper acetate can also be anhydrous copper acetate and / or copper acetate hydrate, the copper sulfate can be anhydrous copper sulfate and / or copper sulfate hydrate, and the copper nitrate can be anhydrous copper nitrate and / or copper nitrate hydrate.

[0046] In the present application, the dispersant is preferably an organic dispersant, preferably includes one or more of polyvinylpyrrolidone, polyethylene glycol and cellulose ether, and more preferably is polyvinylpyrrolidone. In the present application, the molecular weight of the organic dispersant is preferably 5000-10000, and more preferably 6000-8000.

[0047] In the present application, the reducing agent preferably includes one or more of ascorbic acid, citric acid, oxalic acid, phosphoric acid and malic acid, and more preferably is ascorbic acid.

[0048] In the present application, the organic solvent is preferably one or more of alcohol solvents, ether solvents and amide solvents, and more preferably is one or more of benzyl alcohol, phenethyl alcohol, phenylpropanol, dipropylene glycol dimethyl ether, ethylene glycol and dimethylformamide.

[0049] In the present application, the amount of the soluble copper salt is preferably 5-20 parts by mass, and more preferably 10-16 parts; the amount of the telluric acid is preferably 8-30 parts, and more preferably 10-23 parts, and further preferably 15-20 parts; the amount of the dispersant is preferably 100-400 parts, and more preferably 200-300 parts; and the amount of the reducing agent is preferably 30-120 parts, and more preferably 50-100 parts, and further preferably 70-80 parts.

[0050] In the present application, the mass of the soluble copper salt to the volume of the organic solvent is preferably 5-20 mg: 20-80 mL, and more preferably 5-20 mg: 40-60 mL, and further preferably 10-15 mg: 40-60 mL.

[0051] In the present application, the mixing is preferably ultrasonic mixing, and the power of the ultrasonic mixing is preferably 20-40 kHz, and the time is preferably 30-60 min, and more preferably 40-50 min.

[0052] In the present application, the co-precipitation reaction is preferably carried out under the condition of oil bath heating; the temperature of the co-precipitation reaction is preferably 160-200℃, more preferably 170-180℃, and the time is preferably 3-5h, more preferably 4h. By controlling the amount of raw materials and the temperature and time of the co-precipitation reaction, the present application can promote the formation of 3D urchin-like morphology.

[0053] After the co-precipitation reaction, the present application preferably carries out post-treatment on the obtained co-precipitation reaction solution, which preferably comprises the following steps:

[0054] The co-precipitation reaction solution is centrifuged, and the obtained solid is washed and dried.

[0055] In the present application, the centrifugation is preferably high-speed centrifugation, the rate of which is preferably 6000-20000rpm, more preferably 8000-10000rpm, and the time is preferably 5-100min, more preferably 10-80min, more preferably 20-50min. After the centrifugation, the present application discards the supernatant and retains the lower layer precipitate. In the present application, the reagents used for the washing are preferably acetone, ethanol and deionized water in sequence; in the present application, the drying is preferably freeze-drying, the temperature of which is preferably -40--80℃, and the time is preferably 1-200h, more preferably 10-150h, further preferably 50-100h.

[0056] After obtaining the 3D urchin-like copper telluride nanoszyme, the present application mixes an aqueous solution of the 3D urchin-like copper telluride nanoszyme with a hydrogel matrix to carry out gelation, thereby obtaining a 3D urchin-like copper telluride nanoszyme hydrogel. In the present application, the concentration of the aqueous solution of the 3D urchin-like copper telluride nanoszyme is preferably 1-50μg / mL, more preferably 10-40μg / mL, further preferably 20μg / mL. In the present application, the aqueous solution of the 3D urchin-like copper telluride nanoszyme is preferably a uniformly ultrasonically dispersed solution. In the present application, the hydrogel matrix preferably comprises one or several of cationic guar gum, hyaluronic acid, alginate and F127; the mass ratio of the 3D urchin-like copper telluride nanoszyme to the hydrogel matrix (dry weight) is preferably 1:1000-2000, more preferably 1:1500.

