Glycosylated carbon dots functionalized fiber-gel composite dressing and application thereof

Abstract

This invention discloses a glycosylated carbon dot functionalized fiber-gel composite dressing and its applications, belonging to the fields of functional materials and biomedicine. First, this invention develops a novel glycosylated carbon dot CS-ACD using a one-step hydrothermal method. This CS-ACD exhibits significantly enhanced antibacterial and anti-inflammatory activity, primarily killing bacteria through a membrane-dissolving mechanism. It also promotes the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thereby reducing the expression and secretion of inflammatory cytokines. Furthermore, this invention combines electrospun nanofibers loaded with this CS-ACD with an in-situ sprayable fibrin gel to prepare an innovative "sandwich" structured fiber-gel dressing. In an experimental mouse model of infected wounds, this dressing effectively reduces bacterial load, regulates the immune response, alleviates wound inflammation, and promotes collagen deposition and angiogenesis, ultimately accelerating wound healing and tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of functional materials and biomedicine, specifically relating to a glycosylated carbon dot functionalized fiber-gel composite dressing and its application. Background Technology

[0002] Burns, trauma, surgery, and persistent infections pose a serious threat to human health. Infected wounds cause numerous illnesses and deaths worldwide, affecting millions annually. Currently, clinical treatment of infected wounds primarily relies on wound debridement, antibiotic use, and physical dressings to prevent further complications. However, these methods often lead to prolonged treatment times and slow patient recovery. Simultaneously, the widespread overuse of antibiotics and the slow development of new antibiotics are exacerbating the crisis of drug-resistant pathogens. Consequently, the effective clinical management of infected wounds is becoming increasingly challenging. Therefore, novel treatment strategies are needed that can safely and effectively eliminate both susceptible and drug-resistant bacteria and accelerate wound healing.

[0003] Carbon dots (CDs) are nanoscale carbon-based particles with excellent biocompatibility, low cytotoxicity, and inherent antibacterial activity. As novel nanoscale antibacterial agents, CDs exert their bactericidal effects by inhibiting bacterial adhesion, disrupting bacterial cell membranes, and regulating reactive oxygen species (ROS) levels, thereby reducing the likelihood of bacterial resistance. CDs have been reported to effectively kill various pathogens, including Helicobacter pylori, Salmonella, Staphylococcus aureus, and Escherichia coli. Furthermore, they can promote wound healing and repair by inducing macrophages to transition from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thus creating a more regenerative environment.

[0004] Chitosan (CS) is a widely studied natural polysaccharide and an effective antibacterial agent with good biocompatibility, biodegradability, low cytotoxicity, and hemostatic properties. It exerts its bactericidal effect by disrupting bacterial cell walls and increasing cell membrane permeability. These properties have led to the widespread application of chitosan in the treatment of infected wounds. For example, chitosan has been added to antibacterial gels and electrospun fiber membranes for wound dressings. Despite these advances, there are no reports on the synthesis of glycosylated carbon dots from chitosan and their application in the treatment of bacterial infected wounds.

[0005] Nanofibers are highly favored as advanced wound dressings due to their high specific surface area, porosity, and excellent breathability. They are widely used as drug delivery carriers to achieve controlled release and reduce side effects. For example, drug-loaded nanofibers and nanofiber membranes have been shown to accelerate wound healing. Furthermore, fibrin gel is a biomaterial formed by cross-linking fibrinogen with thrombin. It possesses excellent biocompatibility, water retention, and hemostatic properties. A key advantage of fibrin gel is its ability to be sprayed onto the wound site and solidify in situ, enabling rapid application and tissue protection. This property has been used to develop in situ immunotherapy systems and sprayable gels for applications ranging from tumor suppression to wound repair. Recently, researchers have attempted to combine nanofibers with hydrogels to prepare composite fiber-hydrogel dressings, aiming to combine the breathability of fibers with the moisturizing and conforming properties of hydrogels, while achieving sustained drug release and further reducing side effects. Preliminary results show that these composites exhibit a synergistic effect superior to any single component. However, a common limitation of these composites is insufficient inherent antibacterial activity, highlighting the need for further improvements. Summary of the Invention

[0006] To address the aforementioned shortcomings in existing technologies, this invention first developed a novel glycosylated carbon dot, termed CS-ACD, via a one-step hydrothermal method. The combination of chitosan (CS) and aspirin-derived carbon dots (ACD) yielded a nanomaterial with significantly enhanced antibacterial and anti-inflammatory activities, attributed to the synergistic effect between the two components. Mechanistic studies revealed that CS-ACD primarily kills bacteria through a membrane-dissolving mechanism. Furthermore, CS-ACD can induce macrophages to transition from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thereby reducing the expression and secretion of inflammatory cytokines. Based on these findings, this invention further prepared an innovative "sandwich" structured fiber-gel dressing (CS-ACD@Fiber@Gel) by combining CS-ACD-loaded electrospun nanofibers with an in-situ sprayable fibrin gel. In a mouse model of infected wounds, the CS-ACD@Fiber@Gel dressing effectively reduced bacterial load, modulated the immune response, alleviated wound inflammation, and promoted collagen deposition and angiogenesis, ultimately accelerating wound healing and tissue repair. This invention provides a safe and effective glycosylated CD-based therapy, as well as a composite fiber-gel dressing with dual antibacterial and anti-inflammatory functions, offering a promising strategy and foundation for future clinical translation in infected wound management.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a glycosylated carbon dot, the preparation method of which includes: first dissolving aspirin in an alkaline solution and carrying out a hydrothermal reaction, then adding chitosan solution to the reaction system to continue the reaction, and finally purifying and drying the resulting reaction mixture to obtain the glycosylated carbon dot; The chitosan raw material has a degree of deacetylation of 90% and a molecular weight of 150-200 kDa; the chitosan solution has a concentration of 0.01%.

[0008] Furthermore, the ratio of aspirin to chitosan solution is (2~4) grams: (0.5~2) milliliters.

[0009] Furthermore, the hydrothermal reaction is carried out at a temperature of 100-120°C for 4-6 hours; the reaction continues for 0.5-2 hours after the addition of chitosan.

[0010] Furthermore, the purification includes a step of dialysis using a dialysis bag with a molecular weight cutoff of 300-1000 Daltons.

[0011] The second objective of this invention is to provide a glycosylated carbon dot functionalized fiber-gel composite dressing, comprising an electrospun nanofiber layer and a fibrin gel layer; wherein the electrospun nanofiber layer is loaded with the glycosylated carbon dots; and the fibrin gel layer covers at least one surface of the nanofiber layer.

[0012] Furthermore, the method for preparing the electrospun nanofiber layer includes: dissolving a polymer in an organic solvent to obtain a polymer solution; dissolving collagen in an alcohol solvent to obtain a collagen solution; mixing the polymer solution, the collagen solution, and the glycosylated carbon dots to obtain a spinning solution; and electrospinning the spinning solution.

[0013] Furthermore, the polymer is selected from any one or more of polyvinylpyrrolidone, polylactic acid-glycolic acid copolymer, and polycaprolactone; the organic solvent includes hexafluoroisopropanol and / or ethanol; and the alcohol solvent includes ethanol.

