An antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel and its preparation method and use

Abstract

The present invention provides an antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in situ adhesion hydrogel, which is prepared from bio-based carbon dots made from curcumin and carboxymethyl chitosan, mixed with double-bonded collagen and o-nitrobenzene functionalized hyaluronic acid, and chemically cross-linked under ultraviolet light to prepare a hydrogel. The present invention also provides a preparation method and use of the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in situ adhesion hydrogel. The present invention provides a multifunctional bio-inspired hydrogel matrix, which not only has strong tissue adhesion, but also has antibacterial, anti-inflammatory and antioxidant properties, and can be used to repair severely infected corneal wounds. As a corneal substitute, the hydrogel has wound sealing, transparency and appropriate mechanical strength, which are the key characteristics of sutureless hydrogels. In vitro and in vivo experiments have shown that the hydrogel not only has good biocompatibility, but also can reshape the microenvironment through antibacterial infection, antioxidant and anti-inflammatory effects, thereby accelerating wound healing.

Description

Technical Field

[0001] The present invention relates to an antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinking in-situ adhesion hydrogel, a preparation method and application thereof. Background Art

[0002] Bacterial keratitis (BK) is a sight-threatening ocular disease and a leading cause of corneal blindness in developing countries due to rapid bacterial invasion leading to corneal scarring. Notably, the BK microenvironment is characterized by an imbalance between proinflammatory cytokines, matrix metalloproteinases (MMPs), and reactive oxygen species (ROS), which leads to extracellular matrix (ECM) degradation and inhibits the healing process. Traditional treatments, including antibiotics and corticosteroids, not only fail to address the underlying inflammatory cascade that leads to recurrent infection and persistent inflammation, but can also lead to the emergence of antibiotic-resistant strains, further complicating infection management.

[0003] In recent years, zero-dimensional carbon dots (CDs) have been extensively studied and have shown promising potential for antimicrobial applications. Compared to bacterial killing through the generation of ROS, inhibition through physical or mechanical damage may be more effective in reducing CD cytotoxicity. CDs exhibit distinct regulatory abilities towards bacteria and cells depending on their surface charge. Positively charged CDs adhere to and penetrate bacterial cell membranes more readily than negatively charged ones, disrupting bacterial cell integrity. Furthermore, the fascinating aspect of CDs is that their functionality can be tailored to exhibit a wide range of activities by selecting precursors to create specialized CDs with precursor functionality. Numerous studies have demonstrated that CDs exhibit catalytic activities, such as SOD, CAT, and GPx-like enzyme activities, due to their size effects and abundant active sites. These nanozyme activities are exploited to eliminate ROS, thereby achieving cytoprotective, anti-inflammatory, or anti-tumor effects. Therefore, the inventors posed the question: Can the antimicrobial and cellular redox balance of CDs be achieved through the coexistence of positive charge and nanozyme activity? However, current research has focused less on how to engineer the multi-functional balance of CDs to achieve diverse regulatory abilities towards bacteria and cells, and there are few reports on their application in the development of corneal replacements.

[0004] Injectable adhesive hydrogels have been shown to be effective in closing various wounds, including those in the eye, making them an attractive alternative to sutures. The hydrogel's fluid precursor is injected into the target site and cross-linked to adapt to irregular wound shapes, thereby better integrating with native tissue. Therefore, the combination of sutureless hydrogels with nanozyme-active antimicrobial carbon dots is expected to provide a powerful platform for the treatment of severe IK. Furthermore, the biomimetic design of the therapeutic platform to native corneal tissue, in terms of high optical transparency, ECM composition, and mechanical properties, remains to be emphasized.