[0057] In the present application, the gelation is preferably carried out under the condition of stirring, the rate of which is preferably 200-800r / min, more preferably 400-600r / min; in the present application, the temperature of the gelation is preferably 20-40℃, more preferably 25-35℃, and the time is preferably 5-10min, more preferably 6-8min.

[0058] The application provides application of the 3D urchin-like cupric telluride nanoscale enzyme hydrogel in preparation of antibacterial drugs or wound repair drugs.

[0059] In the application, the antibacterial drug preferably comprises an antibacterial drug against gram-negative bacteria, and more preferably one or more of escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and acinetobacter baumannii.

[0060] In the antibacterial process, the 3D urchin-like cupric telluride nanoscale enzyme hydrogel provided by the application can specifically remove gram-negative bacteria and biofilms thereof, and has a good treatment effect on burn wounds infected by pseudomonas aeruginosa, through a multi-modal antibacterial mechanism of consuming glutathione (GSH), physical combination, interfering with lipopolysaccharide (LPS) and flagellum biosynthesis, and enhancing the attack ability of active oxygen (ROS) generated by catalysis. Therefore, the cupric telluride nanoscale enzyme hydrogel of the application has a wide application prospect in specific resistance to gram-negative bacterial infection and burn wound healing.

[0061] The 3D urchin-like cupric telluride nanoscale enzyme hydrogel provided by the application, the preparation method thereof and the application thereof in preparation of antibacterial drugs against gram-negative bacteria will be described in detail in combination with examples below, but they should not be understood as a limitation on the protection scope of the application.

[0062] Example 1

[0063] The 3D urchin-like cupric telluride nanoscale enzyme hydrogel is prepared by the following steps:

[0064] 16 mg of copper chloride, 23 mg of telluric acid, 70 mg of ascorbic acid and 200 mg of polyvinylpyrrolidone are weighed and added into 40 mL of a benzyl alcohol solution to obtain a mixed solution;

[0065] The mixed solution is ultrasonically treated for 30 min to obtain a uniformly mixed solution;

[0066] The uniformly mixed solution is placed in a flask and heated in an oil bath at 160 DEG C for 3 h;

[0067] The heated solution is naturally cooled, centrifuged at a speed of 10,000 rpm for 10 min, and the supernatant is discarded, and the precipitate is left. The obtained precipitate is washed with acetone, ethanol and deionized water for 3 times respectively, and then freeze-dried (the drying temperature is -60 DEG C, and the time is 36 h) to obtain the 3D urchin-like cupric telluride nanoscale enzyme.

[0068] 20 mL of an aqueous solution of the 3D urchin-like cupric telluride nanoscale enzyme with a concentration of 20 μg / mL is prepared, ultrasonically mixed, and then 600 mg of cationic guar gum hydrogel powder is added, and stirred at a speed of 500 r / min for 5 min to obtain the 3D urchin-like cupric telluride nanoscale enzyme hydrogel.

[0069] Figure 1 The image shows a macroscopic photograph of the 3D sea urchin-shaped copper telluride nanozyme prepared in Example 1. As can be seen from the photograph, the copper telluride appears as a black powder.

[0070] Figure 2 The image shows the 3D sea urchin-shaped copper telluride nanozyme prepared in Example 1 in deionized water. As can be seen from the image, the copper telluride nanozyme is uniformly dispersed in water and appears grayish-black.

[0071] Figure 3 The image shows a comparison between the 3D sea urchin-shaped copper telluride nanozyme hydrogel prepared in Example 1 and deionized water and cationic guar gum hydrogels. As can be seen from the image, the 3D sea urchin-shaped copper telluride nanozyme can be uniformly hybridized in the cationic guar gum hydrogel and appears as a light gray color.

