Photoresponse carbon dot nano-enzyme antibacterial material as well as preparation method and application thereof

Abstract

The invention discloses a photoresponse carbon dot nano-enzyme antibacterial material as well as a preparation method and application thereof. Carbon dots are successfully prepared by taking tartaric acid and 3-acetaminophenol as precursors through a hydrothermal method. Under the illumination condition, the prepared carbon dots show excellent oxidase simulation activity, oxygen can be efficiently catalyzed to be converted into superoxide anions, and the carbon dots are endowed with excellent antibacterial performance. In the verification of a staphylococcus aureus infected wound model, the carbon dots can effectively remove bacteria at the wound part, so that inflammation is relieved, angiogenesis and collagenous fiber deposition are promoted, and remarkable treatment performance is shown. No obvious pathological change is found in histopathological analysis of main organs, and the carbon dots are proved to have excellent biocompatibility. Therefore, the carbon dot nano enzyme is a safe and effective antibacterial nano preparation, and is expected to provide a new technical path and application selection for clinical infectious wound treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of photoresponsive nanoscale enzyme and antibacterial nanoscale preparation, and particularly relates to a photoresponsive carbon dot nanoscale enzyme antibacterial material, a preparation method and application thereof. BACKGROUND

[0002] As an important physiological barrier of the body, the skin bears the core protection function of resisting external invasion. Once the skin is damaged, its barrier integrity is destroyed, making the wound an easy area for pathogenic bacteria to colonize, and thus seriously threatening human health. Wound healing is a highly coordinated biological process involving cell proliferation, differentiation and extracellular matrix remodeling. Bacterial infection not only significantly delays the healing period, but also can cause complications such as inflammation and tissue necrosis, thereby greatly increasing the difficulty of treatment. Therefore, the removal of bacteria from the infected site can effectively promote wound healing. At present, the treatment of infected wounds in the clinic still highly depends on antibiotics. However, the overuse of antibiotics has prompted bacteria to evolve drug resistance through various mechanisms such as gene mutation, production of inactivating enzymes, and change of drug action target, leading to the continuous decline of the efficacy of traditional antibiotics. More seriously, the unreasonable use of antibiotics is further accelerating the spread and evolution of drug-resistant strains, posing a serious challenge to global public health security. Therefore, the development of new high-efficiency antibacterial agents that can avoid bacterial drug resistance has become one of the important research topics in the fields of biomedical and public health.

[0003] As a kind of functional nanomaterials that can mimic the activity of natural enzymes, nanoenzymes have become a promising candidate for antibacterial system due to their unique catalytic properties. The core antibacterial mechanism is to generate reactive oxygen species (ROS) through enzyme-catalyzed reactions. These active substances can destroy key biological molecules such as bacterial cell membranes, nucleic acids and proteins through irreversible oxidation, ultimately achieving effective clearance of pathogens. This ROS-based sterilization mechanism not only has a broad-spectrum inhibitory effect on various pathogenic bacteria such as gram-positive and gram-negative bacteria, but also can avoid the problem of bacterial resistance induced by traditional antibiotics due to single target, providing a new idea for the treatment of infected wounds. In recent years, a large number of studies have confirmed that various nanoenzyme systems including metal, metal oxide and carbon-based nanomaterials have shown excellent performance in combating common pathogenic bacteria infections. Among them, carbon dots (CDs) as an important branch of carbon-based nanomaterials stand out due to their small size, rich surface functional groups, low synthesis cost, excellent water solubility and good biocompatibility. More importantly, CDs have intrinsic enzyme activities such as peroxidase-like and oxidase-like activities due to their small size and high-density active sites on the surface, which lay the foundation for their antibacterial function. Through rational selection of precursors, regulation of synthesis conditions and surface functionalization modification strategies, the catalytic behavior of CDs can be precisely controlled and the efficiency of ROS generation can be optimized. In summary, CDs have shown broad application prospects in the field of antibacterial due to their excellent catalytic performance, good biological safety and low cost advantage, providing an effective strategy to solve the problem of wound treatment caused by drug-resistant bacteria infection.

[0004] Specifically, carbon dots with peroxidase or oxidase mimetic activity can generate ROS by catalyzing substrate oxidation reaction, directly destroy the integrity of bacterial cell membrane, and cause irreversible oxidative damage to key biomolecules such as nucleic acids, proteins, and lipids, ultimately leading to bacterial death. Among them, carbon dots with peroxidase mimetic activity rely on hydrogen peroxide (H2O2) as a substrate to catalyze the decomposition of H2O2 into hydroxyl radicals (·OH) and other reactive oxygen species, and use its strong oxidizing property to achieve sterilization. However, this type of carbon dot nanoscale enzyme has obvious limitations: its antibacterial activity not only depends on the supplementation of exogenous H2O2, but also the residual H2O2 will hinder tissue regeneration. In contrast, carbon dots with oxidase mimetic activity can directly catalyze dissolved oxygen to generate reactive oxygen species without the need to provide exogenous H2O2. However, this type of nanoscale enzyme still has deficiencies: it is prone to continuous reaction with oxygen during long-term storage, resulting in a decline in catalytic activity. More importantly, the catalytic activity of these two types of nanoscale enzymes cannot be precisely regulated, and excessive reactive oxygen species are easily generated, which will cause collateral damage to normal tissues, limiting their application potential in the biomedical field. In view of the above limitations, the development of carbon dot nanoscale enzymes with oxidase mimetic activity and controllable activation characteristics is of great significance for precise antibacterial therapy. The core advantage of this material is that it can achieve precise activation of catalytic activity only at the infection site through its responsive activation mechanism, thereby maximizing the efficient elimination of pathogens while minimizing non-specific oxidative damage to healthy tissues. This antibacterial preparation not only addresses the inherent deficiencies of current carbon dot nanoscale enzymes, but also maximizes the bactericidal effect while minimizing adverse reactions, providing a new technical path for the precise treatment of drug-resistant bacterial infections in wounds. SUMMARY