[0014] Furthermore, the method for preparing the fibrin gel layer includes: mixing a fibrinogen solution with a thrombin solution and applying the mixture above and / or below the nanofiber layer to allow it to crosslink and solidify.

[0015] Furthermore, the concentration of the fibrinogen solution is 10-40 mg / mL, and the concentration of the thrombin solution is 10-40 units / mL; the volume ratio of the fibrinogen solution to the thrombin solution is (0.5-2):1.

[0016] A third objective of this invention is to provide the application of the glycosylated carbon dots or the glycosylated carbon dot-functionalized fiber-gel composite dressing in the preparation of products for treating infected wounds.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention develops a multifunctional glycosylated carbon dot (CS-ACD) and its corresponding "sandwich" structured fiber-gel dressing (CS-ACD@Fiber@Gel) for the treatment of infected wounds. In in vitro experiments, CS-ACD exhibits synergistically enhanced antibacterial and anti-inflammatory activity. It primarily kills bacteria through a cell membrane lysis mechanism and promotes the transition of macrophages from a pro-inflammatory M1 state to an anti-inflammatory M2 state. Therefore, CS-ACD effectively reduces the expression and secretion of key inflammatory mediators such as TNF-α, iNOS, and IL-1β. In in vivo experiments, the CS-ACD@Fiber@Gel dressing demonstrated potent efficacy in a mouse model of wound infection, effectively clearing bacteria, modulating immune responses, reducing inflammation, and promoting collagen deposition and angiogenesis. These effects ultimately lead to a significant acceleration of wound healing and tissue repair with minimal scarring. Overall, this invention provides a promising new strategy for the prevention and treatment of infected wounds and shows great potential for clinical translation in wound management. Attached Figure Description

[0018] Figure 1 This document describes the synthesis and characterization of CS-ACD in Example 1 of the present invention. (A) Schematic diagram of the CS-ACD synthesis process. (B) Zeta potential of CS-ACD (mean ± standard deviation, n = 3). (C) Hydrodynamic size distribution of CS-ACD (dynamic light scattering measurement, mean ± standard deviation, n = 3). (D) UV-Vis absorption spectrum of CS-ACD. (E) Fluorescence emission spectra of CS-ACD and ACD under 365 nm excitation. (F) Fourier transform infrared spectra of ACD and CS-ACD.

[0019] Figure 2This invention demonstrates the antibacterial activity of CS-ACD in Example 1. (A) The inhibition zone assay shows the antibacterial effect of CS-ACD compared to CS or ACD alone. (B) Plate count assay of bactericidal activity after treating E. coli with different samples (colonies on representative petri dishes are shown in the figure). (C) Quantitative analysis of bacterial viability obtained from (B), expressed as bactericidal rate (% kill) (mean ± standard deviation, n = 6). (D) Fluorescence microscopy images of E. coli after treatment with PBS (control), CS, ACD, or CS-ACD followed by live / dead staining (live bacteria are green fluorescent, dead bacteria are red). (E) SEM images showing the morphological damage of E. coli under each treatment.

[0020] Figure 3 This is an example of the immunomodulatory effect of CS-ACD on macrophages (RAW 264.7 cells) in Example 1 of this invention. (A and B) Representative fluorescence images obtained after treating macrophages stimulated with lipopolysaccharide (using the samples shown), these cells were immunostained to detect the M1 macrophage marker CD86 (FITC, green) and the M2 macrophage marker CD206 (PE, red). (C) Quantitative analysis of the mean fluorescence intensity (MFI) of CD86 obtained from (A) (mean ± standard deviation, n = 3, based on the unstimulated control group). (D) Quantitative analysis of the CD206 MFI obtained from (B) (mean ± standard deviation, n = 3). (E) TNF-α level in macrophage supernatant as measured by ELISA (mean ± standard deviation, n = 3). (F - H) Relative mRNA expression levels of TNF-α, iNOS and ARG1 in macrophages after different treatments (RT-qPCR analysis, mean ± standard deviation, n = 3).

[0021] Figure 4This document describes the preparation and characterization of the CS-ACD-loaded fiber-gel composite dressing in Example 1 of this invention. (A) Schematic diagram of the process for preparing CS-ACD-loaded nanofibers by electrospinning. (B) Scanning electron microscope image of CS-ACD@Fiber prepared by electrospinning. (C) Fluorescence microscope image of CS-ACD@Fiber, showing the uniformly distributed fluorescent CS-ACD within the fibers. (D) Inverted tube test demonstrating the gelation of fibrin: fibrinogen and thrombin solution (left) are mixed to form a non-flowing gel (right). (E) Scanning electron microscope image of the porous microstructure of the fibrin gel (scale bar: 10 μm). (F) Time-scanning rheological plot of the fibrin gel, showing the storage modulus (G') and loss modulus (G'') changing over time after mixing. (G) Strain scan of the fibrin gel (relationship between G' and G'' and strain %) demonstrating the elastic limit of the gel. Frequency scans of (H) fibrin gels showed stable viscoelastic behavior in the frequency range of (0.1 - 100 radians / second).

[0022] Figure 5 This invention presents the in vivo therapeutic effect of CS-ACD@Fiber@Gel in a mouse model of infected wounds, as described in Example 1. (A) Schematic diagram of experimental design and treatment process. Wounds were infected with Escherichia coli and then treated with PBS, CS-ACD, or CS-ACD@Fiber@Gel, respectively. Wound healing, bacterial load, and tissue analysis were monitored. (B) Representative photographs of wounds in each group over 13 days (the rightmost column shows overlaid wound images generated by ImageJ software). (C) Changes in the percentage of remaining wound area (percentage of initial value) over time in each group (mean ± standard error, n = 4), showing that the wound healing speed in the CS-ACD@Fiber@Gel group was significantly faster. (D) Bacterial content in infected wound tissues after different treatments was determined by homogenization and inoculation. (E) Quantitative results of (D) (mean ± standard error, n = 4). (F) Weight records of mice in each group during the experiment (mean ± standard error, n = 4), indicating that these treatments did not lead to significant weight loss or adverse reactions.

[0023] Figure 6 Histological analysis of wound healing on day 13 post-treatment in Example 1 of this invention. (A) H&E stained sections of wound tissue from each group. (B) Quantitative measurement of scar width in H&E sections of each group (mean ± standard error, n = 3). (C) Quantitative measurement of epidermal thickness of newly formed epidermis in each group (mean ± standard error, n = 3). (D) Masson's trichrome stained sections of wound tissue. (E) Quantitative measurement of collagen deposition as a percentage of tissue area (mean ± standard error, n = 3).

