Zinc-doped antibacterial carbon dots and preparation method and application thereof

Abstract

The present application relates to a kind of zinc-doped antibacterial carbon dots and its preparation method and application, the preparation method of the zinc-doped antibacterial carbon dots is that biomass, anhydrous zinc acetate is dispersed in deionized water according to mass ratio, after ultrasonic stirring, mixed solution is obtained, after hydrothermal reaction, mixed solution is obtained after natural cooling, centrifugal, filtration treatment, carbon dot solution is obtained, carbon dot solution is obtained after freeze-drying treatment, and powder zinc-doped antibacterial carbon dots are obtained.Then zinc-doped antibacterial carbon dots are dissolved in double distilled water with carboxymethyl chitosan, bioactive glass according to proportion, stirring is obtained at room temperature, and composite hydrogel is obtained.The zinc-doped antibacterial carbon dots and composite hydrogel prepared in the present application have good antibacterial effect, the preparation process of composite hydrogel is simple, low in cost, low in cytotoxicity, and zinc-doped antibacterial carbon dots have wide application prospect in the preparation of antibacterial material and the treatment of skin deep lesions, diabetic wound and burn wound dressing material.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a zinc-doped antibacterial carbon dot, its preparation method, and its application. Background Technology

[0002] Nanotechnology offers novel approaches to medical diagnosis and treatment, particularly in biomedicine, providing significant advantages. Nanotherapeutic agents, leveraging the properties of nanomaterials and technologies, have emerged as potential alternatives to antibiotics, demonstrating immense development potential. Nanomaterials are a class of materials with at least one dimension at the nanoscale (1-100 nm) in three-dimensional space, composed of basic units including antimicrobial peptides, metal nanoparticles, semiconductors, polymer nanostructures, and carbon-based nanomaterials. Carbon dots, as a typical form of nanomaterial, are composed of sp... 2 Hybrid carbon nanostructures in sp 3 The hybrid carbon matrix consists of spherical nanoparticles with a diameter of less than 10 nm. Carbon dots possess a wide range of functional properties, including biocompatibility, photostability, low toxicity, ease of synthesis, antibacterial, anti-inflammatory, antioxidant, and anticancer properties, making them widely applicable. However, the preparation of fluorescent carbon dots currently faces several challenges, such as cumbersome preparation steps, difficulty in obtaining some raw materials, insufficient antibacterial activity, and potentially significant toxicity.

[0003] As an important natural barrier, the skin protects internal tissues from mechanical damage, restricts water loss, and prevents pathogens from entering. However, the skin is frequently subjected to external injury, leading to acute or chronic damage. The skin wound healing process is complex, involving multiple stages, including hemostasis, inflammation, cell proliferation, tissue regeneration, repair of damaged tissue, restoration of function, and healing of the affected area. Traditional dressings have limitations in clinical application due to their tendency to increase the risk of infection, cause poor blood circulation, and suffer from poor mechanical properties, poor biocompatibility, poor biodegradability, low water absorption and retention, and high cytotoxicity. Ideal wound dressings should possess good biocompatibility, biodegradability, good water absorption and retention, and be non-cytotoxic. In recent years, hydrogels have proven to be an ideal wound dressing, enabling pain control and controlled drug release, promoting wound repair, and minimizing scarring. Carboxymethyl chitosan, a type of hydrogel, is a chitosan derivative obtained through carboxymethylation. It exhibits good biocompatibility and has wide applications in gene therapy, drug delivery, and tissue engineering. However, in the existing technology, there is little research on developing carbon dots combined with hydrogels as antibacterial materials and wound dressings for treating deep skin lesions, diabetic wounds and burns, which have simple preparation steps, readily available raw materials, good antibacterial activity and low cytotoxicity. Summary of the Invention

[0004] The purpose of this invention is to provide a zinc-doped antibacterial carbon dot, its preparation method, and its application in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] This invention provides a method for preparing zinc-doped antibacterial carbon dots. Biomass and anhydrous zinc acetate are dispersed in deionized water at a mass ratio, and the mixture is ultrasonically stirred to obtain a mixed solution. After hydrothermal reaction, the mixed solution is naturally cooled, centrifuged, and filtered to obtain a carbon dot solution. The carbon dot solution is then freeze-dried to obtain powdered zinc-doped antibacterial carbon dots.