[0005] The technical problem solved by the present application is to provide a light-responsive carbon dot nanoscale enzyme antibacterial material and a preparation method thereof. The method uses tartaric acid and 3-acetylamino phenol as precursors to prepare carbon dots by one-step hydrothermal method. Under light conditions, the carbon dots can specifically catalyze dissolved oxygen to generate superoxide anions, exhibiting excellent oxidase mimetic activity. This property endows the carbon dots with excellent antibacterial performance, and the reactive oxygen species generation capacity of the carbon dots has light activation characteristics, thereby avoiding the excessive accumulation of reactive oxygen species and reducing damage to healthy tissues. Therefore, this light-responsive nanoscale enzyme can precisely treat bacterial-induced wound infections and has great application potential in clinical anti-infection therapy.

[0006] The application adopts the following technical scheme to solve the above technical problems: a light-responsive carbon dot nanoscale enzyme antibacterial material, taking tartaric acid and 3-acetylamino phenol as precursors, is prepared by one-step hydrothermal method to obtain carbon dots, i.e., the light-responsive carbon dot nanoscale enzyme antibacterial material; under light conditions, the prepared carbon dots can efficiently catalyze the conversion of dissolved oxygen into superoxide anion, exhibit excellent light-responsive oxidase simulation activity, and have antibacterial activity, anti-inflammatory ability and collagen fiber deposition promotion effect, which together accelerate the effective healing of bacterial infectious wounds.

[0007] The preparation method of the light-responsive carbon dot nanoscale enzyme antibacterial material comprises the following specific preparation steps: tartaric acid and 3-acetylamino phenol are dissolved in ultrapure water to form a uniform solution, the uniform solution is transferred to a reaction kettle lined with polytetrafluoroethylene, heated at 180 DEG C for 12 hours in a blast drying oven, and after cooling, the mixture is preliminarily purified by centrifugation and membrane filtration, the solution is dialyzed in a cellulose dialysis bag to remove unreacted precursors, and finally the solution is vacuum freeze-dried to obtain carbon dots.

[0008] Further, the mass ratio of tartaric acid to 3-acetylamino phenol is 1:1.

[0009] The light-responsive carbon dot nanoscale enzyme antibacterial material can be used for preparing an antibacterial nanometer preparation.

[0010] Further, the light-responsive carbon dot nanoscale enzyme antibacterial material can be used for preparing an antibacterial nanometer preparation.

[0011] Further, the gram-negative bacteria are Escherichia coli, and the gram-positive bacteria are Staphylococcus aureus.

[0012] Further, when the concentration of the light-responsive carbon dot nanoscale enzyme antibacterial material is 75 mg / mL, the inhibition rate of the carbon dots on Escherichia coli and Staphylococcus aureus reaches 98.4% or above only after 10 minutes of light irradiation, and the carbon dots have excellent antibacterial stability.

[0013] The light-responsive carbon dot nanoscale enzyme antibacterial material can be used for preparing an anti-inflammatory nanometer preparation.

[0014] The light-responsive carbon dot nanoscale enzyme antibacterial material can be used for preparing a nanometer preparation for promoting collagen fiber deposition.

[0015] Further, the light-responsive carbon dot nanoscale enzyme antibacterial material can effectively remove pathogenic bacteria at the infection site, reduce local inflammatory response, promote collagen fiber deposition, and together accelerate wound healing.

[0016] The present application has the following advantages and beneficial effects: the present application designs a carbon dot with light-responsive oxidase mimetic properties and applies it to bacterial infection wound treatment. The carbon dot takes tartaric acid and 3-acetylamino phenol as a precursor and is synthesized by one-step hydrothermal method. Under light conditions, the prepared carbon dot can efficiently catalyze the conversion of dissolved oxygen into superoxide anion, showing excellent light-responsive oxidase mimetic activity. The in vitro experimental results show that the carbon dot has strong antibacterial effect on gram-negative bacteria Escherichia coli ( E. coli ) and gram-positive bacteria Staphylococcus aureus ( S. aureus ). When the concentration of the carbon dot is 75 mg / mL, the inhibition rate of the carbon dot on Escherichia coli and Staphylococcus aureus reaches more than 98.4% only after 10 minutes of irradiation, and the carbon dot has excellent antibacterial stability. In the Staphylococcus aureus infected wound model, the carbon dot effectively removes pathogenic bacteria at the infection site, reduces local inflammatory response, promotes collagen fiber deposition, and accelerates wound healing. This light-responsive nano-enzyme not only has high bactericidal capacity, but also can promote tissue repair, and has no obvious toxicity to healthy tissues. The above research results highlight the great application potential of the carbon dot nano-enzyme in the field of clinical anti-infection treatment and wound management. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Schematic diagram of preparation of light-responsive carbon dot nano-enzyme antibacterial material, in vitro antibacterial and Staphylococcus aureus infected wound treatment.