[0024] Figure 7 Immunofluorescence analysis of wound tissue was performed in Example 1 of this invention to assess angiogenesis and inflammation. (A) CD31 staining (green) of wound sections in each group highlights the formation of new blood vessels. (B) TNF-α staining (red) of wound sections indicates the presence of inflammatory cytokines. (C) iNOS staining (green) of wound sections indicates the activity of inflammatory macrophages. (D) CD31 + Quantitative analysis of the region (percentage of visual field, mean ± standard error, n = 5). (E)TNF-α + Quantitative analysis of the region (percentage of area, mean ± standard error, n = 5). (F)iNOS + Quantification of regions (percentage of area, mean ± standard error, n = 5). Wounds treated with CS-ACD@Fiber@Gel showed significantly increased angiogenesis and significantly decreased inflammatory markers compared to the CS-ACD or PBS control group.

[0025] Figure 8 The fluorescence spectrum characterization of aspirin in Example 1 of this invention under 365 nm excitation.

[0026] Figure 9 Example 1 of the present invention (A) shows the bactericidal activity of CS-ACD evaluated by the plate coating method. (B) shows the quantitative results (mean ± standard deviation, n = 3) in (A).

[0027] Figure 10 The cell viability of Raw 264.7 cells treated with CS, ACS and CS-ACD for 24 hours was evaluated in Example 1 of the present invention (mean ± standard deviation, n = 3).

[0028] Figure 11 The mRNA expression levels of the cell marker CD86 and the inflammatory cytokine IL-1β in macrophages after different treatments were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in Example 1 of this invention (mean ± standard deviation, n = 3). Detailed Implementation

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0030] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0031] Example 1 I. Experimental Methods 1. Cell Culture The cells used in the experiment were RAW 264.7 mouse macrophages. These cells were cultured in high-glucose DMEM medium (containing 4.5 g / L glucose) supplemented with 10.0% fetal bovine serum and 1.0% penicillin-streptomycin. The culture environment was maintained at a constant temperature of 37°C and in a humidified environment containing 5.0% carbon dioxide. When the cells reached approximately 80.0% adherence density, they were passaged or seeded into new culture dishes.

[0032] 2. Preparation of glycosylated carbon dots CS-ACD was synthesized via a one-step hydrothermal reaction using aspirin as the carbon source. The specific procedure was as follows: 3 g of aspirin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A104180-500g) was added to a 50 mL round-bottom flask, along with an appropriate amount of alkaline solution. The flask was heated to 110°C on a thermostatic magnetic stirrer and maintained for 5 hours. Then, 1 mL of chitosan (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number 850125-100g; the degree of deacetylation of the chitosan raw material was 90%, and the molecular weight was 150-200 kDa, 1 x 10⁻⁶ kDa) was added to the reaction mixture. -4 1 g of chitosan was dissolved in 1 mL of ultrapure water (concentration 0.01%), and the mixture was stirred for 1 hour. After the reaction was complete, the mixture was cooled to room temperature, transferred to a dialysis bag (molecular weight cutoff 500 Daltons), and dialyzed with deionized water for 48 hours to remove impurities and free molecules. The dialyzed solution was freeze-dried to obtain CS-ACD solid powder, which was stored at 4°C for subsequent use.

[0033] 3. Preparation of glycosylated carbon dot-loaded fiber gel dressing To prepare CS-ACD-loaded nanofibers (CS-ACD@Fiber), 1 g of polyvinylpyrrolidone (PVP) was dissolved in 10 mL of a hexafluoroisopropanol (HFIP) / ethanol mixture (volume ratio 4:1) and stirred at room temperature for 3 hours to obtain a homogeneous PVP solution. Separately, 0.2 g of collagen (porcine skin collagen, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S25054-10g) was dissolved in 4 mL of ethanol and stirred for 30 minutes to form a homogeneous collagen solution. The collagen (Col) solution was then stirred together with the PVP solution. 25 mg / mL of CS-ACD was added to this PVP / Col mixture, and the mixture was stirred thoroughly to uniformly disperse the carbon dots. The resulting CS-ACD / PVP / Col solution was loaded into a 10 mL plastic syringe equipped with a 22G stainless steel needle and mounted on an electrospinning device. Electrospinning was performed under an applied voltage of 9 kV, with a needle-to-collector distance of 8 cm and a solution flow rate of 0.2 mL / h. A rotating disk (450 rpm) served as the fiber collector to obtain a neatly arranged fiber layer. Electrospinning continued for 6 hours to obtain nanofibers containing CS-ACD (CS-ACD@Fiber). For the fibrin gel fraction, fibrinogen solution (20 mg / mL in PBS, bovine blood fibrinogen, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S12024-1g) was mixed with thrombin solution (bovine plasma thrombin, purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number T8021-1000U, specific activity >2000 U / mg, concentration of 20 units / mL in PBS containing 40 mmol calcium chloride) at a 1:1 (volume ratio). To construct the composite dressing (CS-ACD@Fiber@Gel), fibrinogen and thrombin solutions were sprayed or added before and after the CS-ACD@Fiber material sheet was placed on the wound (in animal experiments). This allowed the mixture to blend and form a fibrin gel with in-situ embedded fibrous material. The fibrin gel was allowed to fully cure for several minutes to form a stable, "sandwich" structured fiber-gel dressing ready for use.

[0034] 4. Characterization of structure and performance The hydrodynamic particle size (diameter) and zeta potential of ACD and CS-ACD were measured using a nanoparticle size analyzer (dynamic light scattering, DLS). UV-Vis absorption spectra were determined using a UV-Vis spectrophotometer, and fluorescence emission spectra were obtained using a fluorescence spectrophotometer. Infrared spectra of dried ACD and CS-ACD powders were determined using a Fourier transform infrared spectrometer to identify characteristic functional groups. CS-ACD@Fiber was observed under UV excitation using a fluorescence microscope to verify the presence of fluorescent carbon dots in the fibers. The morphology of the gels was observed using a scanning electron microscope after freeze-drying the gel samples. Rheological tests were performed on the gels to evaluate their viscoelastic properties and gelation kinetics. For time-scan measurements, fibrinogen and thrombin solutions were mixed on a rheometer plate, and the storage modulus (G') and loss modulus (G'') were recorded over time (frequency 1.0 Hz, strain 1.0%). For strain scanning experiments, the gel was subjected to gradually increasing shear strains (from 0.1% to 1000.0% at a frequency of 1.0 Hz) to determine the critical strain and stability of the gel network. For frequency scanning experiments, the gel was tested at a constant strain of 1.0% from 0.1 to 100 radians / second to evaluate the stability of G' and G'' at different frequency ranges.