[0007] As a further optimization of the present invention, the biomass is corn stalk powder, and the mass ratio of the biomass to anhydrous zinc acetate is 1-3:1.

[0008] As a further optimization of the present invention, the temperature of the hydrothermal reaction is 180℃-260℃ and the time is 2-6h; the centrifugation speed of the centrifugation treatment is 8000rpm and the centrifugation time is 10-20min; the pore size of the filter membrane for the filtration treatment is 0.22μm.

[0009] The present invention also provides a zinc-doped antibacterial carbon dot, which is prepared using the above-described method for preparing zinc-doped antibacterial carbon dots.

[0010] This invention also provides the application of zinc-doped antibacterial carbon dots in the preparation of antibacterial materials and antibacterial wound repair dressings.

[0011] As a further optimization of the present invention, the antibacterial concentration of the zinc-doped antibacterial carbon dots is at least 100 μg / mL, and the bacteria are Gram-positive and Gram-negative bacteria.

[0012] As a further optimization of the present invention, the wound antibacterial repair dressing material is a composite hydrogel.

[0013] The present invention also provides a method for preparing a composite hydrogel, wherein the zinc-doped antibacterial carbon dots, carboxymethyl chitosan, and bioactive glass described above are dissolved in double-distilled water in proportion and stirred at room temperature to synthesize a zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel, i.e., a composite hydrogel.

[0014] As a further optimization of the present invention, the mass ratio of carboxymethyl chitosan, bioactive glass, and zinc-doped carbon dots is 400:5:1-16.

[0015] The beneficial effects of this invention are as follows:

[0016] This invention uses biomass as a carbon source and anhydrous zinc acetate as a dopant to synthesize zinc-doped antibacterial carbon dots via a one-step hydrothermal method. The zinc-doped antibacterial carbon dots exhibit good antibacterial effects against both Gram-positive and Gram-negative bacteria. Subsequently, using carboxymethyl chitosan, bioactive glass, and zinc-doped antibacterial carbon dots as raw materials, and stirring at room temperature, a zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel is synthesized. This composite hydrogel has a simple preparation process, low cost, and low cytotoxicity. The zinc-doped antibacterial carbon dots have broad application prospects in antibacterial materials, as well as in the treatment of deep skin lesions, diabetic wounds, and burn materials. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the preparation process of the composite hydrogel (i.e., zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel) of the present invention.

[0018] Figure 2 These are the characterization diagrams and antibacterial performance test diagrams of the zinc-doped antibacterial carbon dots of the present invention. Figure 2 a is a transmission electron microscope image; Figure 2 b is the fluorescence spectrum; Figure 2 c is the ultraviolet-visible light spectrum; Figure 2 Figure d shows the antibacterial results against Escherichia coli and Staphylococcus aureus. Figure 2 e is a biocompatibility diagram; Figure 2 f and 2g are SEM images before and after treatment with Staphylococcus aureus; Figure 2 h and 2i are SEM images before and after treatment with E. coli.

[0019] Figure 3 a shows the morphology of the composite hydrogel (zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel) of the present invention and its deformation when bent at different angles of the finger. Figure 3 b is a SEM image of the composite hydrogel of the present invention; Figure 3 c is a morphological diagram of the composite hydrogel of the present invention adhering to various material surfaces and body organs and tissues.

[0020] Figure 4 Image a represents the changes in the wound over 12 days; Figure 4 b is a statistical graph showing the wound healing rate over time. Detailed Implementation

[0021] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0022] I. Materials

[0023] Unless otherwise specified, all methods used in this application are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products unless otherwise specified.