[0018] Figure 2 FIG. 1 is a schematic diagram of preparation of light-responsive carbon dot nano-enzyme antibacterial material, in vitro antibacterial and Staphylococcus aureus infected wound treatment.

[0019] Figure 3 FIG. 2 is a schematic diagram of preparation of light-responsive carbon dot nano-enzyme antibacterial material, in vitro antibacterial and Staphylococcus aureus infected wound treatment.

[0020] Figure 4 FIG. 3 is a schematic diagram of preparation of light-responsive carbon dot nano-enzyme antibacterial material, in vitro antibacterial and Staphylococcus aureus infected wound treatment. 1 O2 adducts of electron paramagnetic resonance spectra.

[0021] Figure 5Agar plate images of (a) E. coli and (b) S. aureus under different treatment conditions; (c) Relative survival rates of the two bacteria after different treatments; (d) Growth kinetics curves of the two strains under different carbon dot concentrations after 10 min illumination.

[0022] Figure 6 Live / dead staining microscopy images of (a) E. coli and (b) S. aureus (scale bar: 100 μm); (c) Quantitative analysis of the corresponding fluorescence intensity of E. coli and (d) S. aureus.

[0023] Figure 7 Scanning electron microscopy images of (a) E. coli and (b) S. aureus treated with carbon dots (scale bar: 1 μm); (c) Fluorescence microscopy images of E. coli and (d) S. aureus after staining with reactive oxygen species probes (scale bar: 100 μm).

[0024] Figure 8 Schematic diagram of (a) S. aureus infected wound model construction and treatment strategy; (b) Photographs of infected wounds during treatment (scale bar: 5 mm) and (c) Corresponding healing trajectory simulation diagram; (d) Agar plate images of S. aureus colonies in wound tissues on the 9th day of treatment and (e) Relative survival rates; (f) Relative wound area at different treatment times and (g) Body weight of model mice.

[0025] Figure 9 Hematoxylin-eosin (H&E) staining, (b) Masson staining and (c) immunohistochemical staining of wound sections of the control group, dark treatment group and light treatment group.

[0026] Figure 10 H&E staining of main organ tissue sections (scale bar: 100 μm). DETAILED DESCRIPTION

[0027] The above content of the present application is further described in detail through the following examples, but this should not be understood as limiting the scope of the above subject matter of the present application to only the following examples. Any technology realized based on the above content of the present application falls within the scope of the present application. EXAMPLE

[0028] 1. Experimental methods 1.1 Materials and reagents Tartaric acid (Bide Pharmaceuticals, 98%), 3-acetaminophen (Bide Pharmaceuticals, 99.6%), 3,3',5,5'-tetramethylbenzidine (TMB, Bide Pharmaceuticals, 98%), ethylenediaminetetraacetic acid (EDTA, Sinopharm Group, 99.5%), copper chloride (CuCl2, De'en Chemical, 99%), 1,4-benzoquinone (BQ, Tokyo Chemical Industry, 98%), tryptophan (Trp, Aladdin, 99%), dihydroethidium (DHE, Aladdin... The following reagents were used directly without further purification: 95% (D), circulating tumor DNA (ctDNA, Merck), 5,5-dimethyl-1-pyrrolidone-N-oxide (DMPO, Tongren Chemical Research Institute, 99%), TEMP (Tongren Chemical Research Institute, 99%), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Anaiji Chemical, 98%), paraformaldehyde (Aladdin), and ROS Brite™ 670 (AAT Bioquest). Phosphate buffer and hematoxylin-eosin staining kits were purchased from Wuhan Saiweier Biotechnology Co., Ltd. Bacterial live / dead staining kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Bradford protein colorimetric assay kits were purchased from Wuhan Elayrit Biotechnology Co., Ltd. Immunohistochemistry kits, anti-TNF-α antibody, anti-IL-6 antibody, and anti-VEGF antibody were purchased from Shanghai Sangon Biotech Co., Ltd. Masson trichrome staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. Gram-negative Escherichia coli (ATCC25922) and Gram-positive Staphylococcus aureus (ATCC25923) were purchased from the National Institutes for Food and Drug Control.

[0029] 1.2 Characterization of carbon dots The morphology and size of carbon dots were characterized using transmission electron microscopy (TEM, F200, NEC). Zeta potentials were measured using a Malvern Zetasizer Nano ZS90. X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Fisher Scientific ESCALAB 250Xi X-ray photoelectron spectrometer (aluminum Kα radiation, 200 μm X-ray beam). Fourier transform infrared (FTIR) spectra were recorded using a PE Spectrum 400F spectrometer. UV-Vis absorption and fluorescence emission spectra were recorded using a UV-Vis dual-beam spectrophotometer (UV-2600, Shimadzu) and a fluorescence spectrometer (FS-5, Edinburgh), respectively. Electron paramagnetic resonance (EPR) assays were performed on a Bruker A300 EPR spectrometer. Scanning electron microscopy (SEM) characterization was performed on a Zeiss SUPRA 40 instrument. Cytotoxicity assays were performed using a microplate reader (M2e, Molecular Device).