[0005] Traditional natural products offer advantages such as strong bioactivity, minimal antimicrobial side effects, and low drug resistance. Curcumin is a multifunctional natural product with antioxidant and anti-inflammatory properties, but its poor water solubility significantly limits its bioavailability, bioactivity, and clinical application. Studies have shown that converting curcumin into zero-dimensional carbon can enhance its antimicrobial activity. Lin et al. synthesized quaternized carbon from curcumin and 2,3-epoxypropyltrimethylammonium chloride (GTA), improving its water solubility and enhancing its antimicrobial activity against Escherichia coli. However, GTA, a common cationic surfactant, is generally unstable and exhibits some cytotoxicity. Chitosan, a natural antimicrobial product, holds promise for addressing biocompatibility issues. However, whether CD surfaces made from the natural products chitosan and curcumin can retain the active groups of curcumin and chitosan to enhance their antimicrobial activity, and particularly whether a balance between antimicrobial and cytoprotective properties can be achieved, remains an intriguing question. In addition, natural products collagen and hyaluronic acid are the most important components of corneal tissue and are ideal raw materials for designing seamless hydrogels for the preparation of corneal substitutes.

[0006] Application number: CN202110347174.5, invention name: A method for preparing a nano-modified titanium dioxide-chitosan-curcumin antibacterial complex, which discloses a method for preparing a nano-modified titanium dioxide-chitosan-curcumin antibacterial complex. The method uses glutaraldehyde as a cross-linking agent, and the prepared glutaraldehyde-chitosan-curcumin complex gel is compounded with nano-silane-modified titanium dioxide hydrogel to obtain an antibacterial complex. The present invention uses glutaraldehyde to react with amino groups in chitosan under certain conditions to form a Schiff base, converting the chitosan linear polymer into a substance with a three-dimensional network structure. The antibacterial activity of the Schiff base is also enhanced. Furthermore, the inorganic / organic antibacterial compound formed by the use of nano-modified titanium dioxide and curcumin not only overcomes the shortcomings of curcumin as an antibacterial agent, which is easily oxidized, has poor thermal stability, and has poor antibacterial durability when used alone, but also reduces the cost of titanium dioxide, making it safer and more environmentally friendly. With improved antibacterial durability, it can be used as a multifunctional antibacterial pigment and an antibacterial and mildew-proofing agent for coatings, leather, textiles, etc. This patent discloses an antibacterial and mildew-proofing agent for coatings, leather, textiles, etc. using modified titanium dioxide as the main raw material. In situ hybridization of polymerized curcumin and arginine-derived carbon quantum dots for synergistic treatment of bacterial infections ACS Applied Materials & Interfaces (IF 8.3) Pub Date: 2023-05-28, DOI: 10.1021 / acsami.3c04316 Hong-Jyuan Jian, Anisha Anand, Jui-Yang Lai, Binesh Unnikrishnan, Huan-Tsung Chang, Scott G. Harroun, Chih-Ching Huang” The document discloses the preparation of arginine carbon dots, which are then compounded with curcumin to obtain hybrid nanoparticles, and are used as eye drops for corneal bacterial infections. In situ light-triggered-imine cross-linked composite hydrogel for bone defect repair. Journal of Materials Chemistry B (IF 6.1) Pub Date: 2016-01-11, DOI: 10.1039 / c5tb02377g Jieyuan Zhang 1, Yunlong Yang, Yunfeng Chen, Xiaolin Liu, Shangchun Guo, Linyong Zhu, Yang Wang Affiliation” disclosed that HA-NB, as a photoresponsive component, participates in the cross-linking of the gel.

[0007] With the rapid spread of antibiotic resistance, the development of novel antimicrobial agents has become a key focus in the treatment of BK. However, the cytotoxicity of antimicrobial agents is a major concern in their clinical application. Despite various efforts to balance antimicrobial and biosafety, minimizing adverse reactions and promoting corneal repair during antibiotic use remain challenging. This is primarily due to the fact that these agents destroy bacteria by directly upregulating reactive oxygen species (ROS) levels. However, they are often toxic to both bacteria and cells. Furthermore, while corneal transplantation remains the ultimate means of restoring transparency to infected tissue, the shortage of corneal substitutes severely limits the recovery of vision. Particularly in cases of severe keratitis, not only must the risk of secondary infection be effectively mitigated, but corneal substitutes must also accurately fill the corneal defect with minimal incision. Commonly used sutures can lead to serious potential complications, such as exacerbated infection and corneal neovascularization. Summary of the Invention

[0008] The present invention provides an antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinking in-situ adhesion hydrogel, and also provides a preparation method and use of the hydrogel.