[0035] 5. Antibacterial activity assessment After culturing *E. coli* overnight in LB liquid medium, centrifuge at 12,000 g for 1 minute. Remove the supernatant and resuspend the bacterial pellet in 1 mL of PBS. Repeat this washing step three times to thoroughly rinse the bacteria. The washed bacteria are then diluted to 1 × 10⁻⁶. 7 CFU / mL is used for inhibition zone assays. For disk diffusion (agar well) assays, prepare LB agar plates and dilute the bacterial suspension (approximately 2 × 10⁻⁶) to a suitable level. 5 CFU was evenly spread on each plate. Wells approximately 1 mm in diameter were then punched into the agar using a sterile pipette tip. 20 μL of sample solution (CS, ACD, or CS-ACD, all at the same concentration of 20 mg / mL in PBS) was added to each well. PBS was added to one well as a control. The plates were incubated overnight at 37°C, and the diameter of the bacterial inhibition zone around each well was observed and measured. For bactericidal activity (viable count), the washed E. coli suspension was diluted to approximately 1 × 10⁻⁶. 6In M9 medium containing CFU / mL, aliquots of the bacterial suspension (usually 200 μL) were incubated with CS, ACD, or CS-ACD (5 mg / mL or 10 mg / mL) at 37°C for 24 hours. After incubation, the suspension was serially diluted in PBS, and 100 μL of each dilution was plated on LB agar plates (repeated three times). Bacteria treated with PBS (without sample) served as a control. After overnight incubation at 37°C, colonies were counted, and the bactericidal efficiency (sterilization rate) was calculated by comparing the CFU of the treated group with that of the PBS control group. All experiments were performed at least three independent replicates.

[0036] 6. Staining and imaging of dead / live bacteria Samples were taken from E. coli cultured overnight and diluted with M9 medium to approximately 1 × 10⁻⁶. 6 CFU / mL. The bacterial suspension was then co-cultured with CS, ACD, or CS-ACD for 24 hours (PBS as a control; CS, ACD, or CS-ACD used was 10 mg / mL) at 37°C. Afterward, the suspension was incubated in the dark at room temperature for 30 minutes, and stained with 5 μM calcein-AM (which gives live bacteria a green color) and 0.5 μg / mL propidium iodide (PI, which gives dead bacteria a red color). Each stained bacterial sample was 0.2 mL and washed three times with PBS in a dark room to remove excess dye. A drop of the stained bacterial suspension was placed on a glass slide and covered with a coverslip. Fluorescence images were immediately captured using a fluorescence microscope.

[0037] 7. Bacterial morphology is observed using a scanning electron microscope. E. coli suspension (approximately 1 × 10⁻⁶) 6 CFU / mL in M9 was co-cultured with CS, ACD, or CS-ACD for 24 hours (PBS as a control, CS, ACD, or CS-ACD at 10 mg / mL), similar to the sterilization test conditions. After treatment, 500 μL of each bacterial suspension was fixed with an equal volume of 3.0% glyceraldehyde in PBS and incubated at room temperature for at least 2 hours (or overnight at 4°C). The fixed bacteria were then gently precipitated (12,000 rpm for 1 minute) and washed with PBS. For dehydration, the bacterial precipitate was sequentially immersed in a series of ethanol concentrations (30.0%, 50.0%, 70.0%, 90.0%, and 100.0%; immersion for 10 minutes each). Finally, a 100.0% ethanol suspension containing the bacteria was dropped onto a clean silicon wafer and allowed to air dry. The dried sample was sputtered with a thin layer of gold and observed under a scanning electron microscope to assess the structural integrity of the bacteria.

[0038] 8. Cell viability assay The cytotoxicity of CS-ACD, ACD, and CS against RAW 264.7 macrophages was evaluated using the CCK-8 assay. Cells were cultured at 2.0 × 10⁶ cells / year. 4 Cells were seeded at a density of cells / well in 96-well plates, with 100 μL of DMEM medium added to each well, and allowed to adhere for 12 hours. The medium was then replaced with 100 μL of fresh medium containing different concentrations (0, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 μg / mL) of CS-ACD, ACD, or CS. Cells treated with PBS served as a negative control (100.0% cell viability reference value). After 24 hours of treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 3 hours. The absorbance at 450 nm was measured using a microplate reader. Cell viability (%) was calculated by dividing the absorbance of the treated wells by the absorbance of the control wells.

[0039] 9. Anti-inflammatory activity assessment 1.0×10 5 RAW 264.7 cells were seeded in 24-well plates and cultured for 12 hours. To simulate an inflammatory environment, cells were stimulated with 100 ng / mL LPS for 24 hours to induce M1 polarization. The medium was then replaced with fresh medium containing CS, ACD, or CS-ACD (50 μg / mL of CS, ACD, or CS-ACD). Cells were incubated in these media for 24 hours. To analyze secreted cytokines, the supernatant was collected and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected using an ELISA kit, following the manufacturer's instructions. To assess macrophage polarization, cells in each well were gently washed with PBS and then fixed with 4.0% paraformaldehyde. After fixation, cells were blocked in PBS with 3.0% BSA for 30 minutes to prevent nonspecific binding. Cells were then incubated for 2 hours at room temperature in the dark with FITC-conjugated anti-mouse CD86 antibody (1:200 dilution, a marker for M1 macrophages) and PE-conjugated anti-mouse CD206 antibody (1:200 dilution, a marker for M2 macrophages). Cells were washed three times with PBS to remove unbound antibodies. Finally, fluorescence images were captured using a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software to obtain the mean fluorescence intensity (MFI) for each marker.

[0040] 10. RNA extraction and RT-qPCR RAW 264.7 cells were seeded into 24-well plates (1.0 × 10⁶ cells per well). 5Cells were allowed to attach for 12 hours. Cells were then stimulated with 100 ng / mL LPS for 24 hours to induce an inflammatory state. After LPS induction, cells were treated with PBS, CS, ACD, or CS-ACD for 24 hours (CS, ACD, or CS-ACD were used at 50 μg / mL). Total RNA was extracted from each well using TRIzol reagent, following the manufacturer's instructions. An equal amount of RNA was extracted from each sample and reverse transcribed into cDNA using a reverse transcription kit. The resulting cDNA was used as a template for quantitative PCR, which was performed on a QuantStudio 5 real-time PCR instrument using 2×SYBR Green qPCR Master Mix. Specific primers for TNF-α, iNOS, ARG1, and actin (host marker control) were used (specific primer sequences are shown in Table 1 below).

[0041] Table 1 Primer sequences used in real-time quantitative PCR experiments (SEQ ID NO. 1~12) 11. Animal wound infection models and treatment The in vivo efficacy of these treatments was evaluated using 6-8 week old female BALB / c mice. Mice were anesthetized with sodium pentobarbital solution, their dorsal hair was shaved, and the wounds were disinfected. A full-thickness excision wound approximately 10 mm in diameter was created on the back of each mouse. To establish an infected wound model, 20 μL of E. coli suspension (at a concentration of 1 × 10⁻⁶ in PBS) was used. 7CFU / mL was evenly applied to the wound surface. The inoculated wounds were left in place for 6 hours to observe infection status, at which point slight redness and exudate were visible. Mice were then randomly assigned to three groups: PBS, CS-ACD, and CS-ACD@Fiber@Gel (CS-ACD dosage: 200 μL, 25 mg / mL; CS-ACD@Fiber@Gel dosage: 0.03 g). Wounds were photographed on days 0, 3, 6, 10, and 13 post-treatment to monitor wound healing. Throughout the experiment, the weight of each mouse was recorded as an indicator of overall health and potential systemic effects of treatment. To assess bacterial load at the wound site, on day 5, some mice from each group were euthanized, and wound tissue (including scab and a small piece of underlying tissue) was collected under aseptic conditions. The tissue was homogenized in sterile PBS using a tissue homogenizer. The homogenate was serially diluted, and 100 μL of each dilution was spread onto LB agar plates. After incubation overnight at 37°C, bacterial colonies were counted. To analyze tissue repair and immune response, the remaining mice were euthanized on day 14, and wound areas (including healed tissue and normal skin margins) were harvested. Tissue samples were fixed in 4.0% formaldehyde, embedded in paraffin, and sectioned (5 μm thick) for histological staining. H&E staining was performed to assess overall morphology and to measure epidermal thickness and scar width. Masson's trichrome staining was performed to assess collagen deposition. After dewaxing and antigen retrieval, the dewaxed, antigen-retrieval-treated sections were stained with immunofluorescence. Sections were incubated with primary antibodies against CD31 (a vascular endothelial marker), TNF-α, and iNOS, followed by detection with the corresponding fluorescent secondary antibodies. Cell nuclei were counterstained with DAPI. Stained sections were observed under a fluorescence microscope. Image analysis was performed using ImageJ to quantify parameters such as the proportion of positively stained areas for CD31, TNF-α, and iNOS, average scar width, epidermal thickness, and percentage of collagen area (from Masson's staining).

