Defect-rich CoS / CoS2 heterojunction nanozymes, their preparation methods and applications

Abstract

This invention aims to provide a defect-rich S-type CoS / CoS2 heterojunction multifunctional nanozyme, prepared from a polar organic solvent, a soluble cobalt salt, a surfactant, and an organic sulfur source. This nanozyme possesses both peroxidase (POD) and catalase (CAT) properties. The invention also explores the application of this defect-rich CoS / CoS2 heterojunction nanozyme in the preparation of antibacterial drugs and wound-healing drugs. Benefiting from the introduction of S-type heterojunctions and suitable defects, this nanozyme can spontaneously and abundantly generate reactive oxygen species (ROS) such as hydroxyl radicals (·OH), superoxide anions (O2−), and singlet oxygen (1O2) at room temperature without external stimulation such as light.

Description

Technical Field

[0001] This invention relates to the field of interdisciplinary technology of nanomaterials and biomedicine, and in particular to a defect-rich CoS / CoS2 heterojunction nanozyme. Background Technology

[0002] The overuse of antibiotics in clinical and agricultural fields has led to the emergence of numerous multidrug-resistant pathogens (such as Pseudomonas aeruginosa), triggering a serious public health crisis. In recent years, nanomaterials, due to their powerful antibacterial properties and low susceptibility to bacterial resistance, have become an effective alternative to antibiotics. Among them, nanozymes, as a new generation of nanomaterials that mimic the catalytic activity of natural enzymes, offer advantages such as good stability, low cost, and ease of large-scale production.

[0003] However, existing peroxidase (POD) and catalase (CAT) nanozymes face two major objective drawbacks in practical applications: (1) The rapid recombination of photogenerated electron-hole pairs and the slow reaction kinetics limit the catalytic efficiency; (2) Existing high-efficiency nanozyme systems usually require external stimuli (such as light, ultrasound, electricity or magnetic field) to trigger their enzyme-like activity. In particular, photocatalytic nanozymes often cannot spontaneously generate enough free radicals to inactivate drug-resistant bacteria in dark environments (such as deep infection in the body or no light conditions), which greatly limits their convenience for practical application in clinical in vivo. Summary of the Invention

[0004] In view of the above technical problems, the present invention aims to provide a defect-rich S-type CoS / CoS2 heterojunction multifunctional nanozyme, which is prepared by polar organic solvent, soluble cobalt salt, surfactant and organic sulfur source, with a mass ratio of 40:1:0.9:0.8; the nanozyme has both peroxidase (POD) and catalase (CAT) characteristics.

[0005] The preparation method involves the following steps: S1: Mix the polar organic solvent, soluble cobalt salt and surfactant, and stir until homogeneous to form a uniform solution; S2: Add an organic sulfur source to the homogeneous solution and continue stirring to obtain a precursor mixture; S3: Transfer the precursor mixture to a closed reaction vessel for a solvothermal reaction; S4: After the reaction is complete, cool to room temperature, and then separate the solid and liquid components, wash and dry to obtain the defect-rich CoS / CoS2 heterojunction nanozyme.

[0006] In step S1, the polar organic solvent is an amide-based polar solvent, preferably N,N-dimethylacetamide; The soluble cobalt salt is a water-soluble divalent cobalt salt, preferably cobalt chloride hexahydrate; The surfactant is a nonionic polymeric surfactant, preferably polyethylene glycol 2000.

[0007] In step S2, the organic sulfur source is a slow-release sulfur-containing compound, preferably thioacetamide.

