Copper-doped tricobalt tetraoxide nano-antibacterial agent and application thereof in preparation of product for treating keratitis

Abstract

The present application relates to a kind of copper-doped tricobalt tetraoxide (Cu-Co3O4) nanometer antibacterial agent and its application in the preparation of keratitis treatment product, belong to the field of biotechnology.The present application uses cobalt nitrate hexahydrate and copper nitrate trihydrate as raw material, uses isopropyl alcohol and glycerol as solvent, and successfully prepares copper-doped tricobalt tetraoxide nanometer antibacterial agent by solvothermal method.Experiment shows that under the near-infrared laser irradiation of wavelength 808nm, power density 0.5W / cm 2 , copper-doped tricobalt tetraoxide nanometer antibacterial agent has good photo-thermal, photodynamic performance and broad-spectrum antibacterial activity.In vitro experiment verifies that it can efficiently kill pathogenic bacteria, and is not prone to cause drug resistance;In vivo experiment verifies that it can effectively treat MRSA infected bacterial keratitis.The present application lays a theoretical foundation for the preparation of keratitis treatment, effective nanometer antibacterial agent and drug.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a copper-doped cobalt tetroxide nano-antibacterial agent and its application in the preparation of products for treating keratitis. Background Technology

[0002] The cornea, located at the front of the fibrous membrane of the eyeball, is the eye's first line of defense against external infections. Due to its fragile structure, the cornea is highly susceptible to invasion by pathogens such as bacteria and fungi. Improper or ineffective treatment of bacterial keratitis can lead to corneal perforation, intraocular infection, and even irreversible vision loss or blindness. Currently, clinical treatment mainly relies on antibiotic eye drops (such as levofloxacin eye drops), but due to the low bioavailability of these drugs on the ocular surface, frequent administration is often required, resulting in less than ideal treatment outcomes. Furthermore, with the widespread use of antibiotics, the emergence of drug-resistant strains (such as methicillin-resistant Staphylococcus aureus (MRSA)) and the formation of their biofilms further complicate the treatment of bacterial keratitis. Therefore, developing an efficient and convenient treatment strategy and formulation for keratitis infections is of great significance.

[0003] Based on the unique advantage of the eye's translucency, a phototherapy strategy is proposed. Currently, photothermal nanomaterials are a novel antibacterial nanotechnology, attracting widespread attention in the antibacterial field due to their good stability, simple preparation, stable photothermal properties, and avoidance of drug resistance. However, photothermal nanomaterials still have some limitations in practical applications. For example, single-mode photothermal therapy may lead to bacterial heat resistance, and continuous heating may damage normal cells and tissues. These defects limit their widespread application. Copper (Cu) is an essential trace element for the human body. Its ionic form (Cu2+) plays a crucial role in regulating various cytokines and growth factors, especially in promoting wound healing. Furthermore, Co3O4 can also act as a photosensitizer, possessing good membrane permeability and excellent biocompatibility, and is less likely to induce drug resistance. It can also catalyze the generation of reactive oxygen species (ROS) in aqueous media, disrupting the integrity of pathogenic bacteria through oxidative stress. Copper-doped cobalt tetroxide (Cu-Co3O4) nano-antibacterial agents provide a new approach to solving the above problems. It combines good photothermal and photodynamic properties. This method utilizes photodynamic therapy to generate ROS in synergistic photothermal effects, achieving efficient sterilization and biofilm control at a gentle temperature with low risk of drug resistance. It overcomes the problems of traditional photothermal therapy, such as the development of bacterial heat resistance due to single-mode photothermal treatment, damage to normal cells and tissues caused by excessively high temperatures, and local tissue damage due to high temperatures. This provides a safe and efficient solution for the treatment of bacterial keratitis. Summary of the Invention

[0004] The purpose of this invention is to provide a copper-doped cobalt tetroxide nano-antibacterial agent and its application in the preparation of products for treating keratitis, so as to solve the problems existing in the prior art. This copper-doped cobalt tetroxide nano-antibacterial agent, in conjunction with light irradiation, can significantly alleviate the symptoms of keratitis, laying the foundation for the treatment of keratitis.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing a copper-doped cobalt tetroxide nano-antibacterial agent, comprising the following steps:

[0007] (1) Under room temperature conditions, using Co(NO3)2·6H2O and Cu(NO3)2·3H2O as raw materials and glycerol and isopropanol as solvents, the raw materials were added to the mixed solvent and stirred evenly. Then, the mixture was reacted at high temperature, cooled, centrifuged, washed and dried to obtain the precursor of Cu-Co3O4 nanomaterials.