[0042] 12. Statistical Analysis All quantitative data are presented as mean ± standard deviation (SD) or mean ± standard error of mean (SEM), as explained at the end of the document. Statistical analysis was performed using GraphPad Prism 8.0 software. Unpaired two-tailed t-tests were used for comparisons between two groups. For comparisons among multiple groups, one-way or two-way ANOVA was performed first, followed by Tukey's post-hoc test for pairwise comparisons. In all analyses, p < 0.05 was considered statistically significant.

[0043] II. Experimental Results and Discussion 1. Preparation and characterization of CS-ACD First, a one-step hydrothermal method was used to synthesize glycosylated carbon dots (CS-ACD). Figure 1 A), and then its physicochemical properties were characterized. Dynamic light scattering (DLS) measurements showed that the hydrodynamic diameter of CS-ACD was approximately 37.6 nm, significantly larger than the diameter of the precursor ACD (approximately 3 nm), confirming the successful chitosan modification. Zeta potential measurements showed a change from -18 mV for ACD to +29.9 mV for CS-ACD, further demonstrating the successful binding with chitosan. Figure 1 B, C). UV-Vis and fluorescence spectroscopy showed that CS-ACD and ACD had similar characteristic absorption peaks at approximately 230 nm and 296 nm, respectively, and both exhibited maximum fluorescence emission at 405 nm. In contrast, chitosan and aspirin did not show obvious characteristic absorption peaks or fluorescence spectra at the same positions. Figure 1 D, E and Figure 8 This indicates that chitosan modification did not alter the fundamental photophysical properties of the carbon dots. Finally, Fourier transform infrared (FTIR) spectroscopy provided further evidence of successful synthesis. CS-ACD at 1580–1590 cm⁻¹ -1 A characteristic peak is observed at 1650–1660 cm⁻¹, corresponding to the C–N stretching vibration of chitosan, while a peak is observed at 1650–1660 cm⁻¹. -1 There is a peak at this point, attributed to the C=O stretching vibration of ACD ( Figure 1 F). In summary, these results confirm the successful preparation of glycosylated carbon dot CS-ACD.

[0044] 2. Antibacterial activity and mechanism of CS-ACD Next, the antibacterial activity of CS-ACD was evaluated, and the potential synergistic effect between CS and ACD was investigated. Disc diffusion assays showed that, at the same concentration, the bacterial inhibition zone produced by CS-ACD was significantly larger than that produced by ACD or CS alone. Figure 2 A). This indicates that the antibacterial activity is synergistically enhanced when the two components are combined. To further quantify this effect, a bactericidal test was conducted, in which bacteria were incubated with each material and the resulting colony-forming units (CFU) were counted. The results showed that CS-ACD treatment produced the fewest bacterial colonies, followed by ACD, CS, and PBS (control group), with similar trends at different concentrations. Figure 2 B and Figure 9 , Figure 2 B and Figure 9 The concentrations used were 10 mg / mL and 5 mg / mL, respectively. Quantitative analysis showed that the sterilization rate of the CS-ACD group reached approximately 97.3%, while that of the ACD group was 89.9% and that of the CS group was 59.9%. Figure 2C). These findings confirm that CS and ACD work synergistically, resulting in CS-ACD having a superior antibacterial effect.

[0045] To elucidate the antibacterial mechanism of CS-ACD, live / dead fluorescence staining and scanning electron microscopy (SEM) were used to examine bacterial viability and morphology. Live / dead staining showed that almost all bacteria were viable (green) in the PBS group, while the proportion of dead bacteria increased in the CS and ACD groups, reaching almost 100.0% in the CS-ACD group. Figure 2 D). Consistent with these results, SEM imaging also showed that bacteria in the CS-ACD group exhibited severe structural damage and complete cell rupture. In contrast, bacteria in the PBS group remained intact, while bacteria treated with ACD or CS showed only partial membrane rupture (D). Figure 2 E). These results indicate that CS-ACD produces a synergistically enhanced bactericidal effect and kills bacteria primarily through a membrane disruption mechanism. Specifically, the positively charged amino acid groups on chitosan can bind to the negatively charged bacterial membrane through electrostatic interactions, leading to membrane disruption. Meanwhile, aspirin-derived carbon dots (ACDs) have been reported to enhance bacterial outer membrane permeability and significantly inhibit bacterial growth and colony formation. These properties may help explain the strong antibacterial activity of ACDs and their enhanced effect when used in combination with chitosan. Notably, this physical disruption mechanism is less likely to induce bacterial resistance, consistent with the theoretical basis for using non-antibiotic nanomaterials.