[0030] 1.3 Preparation of carbon dots First, 0.25 g of tartaric acid and 0.25 g of 3-acetaminophen were dissolved in 10 mL of ultrapure water to form a homogeneous solution. This homogeneous solution was then transferred to a Teflon-lined reactor and heated at 180°C for 12 hours in a forced-air drying oven. After cooling, the mixture was preliminarily purified by centrifugation and membrane filtration. To further purify the product, the solution was dialyzed in a cellulose dialysis bag (100-500 Da) to remove unreacted precursors. Finally, the solution was freeze-dried under vacuum to obtain carbon dots.

[0031] 1.4 Assay for the light-responsive oxidase activity of carbon dots The oxidase-mimicking activity of carbon dots was assessed using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). 20 μL of carbon dot solution (2 mg / mL) and 40 μL of TMB solution (10 mM) were added to HAc-NaAc buffer (0.2 M, pH 3.5), resulting in a total mixed volume of 1.0 mL. The mixture was then placed under an LED lamp (24 mW / cm²) at room temperature. 2 Irradiation was performed under [a specific irradiation method], and the absorption spectrum at 500-800 nm and absorbance at 652 nm were recorded. The optimization experiment followed a similar procedure: pH was tested in the range of 3.0-5.5 at 0.5 intervals; temperature was optimized in the range of 25-50 ℃ at 5 ℃ intervals. Each experiment was repeated at least three times to ensure the reliability of the results.

[0032] 1.5 In vitro reactive oxygen species detection To investigate the catalytic mechanism of carbon dots, reactive oxygen species (ROS) capture experiments were conducted. Under optimal conditions, various capture agents were added to the carbon dot-TMB system, including EDTA (hole h⁺ capture agent), CuCl₂ (electron e⁻ capture agent), and BQ (superoxide anion O₂). •− scavengers), DMPO (hydroxyl radical ·OH scavenger), and Trp (singlet oxygen). 1 O2 scavenger). The mixed solution was then irradiated with an LED lamp for 10 minutes, and the absorption spectrum at 500-800 nm was recorded. Dihydroethidium (DHE) was used as a fluorescent probe to detect superoxide anions. Carbon dots (2 mg / mL, 10 μL), DHE (40 μM), and ctDNA (500 μg / mL) were added to PBS buffer (1 mL, pH=7.4), and the fluorescence intensity at 600 nm was recorded after 10 minutes of illumination. Electron paramagnetic resonance was used to identify the generation of reactive oxygen species: after light treatment, DMPO was used as a scavenger to detect ·OH and O2. •− TEMP reagent was used to capture 1O2. For the detection of ·OH, DMPO (10 μL) was added to the carbon dots (2 mg / mL, 20 μL) in HAc-NaAc buffer (120 μL, 0.2 M, pH=3.5); for the detection of O2 •− , DMPO (10 μL) was mixed with the carbon dots (2 mg / mL, 20 μL) in methanol solution (120 μL).

[0033] 1.6 In vitro antibacterial experiments Escherichia coli and Staphylococcus aureus were chosen as model bacteria to evaluate the antibacterial performance of the carbon dots. The carbon dot concentration and light exposure time were used as variables, and the light source was an LED lamp (24 mW / cm 2 ). First, the two bacteria were cultured to the logarithmic growth phase, centrifuged at 6000 rpm for 5 min, and washed with PBS three times. Subsequently, the bacterial suspension was treated differently: different concentrations of carbon dots (0, 25, 50, 75, 100, 125, 150 μg / mL) and different light exposure times (0, 5, 10 min). 100 μL of the bacterial diluent (10 4 CFU / mL) was inoculated on nutrient broth (NB) agar plates, which were incubated at 37 °C for 15 h. The relative survival rate of the bacteria was determined by colony counting.

[0034] In the nutrient broth liquid medium, the bacterial suspension was treated with different concentrations of carbon dots (0, 25, 50, 75, 100, 125, 150 μg / mL) for 10 min; in another set of experiments, the bacterial suspension was treated with a fixed concentration of carbon dots (150 μg / mL) and exposed to light for different lengths of time (0, 5, 10 min). The optical density (OD 600 ) at 600 nm was measured at 0, 2, 4, 6, 8, 10, and 12 h, respectively.

[0035] 1.7 Bacterial morphological characterization The bacterial solution (1×10 8 CFU / mL) was incubated with the carbon dots in a 37 °C shaker for 30 min, followed by light treatment or placement in the dark for 10 min. The mixture was centrifuged at 6000 rpm for 5 min, washed with PBS (0.1 M, pH=7.4) three times, and fixed with 4 wt% paraformaldehyde for 2 h. Subsequently, the suspension was dehydrated with different concentrations of ethanol (30%, 50%, 70%, 90%, and 100%) in a gradient, each for 15 min. After centrifugation, the bacteria were resuspended in anhydrous ethanol and dropped onto a silicon wafer. After drying and gold spraying, the silicon wafer was observed by field emission scanning electron microscopy to observe the bacterial morphology.