[0008] (2) The precursor of the Cu-Co3O4 nanomaterial is annealed at a slow heating rate in a high temperature environment for 2 hours to obtain a black powder Cu-Co3O4 nanomaterial, which is the copper-doped cobalt tetroxide nano antibacterial agent.

[0009] Optionally, the ratio of Co(NO3)2·6H2O, Cu(NO3)2·3H2O, glycerol and isopropanol is (0.1-0.3)mol∶(0.1-0.2)mol∶(5-10)mL∶(40-50)mL.

[0010] Optionally, the conditions for uniform stirring are: stirring the raw materials and mixed solvent at a rate of 300-500 r / min for 30-50 min; and the high-temperature reaction is: after uniform stirring of the raw materials and mixed solvent, heating at 150-200℃ for 5-8 h.

[0011] Optionally, the centrifugation is performed with a centrifugal force of 6000-8000g and a centrifugation time of 4-6min.

[0012] Optionally, the washing process involves washing with ethanol 3-5 times, each time for 3-5 minutes; the drying temperature is 50-70℃.

[0013] Optionally, the precursor of the Cu-Co3O4 nanomaterial is annealed in a high-temperature environment of 300-400℃ at a heating rate of 0.5-2℃ / min.

[0014] Optionally, the Cu-Co3O4 nano-antibacterial agent has a double-shell hollow sphere structure with a size of 529.7±2.8nm.

[0015] The present invention also provides the application of the copper-doped cobalt tetroxide nano-antibacterial agent or its synergistic illumination in the preparation of antibacterial products, wherein the illumination is near-infrared illumination, and the antibacterial effect includes killing methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and / or Salmonella typhimurium.

[0016] The present invention also provides the application of the copper-doped cobalt tetroxide nano-antibacterial agent or its synergistic illumination in the preparation of products for treating keratitis, wherein the illumination is near-infrared illumination and the keratitis is bacterial keratitis caused by MRSA infection.

[0017] Optionally, the near-infrared illumination conditions are: laser wavelength of 700-1000 nm and irradiation intensity of 0.4-0.6 W / cm². 2 The irradiation time is 10-20 minutes.

[0018] The present invention discloses the following technical effects:

[0019] 1. This invention prepares a copper-doped cobalt tetroxide nanoparticle antibacterial agent, which possesses both excellent photodynamic and photothermal activity. At a wavelength of 808 nm and a power density of 0.5 W / cm², it exhibits superior performance. 2 Under near-infrared laser irradiation for 15 minutes, in vitro experiments verified that the temperature rise can reach 45℃, which can achieve mild photothermal / photodynamic synergistic antibacterial effect within the safe range acceptable to the human body. At the same time, the hemolysis rate of the copper-doped cobalt tetroxide nano-antibacterial agent in the range of 41.7-166μg / mL is lower than the 5% safety threshold specified in the pharmacopoeia, which shows good biosafety and clinical application potential.

[0020] 2. This invention prepares a copper-doped cobalt tetroxide nano-antibacterial agent, which can effectively treat bacterial keratitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Compared with traditional antibiotic eye drops (levofloxacin), it can not only effectively kill drug-resistant bacteria, but also avoid the development of drug resistance.

[0021] 3. This invention prepares a copper-doped cobalt tetroxide nano-antibacterial agent, which has strong broad-spectrum bactericidal ability against Gram-positive and Gram-negative bacteria. It includes, but is not limited to, methicillin-resistant Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Characterization results for Cu-Co3O4; A: Scanning electron microscope image, B: Transmission electron microscope image, C: X-ray diffraction pattern, D: Energy dispersive spectroscopy image;

[0024] Figure 2 To determine the formation of O2 in Cu-Co3O4 under both illumination and no illumination for 15 minutes. 2- , 1 Electron paramagnetic resonance analysis results of O2 and ·OH; A: ·O 2- B: 1 O2, C:·OH;

[0025] Figure 3 Image of Cu-Co3O4 photothermal infrared imager;

[0026] Figure 4 The results show the in vitro antibacterial effects of Cu-Co3O4; A: Colony growth of MRSA on solid culture plates after Cu-Co3O4 light treatment and without light treatment; B: Bar charts of antibacterial rates of MRSA after Cu-Co3O4 light treatment and without light treatment; Values ​​are expressed as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001;