[0008] The preparation method and raw material ratio of a defect-rich CoS / CoS2 heterojunction nanozyme are as follows: Step 1: Add 40ml of N,N-dimethylacetamide, 1.0g of cobalt chloride hexahydrate (CoCl2·6H2O) and 0.9g of surfactant polyethylene glycol 2000 (PEG2000) to a 50ml beaker; Step 2: Perform magnetic stirring for 0.5 hours (0.5h) to form a homogeneous precursor mixture solution; Step 3: Add 0.8g of thioacetamide to the above homogeneous solution. Step 4: Transfer the mixture to a 50 ml PTFE-lined stainless steel high-pressure reactor and perform solvothermal treatment at 180°C for 20 hours (20 h). Step 5: After the reaction vessel has cooled to room temperature, separate the precipitate by centrifugation; Step 6: Wash the precipitate with water and anhydrous ethanol to remove impurities, and finally obtain the CoS / CoS2 heterojunction multifunctional nanozyme.

[0009] Application of the defect-rich CoS / CoS2 heterojunction nanozyme in the preparation of antibacterial drugs.

[0010] Application of the defect-rich CoS / CoS2 heterojunction nanozyme in the preparation of drugs that promote wound healing.

[0011] The beneficial effects of this invention are as follows:

[0012] 1. A novel one-step method for preparing biphase co-occurring nanosheets is provided.

[0013] This application overcomes the challenge of simultaneously and uniformly controlling the coexistence of two different crystalline phases, CoS and CoS2, in a one-step reaction by combining a specific precursor (cobalt chloride and thioacetamide) with a soft template agent (surfactant polyethylene glycol 2000). The present invention abandons the conventional thiourea sulfur source, creatively selecting thioacetamide and precisely matching it with polyethylene glycol 2000 of a specific molecular weight and DMAc solvent. This specific combination forms a unique microscopic reaction kinetic environment at 180°C, successfully overcoming the thermodynamic barrier of two-phase coexistence and inducing the product to grow into "regular polygonal nanosheets" with a high specific surface area in one step, avoiding the batch-to-batch instability problems caused by traditional multi-step deposition or physical mixing.

[0014] 2. A simple process for in-situ defect engineering using the "one-pot method" is provided.

[0015] Existing defect-type nanomaterials, such as vacancy-rich catalysts, typically rely on demanding and expensive post-processing techniques to create lattice defects. This multi-step method is not only cumbersome and energy-intensive, but also makes it difficult to precisely control the concentration and location of defects. This invention employs a simplified "one-pot solvothermal method." Under specific reaction conditions at 180°C, while the material assembles a coherent heterojunction, a large number of specific metal / sulfur double-vacancy defects (i.e., the hete-V structure of VSCoCo and VSCoSSSS) are spontaneously and in situ generated within its lattice. This "in-situ growth with inherent defects" process significantly simplifies the manufacturing process and reduces the cost of industrial production.

[0016] 3. This invention overcomes environmental limitations, eliminating the need for light-driven processes and achieving efficient room-temperature generation of reactive oxygen species without the need for light.

[0017] This invention benefits from the introduction of S-type heterojunctions and suitable defects. This nanozyme can spontaneously and massively generate reactive oxygen species (ROS) such as hydroxyl radicals (·OH), superoxide anions (O2-), and singlet oxygen (1O2) at room temperature without the need for external stimulation such as light.

[0018] 4. The nanozyme proposed in this invention has excellent antibacterial efficiency.

[0019] The product of this invention exhibits excellent peroxidase (POD) and catalase (CAT) activities. At a low concentration of 50 ppm, it can achieve a kill rate of up to 99.99% against Pseudomonas aeruginosa after 30 minutes of treatment, and can significantly damage the bacterial cell membrane and induce DNA damage.

[0020] 5. The nanozyme proposed in this invention has a significant wound healing promoting effect.

[0021] In an in vivo wound model of drug-resistant bacterial infection, the wound healing rate reached approximately 51.9% after 6 days of treatment with the nanozyme of this invention (far exceeding the 4.8% of the control group). It can effectively reduce the accumulation of immune cells at the wound site and significantly reduce bacterial load, demonstrating extremely high clinical translational value. Attached Figure Description

[0022] Figure 1 This is a scanning electron microscope (SEM) image of the CoS / CoS2 multifunctional heterojunction nanozyme of the present invention.