[0009] The present invention provides an antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel, which is prepared from bio-based carbon dots prepared from curcumin and carboxymethyl chitosan, mixed with double-bonded collagen and o-nitrobenzene functionalized hyaluronic acid, and chemically cross-linked under ultraviolet light to prepare a hydrogel.

[0010] The weight ratio of curcumin, carboxymethyl chitosan, double-bonded collagen, and o-nitrobenzene functionalized hyaluronic acid is:

[0011] 1-3 parts of curcumin, 1-3 parts of carboxymethyl chitosan, 1-4 parts of double-bonded collagen, and 1-4 parts of o-nitrobenzene functionalized hyaluronic acid.

[0012] Preferably, the weight ratio of curcumin, carboxymethyl chitosan, double-bonded collagen, and o-nitrobenzene functionalized hyaluronic acid is:

[0013] 1 part of curcumin, 2 parts of carboxymethyl chitosan, 2 parts of double-bonded collagen, and 2 parts of o-nitrobenzene functionalized hyaluronic acid.

[0014] The preparation method of the bio-based carbon dots is as follows:

[0015] Curcumin and carboxymethyl chitosan were weighed, placed in a beaker, and stirred rapidly until they were evenly dispersed to form a dark yellow suspension; the mixture was transferred to a reactor lined with polytetrafluoroethylene and placed in an oven at 180°C for 8 hours; after the reaction, the mixture was cooled to room temperature; the reaction solution was centrifuged at 8000 r / min for 10 minutes to remove insoluble precipitates, and the supernatant was placed in a 500 Dalton dialysis bag and dialyzed for 3 days, with the water changed every day; the dialyzed liquid was transferred to a culture dish for freeze-drying, and the black powder obtained was the bio-based carbon dots CC-CDs.

[0016] Wherein, the preparation method of the double-bonded collagen is:

[0017] Use acetic acid solution to dissolve type I collagen at a concentration of 1% w / v; add methacrylic acid under magnetic stirring, and use NaOH to adjust the pH value to 7.4, and react for 4 hours; transfer the solution into an 8000-14000Da dialysis bag to remove unreacted small molecules, freeze-dry, and store in the dark to obtain the product.

[0018] Wherein, the preparation method of the o-nitrobenzene functionalized hyaluronic acid is:

[0019] Hyaluronic acid and amino-nitrobenzene were dissolved in deionized water, and 1-hydroxybenzotriazole (HOBt) was added. The pH of the mixed solution was adjusted to 4.5, and 0.2 g of EDC hydrochloride was added. The mixture was reacted at room temperature for 48 hours. After the reaction was completed, the solution was transferred to an 8000-14000 Da dialysis bag to remove unreacted small molecules, and then freeze-dried and stored in the dark.

[0020] The present invention also provides a method for preparing the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel, which comprises the following steps:

[0021] a. Prepare a 0.05% photoinitiator solution with 0.1M acetic acid;

[0022] b. using the initiator solution to dissolve the double-bonded collagen and o-nitrobenzene-functionalized hyaluronic acid, maintaining the final concentration of the double-bonded collagen at 1%, and the final concentrations of the o-nitrobenzene-functionalized hyaluronic acid at 0.5%, 1%, and 2%, respectively;

[0023] c. The above solution was mixed with 100 μg / ml of bio-based carbon dots, the pH was adjusted to 7, and the mixture was incubated at 37°C for 30 min. The mixture was then irradiated with UV light for 60 s to achieve chemical cross-linking. Finally, the antibacterial and anti-inflammatory bio-based carbon dots-photocross-linked in situ adhered hydrogel CHB@CC was obtained.