[0036] 1.8 Bacterial live / dead fluorescent staining The bacterial solution (1×108 CFU / mL) with carbon dots for 5 min at 37 °C, followed by light treatment or placed in the dark for 10 min. The treated bacteria were incubated with DMAO and PI staining reagents for 30 min at 37 °C in the dark. Subsequently, the bacterial suspension was centrifuged (6000 rpm, 5 min) and washed with PBS (0.1 M, pH=7.4) for three times. The images of each group of bacteria were taken by laser scanning confocal microscope.

[0037] 1.9 Intrabacterial reactive oxygen species detection ROS Brite 670 was used as a fluorescent probe to detect the generation of reactive oxygen species. First, a solution of ROS Brite 670 with a concentration of 10 μM was prepared. The bacterial solution (1 x 10 8 CFU / mL) with 10 μL ROS Brite 670 was incubated for 30 min at 37 °C in the dark. After incubation, centrifugation was performed at 6000 rpm for 5 min, and washing with PBS (0.1 M, pH=7.4) was performed for three times. Subsequently, the bacterial suspension was incubated with carbon dots for 5 min at 37 °C, and after light treatment or placed in the dark for 10 min, centrifugation, washing and redispersion were performed. The fluorescence images of each group were recorded by laser scanning confocal microscope, with an excitation wavelength of 640 nm and a receiving wavelength range of 650-720 nm.

[0038] 1.10 In vivo antibacterial and wound healing evaluation All animal experiments were conducted in accordance with the "Regulations on the Management of Experimental Animals in China" and were approved by the Animal Experiment Committee of Xinxiang Medical College. To explore the in vivo antibacterial performance of carbon dots, a Staphylococcus aureus infection wound model was established in 6-8-week-old female Kunming mice. First, a wound with a diameter of about 8 mm was created on the back of each mouse, and 50 μL of Staphylococcus aureus suspension (10 8 CFU / mL) was added to the wound. After 24 hours of infection, the mice were randomly divided into three groups: group I (control group), group II (dark treatment group, treated only with carbon dots), and group III (light treatment group, treated with carbon dots + light). HAc-NaAc buffer (0.2 M, pH=5.5) was used as a dispersion liquid, and the control group was only treated with the buffer. In the dark treatment group, HAc-NaAc buffer containing carbon dots (200 μg / mL) was added to the wound and placed in the dark for 10 min; in the light treatment group, the wound was irradiated with an LED lamp for 10 min after adding the carbon dot solution.

[0039] Wound status and mouse body weight were recorded daily until day 9. Wound size was analyzed using Image J software and the corresponding healing trajectory simulation plot was drawn. On day 9 of treatment, bacteria were collected from the wound site and inoculated onto agar plates, agar plate images were recorded and relative survival rate was calculated. After day 9 of treatment, mice were sacrificed, major organs such as heart, liver, spleen, lung, kidney, and wound tissue were collected, and these tissues were fixed in 4 wt% paraformaldehyde overnight, dehydrated and embedded in paraffin for subsequent histological analysis.

[0040] 1.11 Hematoxylin-eosin (H&E), Masson's trichrome and immunohistochemical staining Wound tissue of mice was cut into 4 pm-thick sections and subjected to stepwise deparaffinization: first soaked in xylene for 15 min, then sequentially soaked in 100%, 85% and 75% ethanol for 5 min, and finally soaked in water for 5 min. Hematoxylin-eosin (H&E) staining, Masson's trichrome staining, and TNF-a, IL-6 and VEGF immunohistochemical staining were then performed. Staining images were recorded using an optical microscope for histological analysis.

[0041] 2. Results and discussion 2.1 Characterization of carbon dots The preparation process of carbon dot nanoszyme is shown in Figure 1 . First, the morphology and size of carbon dots were characterized by transmission electron microscopy (TEM). As shown in Figure 2 a-c, carbon dots exhibited monodisperse near-spherical morphology with an average particle size of 4.02 nm. High-resolution transmission electron microscopy (HRTEM) images showed that the lattice fringe spacing of carbon dots was 0.21 nm, corresponding to the (100) plane of graphite carbon, confirming its graphitic structure. X-ray photoelectron spectroscopy (XPS) survey analysis showed that carbon dots were mainly composed of carbon (60.85%), oxygen (33.44%) and nitrogen (5.71%) elements (Fig. Figure 2 d). Peak fitting of carbon 1s spectrum (Fig. Figure 2 e) showed three characteristic peaks at 284.8 eV, 286.6 eV and 288.9 eV, corresponding to C-C / C=C, C-O / C-N and C=0, respectively. Peak fitting of nitrogen 1s spectrum (Fig. Figure 2 f) identified three nitrogen species: pyridine nitrogen (399.6 eV), pyrrole nitrogen (400.3 eV) and graphite nitrogen (402.0 eV).