[0023] Figure 2 The image shown is a high-resolution transmission electron microscope (HR-TEM) image of the CoS / CoS2 multifunctional heterojunction nanozyme of this invention, indicating the lattice spacing of CoS and CoS2.

[0024] Figure 3 This is the EPR spectrum of free radicals generated by the nanozyme of the present invention under light-free conditions.

[0025] Figure 4 This is a comparison chart of the in vitro antibacterial performance (plate count method) of the nanozyme of the present invention against Pseudomonas aeruginosa.

[0026] Figure 5 This is a comparison of the healing effects of the nanozyme of the present invention on a mouse model of wound infection caused by drug-resistant bacteria. Detailed Implementation

[0027] The technical solution of the present invention will be described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0028] Example 1

[0029] The preparation method of defect-rich CoS / CoS2 heterojunction multifunctional nanozymes is as follows.

[0030] First, add 40 ml of N,N-dimethylacetamide, 1.0 g of cobalt chloride hexahydrate, and 0.9 g of surfactant polyethylene glycol 2000 to a 50 ml beaker, and stir magnetically for 0.5 hours at room temperature to form a completely homogeneous solution.

[0031] Subsequently, 0.8 g of thioacetamide was added to the homogeneous solution and stirred thoroughly. Then, the entire mixture was transferred to a 50 mL polytetrafluoroethylene-lined stainless steel high-pressure reactor, sealed, and placed in an oven for solvothermal reaction at 180°C for 20 hours.

[0032] After the reaction was completed, the reaction vessel was allowed to cool naturally to room temperature. The resulting mixture was removed and centrifuged, and the bottom precipitate was collected. The precipitate was washed several times with deionized water and anhydrous ethanol to thoroughly remove residual impurities. Finally, it was dried to obtain a CoS / CoS2 heterostructured multifunctional nanozyme product in the form of regular polygonal nanosheets.

[0033] Example 2

[0034] The preparation method of single-phase CoS nanozymes is as follows.

[0035] Take a 50 mL beaker and add 40 mL of anhydrous ethanol, 0.8 g of cobalt nitrate hexahydrate (Co(NO3)2·6H2O), and 200 μL of aniline in sequence. Stir on a magnetic stirrer for 0.5 hours to form a homogeneous solution. Then add 0.4 g of thiourea to the above solution and continue stirring until completely dissolved.

[0036] The resulting mixture was transferred to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene, sealed, and placed in an oven for a solvothermal reaction at 170 °C for 14 hours.

[0037] After the reaction was complete, the reaction vessel was allowed to cool naturally to room temperature. The reaction product was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 minutes, discarding the supernatant. The resulting precipitate was washed three times each with deionized water and anhydrous ethanol, and centrifuged under the same conditions after each wash.

[0038] The washed product was placed in a vacuum drying oven and dried at 60 °C for 12 hours until constant weight was obtained, thus obtaining the monophase CoS nanozyme.

[0039] Example 3

[0040] The preparation method of single-phase CoS2 nanozymes is as follows.

[0041] Take a 50 mL beaker and add 40 mL of N,N-dimethylacetamide, 1.0 g of cobalt chloride hexahydrate (CoCl2·6H2O), and 0.9 g of polyethylene glycol 4000 (PEG-4000) in sequence. Stir on a magnetic stirrer for 0.5 hours to form a homogeneous solution. Then add 0.8 g of thiourea to the above solution and continue stirring until completely dissolved.

[0042] The resulting mixture was transferred to a 50 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene, sealed, and placed in an oven for a solvothermal reaction at 150 °C for 20 hours.

[0043] After the reaction was complete, the reaction vessel was allowed to cool naturally to room temperature. The reaction product was transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 minutes, discarding the supernatant. The resulting precipitate was washed three times each with deionized water and anhydrous ethanol, and centrifuged under the same conditions after each wash.