[0024] The present invention also provides the use of the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel described in the section in preparing an external-use medicine for treating infectious keratitis.

[0025] Wherein, the medicine is an external eye medicine.

[0026] The present invention also provides use of the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinking in-situ adhesion hydrogel in the preparation of a corneal substitute.

[0027] This invention utilizes the natural herbal ingredient curcumin and amino-rich chitosan to successfully synthesize positively charged nanozyme-activated carbon dots (CDs) via a simple hydrothermal method. Experiments have demonstrated that, on the one hand, the CDs, due to their moderate positive charge, can effectively disrupt bacterial membranes and induce the excretion of cytoplasmic DNA and RNA, thereby killing bacteria. On the other hand, the retention of curcumin functional groups endows the CDs with potent superoxide dismutase (SOD) and catalase (CAT) activities, thereby regulating ROS levels and achieving cellular protection. Furthermore, the invention provides an injectable adhesive hydrogel with photostretchability that can mimic extracellular matrix (ECM) components. A novel nitrobenzene-grafted hyaluronic acid (HA-NB) is innovatively incorporated into methacrylated collagen. Under ultraviolet (UV) irradiation, the methacrylated collagen photopolymerizes to form a hydrogel, thereby providing material stability and controlling the hydrogel's mechanical properties. Simultaneously, the NB-HA acquires in situ adhesion to tissue surfaces through a phototriggered imine cross-linking (PIC) reaction. After adding curcumin-chitosan CDs (CC-CDs) to photostrictive Col-MA / HA-NB hydrogel (CHB@Gel), a composite hydrogel system (CHB@CC) was obtained, which can noninvasively fill corneal defects and effectively treat severe bacterial keratitis.

[0028] Furthermore, the composite hydrogel's in vitro antibacterial, cytoprotective, and macrophage polarization-regulating properties were evaluated. After implantation into bacterially infected rat corneal defects, the CHB@CC's ability to eliminate bacteria and inflammation and promote corneal repair was evaluated in vivo. Finally, the potential mechanism by which CHB@CC promotes the treatment of bacterial keratitis through balanced antibacterial and cytoprotective properties was explored. This innovative, all-natural hydrogel product has the potential to become a clinical material for the treatment of infectious keratitis, particularly bacterial keratitis.

[0029] This invention combines strongly positively charged carboxymethyl chitosan and curcumin in a one-pot process to prepare carbon dots. This method aims to combine the antibacterial properties of carboxymethyl chitosan with the antioxidant properties of curcumin, while also providing a certain degree of enzyme-mimicking activity. This invention also designs an adhesive hydrogel matrix to address the corneal transplantation challenges faced by patients with bacterial keratitis. The composite of carbon dots and gel not only enables matrix filling but also provides effective antibacterial and antioxidant properties. The invention utilizes HA-NB not only as an adhesive component but also as a component capable of undergoing an imine cross-linking reaction with MA-Col. Ultraviolet light not only excites HA-NB to expose aldehyde groups but also enables chemical cross-linking of MA-Col.

[0030] In severe viral keratitis, the infected corneal tissue needs to be removed. The hydrogel of the present invention can be filled in situ at the corneal defect and adhere to the corneal tissue in situ under ultraviolet light to form a tight connection.