[0046] 3. Anti-inflammatory activity and mechanism of CS-ACD Next, the anti-inflammatory activity of CS-ACD was evaluated to determine whether it also had a synergistic effect. Cytotoxicity testing showed that CS-ACD had no significant cytotoxicity. Figure 10 Subsequently, RAW 264.7 macrophages were first stimulated with 100 ng / mL lipopolysaccharide (LPS) for 24 hours to induce a pro-inflammatory M1 phenotype, followed by treatment with CS, ACD, or CS-ACD for 24 hours. Macrophage phenotype transitions were analyzed by immunofluorescence staining; M1 macrophages were labeled with FITC-anti-CD86 (green), and M2 macrophages were labeled with PE-anti-CD206 (red). Fluorescence imaging showed that LPS stimulation significantly increased green fluorescence (M1 marker CD86) compared to untreated control cells. This green fluorescence signal subsequently decreased after treatment with CS, ACD, or CS-ACD, and notably, the fluorescence intensity in the CS-ACD group almost recovered to the control level. Figure 3A). Quantitative analysis showed that after stimulation with lipopolysaccharide (LPS), the mean fluorescence intensity (MFI) of CD86 increased by 2.92-fold (higher than the control group), while treatment with CS-ACD brought it close to baseline (a 2.87-fold decrease relative to the increase caused by LPS). In contrast, CS and ACD treatments only reduced the MFI of CD86 by 0.94-fold and 1.23-fold, respectively. Figure 3 C). Conversely, LPS stimulation led to a significant decrease in red fluorescence (M2 marker CD206), which was subsequently restored by CS, ACD, or CS-ACD treatment. Among these, CS-ACD produced the strongest red fluorescence. Figure 3 B). Quantitative analysis showed that LPS exposure reduced the MFI of CD206 to approximately 15.2% of the control group, while CS-ACD treatment increased it by 13.59 times compared to LPS levels. This increase was significantly greater than that achieved by CS (2.69 times) or ACD (7.62 times), and the CS-ACD group was significantly different from other groups. Figure 3 D). These results indicate that CS-ACD possesses potent immunomodulatory effects, promoting the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type. Furthermore, the anti-inflammatory effect of CS-ACD is significantly stronger than that of CS or ACD alone, suggesting a synergistic enhancement in immunomodulation. To further investigate the anti-inflammatory mechanism, the levels of inflammatory cytokines in cell supernatants and the mRNA expression of related genes in macrophages were measured under different treatment conditions. Enzyme-linked immunosorbent assay (ELISA) results showed that LPS stimulation increased the secretion of tumor necrosis factor-α (TNF-α) from 69.17 pg / mL to 94.17 pg / mL. Treatment with CS-ACD effectively reduced TNF-α secretion to 76.86 pg / mL, a reduction of 17.30 pg / mL compared to the LPS group, while CS and ACD only reduced TNF-α by 5.55 and 10.96 pg / mL, respectively. Figure 3 E). Similarly, LPS stimulation upregulated TNF-α mRNA expression by approximately 1.84-fold relative to the control group, while CS-ACD treatment inhibited TNF-α mRNA expression by approximately 9.85-fold (relative to LPS conditions). In contrast, CS and ACD reduced TNF-α mRNA expression by only 1.27-fold and 1.80-fold, respectively. Figure 3 F). LPS also increased iNOS (M1 marker) mRNA by 2.49-fold, while CS-ACD decreased iNOS expression by 4.40-fold. In comparison, CS only reduced the effect by 1.31-fold, and ACD reduced it by 2.16-fold. Figure 3G). Furthermore, LPS upregulated CD86 (M1 marker) mRNA by 1.57-fold, while CS-ACD reduced CD86 expression by 3.12-fold (compared to LPS), compared to a 1.04-fold reduction in CS and a 1.86-fold reduction in ACD. Figure 11 A). LPS stimulation also increased IL-1β mRNA by approximately 4.36-fold, while CS-ACD treatment inhibited IL-1β mRNA by 1.96-fold. CS and ACD did not inhibit IL-1β mRNA. Figure 11 B).

[0047] Conversely, LPS downregulated ARG1 (M2 marker) mRNA to 0.92-fold in the control group, while CS-ACD treatment increased ARG1 expression by 3.44-fold (compared to LPS), which was superior to CS (1.54-fold) and ACD (1.80-fold). Figure 3 (H). The differences observed with CS-ACD compared to other groups were statistically significant. These results further indicate that CS-ACD possesses superior immunomodulatory activity and can effectively inhibit the expression of multiple inflammatory mediators. Notably, the anti-inflammatory effect of CS-ACD is much stronger than that of CS or ACD alone, highlighting a synergistic effect. The mechanism behind this effect may involve the inhibitory effect of CS-ACD on pro-inflammatory signaling pathways. For example, CS-ACD can downregulate the expression of iNOS and TNF-α in macrophages by interfering with the IκK / IκB / NF-κB pathway and the COX-2 / PGE2 / EP2 / NF-κB feedback loop. This is consistent with previous findings that carbon-based nanomaterials can modulate macrophage activation and inhibit the NF-κB signaling pathway, thereby alleviating inflammation.

[0048] 4. Construction and characterization of CS-ACD@Fiber@Gel To translate in vitro research results into practical wound treatment methods, this invention develops a "sandwich" structured fiber-gel composite dressing loaded with CS-ACD (CS-ACD@Fiber@Gel). This composite dressing aims to maintain a breathable and moist wound environment and achieve sustained release of CS-ACD, thereby improving therapeutic efficacy and reducing side effects. This fiber-gel dressing utilizes the complementary advantages of nanofibers and hydrogels (the electrospun fiber layer provides a large surface area and porosity for gas exchange), while the fibrin gel layer provides moisture, adheres to the wound, and aids in hemostasis. These properties together create a favorable environment for wound healing. Firstly, CS-ACD-filled nanofibers (CS-ACD@Fiber) were prepared using electrospinning technology. Figure 4A). Fiber layers were prepared through an electrospinning process under optimized conditions. The morphology of the obtained nanofibers was characterized by scanning electron microscopy (SEM) and fluorescence microscopy. SEM images showed that CS-ACD@Fiber had a uniform fiber morphology, a smooth surface, and an average fiber diameter of approximately 1.5 μm, comparable to the control group fibers without CS-ACD. Figure 4 B). This indicates that the incorporation of CS-ACD did not significantly alter the fiber formation or structure. Fluorescence microscopy confirmed that CS-ACD was successfully loaded into the fibers, as CS-ACD@Fiber exhibited the typical blue fluorescence of ACD throughout the fiber network, a characteristic not found in the control fibers without CS-ACD loading. Figure 4 C). These results demonstrate that the preparation of CS-ACD@Fiber was successful and that the encapsulation of carbon dots in nanofibers was effective. Next, this invention incorporates CS-ACD@Fiber into fibrin gel to prepare a composite dressing. Fibrin gel was chosen as the matrix due to its excellent biocompatibility and ability to form protective blood clots in vivo. To construct CS-ACD@Fiber@Gel, CS-ACD-loaded fiber sheets were embedded in a fibrinogen solution and then cross-linked by adding thrombin. The fibrinogen / thrombin mixture rapidly gelled after mixing, as confirmed by a tube inversion test. These solutions were liquid before mixing and formed a stable gel within seconds of mixing. Figure 4 D). Scanning electron microscopy imaging of fibrin gels revealed a rich porous network with pore sizes typically exceeding 2.5 micrometers. Figure 4 E), indicating that this is a scaffold with a high surface area, capable of accommodating fibrous materials and promoting nutrient exchange. Rheological measurements show that the fibrin gel exhibits rapid gelation kinetics and strong mechanical properties. After mixing, the storage modulus (G') quickly exceeds the loss modulus (G''), reflecting the formation of a cross-linked network ( Figure 4 F). The gel maintains its integrity even under strain scanning up to large deformations and exhibits stable viscoelastic behavior across a wide frequency range. Figure 4 (G, H). These results demonstrate that the fibrin gel provides a suitable and robust matrix for the fibrous components. The final CS-ACD@Fiber@Gel composite fully leverages the advantages of both components. The nanofibers reinforce the gel and allow for drug loading, while the gel provides conformability and a moist environment.