[0044] The washed product was placed in a vacuum drying oven and dried at 60 °C for 12 hours until constant weight was obtained, thus obtaining the single-phase CoS2 nanozyme.

[0045] Example 4

[0046] The in vitro antibacterial properties and bacterial cell membrane damage evaluation methods of the nanozyme of this invention are as follows: Pseudomonas aeruginosa was selected as a representative pathogenic bacterium.

[0047] The bacteria were inoculated into LB medium and cultured overnight with shaking at 37 degrees Celsius and 200 rpm. The next day, the bacterial culture was diluted 50 times in fresh LB medium and incubated until the logarithmic growth phase, so that the absorbance value at a wavelength of 600 nanometers reached about 1.0.

[0048] Collect bacterial cells in the logarithmic growth phase and dilute with physiological saline.

[0049] Take 3 ml of bacterial suspension containing 10^6 colony-forming units, mix it with 50 ppm of the above nanozyme, and incubate with shaking at 37 degrees Celsius for 30 minutes.

[0050] The bacterial culture was then serially diluted with physiological saline, and samples were spread onto agar plates and incubated at 37 degrees Celsius for 24 hours for colony counting.

[0051] The results showed that the kill rate of Pseudomonas aeruginosa reached approximately 99.99% after treatment with CoS / CoS2 heterojunction nanozymes.

[0052] Further assessment of cell membrane damage was conducted using dual-fluorescence staining.

[0053] After centrifugation and washing, the treated Pseudomonas aeruginosa was resuspended in physiological saline, and 1 µM green nucleic acid dye and 10 µM red fluorescent nucleic acid dye were added. The mixture was incubated at room temperature in the dark for 15 min, and then observed and imaged under a fluorescence microscope.

[0054] The results showed that the control group had almost no staining, while the red fluorescence was significantly enhanced after treatment with the nanozyme of this invention, indicating that the bacterial cell membrane was severely damaged.

[0055] Example 5

[0056] Evaluation of the effect of the nanozyme of the present invention on promoting wound healing in vivo by Pseudomonas aeruginosa infection.

[0057] Animal model establishment: Six-week-old SPF-grade female BALB / c mice were selected to establish a dorsal skin wound infection model. Before the experiment, the hair on the back of the mice was shaved, and a full-thickness circular incision of about 0.5 cm in diameter was made in the midline area of ​​the mouse's back using sterile surgical scissors.

[0058] Bacterial inoculation: Pseudomonas aeruginosa was cultured to the logarithmic growth phase, and the absorbance of the bacterial suspension at 600 nm was adjusted to 1.0. The cells were collected by centrifugation, resuspended in sterile phosphate-buffered saline (PBS), and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 CFU / mL. Apply 0.1 mL of the above bacterial suspension to each wound (i.e., 1 × 10⁻⁶ CFU / mL per wound). 8 CFU), to establish a local infection model.

[0059] Grouping and drug administration: One hour after infection establishment, mice were randomly divided into two groups. Mice in the experimental group were uniformly coated with 50 ppm of the CoS / CoS2 heterojunction multifunctional nanozyme (dispersed in PBS) prepared in Example 1 at the wound site; mice in the control group were coated with an equal volume of PBS solution at the wound site.

[0060] Therapeutic effect evaluation:

[0061] (1) Wound healing rate: The wound area of ​​mice in each group was measured on day 6 of treatment. The results showed that the wound healing rate of the experimental group was about 51.9%, while the healing rate of the control group was only 4.8%.

[0062] (2) Tissue bacterial load: Wound tissues from mice in each group were collected, homogenized, and then plated for bacterial count. The results showed that the tissue bacterial load of the experimental group mice was significantly lower than that of the control group.