[0072] Figure 4 The image shows a transmission electron microscope (TEM) image of the 3D sea urchin-shaped copper telluride nanozyme prepared in Example 1. The image shows that the copper telluride nanozyme is 3D sea urchin-shaped with a diameter of about 200 nm and has a uniform morphology.

[0073] Figure 5 The image shows the elemental mapping of the 3D sea urchin-shaped copper telluride nanozyme prepared in Example 1. As can be seen from the image, copper and tellurium elements are uniformly distributed in the 3D sea urchin-shaped copper telluride nanozyme.

[0074] Figure 6 The image shows an X-ray diffraction (XRD) analysis of the 3D urchin-shaped copper telluride nanozyme prepared in Example 1. The image shows that the diffraction peak positions of the 3D urchin-shaped copper telluride nanozyme match those of CuTe (PDF#04--008--8274) and Cu2Te (PDF#04--007--6583), thus indicating that the chemical formula is Cu. x Te.

[0075] Figure 7 X-ray photoelectron spectroscopy (XPS) of the 3D urchin-shaped copper telluride nanozyme prepared in Example 1. x Cu is present in Te 2+ and Cu + The mixed valence state of Te, at the same time 2- and Te 4+ It also exists in mixed valence states.

[0076] Figure 8Enzymatic activity verification picture of 3D urchin-like cupric telluride nanoszyme prepared in Example 1. 3D urchin-like cupric telluride nanoszyme can catalyze oxygen molecules to generate superoxide anion through peroxidase-like activity, and superoxide anion makes OPD reaction system present yellow product and has absorption peak at ultraviolet absorption peak 430 nm. At the same time, electron spin resonance (ESR) is further used to prove that 3D urchin-like cupric telluride has peroxidase-like activity. Moreover, 3D urchin-like cupric telluride nanoszyme has glutathione peroxidase-like activity, so that it has the ability to consume glutathione.

[0077] Figure 9 Bacterial plating picture after 3D urchin-like cupric telluride nanoszyme prepared in Example 1 is treated with MRSA, E. coli and P. aeruginosa. A certain volume of MRSA bacterial suspension is taken, and the OD value is adjusted to about 0.6 (1.0×10 7 CFU / mL), and gradient concentrations of 3D urchin-like cupric telluride nanoszyme working solution (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL) are prepared. 20 μL of the above three kinds of bacterial suspensions are added to 96-well plates, and 180 μL of 3D urchin-like cupric telluride nanoszyme working solution of corresponding concentration is added to each well, and incubated at 37℃ for 4 h. The bacterial suspension after incubation is diluted 10 3 times, 20 μL of the diluted bacterial suspension is added to a meat agar plate, and the plate is placed in a 37℃ incubator for 12 h. The agar plate is taken out and photographed. When the concentration of 3D urchin-like cupric telluride nanoszyme working solution reaches 20 μg / mL, the activity of E. coli and P. aeruginosa can be ignored. From the pictures, it can be seen that 3D urchin-like cupric telluride nanoszyme has the ability to kill gram-negative bacteria at the same concentration. Figure 9 It can be seen that 3D urchin-like cupric telluride nanoszyme can specifically kill gram-negative bacteria at the same concentration.

[0078] Figure 10 Bacterial growth curve statistics after 3D urchin-like cupric telluride nanoszyme prepared in Example 1 is treated with gram-positive bacteria MRSA, S. aureus and E. faecalis and gram-negative bacteria E. coli, P. aeruginosa and K. pneumoniae. A certain volume of gram-positive bacteria MRSA, S. aureus and E. faecalis and gram-negative bacteria E. coli, P. aeruginosa and K. pneumoniae bacterial suspension is taken, and the OD value is adjusted to about 0.6 (1.0×10 73D urchin-shaped copper telluride nanozyme working solutions of varying concentrations (5 μg / mL, 10 μg / mL, and 20 μg / mL) were prepared. 20 μL of each of the six bacterial suspensions was added to a 96-well plate, followed by 180 μL of the corresponding concentration of 3D urchin-shaped copper telluride nanozyme working solution. The plates were incubated at 37°C, and the absorbance at OD 600 nm was measured at different time points using a microplate reader. The results indicate that the 3D urchin-shaped copper telluride nanozyme can specifically kill Gram-negative bacteria.