[0049] 5. The in vivo therapeutic effect of CS-ACD@Fiber@Gel on infected wounds Next, this invention evaluated the in vivo therapeutic effect of CS-ACD@Fiber@Gel using a mouse model of infected wounds. A full-thickness skin wound (approximately 10 mm in diameter) was created on the back of each mouse and inoculated with *E. coli* to establish an infected wound. Six hours after infection, the mice were divided into three groups and treated with PBS (control group), CS-ACD (solution), or CS-ACD@Fiber@Gel dressing (by spraying fibrin solution onto the wound covered with a fibrous membrane), respectively. The wound healing process was monitored by photographing the wounds and periodically measuring the wound area. Figure 5 A).

[0050] Over time, photographs of the wounds showed that all groups exhibited a gradual healing trend, but the CS-ACD@Fiber@Gel group healed significantly faster. Figure 5 B). By day 13, wounds treated with CS-ACD@Fiber@Gel were almost completely closed, while wounds treated with CS-ACD or PBS still showed significant signs of non-healing. Quantitative analysis of wound area confirmed this difference. On day 13, the mean wound area in the CS-ACD@Fiber@Gel group was only about 1.0%, significantly smaller than that in the CS-ACD group (11.6%) or the PBS group (18.4%). Figure 5 C). These results indicate that CS-ACD@Fiber@Gel effectively promotes wound healing, and that the sustained release and local retention of CS-ACD in the fiber-gel matrix are more effective than a single application of CS-ACD solution alone.

[0051] To further elucidate the mechanism of improved healing, this invention assessed the bacterial load in the wound and monitored systemic effects. Three days after treatment, wound tissue was collected, homogenized, and inoculated with cultures to quantify the number of viable bacteria. Culture counts showed that the bacterial load in wounds treated with CS-ACD@Fiber@Gel was significantly lower than that in wounds treated with CS-ACD or PBS alone. Figure 5 D). On average, each tissue in the CS-ACD@Fiber@Gel group had 3.07 × 10⁻⁶ tissue samples. 5 The live bacteria in CFU, compared with the PBS group (4.68 × 10⁻⁶). 6 The CFU (Cost Per Quantity) was reduced by approximately 15.26 times. This antibacterial effect is significantly stronger than using CS-ACD solution alone, which only achieved a reduction of approximately 1.64 times in CFU. Figure 5E). The significant reduction in bacterial counts in the CS-ACD@Fiber@Gel group indicates that this composite dressing effectively controls infection at the wound site, which may be a key factor in its enhanced wound healing performance. Throughout the study, the weight of the mice was also monitored to check for any systemic toxicity or adverse reactions. No significant differences in weight were observed among the three groups throughout the study period (except for a brief, slight decrease in weight in all groups around day 3, due to the initial infection). Figure 5 F). This indicates that the CS-ACD@Fiber@Gel dressing did not cause observable systemic toxicity in vivo.

[0052] 6. The in vivo therapeutic mechanism of CS-ACD@Fiber@Gel To gain a deeper understanding of the tissue-level impact and healing quality, histological and immunofluorescence analyses were performed on wound tissue collected 13 days post-treatment. H&E staining of wound sections showed that tissue regeneration was more mature in the CS-ACD@Fiber@Gel group compared to the control group. Figure 6 A). Notably, the residual scar width was significantly smaller in the CS-ACD@Fiber@Gel group. Quantitative measurements showed that the mean scar width in the CS-ACD@Fiber@Gel treatment group was 0.92 mm, while the scar widths in the PBS and CS-ACD groups were 2.55 mm and 2.25 mm, respectively. Figure 6 B). Furthermore, the newly formed epidermis in the CS-ACD@Fiber@Gel group was thinner and had a more normal appearance (average thickness 0.05 mm), while the epidermis in the PBS group (0.19 mm) and the CS-ACD group (0.18 mm) was thicker. Figure 6 C). Masson's trichrome staining further demonstrated the improved healing. Wounds treated with CS-ACD@Fiber@Gel showed extensive collagen deposition in the granulation tissue (stained blue), while other groups had less collagen content ( Figure 6 D). In the CS-ACD@Fiber@Gel wound, the collagen-rich area accounted for 47.0% of the total tissue volume, significantly higher than that in the PBS group (16.6%) or the CS-ACD group (24.0%). Figure 6 E). These findings suggest that this composite dressing promotes improved wound healing quality, characterized by reduced scarring, thinner (closer to normal) epidermis, and increased collagen deposition, which should mean that the healed tissue will be stronger and more functional.

[0053] Next, this invention examined angiogenesis and inflammatory markers in wound tissue to understand how CS-ACD@Fiber@Gel promotes healing at the molecular level. CD3 immunofluorescence staining (labeling endothelial cells and new microvessels) showed significantly enhanced new angiogenesis in the CS-ACD@Fiber@Gel group. Wound sections from CS-ACD@Fiber@Gel-treated mice showed numerous CD31-positive microvessels (green fluorescence), while the PBS and CS-ACD groups showed relatively sparse signal. Figure 7 A). Image analysis confirmed that the CD31-positive area in the CS-ACD@Fiber@Gel wound (accounting for 6.3% of the field of view) was significantly larger than that in the PBS group (3.0%) or the CS-ACD group (3.4%). Figure 7 (D) indicates that this composite dressing promotes angiogenesis. However, staining for inflammatory proteins showed the opposite trend. In the wound of the PBS control group, TNF-α (an inflammatory cytokine) showed strong red fluorescence (D). Figure 7 B). The signal was slightly lower in CS-ACD-treated wounds and even lower in CS-ACD@fiber@gel-treated wounds, indicating that the dressing significantly reduced local TNF-α levels. Quantitative analysis showed that the TNF-α positive area decreased from 21.2% in PBS-treated wounds to 19.4% in CS-ACD-treated wounds, and only 8.4% in CS-ACD@fiber@gel-treated wounds. Figure 7 E). A similar pattern was observed for iNOS (an inflammatory enzyme primarily produced by M1 macrophages). The iNOS signal (green) was strong in PBS-treated wounds, decreased after CS-ACD treatment, and almost zero in wounds treated with CS-ACD@Fiber@Gel. Figure 7 C). The measured values ​​of iNOS positive regions were 19.5% (PBS), 14.9% (CS-ACD), and only 7.3% (CS-ACD@Fiber@Gel). Figure 7 (F). These results indicate that the wound environment created by this composite dressing significantly reduces inflammation levels and promotes higher levels of angiogenesis, demonstrating a clear advantage over the control group. By clearing bacteria and releasing the immunomodulatory CS-ACD, the dressing appears to disrupt the infection and inflammation cycle that inhibits healing. Consequently, tissue repair processes such as collagen deposition and angiogenesis are enhanced, resulting in faster closure and superior healing outcomes.