[0063] (3) Histopathological analysis: H&E staining showed that there was obvious immune cell infiltration and severe inflammatory reaction in the wound tissue of the control group; while the wound tissue of the experimental group had intact boundaries and the phenomenon of immune cell aggregation was significantly reduced.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. The various components mentioned in this invention are common technologies in the existing field. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A defect-rich S-shaped CoS / CoS2 heterojunction multifunctional nanoszyme, characterized in that, A polar organic solvent, a soluble cobalt salt, a surfactant, and an organic sulfur source are mixed in a mass ratio of 40:10:0.9:0.

8. The nanoscale enzyme has the characteristics of peroxidase (POD) and catalase (CAT); The operation steps of the preparation method are: S1: the polar organic solvent, the soluble cobalt salt, and the surfactant are mixed and stirred uniformly to form a uniform solution; S2: the organic sulfur source is added to the uniform solution, and stirring is continued to obtain a precursor mixture; S3: the precursor mixture is transferred to a sealed reaction container for solvothermal reaction; S4: after the reaction is completed, the temperature is cooled to room temperature, and the solid-liquid separation, washing, and drying are performed to obtain the CoS / CoS2 heterojunction nanoscale enzyme rich in defects.

2. A defective S-type CoS / CoS2 heterojunction multifunctional nanoszyme according to claim 1, characterized in that, In step S1, the polar organic solvent is an amide polar solvent, preferably N,N-dimethylacetamide; The soluble cobalt salt is a water-soluble divalent cobalt salt, preferably cobalt chloride hexahydrate; The surfactant is a non-ionic high-molecular surfactant, preferably polyethylene glycol 2000.

3. The preparation method of claim 1, wherein: In step S2, the organic sulfur source is a slow-release sulfur-containing compound, preferably thioacetamide.

4. A defective S-type CoS / CoS2 heterojunction multifunctional nanoszyme according to claim 1, wherein, The operation steps and raw material ratios of the preparation method are: A CoS / CoS2 heterojunction nanoscale enzyme rich in defects, the operation steps and raw material ratios of the preparation method are: Step 1: 40 ml of N,N-dimethylacetamide, 1.0 g of cobalt chloride hexahydrate (CoCl2·6H2O), and 0.9 g of surfactant polyethylene glycol 2000 (PEG2000) are added to a 50 ml beaker; Step 2: magnetic stirring is performed for 0.5 hours (0.5 h) to form a uniform precursor mixture solution; Step 3: 0.8 g of thioacetamide is added to the uniform solution; Step 4: the mixture is transferred to a polytetrafluoroethylene-lined stainless steel high-pressure reaction kettle with a capacity of 50 ml, and solvothermal treatment is performed at 180°C for 20 hours (20 h); Step 5: after the reaction kettle is cooled to room temperature, the precipitate is separated by centrifugation; Step 6: the precipitate is washed with water and anhydrous ethanol to remove impurities, and finally a CoS / CoS2 heterojunction multifunctional nanoscale enzyme is obtained.

5. A defective S-type CoS / CoS2 heterojunction multifunctional nanoszyme according to claim 1, wherein, In step S3, the temperature of the solvothermal reaction is 160°C to 200°C, preferably 180°C, and the reaction time is 12 h to 24 h.

6. A defective S-type CoS / CoS2 heterojunction multifunctional nanoszyme according to claim 1, wherein, In step S4, the washing is performed with deionized water and anhydrous ethanol for 2 to 4 times each; the drying is vacuum drying at a temperature of 50°C to 70°C for 8 h to 12 h.

7. A defective S-type CoS / CoS2 heterojunction multifunctional nanoszyme according to any one of claims 1-6, characterized in that The CoS / CoS2 heterojunction nanoscale enzyme rich in defects is used in the preparation of an antibacterial drug.

8. A defective S-type CoS / CoS2 heterojunction multifunctional nanoszyme according to any one of claims 1-6, characterized in that The CoS / CoS2 heterojunction nanoscale enzyme rich in defects is used in the preparation of a wound healing promoting drug.

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