[0024] II. Methods

[0025] 2.1 Preparation of Zinc-Doped Antibacterial Carbon Dot-Based Carboxymethyl Chitosan Hydrogel

[0026] 2.1.1 Preparation of zinc-doped antibacterial carbon dots: Corn stalk powder and anhydrous zinc acetate were dispersed in deionized water at a mass ratio and ultrasonically stirred to obtain a mixed solution; the mixed solution was placed in a polytetrafluoroethylene reactor and subjected to hydrothermal reaction at 240℃ for 4 hours; the mixed solution after hydrothermal reaction was naturally cooled, centrifuged at 8000 rpm for 15 min, and filtered through a 0.22 μm filter membrane to obtain a carbon dot solution, which was finally freeze-dried to obtain powdered zinc-doped antibacterial carbon dots;

[0027] The mass ratio of the biomass to anhydrous zinc acetate is 1-3:1;

[0028] It should be noted that the zinc-doped antibacterial carbon dot sample used in the following tests consisted of 0.5g of corn stalk powder and 0.5g of anhydrous zinc acetate dispersed in 25mL of deionized water at a mass ratio.

[0029] 2.1.2 Obtaining zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel: The powdered zinc-doped antibacterial carbon dots prepared in step 2.1.1 above are dissolved in double-distilled water in proportion with carboxymethyl chitosan and bioactive glass, and stirred at room temperature for 6 hours to obtain zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel.

[0030] The mass ratio of carboxymethyl chitosan, bioactive glass, and zinc-doped carbon dots is 400:5:1-16.

[0031] It should be noted that the zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel sample used in the following tests consisted of 0.02 g of zinc-doped antibacterial carbon dots, 8 g of carboxymethyl chitosan, and 0.1 g of bioactive glass dispersed in 100 mL of double-distilled water.

[0032] 2.2 Characterization and evaluation of antibacterial properties of zinc-doped antibacterial carbon dots

[0033] 2.2.1 Characterization of Zinc-Doped Antibacterial Carbon Dots

[0034] The zinc-doped antibacterial carbon dots prepared according to step 2.1 above were uniformly dispersed in water, exhibiting good water dispersibility. Figure 2 The transmission electron microscope image of a shows that the zinc-doped antibacterial carbon dots are not standard spherical, and the particle size distribution is 1.5-5 nm. Figure 2The fluorescence spectrum of b shows the fluorescence spectrum of the zinc-doped antibacterial carbon dot solution. The fluorescence spectrum of typical carbon dots can be observed in the figure. In the excitation wavelength range of 300-380 nm, the fluorescence emission peak intensity of zinc-doped antibacterial carbon dots first increases and then decreases with the excitation wavelength, and the maximum emission spectrum is obtained at the excitation wavelength of 340 nm. Figure 2 The UV-Vis spectrum of c shows the blue fluorescence emitted by the zinc-doped antibacterial carbon dots under UV light irradiation.

[0035] 2.2.2 Evaluation of the antibacterial properties of zinc-doped antibacterial carbon dots

[0036] The minimum inhibitory concentration (MIC) of the zinc-doped antibacterial carbon dots prepared in step 2.1 above against Staphylococcus aureus and Escherichia coli (with Escherichia coli as the model bacterium for Gram-negative bacteria and Staphylococcus aureus as the model bacterium for Gram-positive bacteria) was tested as follows:

[0037] Dilute the bacterial culture with PBS to a concentration of 1 × 10⁻⁶ for E. coli. 6 CFU / mL) and Staphylococcus aureus (1×10⁻⁶ CFU / mL) 5 The diluted bacterial suspension was mixed with zinc-doped antibacterial carbon dot solution (final concentrations of 0.8, 0.4, 0.2, 0.1, and 0.05 mg / mL) at a 1:1 ratio. The control group was treated with sterile PBS. The mixtures were incubated at 37°C for 24 hours. Subsequently, 100 μL of the bacterial suspension was evenly spread on LB solid agar plates and incubated at 37°C for 24 hours. Finally, the plates were photographed and the relative bacterial activity was obtained by calculating the number of colonies.