[0027] Figure 5 Results of broad-spectrum antibacterial activity of Cu-Co3O4: A: Colony growth of *Escherichia coli*, *Enterococcus faecalis*, *Staphylococcus aureus*, and *Salmonella typhimurium* on solid culture plates after Cu-Co3O4 light treatment and without light treatment; B: Bar chart of antibacterial rates of *Escherichia coli*, *Enterococcus faecalis*, *Staphylococcus aureus*, and *Salmonella typhimurium* after Cu-Co3O4 light treatment and without light treatment; Values ​​are expressed as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001;

[0028] Figure 6 Representative slit-lamp micrographs of BALB / c mice with MRSA-infected keratitis after different treatments at different days;

[0029] Figure 7 The image shows the growth of bacterial colonies in mouse eye homogenates treated with PBS, LVFX (levofloxacin eye drops), Cu-Co3O4 light, and no light.

[0030] Figure 8 Representative histopathological observation of paraffin sections of cornea from mice with bacterial keratitis stained with H&E and Masson staining on day 9;

[0031] Figure 9 Color changes and hemolysis rate determination results of erythrocytes treated with different concentrations of Cu-Co3O4 (41.7, 83.3, 104, 125, 166 μg / mL);

[0032] Figure 10 The mechanism of Cu-Co3O4 against biofilm; A: biofilm clearance experiment, B: biofilm inhibition experiment. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] Example 1: Preparation and Characterization of Cu-Co3O4

[0039] 1. Preparation method of Cu-Co3O4

[0040] Cu-Co3O4 nanomaterials were synthesized using a self-template strategy. At room temperature, 0.073 g of Co(NO3)2·6H2O (0.25 mmol) and 0.03 g of Cu(NO3)2·3H2O (0.125 mmol) were added to a mixed solvent of 8 mL glycerol and 40 mL isopropanol, and stirred at 400 rpm for 40 min. The resulting solution was transferred to a 100 mL autoclave and heated at 180 °C for 6 h. After natural cooling to room temperature, the product was centrifuged, washed three times with ethanol (5 min, 6976 g), and finally dried in a 60 °C oven to obtain the precursor of Cu-Co3O4 nanomaterials, namely CuCo-glyceric acid solid spheres. The prepared CuCo-glyceric acid solid spheres were then annealed in air at 350 °C with a slow heating rate of 1 °C / min. After 2 h, black powdery Cu-Co3O4 nanomaterials were obtained, which is the copper-doped cobalt tetroxide nano-antibacterial agent.

[0041] 2. Nanoparticle characterization

[0042] (1) The results were observed using a scanning electron microscope as follows: Figure 1 As shown in Figure A, Cu-Co3O4 exhibits a spherical structure with a smooth surface and uniform size.

[0043] (2) Observation by transmission electron microscopy, the results are as follows: Figure 1 As shown in Figure B, Cu-Co3O4 exhibits a double-shell hollow structure.

[0044] (3) Analysis was performed using X-ray diffraction experiments, and the results are as follows: Figure 1 As shown in Figure C, typical diffraction peaks corresponding to the cubic spinel Co3O4 (PDF42-1467) were observed, but no diffraction peaks related to Cu were found, indicating that Cu exists in a doped manner.

[0045] (4) Analysis using energy-dispersive spectroscopy yielded the following results: Figure 1 As shown in Figure D, no phase separation or elemental segregation was observed, indicating that Cu, Co, and O are highly dispersed throughout the nanoparticle structure.

[0046] The above experimental results demonstrate that Cu-Co3O4 was successfully prepared.

[0047] (5) Electron paramagnetic resonance data of Cu-Co3O4

[0048] Electron paramagnetic resonance (EPR) was used to measure the performance of Cu-Co3O4 liquid material under dark conditions for 15 min and at a power of 808 nm and a power density of 0.5 W / cm².2 Types of free radicals generated by near-infrared laser irradiation for 15 minutes: hydroxyl radicals, superoxide anion radicals, and singlet oxygen.

[0049] like Figure 2 As shown, under 15 min of darkness, superoxide anions (O2) 2- ), singlet oxygen ( 1 O2), hydroxyl radicals (OH) - The peak value of reactive oxygen species generated by Cu-Co3O4 under light irradiation for 15 minutes is higher than that under dark conditions for 15 minutes, indicating that the generation of reactive oxygen species can be promoted under near-infrared light irradiation. Cu-Co3O4 material has photodynamic effect (PDT).