[0031] The present invention provides a multifunctional bioinspired hydrogel matrix that not only exhibits strong tissue adhesion but also possesses antibacterial, anti-inflammatory, and antioxidant properties, making it suitable for repairing severely infected corneal wounds. As a corneal substitute, the CHB@CC hydrogel exhibits wound sealing, transparency, and adequate mechanical strength—key properties for sutureless hydrogels. In vitro and in vivo experiments demonstrated that the CHB@CC hydrogel not only exhibits good biocompatibility but also reshapes the microenvironment through antibacterial, anti-oxidant, and anti-inflammatory effects, thereby accelerating wound healing. In summary, the application of this multifunctional hydrogel loaded with antibacterial and anti-inflammatory CDs offers a promising approach for treating infectious keratitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 (A) Schematic diagram of carbon dot synthesis; TEM (B), particle size distribution (C), UV-Vis absorption spectrum (D), and fluorescence excitation and emission spectra (E) of carbon dots; (F) DPPH scavenging ability test of carbon dots; (G) hydroxyl radical scavenging ability test of carbon dots; (H) hydrogen peroxide scavenging ability test of carbon dots; (I) total antioxidant capacity test of carbon dots; SOD enzyme activity test of carbon dots (J) and schematic diagram (K); CAT enzyme activity test of carbon dots (L) and schematic diagram (M);

[0033] Figure 2 (A) Digital photos of carbon dot plating experiments at different concentrations; (B) SEM images of Escherichia coli and Staphylococcus aureus after treatment with 100 μg / ml carbon dots; (C) Zeta potential measurement of bacteria and carbon dots; (D) Statistical analysis of nucleic acid concentrations in culture medium after treatment with 100 μg / ml carbon dots; (E) Schematic diagram of carbon dots killing bacteria; Confocal image of co-culture of carbon dots and corneal epithelial cells (F) and schematic diagram of their interaction (G);

[0034] Figure 3 Frequency sweep (A), stress-strain curve (B), elongation at break (C), Young's modulus (D), optical image (E), transmittance (F), transmittance at 365 nm (G), and transmittance at 550 nm of gels at different HA-NB concentrations; schematic diagram of lap shear adhesion experiment and adhesion strength statistics (I); schematic diagram of burst pressure test and numerical statistics (J); gel swelling (K), degradation (L), and carbon dot release curve (M);

[0035] Figure 4:(A) Live-dead and cytoskeleton staining of Conrol, CHB-Gel, CC-CDs, and CHB@CC; (B) Cell proliferation data; (C) Reactive oxygen species scavenging capacity of different groups; (D) Immunofluorescence staining of TNF-α, IL-β, and IL-6. Fluorescence quantification of TNF-α (E), IL-β (E), and IL-6 (E). (H) Flow cytometry data of Conrol, CHB-Gel, CC-CDs, and CHB@CC;

[0036] Figure 5 (A) Timeline of animal experiments on bacterial keratitis; (B) Optical and sodium fluorescein-stained corneal images of the Conrol, CHB-Gel, CC-CDs, and CHB@CC groups at 1, 4, 7, and 28 days. Corneal epithelialization rate (C) and stromal thickness (D) at different time periods; (E) OCT images of the corneas of the Conrol, CHB-Gel, CC-CDs, and CHB@CC groups at 1, 4, 7, and 28 days. DETAILED DESCRIPTION

[0037] Example 1 Preparation of the Antibacterial and Anti-inflammatory Bio-based Carbon Dot-Photocrosslinked In-situ Adhesion Hydrogel of the Present Invention

[0038] a. Preparation of Bio-based Carbon Dots

[0039] First, 30 mg of curcumin and 60 mg of carboxymethyl chitosan (CS) were weighed and placed in a beaker containing 40 ml of deionized water. The mixture was rapidly stirred for 30 minutes until uniformly dispersed and a dark yellow suspension formed. The mixture was then transferred to a polytetrafluoroethylene-lined reactor and placed in an oven at 180°C for 8 hours.

[0040] After the reaction is complete, cool to room temperature. Centrifuge the reaction solution at 8000 rpm for 10 minutes to remove insoluble precipitates. The supernatant is then placed in a 500-Dalton dialysis bag and dialyzed for three days, changing the water three times daily. The dialyzed solution is transferred to a Petri dish and freeze-dried to obtain a black powder, which is CC-CDs.