[0038] Experimental Conclusions: Using *Escherichia coli* and *Staphylococcus aureus* as model bacteria for Gram-negative and Gram-positive bacteria, respectively, different concentrations of zinc-doped antibacterial carbon dot solutions showed the following antibacterial effects against *Escherichia coli* and *Staphylococcus aureus*: Figure 2 As shown in d, by Figure 2 As can be seen, with the increase of the concentration of zinc-doped antibacterial carbon dot solution, the number of colony units on the agar plates of Escherichia coli and Staphylococcus aureus decreased significantly. It can be seen that zinc-doped antibacterial carbon dots have a good antibacterial effect on both Gram-negative and Gram-positive bacteria. The inhibitory concentration of zinc-doped antibacterial carbon dots against Escherichia coli and Staphylococcus aureus is at least 100 μg / mL.

[0039] To determine the morphological changes of *Escherichia coli* and *Staphylococcus aureus* after treatment with the zinc-doped antibacterial carbon dots prepared in step 2.1 above, scanning electron microscopy was performed. Specifically, the zinc-doped antibacterial carbon dot solution (0.8 mg / mL) was added to *Escherichia coli* (1 × 10⁻⁶ mg / mL) and then to *S. coli* (1 × 10⁻⁶ mg / mL). 6 CFU / mL) and Staphylococcus aureus (1×10⁻⁶ CFU / mL) 5All bacterial suspensions were incubated at 37°C for 8 h at CFU / mL, followed by centrifugation at 5000 rpm for 5 min at 4°C. Bacteria were washed three times with 0.1 M PBS (pH 7.4) for 15 min each time and fixed in 2.5% (v / v) glutaraldehyde solution for 2 h. Dehydration was performed with 30%, 50%, 70%, 80%, 90%, and 100% ethanol for 10 min each. After dehydration, the samples were dried with CO2 and sputtered with gold in an ion-coating system for 2 min. Finally, the morphology of the bacterial cells was observed using scanning electron microscopy.

[0040] Experimental Conclusion: To more intuitively compare the differences in cell membrane damage caused by zinc-doped antibacterial carbon dot solutions to Gram-negative and Gram-positive bacteria, *Escherichia coli* was used as the model bacterium for Gram-negative bacteria, and *Staphylococcus aureus* as the model bacterium for Gram-positive bacteria. Scanning electron microscopy was used to observe the morphology of the bacteria, such as... Figure 2 As shown at f and 2h, the untreated (blank group) *E. coli* and *Staphylococcus aureus* bacteria had intact structures, smooth cell membranes, and no damage; as shown in f and 2h... Figure 2 As shown in g and 2i, SEM images of Escherichia coli and Staphylococcus aureus treated with 800 μg / mL zinc-doped antibacterial carbon dot solution show that their cell morphology is incomplete, cell membranes are damaged and shrunken, and some cells even show complete rupture and leakage of contents.

[0041] Cell viability assessment: Following standard protocol, the cytotoxicity of zinc-doped antibacterial carbon dot solution to 3T3 cells was assessed using the MTT assay. 3T3 cells were seeded in 96-well plates (blank wells were used as zeroing wells) and cultured for 24 h. Subsequently, the original culture medium was discarded, and different concentrations of zinc-doped antibacterial carbon dot solution (final concentrations of 0, 50, 100, and 200 μg / mL, respectively) dispersed in PBS were added to the 96-well plates. The plates were then returned to the incubator for another 24 h. The next day, the mixed culture medium was aspirated, and cytotoxicity was determined using the MTT assay. Four hours later, 150 μL of DMSO was added to each well to dissolve the purple crystals at the bottom of the plate. The plates were shaken at 150 rpm for 10 minutes to completely dissolve the crystals. Finally, the absorbance of the purple solution in the 96-well plate was measured at 490 nm using a microplate reader. Ten replicates were set up for each concentration, and cell viability was assessed using the statistical mean and standard deviation.