[0050] 3. Photothermal infrared imager diagram of Cu-Co3O4

[0051] Different concentrations of Cu-Co3O4 (50, 100, 200, 300, 400 μg / mL) were used at 0.5 W / cm². 2 The Cu-Co3O4 was irradiated with near-infrared light for 15 minutes. Real-time infrared images of Cu-Co3O4 were captured at different times (0, 3, 6, 9, 12, 15 minutes) using a near-infrared imager. The real-time temperature was recorded every 20 seconds using an electronic thermometer.

[0052] The results are as follows Figure 3 As shown, images captured by a near-infrared imager at different time points (0, 3, 6, 9, 12, and 15 min) clearly demonstrate the temperature change trend of different concentrations of Cu-Co3O4. With increasing time and concentration, the temperature shows a significant upward trend, indicating that the copper-doped cobalt tetroxide nano-antibacterial agent provided by this invention has excellent photothermal properties.

[0053] 4. Study on the in vitro antibacterial effect of Cu-Co3O4

[0054] MRSA bacterial culture to OD 600 After 0.5 (10 8 CFU / mL), diluted 100 times with LB broth. 6 CFU / mL, for later use. Final concentration diluted 200-fold. Set Cu-Co3O4 concentration gradients as follows: 5, 12.5, 25, 37.5, 50, 62.5, 75, 87.5 μg / mL; PBS control group.

[0055] Set the group as follows:

[0056] NIR(-): PBS, 5, 12.5, 25, 37.5, 50, 62.5, 75, 87.5μg / mL Cu-Co3O4;

[0057] NIR(+): PBS, 5, 12.5, 25, 37.5, 50, 62.5, 75, 87.5μg / mL Cu-Co3O4;

[0058] More than 100 μL of solution was mixed with 100 μL of MRSA bacterial culture. The NIR(-) group was placed in the dark for 15 min, and the NIR(+) group was subjected to 808 nm, 0.5 W / cm². 2 Irradiate with laser light for 15 min. Transfer 10 μL onto solid agar medium and incubate at 37°C for 16 h.

[0059] like Figure 4 As shown, regardless of whether light was added, a large number of colonies grew on the solid culture medium plates in the PBS group, indicating that the PBS group had no antibacterial ability. Secondly, under no light conditions, as the concentration of Cu-Co3O4 gradually increased, the number of colonies decreased accordingly. When the Cu-Co3O4 concentration reached 87.5 μg / mL, almost no colonies grew on the solid culture medium plates, indicating that Cu-Co3O4 has antibacterial ability. In contrast, under light conditions (808 nm, 0.5 W / cm²),... 2 After 15 minutes of irradiation, a Cu-Co3O4 concentration of 62.5 μg / mL was sufficient to achieve no colony growth on solid culture plates, indicating that the amount of photosensitizer required under light conditions can be shortened and synergistic antibacterial effects can be achieved. Therefore, 62.5 μg / mL is considered the minimum bactericidal concentration of Cu-Co3O4 under light conditions.

[0060] To further evaluate the antibacterial properties, this invention measured the optical density (OD) of the bacterial suspension at 600 nm. 600 Regardless of whether near-infrared irradiation is applied, the antibacterial rate increases with the concentration of Cu-Co3O4. Without light irradiation, the Cu-Co3O4 concentration reaches 87.5 μg / mL, with an antibacterial rate close to 90%, indicating that Cu-Co3O4 itself can inhibit bacteria by generating active oxygen. In contrast, under light irradiation, the antibacterial rate reaches 90% when the Cu-Co3O4 concentration is 62.5 μg / mL, indicating that Cu-Co3O4 can not only achieve antibacterial effects through photothermal properties, but also synergistically kill bacteria with photodynamic properties.

[0061] 5. Study on the broad-spectrum antibacterial effect of Cu-Co3O4

[0062] Cultures of four common clinical pathogens—Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Salmonella typhimurium—to OD200. 600nm After reducing the concentration to 0.5, dilute it 100 times with broth and set aside. Prepare Cu-Co3O4 with a concentration of MBC (62.5 μg / mL) and set aside.

[0063] 100 μL of Cu-Co3O4 solution was mixed with more than 100 μL of the four bacterial cultures. The NIR(-) group was placed in the dark for 15 min, and the NIR(+) group was exposed to a laser at 808 nm and 0.5 W / cm². 2 Irradiate for 15 minutes. After the above operations are completed, transfer 10 μL onto an agar plate and incubate at 37°C for 16 hours.