[0079] Figure 11 Scanning electron microscopy (SEM) images of MRSA, E. coli, and P. aeruginosa killed by the 3D sea urchin-shaped copper telluride nanozyme prepared in Example 1 are shown. For morphological analysis, bacteria treated with the 3D sea urchin-shaped copper telluride nanozyme were collected by centrifugation, fixed with 2.5% glutaraldehyde, and incubated at 4°C for 12 h. The glutaraldehyde was removed by centrifugation. The bacteria were then treated sequentially with a series of ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 10 minutes each. They were then drop-coated onto silicon wafers and freeze-dried. The morphology of the bacteria was observed using field emission scanning electron microscopy (FETS). SEM revealed that the cell wall and cell membrane structures of Gram-negative bacteria were extensively disrupted, while Gram-positive bacteria showed no significant disruption.

[0080] Figure 12 The image shown is an image taken using a field emission scanning electron microscope (FET) of the 3D urchin-shaped copper telluride nanoenzyme hydrogel prepared in Example 1. After freeze-drying, the prepared 3D urchin-shaped copper telluride nanoenzyme hydrogel was observed on a silicon wafer, revealing a porous microstructure.

[0081] Figure 13 A bacterial plating image of the 3D sea urchin-shaped copper telluride nanoenzyme hydrogel prepared in Example 1. The specific process is as follows: The cultured bacterial solution was diluted to 1×10⁻⁶. 6 CFU / mL, then four test materials (PBS as blank control, guar gum hydrogel as matrix control, copper telluride as active ingredient control, and copper telluride nanozyme hydrogel as experimental group) were mixed with three bacterial suspensions at a 1:1 volume ratio (total volume 200 μL / group); the mixtures were incubated in a 37°C constant temperature shaker at 200 rpm for 4 hours. After co-incubation, the reaction was quickly terminated: the bacterial pellet was collected by centrifugation at 4°C and 8000 rpm for 5 minutes, resuspended in sterile PBS and washed twice to remove unbound material fragments or residual culture medium components. Finally, the bacterial pellet was resuspended in 1 mL of sterile PBS, and the OD was calibrated again using a spectrophotometer. 600 Approximately 0.1 (corresponding to approximately 1 × 10⁻⁶) 6CFU / mL), then 100 μL of the bacterial solution was diluted by 10 times gradient, and 200 μL of the bacterial solution of different dilutions was uniformly coated on LB solid medium plate.

[0082] It can be seen from the results that the 3D urchin-like cupric telluride nanoszyme hydrogel has excellent antibacterial ability to gram-negative bacteria.

[0083] Figure 14 The mouse burn wound infected with Pseudomonas aeruginosa after treatment with the 3D urchin-like cupric telluride nanoszyme hydrogel prepared in Example 1 was healed, and a statistical diagram thereof is shown. The specific process is as follows: C57BL / 6 mice with a body weight of 20-25 g were selected to construct a burn model to form a burn wound with a depth of about II-III. 24 hours after the burn, 50 μL of Pseudomonas aeruginosa solution with a concentration of 1 x 10 7 CFU / mL was uniformly dropped on the wound, and 48 hours after the burn, the mice were randomly divided into 4 groups (n≥5 for each group): the PBS group (100 μL of PBS was dropped on the wound), the guar gum hydrogel group (50 μL of guar gum hydrogel with a concentration of 2% was uniformly applied), the cupric telluride group (5 mg of cupric telluride powder was directly applied on the wound and covered with gauze), and the cupric telluride nanoszyme hydrogel group (50 μL of cupric telluride nanoszyme composite hydrogel containing 2% guar gum was applied). The wound was photographed every day after the burn, and the mice were sacrificed after the last photograph on the 14th day (or when the wound was basically healed), and the wound tissue was subjected to pathological examination. ImageJ software was used to analyze the wound healing.