[0042] Experimental conclusions: such as Figure 2 As shown in e, Figure 2 The image shows the viability of 3T3 cells after treatment with different concentrations of zinc-doped antibacterial carbon dot solutions for 24 hours. Cytotoxicity data indicate that different concentrations of zinc-doped antibacterial carbon dot solutions do not harm the cells and may even promote cell proliferation to some extent. These data strongly suggest that zinc-doped antibacterial carbon dots exhibit low cytotoxicity and have significant application potential in the antibacterial field.

[0043] 2.3 Performance Evaluation of Zinc-Doped Antibacterial Carbon Dot-Based Carboxymethyl Chitosan Hydrogel

[0044] The study tested the adhesion of zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel to various material surfaces and body organs and tissues, as well as the deformation of the zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel as it was bent at different angles with a finger.

[0045] Experimental conclusion: Figure 3 a and 3b show that the zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel is completely transparent and exhibits an interconnected macroporous structure. This range of pore sizes promotes the proliferation and migration of epidermal cells. Figure 3 A and 3C show that the zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel can adhere to various surfaces, such as skin, glass, Teflon, rubber, and steel, exhibiting broad-spectrum adhesion. Furthermore, the hydrogel demonstrates high adhesion to biological tissues such as the heart, liver, spleen, lungs, and kidneys, which is crucial for biomedical applications. It was also found that the hydrogel possesses strong adhesion and can stretch / contract when the finger is bent, which is beneficial for wound healing.

[0046] The experiment used male BALA / c5 mice weighing between 18-22g, purchased from SPF (China) Hangzhou Ziyuan Experimental Technology Co., Ltd. Under general anesthesia, the dorsal fur of these animals was removed with depilatory cream. A deep, full-thickness skin defect with a diameter of 8 mm was carefully created using a dermal biopsy punch. Then, 50 μL of bacterial suspension (Staphylococcus aureus, 1×10⁻⁶) was applied to the wound. 6 CFUmL-1) was covered with sterile zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel and fixed with 3M tape; an untreated wound was used as a control; wound healing was recorded every 2 days; wound healing rate was analyzed by comparing wound area.

[0047] Experimental conclusion: Figure 4 Studies a and 4b investigated the effect of zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel dressings on full-thickness wound healing. The blank control group showed typical spontaneous wound repair, while the zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel dressing significantly improved wound healing progress. After 12 days of treatment, the wounds of mice in the zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel dressing group healed completely, demonstrating a faster healing rate compared to spontaneous wound repair.

[0048] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of zinc-doped antibacterial carbon dots in the preparation of antibacterial wound repair dressing materials, characterized in that: The method for preparing the zinc-doped antibacterial carbon dots is as follows: corn stalk powder and anhydrous zinc acetate are dispersed in deionized water at a mass ratio of 1-3:1, and a mixed solution is obtained after ultrasonic stirring. The mixed solution after hydrothermal reaction is naturally cooled, centrifuged, and filtered to obtain a carbon dot solution. The carbon dot solution is freeze-dried to obtain powdered zinc-doped antibacterial carbon dots. The hydrothermal reaction is carried out at a temperature of 180℃-260℃ for 2-6 hours; the centrifugation process is carried out at a speed of 8000 rpm for 10-20 minutes; and the filter membrane used for filtration has a pore size of 0.22 μm. The wound antibacterial repair dressing material is a composite hydrogel. The preparation method of the composite hydrogel is to dissolve the zinc-doped antibacterial carbon dots, carboxymethyl chitosan, and bioactive glass in double-distilled water in proportion and stir at room temperature to synthesize zinc-doped antibacterial carbon dot-based carboxymethyl chitosan hydrogel, i.e., composite hydrogel. The mass ratio of carboxymethyl chitosan, bioactive glass, and zinc-doped antibacterial carbon dots is 400:5:1-16.

2. The application of zinc-doped antibacterial carbon dots according to claim 1 in the preparation of antibacterial wound repair dressing materials, characterized in that: The antibacterial concentration of the zinc-doped antibacterial carbon dots is at least 100 μg / mL, and the bacteria are Gram-positive and Gram-negative bacteria.

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