[0064] like Figure 5 As shown, dense bacterial colonies were observed on the solid culture medium plates of the PBS group regardless of whether near-infrared laser irradiation was applied, indicating that PBS has no antibacterial ability. In contrast, Cu-Co3O4 also produced a large number of colonies of the four pathogenic bacteria on the solid culture medium plates without light irradiation, indicating that Cu-Co3O4 has no antibacterial effect against the four common clinical pathogenic bacteria. On the contrary, under light irradiation, the bacterial colonies of the four pathogenic bacteria on the solid culture medium plates were almost eradicated, and the antibacterial efficiency against all tested bacteria exceeded 90%, indicating that Cu-Co3O4+NIR has broad-spectrum antibacterial activity.

[0065] 6. Therapeutic effect of MRSA-induced bacterial keratitis in mice

[0066] 6.1 Establishment of a mouse model of bacterial keratitis:

[0067] Six-week-old female BALB / c mice were selected and anesthetized intraperitoneally with 2.5% chloral hydrate, followed by local anesthesia with 0.5% promecaine hydrochloride eye drops. Using a sterile needle, 3-5 parallel incisions, each 1 mm long, were gently made on the corneal surface. Logarithmic growth phase MRSA bacterial solution (0.75 × 10⁻⁶) was then injected. 6 CFU / mL (30 μL) was instilled into the scratched area of ​​the eye and left to stand for several seconds. After 24 hours of feeding, all mice showed varying degrees of corneal opacity and decreased corneal transparency, indicating successful establishment of a bacterial keratitis mouse model.

[0068] 6.2 Treatment of ocular infections in mice with bacterial keratitis

[0069] Mice were randomly divided into 6 groups: healthy group, PBS group, PBS+NIR group, LVFX (levofloxacin) group, Cu-Co3O4 group, and Cu-Co3O4+NIR group, with 3 mice in each group. All groups received the same drug dosage (60 μL, 62.5 μg / mL). The laser irradiation conditions for the PBS+NIR group and the Cu-Co3O4+NIR group were the same (808 nm, 0.5 W / cm²). 2 (10 min). The severity and treatment of the eye infection were continuously observed from day 1, and all observations were conducted under a slit lamp.

[0070] like Figure 6 As shown, 24 hours after infection, all mice exhibited varying degrees of corneal opacity and decreased corneal transparency. In the PBS and PBS+NIR groups, corneal opacity increased over time, eventually spreading to the entire eyeball. Compared to the PBS group, the LVFX and Cu-Co3O4 groups showed a slight reduction in corneal opacity on day 9. In contrast, the Cu-Co3O4+NIR group showed a significantly different treatment effect compared to the PBS, PBS+NIR, LVFX, and Cu-Co3O4 groups on day 7, indicating excellent infection control. Compared to the healthy group, the cornea regained transparency, and the iris texture and pupil were clearly visible by day 9. These results demonstrate that Cu-Co3O4+NIR treatment can significantly cure bacterial keratitis in mice, confirming that Cu-Co3O4+NIR has good in vitro and in vivo bactericidal and anti-infective efficacy.

[0071] 6.3 Bacterial Culture Experiment

[0072] The dissected mouse eyeballs were homogenized in 4-5 mL of sterile broth, diluted 10,000 times with PBS, and 10 μL was transferred onto LB agar plates. The mixture was spread evenly with a cotton swab, and the plates were incubated for 16 hours. Colony formation was then observed, and photographs were taken. Colony counting was performed using ImageJ.

[0073] Colonies on solid culture media were counted using the plate count method, and the results are as follows: Figure 7 As shown, regardless of whether light exposure was applied, the PBS group exhibited a large number of colonies on its solid culture medium plates, indicating that PBS had no antibacterial effect. Compared with the PBS group and the PBS+NIR group, the colony counts in the other three groups showed a significant decreasing trend. In contrast, the Cu-Co3O4+NIR group had the fewest colonies on the solid culture medium of mouse eyeball homogenate, showing a significant difference compared to the eyeballs of mice treated with levofloxacin (LVFX) and Cu-Co3O4. This indicates that Cu-Co3O4+NIR can effectively treat bacterial keratitis in mice.