[0041] b. Preparation of gel:

[0042] Preparation of double-bonded collagen (MA-Col): Dissolve type I collagen in 10 ml of 0.1 M acetic acid solution to a concentration of approximately 1% (w / v). Add 0.5 ml of methacrylic acid under magnetic stirring, adjust the pH to approximately 7.4 with NaOH, and allow to react for 4 hours. Transfer the solution to an 8,000-14,000 Da dialysis bag to remove unreacted small molecules, then lyophilize and store in the dark.

[0043] Preparation of o-Nitrobenzene-Functionalized Hyaluronic Acid (HA-NB): Dissolve 0.408g of hyaluronic acid and 0.224g of amino-o-nitrobenzene in 50ml of deionized water, then add 0.153g of HOBt. Adjust the pH of the mixture to 4.5, add 0.2g of EDC hydrochloride, and allow to react at room temperature for 48 hours. After the reaction is complete, transfer the solution to an 8000-14000Da dialysis bag to remove unreacted small molecules, then lyophilize and store in the dark.

[0044] Preparation of the composite hydrogel (CHB@CC): First, a 0.05% photoinitiator solution was prepared using 0.1M acetic acid. This initiator solution was then used to dissolve MA-Col and HA-NB, maintaining a final concentration of 1% for MA-Col and 0.5%, 1%, and 2% for HA-NB, respectively. This solution was mixed with 100 μg / ml of CC-CDs, the pH was adjusted to 7, and the mixture was incubated at 37°C for 30 minutes. The mixture was then irradiated with UV light for 60 seconds to achieve chemical crosslinking, resulting in the CHB@CC composite hydrogel.

[0045] The beneficial effects of the present invention are demonstrated by the following pharmacodynamic tests.

[0046] Experimental Example 1. Characterization of Antioxidant and Enzyme Activities of CC-CDs

[0047] Curcumin (Cur) and carboxymethyl chitosan (CS) were reacted at 180℃ for 8 hours to synthesize CC-CDs ( Figure 1 A). Figure 1 B depicts the TEM and HRTEM images of the obtained CC-CDs, indicating their good monodispersity and uniform size, with an average size of 2.9 ± 0.9 nm ( Figure 1 C). Figure 1 The inset in B shows that CC-CDs have a graphene-like layered structure with a lattice spacing of 0.21 nm. Next, the optical properties of CC-CDs were further studied. The absorption spectrum of CC-CDs shows a wide absorption range from 350 nm to 800 nm, with a clear peak at 416 nm ( Figure 1 D). In addition, the prepared CC-CDs have strong fluorescence properties under the excitation wavelength of 366nm, and the optimal emission wavelength is 450nm ( Figure 1 E).