[0042] 2.2 Photoresponsive oxidase activity of carbon dots The photoresponsive catalytic behavior of carbon dots was systematically evaluated using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate. Under illumination, the carbon dots catalyze the oxidation of TMB by reactive oxygen species mediated by dissolved oxygen, producing a blue product with characteristic absorption at 652 nm. Figure 3 As shown in Figure a, under illumination, only the carbon dot-TMB system exhibited significant color changes and increased absorbance. In the control experiment, neither TMB nor the carbon dot solution showed significant changes under the same conditions, indicating that the observed transformation is a result of carbon dot-specific catalytic oxidation of TMB. Furthermore, under alternating on and off illumination conditions, the absorbance of the carbon dots at 652 nm showed a stepwise increasing trend, further confirming the photoactivated nature of its catalytic activity. Figure 3 (b) Similar to natural oxidases, the catalytic activity of carbon dots depends on pH, temperature, and catalyst concentration. Optimization experiments showed that pH 3.5 and 30 °C were the optimal catalytic conditions. Figure 3 c and Figure 3 (d). Furthermore, the catalytic activity of carbon dots also exhibits concentration- and laser power-dependent behavior ( Figure 3 (e). To evaluate the catalytic stability of the carbon points, their performance was tested under long-term storage conditions. Figure 3 As shown in Figure f, the oxidase-simulated activity of the carbon dots remained almost unchanged after 50 days of storage, which fully demonstrates their excellent catalytic stability.

[0043] 2.3 Catalytic mechanism of carbon dots A series of verification experiments revealed the catalytic mechanism of carbon dots. Compared with air-saturated conditions, the absorbance of the CDs-TMB system was significantly reduced under a nitrogen atmosphere, indicating that dissolved oxygen is an essential reactant for the generation of reactive oxygen species. Figure 4 (a) Multiple active species may be involved in the catalytic process, including electrons (e⁻) and photogenerated holes (h⁻). + ), superoxide anion (O2) •− ), hydroxyl radicals (·OH) and singlet oxygen ( 1 O2). To identify the active species, validation was performed using specific scavengers: EDTA was used to remove h. + CuCl2 is used to remove e⁻, and benzoquinone (BQ) is used to remove O₂. •− 5,5-Dimethyl-1-pyrrolidone-N-oxide (DMPO) is used to scavenge ·OH, and tryptophan (Trp) is used to scavenge ·OH. 1 O2. For example... Figure 4 As shown in Figure b, the experimental results indicate that CuCl2 and BQ significantly inhibit the oxidation of TMB, suggesting that e⁻ and O₂·⁻ are the main catalytic mediators. Subsequently, dihydroethidium (DHE) fluorescent probes were used to further detect O₂. •− The generation of . For exampleFigure 4 As shown in Figure c, the fluorescence of the DHE system was significantly enhanced after co-treatment with carbon dots and light, confirming the presence of O2. •− The generation of O2. Furthermore, the EPR spectrum only detected O2 under illumination. •− The characteristic signal was observed, but no ·OH or 1 O2 signal ( Figure 4 (df). In summary, this catalytic system mainly generates O2 via a photogenerated electron-mediated oxygen reduction pathway. •− This further drives the oxidation of TMB.

[0044] 2.4 Antibacterial activity of carbon dots Given that carbon dots have been shown to possess photoresponsive oxidase mimicry activity, we further evaluated their antibacterial potential. Using Gram-negative *Escherichia coli* and Gram-positive *Staphylococcus aureus* as representative strains, the antibacterial activity of the carbon dots was assessed using a plate count method. The results are as follows: Figure 5 As shown in Figures a and b, neither carbon dot treatment alone nor light treatment alone had a significant effect on the survival of either bacterium. However, a large number of bacteria died after combined carbon dot and light treatment, and the bacterial colony count decreased significantly with increasing carbon dot concentration and prolonged light exposure. Figure 5 The results showed that under light irradiation, bacterial survival rates gradually decreased with increasing carbon dot concentration. When the carbon dot concentration was 50 μg / mL and the irradiation time was 10 minutes, the survival rates of *E. coli* and *Staphylococcus aureus* decreased by 85.5% and 89.6%, respectively. Further increasing the carbon dot concentration to 100 μg / mL almost completely inhibited both bacteria. In addition, we investigated the effect of irradiation time on the antibacterial effect of carbon dots. When the carbon dot concentration was 75 μg / mL, as the irradiation time increased from 5 minutes to 10 minutes, the survival rate of *E. coli* decreased from 11% to 1.6%, and the survival rate of *Staphylococcus aureus* decreased from 18.6% to 0.8%. These results indicate that carbon dots exhibit excellent antibacterial activity, and their antibacterial performance is highly dependent on their concentration and irradiation time. To further evaluate the antibacterial activity of carbon dots, the growth kinetics of the two bacteria after treatment with different concentrations of carbon dots were examined. Bacterial suspensions were treated with carbon dots of varying concentrations (0-150 μg / mL) and exposed to light for 10 minutes. The absorbance (OD) at 600 nm was then recorded. 600 To examine the degree of bacterial growth inhibition. For example... Figure 5 As shown in Figure d, the inhibitory effect on both bacterial strains gradually increased with increasing carbon dot concentration. After exposure to 150 μg / mL carbon dots, both strains showed significant growth inhibition after 12 hours of incubation. These results indicate a positive correlation between bacterial growth inhibition and carbon dot concentration, consistent with observations using the plate count method.