[0074] 6.4 Observation of H&E staining and Masson histopathological tissue section staining of corneal tissue in mice with bacterial keratitis

[0075] After treatment, the mice were euthanized, the entire eyeball was removed, the eyeball was fixed and stained with H&E and Masson staining, mounted, and subjected to pathological histological analysis.

[0076] like Figure 8 As shown, the histopathological results of H&E and Masson sections revealed that, compared with the healthy group, the corneal structure of MRSA-infected individuals in the PBS and PBS+NIR groups was irregular, with concave areas, accompanied by significant interstitial edema and abundant inflammatory cell infiltration. In the LVFX and Cu-Co3O4 groups, although the inflammatory response was somewhat alleviated, considerable inflammatory cell infiltration and interstitial edema were still observed. In contrast, the corneal structure in the Cu-Co3O4+NIR group was essentially restored to normal, with clear tissue structure and a significantly reduced inflammatory response.

[0077] 7. Biosafety evaluation of Cu-Co3O4

[0078] One mL of whole blood from a healthy mouse was added to 7 mL of 0.9% NaCl solution for resuscitation. The mixture was centrifuged at 8000 rpm for 3 min, and the supernatant was discarded. The supernatant was washed repeatedly with 0.9% NaCl until it was no longer red or slightly yellowish. The lower layer of red blood cells was resuspended in four times the volume of 0.9% NaCl to obtain a red blood cell suspension. Experimental groups were set up with Cu-Co3O4 (41.7, 83.3, 104, 125, 166 μg / mL), a positive control (H2O), and a negative control (0.9% NaCl). 500 μL of different concentrations of Cu-Co3O4, H2O, and 0.9% NaCl were mixed with 100 μL of the red blood cell suspension, and incubated in a 37℃ water bath for 1 h. After incubation, the mixture was centrifuged at 6000 rpm for 5 min. The color of the supernatant and the UV absorbance at 545 nm were observed, and the hemolysis rate was calculated.

[0079] Hemolysis rate (%) = 100 × (Asample - A) 0.9％NaCl ) / (A H2O -A 0.9％NaCl )

[0080] like Figure 9As shown, the blood safety of Cu-Co3O4 was evaluated using an in vitro hemolysis experiment. The absorbance at 545 nm of the supernatant after incubation of Cu-Co3O4 with erythrocyte suspension at different concentrations (41.7, 83.3, 104, 125, and 166 μg / mL) was measured using ultraviolet absorption spectroscopy. The results showed that the absorbance values ​​of the supernatant in each concentration group were similar to those of the negative control group (0.9% NaCl solution), and the supernatant remained clear without obvious red color (as shown in the inset). Further calculations indicated that the hemolysis rate of Cu-Co3O4 at all concentrations was below the 5% safety threshold specified in the 2020 edition of the Chinese Pharmacopoeia. These results fully demonstrate that Cu-Co3O4 has good blood safety.

[0081] 8. Anti-biofilm mechanism of Cu-Co3O4

[0082] Biofilm removal experiment: MRSA bacterial culture was cultured to (10) 6 Add 100 μL of bacterial culture to each well of a 96-well plate (CFU / mL). Incubate the plate in a 37℃ biochemical incubator for 48 h. After 48 h of incubation following different treatments, a viscous substance floating in the wells is observed, which is the biofilm formed. Aspirate the culture medium from the wells and wash twice with PBS. Add 100 μL of PBS / Cu-Co3O4 to each well and administer different treatments. After standing for 1 h, aspirate the liquid from the wells and wash twice with PBS. Add 200 μL of methanol to fix the biofilm for 15 min, then aspirate the methanol with a syringe and stain the biofilm with 0.05% (w / v) crystal violet solution prepared with 100 μL of PBS. Aspirate the dye after 30 min and wash three times with PBS. Add 100 μL of 95% ethanol solution to each well to dissolve the dye. Finally, measure the absorbance of each group of samples at 590 nm using a microplate reader to measure the amount of bacterial biofilm formation. Biofilm inhibition experiment: Cultivate MRSA bacterial culture to (10 6 After confirming the bacterial concentration (CFU / mL), add 100 μL of bacterial culture to each well of a 96-well plate. Add more than 100 μL of solution to each 100 μL of bacterial culture and mix for different treatments. Incubate the plates at 37°C for 48 h. After 48 h, aspirate the liquid from the wells (avoiding biofilm aspiration) and wash twice with sterile PBS. Add 200 μL of methanol to fix the biofilm for 15 min, then aspirate the methanol with a syringe and stain the biofilm with 0.05% (w / v) crystal violet solution prepared with 1 mL PBS. After 30 min, aspirate the dye and wash three times with PBS. Add 100 μL of 95% ethanol solution to each well to dissolve the dye. Measure the absorbance of each sample at 590 nm using a microplate reader to determine the amount of bacterial biofilm formation.