[0054] In summary, this invention develops a glycosylated carbon dot (CS-ACD) that combines the inherent antibacterial properties of aspirin-derived carbon dots with the biocompatibility and antibacterial properties of chitosan. The results of this invention demonstrate that the antibacterial and anti-inflammatory activities of CS-ACD are significantly stronger than any single component, highlighting a significant synergistic effect. CS-ACD exhibits a bactericidal effect of over 97.0% against *E. coli* in vitro, while the effects of using chitosan or carbon dots alone are significantly weaker. This synergistic enhancement effect can be attributed to the complementary mechanism of the two components: the polycationic chains of chitosan bind to and disrupt the bacterial cell membrane, while the aspirin-derived carbon core can penetrate and further disrupt the bacterial membrane and intracellular structures. These physical disruption mechanisms differ from those of traditional antibiotics and are unlikely to induce bacterial resistance. Therefore, the glycosylated carbon dot of this invention provides a promising non-antibiotic strategy for eliminating pathogens, including drug-resistant strains. Importantly, CS-ACD not only directly kills bacteria but also actively modulates the host's immune response. This invention observes that CS-ACD induces the conversion of macrophages to the anti-inflammatory M2 phenotype, while simultaneously decreasing levels of pro-inflammatory cytokines (TNF-α, iNOS) and increasing levels of anti-inflammatory markers (ARG1, CD206). This immunomodulatory function is highly beneficial for promoting healing, as excessive M1 inflammation has been found to delay tissue repair. The findings of this invention are consistent with previous reports that certain carbon nanomaterials can promote the conversion to the M2 phenotype and tissue regeneration. Molecular data suggest that CS-ACD may exert its effects by inhibiting NF-κB pathway signaling (confirmed by reduced expression of iNOS and TNF-α), possibly through interference with IκK / IκB activation or feedback loops involving COX-2 and PGE2. By simultaneously eliminating bacteria and suppressing the inflammatory response, CS-ACD addresses two key obstacles in the healing process of infected wounds.

[0055] By integrating CS-ACD into a composite fiber-gel dressing, this invention translates these synergies into a practical form of treatment. Advanced wound dressings typically aim to optimize the wound microenvironment, maintain moisture, promote gas exchange, and deliver medication in a controlled manner. The "sandwich" structure of the CS-ACD@Fiber@Gel dressing of this invention achieves these goals and also possesses potent antibacterial and anti-inflammatory properties. The electrospun nanofiber layer in the dressing provides a large drug-carrying surface area and allows airflow to the wound site, which is crucial for tissue oxygenation. On the other hand, the fibrin gel layer conforms to the wound bed, preventing dryness and providing hemostasis. The rapid in-situ gelation of fibrin allows the dressing of this invention to be used in a spray form, which may be advantageous for covering irregularly shaped or large wounds. Furthermore, the fiber-gel composite material, as a continuous release system for CS-ACD, can gradually deliver carbon dots to the wound site over time. This continuous release system has been shown to accelerate wound healing while reducing side effects. This invention reveals that CS-ACD@Fiber@Gel dressings are significantly more effective than single-use CS-ACD solutions in promoting wound healing and reducing bacterial counts. Previous studies combining nanofibers with hydrogels in wound dressings have shown improved therapeutic efficacy due to their moist and bioactive environment, but often lack strong antibacterial capabilities. By encapsulating CS-ACD within a fiber-gel matrix, the dressing of this invention directly addresses this limitation, providing broad-spectrum antibacterial action without relying on traditional antibiotics or disinfectants. This comprehensive approach ensures infection control throughout the healing process, thereby preventing complications and further accelerating tissue repair.

[0056] The in vivo performance of the CS-ACD@Fiber@Gel dressing highlights the importance of simultaneously managing infection and inflammation in wound treatment. Wounds treated with the composite dressing of this invention showed significantly lower bacterial loads and levels of inflammatory markers than untreated wounds or wounds treated with CS-ACD alone. This is consistent with significantly improved healing indicators. Wounds treated with the composite healed faster, with thinner scars and more regenerated tissue (such as collagen and new blood vessels) than the control group. The reduction in early inflammation may lay the foundation for better healing. Excessive TNF-α and iNOS in infected wounds lead to tissue damage and fibrosis, while inhibiting them helps protect healthy tissue and promote regeneration. The present invention observed robust angiogenesis (higher CD31 density) in the CS-ACD@Fiber@Gel group, which is crucial for nutrient delivery.

[0057] 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. A glycosylated carbon dot, characterized in that, The preparation method includes: first, dissolving aspirin in an alkaline solution and carrying out a hydrothermal reaction; then, adding chitosan solution to the reaction system to continue the reaction; and finally purifying and drying the resulting reaction mixture to obtain the glycosylated carbon dots. The chitosan raw material has a degree of deacetylation of 90% and a molecular weight of 150-200 kDa; the chitosan solution has a concentration of 0.01%.

2. The glycosylated carbon dots according to claim 1, characterized in that, The ratio of aspirin to chitosan solution is (2~4) g: (0.5~2) ml.

3. The glycosylated carbon dots according to claim 1, characterized in that, The hydrothermal reaction is carried out at a temperature of 100-120°C for 4-6 hours; the reaction continues for 0.5-2 hours after the addition of chitosan.

4. The glycosylated carbon dots according to claim 1, characterized in that, The purification includes a step of dialysis using a dialysis bag with a molecular weight cutoff of 300-1000 Daltons.

5. A glycosylated carbon dot functionalized fiber-gel composite dressing, characterized in that, It includes an electrospun nanofiber layer and a fibrin gel layer; the electrospun nanofiber layer is loaded with glycosylated carbon dots as described in any one of claims 1 to 4; the fibrin gel layer covers at least one surface of the nanofiber layer.

6. The glycosylated carbon dot functionalized fiber-gel composite dressing according to claim 5, characterized in that, The method for preparing the electrospun nanofiber layer includes: dissolving a polymer in an organic solvent to obtain a polymer solution; dissolving collagen in an alcohol solvent to obtain a collagen solution; mixing the polymer solution, the collagen solution, and the glycosylated carbon dots according to any one of claims 1 to 4 to obtain a spinning solution; and performing electrospinning on the spinning solution.

7. The glycosylated carbon dot functionalized fiber-gel composite dressing according to claim 6, characterized in that, The polymer is selected from any one or more of polyvinylpyrrolidone, polylactic acid-glycolic acid copolymer, and polycaprolactone; the organic solvent includes hexafluoroisopropanol and / or ethanol; and the alcohol solvent includes ethanol.

8. The glycosylated carbon dot functionalized fiber-gel composite dressing according to claim 5, characterized in that, The method for preparing the fibrin gel layer includes: mixing a fibrinogen solution with a thrombin solution and applying the mixture above and / or below the nanofiber layer to allow it to crosslink and solidify.

9. The glycosylated carbon dot functionalized fiber-gel composite dressing according to claim 8, characterized in that, The concentration of the fibrinogen solution is 10-40 mg / mL, and the concentration of the thrombin solution is 10-40 units / mL; the volume ratio of the fibrinogen solution to the thrombin solution is (0.5-2):

1.

10. The use of the glycosylated carbon dots according to any one of claims 1 to 4 or the glycosylated carbon dot functionalized fiber-gel composite dressing according to any one of claims 5 to 9 in the preparation of products for treating infected wounds.

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