[0045] Further verification of the photoresponsive bactericidal effect of carbon dots was achieved through live / dead bacterial staining experiments. N,N-dimethylaniline N-oxide (DMAO) specifically labeled live bacteria, emitting green fluorescence; while dead bacteria were co-stained with both DMAO and propidium iodide (PI), exhibiting both green and red fluorescence. Figure 6 a and Figure 6 As shown in Figure c, both bacterial strains exhibited significant green fluorescence in the dark-treated groups, indicating good bacterial survival. In contrast, the light-treated groups showed bright red fluorescence, indicating that a large number of bacteria had died. Quantitative analysis of the relative fluorescence intensity using ImageJ software showed that, compared with the dark control group, the red fluorescence signal in the light-treated groups of both strains was significantly increased (…). Figure 6 b and Figure 6 (d). The above experimental results collectively demonstrate that carbon dots exhibit excellent antibacterial activity under light conditions.

[0046] 2.5 Antibacterial mechanism of carbon dots To elucidate the antibacterial mechanism of carbon dots, bacterial morphology was first characterized using scanning electron microscopy. For example... Figure 7 a and Figure 7 As shown in Figure b, in the dark treatment group, *Escherichia coli* and *Staphylococcus aureus* maintained their typical rod-shaped and spherical morphologies, respectively, with smooth bacterial surfaces and intact structures. However, in the light-treated group, both bacteria showed significant morphological changes, with *E. coli* exhibiting obvious wrinkles and depressions on its surface. Figure 7 (a, white arrow) while Staphylococcus aureus suffered more severe damage, with some cells completely ruptured ( Figure 7 (b, white arrow). Subsequently, the level of reactive oxygen species (ROS) within the bacterial cells was quantitatively detected using the ROS Brite 670 fluorescent probe. Figure 7 As shown in Figure 1, both bacterial groups exhibited significantly higher red fluorescence signals under light than their control groups, indicating that carbon dots can effectively induce the generation of large amounts of reactive oxygen species (ROS) under light. These results demonstrate that carbon dots can adsorb onto the bacterial surface, generate ROS under light, and subsequently induce cell membrane structure damage and bacterial death through oxidative damage.

[0047] 2.6 In vivo antibacterial properties and wound healing promoting effect of carbon dots Based on the superior in vitro bactericidal properties of carbon dots, we further evaluated their in vivo antibacterial activity and wound treatment potential using a mouse wound model infected with Staphylococcus aureus. The experimental design and treatment process included wound creation, bacterial inoculation, infection establishment, and treatment, as follows: Figure 8 As shown in Figure a. First, a circular wound with a diameter of 8 mm was created on the back of the mouse, and 1×10⁻⁶ cells were injected. 8CFU / mL Staphylococcus aureus was incubated for 24 hours to establish stable infection. Mice were randomly divided into three groups: control group (treated with HAc-NaAc buffer), dark treatment group (treated with carbon dots without light), and light treatment group (treated with both carbon dots and light). Wound healing progress was recorded at predetermined time points. Figure 8 The results showed that all groups exhibited clear infection characteristics, including severe swelling and purulent exudate, 24 hours after infection, confirming the successful establishment of the infection model. Three days after treatment, the wound healing rate in the light-treated group was significantly faster than that in the control and dark-treated groups. On the ninth day of treatment, the difference was even more significant; the wounds in the light-treated group almost completely healed (area reduction of 94.2%), while the control group (area reduction of 77.2%) and the dark-treated group (area reduction of 82.3%) still showed significant unhealed areas. Figure 8 (f). The simulated wound healing trajectory further confirms that the healing in the light-exposed group was significantly faster ( Figure 8 (c) To assess in vivo antibacterial activity, wound tissue was harvested on day 9 of treatment, ground, and then cultured for bacteria. Figure 8 As shown in d and e, the bacterial survival rate at the wound site in the light-irradiated group was significantly lower than that in the other two groups, with a bacterial clearance rate as high as 97.3%. This indicates that carbon dots can effectively remove Staphylococcus aureus from infected wound sites under light conditions and promote wound healing.