[0084] It can be seen from the results that the cupric telluride nanoszyme hydrogel has excellent therapeutic effect on the mouse burn wound infected with Pseudomonas aeruginosa.

[0085] Figure 15 The bacterial plating and statistical diagram of the mouse burn wound infected with Pseudomonas aeruginosa after treatment in Example 1 are shown. On the last day, the burn wound of each group was collected and placed in physiological saline, and the physiological saline of each group was diluted and plated to evaluate the growth of Pseudomonas aeruginosa in the wound of each treatment group, and imageJ was used for statistical analysis of the number of bacteria. It can be seen from the results that the cupric telluride nanoszyme hydrogel has excellent killing effect on Pseudomonas aeruginosa infected in the mouse burn wound.

[0086] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A 3D urchin-shaped copper telluride nanoenzyme hydrogel, characterized in that, It includes a hydrogel matrix and a 3D urchin-shaped copper telluride nanozyme dispersed in the hydrogel matrix.

2. The 3D urchin-shaped copper telluride nanoenzyme hydrogel according to claim 1, characterized in that, The 3D urchin-shaped copper telluride nanozyme has a particle size of 200–300 nm.

3. The 3D urchin-shaped copper telluride nanoenzyme hydrogel according to claim 1 or 2, characterized in that, The hydrogel matrix includes one or more of cationic guar gum hydrogel, hyaluronic acid hydrogel, alginate hydrogel and F127 hydrogel; The mass ratio of the 3D urchin-shaped copper telluride nanozyme to the hydrogel matrix is ​​1:1000-2000.

4. The method for preparing the 3D urchin-shaped copper telluride nanoenzyme hydrogel according to any one of claims 1 to 3, characterized in that, Includes the following steps: A soluble copper salt, telluric acid, dispersant, reducing agent and organic solvent were mixed and co-precipitated to obtain 3D sea urchin-shaped copper telluride nanozymes. The aqueous solution of the 3D sea urchin-shaped copper telluride nanozyme was mixed with a hydrogel matrix and gelled to obtain a 3D sea urchin-shaped copper telluride nanozyme hydrogel.

5. The preparation method according to claim 4, characterized in that, The soluble copper salt includes one or more of copper chloride, copper acetate, copper sulfate, and copper nitrate; The dispersant includes one or more of polyvinylpyrrolidone, polyethylene glycol, and cellulose ether; The reducing agent includes one or more of ascorbic acid, citric acid, oxalic acid, phosphoric acid, and malic acid.

6. The preparation method according to claim 4 or 5, characterized in that, The amount of the soluble copper salt is 5-20 parts by mass, the amount of the telluric acid is 8-30 parts, the amount of the dispersant is 100-400 parts, and the amount of the reducing agent is 30-120 parts.

7. The preparation method according to claim 4, characterized in that, The coprecipitation reaction is carried out at a temperature of 160–200°C for 3–5 hours.

8. The preparation method according to claim 4 or 7, characterized in that, The gelation is carried out under stirring conditions, and the gelation time is 5 to 10 minutes.

9. The application of the 3D urchin-shaped copper telluride nanoenzyme hydrogel according to any one of claims 1 to 3 or the 3D urchin-shaped copper telluride nanoenzyme hydrogel prepared by the preparation method according to any one of claims 4 to 8 in the preparation of antibacterial drugs or wound repair drugs.

10. The application according to claim 9, characterized in that, The antibacterial drugs include those against Gram-negative bacteria.

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