[0083] like Figure 10As shown, the PBS group and the PBS+NIR group showed the darkest crystal violet staining and the highest biofilm formation, indicating that PBS had no biofilm inhibitory effect under both light and no light conditions. The Cu-Co3O4 group showed a lighter crystal violet staining than the PBS group, and the Cu-Co3O4 group also showed a lower biofilm formation, indicating that the Cu-Co3O4 group could remove some biofilm. In contrast, the Cu-Co3O4+NIR group showed the lightest crystal violet staining, showing a significant difference from the other three groups, and the lowest biofilm formation, indicating that the Cu-Co3O4+NIR group could effectively inhibit biofilm growth. Further investigation was then conducted to explore the ability of Cu-Co3O4 to inhibit biofilm formation. The crystal violet staining images of both the PBS-lit and unlit groups were darker, and a large amount of biofilm was produced in the PBS group, indicating that PBS had no biofilm inhibitory effect. In contrast, the crystal violet staining images of the Cu-Co3O4 group were lighter than those of the PBS group, and the amount of biofilm formed was lower in the Cu-Co3O4 group, indicating that the Cu-Co3O4 group could inhibit some biofilm growth. In comparison, the crystal violet staining images of the Cu-Co3O4+NIR group were the lightest, showing a significant difference from the other three groups, and the amount of biofilm formed was significantly reduced, indicating that the Cu-Co3O4+NIR group could effectively inhibit biofilm growth.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of copper-doped cobalt tetroxide nano-antibacterial agent in the preparation of antibacterial products, characterized in that, The preparation method of the copper-doped cobalt tetroxide nano-antibacterial agent includes the following steps: (1) Under room temperature conditions, using Co(NO3)2∙6H2O and Cu(NO3)2∙3H2O as raw materials and glycerol and isopropanol as solvents, the raw materials were added to the mixed solvent and stirred evenly. Then, the mixture was reacted at high temperature, cooled, centrifuged, washed and dried to obtain the precursor of Cu-Co3O4 nanomaterials. (2) The precursor of the Cu-Co3O4 nanomaterial is annealed in a high-temperature environment for 2 hours to obtain a black powder Cu-Co3O4 nanomaterial, which is the copper-doped cobalt tetroxide nano antibacterial agent. The ratio of Co(NO3)2∙6H2O, Cu(NO3)2∙3H2O, glycerol and isopropanol is (0.1-0.3) mol : (0.1-0.2) mol : (5-10) mL : (40-50) mL; The precursor of the Cu-Co3O4 nanomaterial was annealed in a high-temperature environment of 300-400℃ at a heating rate of 0.5-2℃ / min; The antibacterial properties include killing methicillin-resistant Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and / or Salmonella typhimurium; The copper-doped cobalt tetroxide nano-antibacterial agent exhibits broad-spectrum antibacterial activity under near-infrared light irradiation. The Cu-Co3O4 has a double-shell hollow spherical structure with a size of 529.7±2.8 nm.

2. The application as described in claim 1, characterized in that, The conditions for uniform stirring are as follows: the raw materials and mixed solvent are stirred at a rate of 300-500 r / min for 30-50 min; the high-temperature reaction is as follows: after the raw materials and mixed solvent are stirred evenly, they are heated at 150-200 ℃ for 5-8 h.

3. The application as described in claim 1, characterized in that, The centrifugation process involves a centrifugal force of 6000-8000 g and a centrifugation time of 4-6 min.

4. The application as described in claim 1, characterized in that, The washing process involves washing with ethanol 3-5 times, each time for 3-5 minutes; the drying temperature is 50-70 ℃.

5. The application as described in claim 1, characterized in that, The near-infrared illumination conditions are: laser wavelength of 700-1000 nm and power density of 0.4-0.6 W / cm². 2 The irradiation time is 10-20 minutes.

6. The application as described in claim 1, characterized in that, The antibacterial products include products for treating keratitis, specifically bacterial keratitis caused by methicillin-resistant Staphylococcus aureus (MRSA).

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