[0048] Histopathological analysis further assessed the wound healing process and inflammatory status. H&E staining results showed ( Figure 9 In Figure a), the control and dark treatment groups showed extensive infiltration of neutrophils and lymphocytes, indicating persistent inflammation caused by infection. In contrast, the wound treated with combined carbon dot and light irradiation showed significantly reduced inflammatory cell infiltration, along with abundant fibroblasts and new hair follicles, indicating enhanced tissue regeneration. Masson trichrome staining revealed sparse collagen fiber deposition in the control and dark treatment groups, while the light irradiation group exhibited denser collagen fiber deposition, further demonstrating good tissue repair progress. Figure 9 (b) Subsequently, immunohistochemical staining was used to assess the expression of two pro-inflammatory cytokines (interleukin-6, IL-6; tumor necrosis factor-α, TNF-α) and vascular endothelial growth factor (VEGF). IL-6 and TNF-α are inflammatory markers, while VEGF is a marker of angiogenesis. Figure 9 As shown in Figure c, compared with the other two groups, the expression of IL-6 and TNF-α was significantly reduced in the phototherapy group, while the expression of VEGF was significantly upregulated, indicating that the combined treatment of carbon dots and phototherapy can effectively reduce inflammation, promote angiogenesis, and jointly accelerate wound healing. To assess the biosafety of carbon dots, the body weight of mice was monitored during treatment, and H&E staining analysis was performed on tissue sections of major organs. Figure 8As shown in Fig. 5, the weight change trends of mice in each group were consistent during the treatment period, indicating that the carbon dots had no obvious toxicity. In addition, the H&E staining results of major organs showed that no obvious pathological damage or structural abnormalities occurred after 9 days of treatment Figure 10 ), confirming that the carbon dots had good in vivo biocompatibility. In summary, the in vivo experimental results showed that the carbon dots combined with light treatment could effectively clear bacteria at the infection site, relieve inflammation, promote angiogenesis, and enhance collagen fiber deposition, which collectively promoted wound healing.

[0049] 3. Conclusion In summary, the present application developed a carbon dot nanoscale enzyme with light-responsive oxidase mimetic activity and successfully used it for the treatment of infected wounds. The carbon dots were prepared by a hydrothermal method using tartaric acid and 3-acetylamino phenol as precursors. Under light conditions, the prepared carbon dots exhibited excellent oxidase mimetic activity and could efficiently catalyze the conversion of oxygen to superoxide anion, endowing the carbon dots with strong antibacterial ability. In the wound model infected with Staphylococcus aureus, the carbon dots could significantly accelerate wound healing, which was attributed to their strong antibacterial activity, inflammation relief, angiogenesis promotion, and collagen fiber deposition capacity. At the same time, the histopathological analysis results of major organs confirmed that the carbon dots had good biocompatibility. The carbon dot nanoscale enzyme has the characteristics of light-controllable and on-demand activation, and is a safe antibacterial nanoscale preparation, which is expected to provide a new technical path and application choice for the treatment of clinical infected wounds.

[0050] The above shows and describes the basic principles, main features and advantages of the present application. Without departing from the spirit and scope of the present application, there are various changes and improvements of the present application, which fall within the scope of the present application.

Claims

1. A photoresponsive carbon dots nanoszyme antibacterial material, characterized in that: A carbon dot, i.e., a light-responsive carbon dot nanoscale enzyme antibacterial material, is prepared by using a one-step hydrothermal method with tartaric acid and 3-acetylamino phenol as precursors. Under light conditions, the prepared carbon dot can efficiently catalyze the conversion of dissolved oxygen into superoxide anion, exhibit excellent light-responsive oxidase mimetic activity, and simultaneously have antibacterial activity, anti-inflammatory ability and collagen fiber deposition promotion effect.

2. The preparation method of the light-responsive carbon dots nanosenzyme antibacterial material according to claim 1, characterized in that The specific preparation steps are as follows: tartaric acid and 3-acetylamino phenol are dissolved in ultrapure water to form a uniform solution, the uniform solution is transferred to a reaction kettle lined with polytetrafluoroethylene, heated at 180 ℃ in a forced air drying oven for 12 hours, cooled, and then the mixture is preliminarily purified by centrifugation and membrane filtration, the solution is dialyzed in a cellulose dialysis bag to remove unreacted precursors, and finally the solution is vacuum freeze-dried to obtain the carbon dot.

3. The method according to claim 2, wherein the method comprises the following steps: 3.

1. preparing a solution of the carbon dots; 3.

2. preparing a solution of the enzyme; 3.

3. mixing the solutions of the carbon dots and the enzyme to obtain the photoresponsive carbon dots nanoszyme antibacterial material. The mass ratio of tartaric acid to 3-acetylamino phenol is 1:

1.

4. The light-responsive carbon dot nanoscale enzyme antibacterial material of claim 1 is used in the preparation of an antibacterial nanoscale preparation.

5. The light-responsive carbon dot nanoscale enzyme antibacterial material of claim 1 is used in the preparation of an antibacterial drug against gram-negative bacteria or / and gram-positive bacteria.

6. Use according to claim 5, characterized in that: The gram-negative bacteria is Escherichia coli, and the gram-positive bacteria is Staphylococcus aureus.

7. Use according to claim 6, characterized in that: When the concentration of the light-responsive carbon dot nanoscale enzyme antibacterial material is 75 mg / mL, the inhibition rate of the carbon dot on Escherichia coli and Staphylococcus aureus reaches more than 98.4% only after 10 minutes of light irradiation, and the carbon dot has excellent antibacterial stability.

8. The light-responsive carbon dot nanoscale enzyme antibacterial material of claim 1 is used in the preparation of an anti-inflammatory nanoscale preparation.

9. The light-responsive carbon dot nanoscale enzyme antibacterial material of claim 1 is used in the preparation of a nanoscale preparation for promoting collagen fiber deposition.

10. Use according to claim 9, characterized in that: The light-responsive carbon dot nanoscale enzyme antibacterial material effectively removes pathogenic bacteria at the infection site, reduces local inflammatory response, promotes collagen fiber deposition, and collectively accelerates wound healing.

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