[0048] Curcumin is a natural polyphenol that has been widely studied as an antioxidant and anti-inflammatory molecule due to its excellent biocompatibility. However, curcumin has low bioavailability due to its strong hydrophobicity and rapid degradation and elimination. After converting curcumin into carbon dots, its water solubility is expected to be enhanced while retaining its antioxidant properties. Therefore, a series of experiments were designed to investigate the antioxidant properties of these carbon dots. It is well known that reactive oxygen and nitrogen species (RONS) mainly include nitric oxide (NO-), hydroxyl radicals (-OH) and hydrogen peroxide (H2O2). As a representative of nitrogen-centered free radicals, DPPH was first used to test the efficiency of CC-CDs in scavenging free radicals. As Figure 1 As shown in Figure F, when DPPH comes into contact with CC-CDs, the characteristic absorption peak at 515 nm decreases significantly due to the scavenging effect of nitrogen-centered free radicals. It is worth noting that at a concentration of 100 μg / ml, the scavenging efficiency is as high as 98%. At the same time, the ability of CC-CDs to scavenge -OH was studied using 3,3',5,5'-tetramethylbenzidine (TMB) as an indicator. Using the classic Fe 2+ The Fenton reaction of H2O2 generates -OH in situ in aqueous solution. When hydroxyl radical (-OH) exists, it can react with TMB to generate oxidized TMB (oxTMB), which shows characteristic absorption peaks at 370 nm and 652 nm. Figure 1 As shown in Figure G, after the addition of CC-CDs, the TMB+-OH complex showed no characteristic absorption peaks at 370 nm and 652 nm, indicating that CC-CDs have a strong scavenging ability for hydroxyl radicals. Hydrogen peroxide (H2O2) is also a major ROS in organisms. Therefore, the ability of CC-CDs to scavenge H2O2 was also studied using a H2O2 detection kit. After 20 minutes of incubation, CC-CDs could scavenge 90% of H2O2 (1.0 mM), demonstrating a high scavenging efficiency ( Figure 1 H). In addition, the total antioxidant capacity (TAC) of CC-CDs at different concentrations was evaluated, and the results were shown in Figure 2. Figure 1 As shown in Figure 1, the antioxidant activity of CC-CDs is concentration-dependent to some extent. Specifically, at a concentration of 100 μg / mL, the total antioxidant capacity of CC-CDs matched that of a standard solution containing 6 mM FeSO4, demonstrating its potent and broad-spectrum ROS scavenging ability. It is particularly noteworthy that carbon dots generally exhibit more efficient catalytic activity due to their large surface area and abundant active groups. To further investigate the source of the antioxidant properties of carbon dots, enzymatic activity assays were performed on CC-CDs. As a key antioxidant enzyme in cells against ROS, SOD catalyzes the oxidation of -O 2- Disproportionation to generate O2 and H2O2, thereby reducing oxidative stress ( Figure 1K). Therefore, SOD mimetics are used as potential therapeutic agents to combat inflammatory diseases caused by oxidative stress. Figure 1 J showed that the catalytic performance increased significantly with the increase of CC-CDs concentration. When the concentration was 100 μg / ml, its SOD-like activity reached 80%. CAT enzyme can convert hydrogen peroxide into water and oxygen and is also an important antioxidant enzyme ( Figure 1 M). According to the determination of CAT enzyme activity kit, CC-CDs also have strong CAT activity, and its activity is related to concentration to a certain extent ( Figure 1 L).

[0049] In conclusion, curcumin and chitosan were combined via a hydrothermal method to synthesize CC-CDs that not only possessed potent antioxidant activity but also SOD-like and CAT-like activities, which would play a great role in inflammation-related environments.

[0050] Experimental Example 2. Evaluation of the Antibacterial Performance and Bacterial-Cell Compatibility of Carbon Dots

[0051] Another possible benefit of synthesizing carbon dots using curcumin and chitosan as raw materials is their excellent antibacterial properties. To investigate whether CC-CDs possess universal antibacterial effects, Escherichia coli and Staphylococcus aureus were selected as model Gram-negative and Gram-positive bacteria, respectively, and the antibacterial activity of the carbon dots was evaluated through bacterial growth inhibition experiments.

[0052] like Figure 2 As shown in A, the growth of E. coli and S. aureus was affected by the dose of CC-CDs. As the concentration increased from 0 μg / mL to 10, 50, and 100 μg / mL, the bacterial viability gradually decreased. In addition, the morphological changes of E. coli and S. aureus after treatment with 100 μg / mL CC-CDs were observed using scanning electron microscopy. Figure 2 As shown in B, the bacteria in the untreated group maintained a smooth, undamaged membrane. In contrast, the cell walls and membranes of the bacteria treated with CC-CDs showed obvious wrinkles and damage. Zeta potential analysis further confirmed that both S. aureus and E. coli were negatively charged (-15±1.34mV and -20±1.4mV), while the carbon dots were significantly positively charged (-21±3.4mV). After 30 minutes of co-culture, the surface charge of the bacteria increased significantly, reaching (-4.8±1.15mV and -5.4±1.28mV) ( Figure 2 C). In addition, the bacterial nucleic acids (DNA and RNA) after incubation with CC-CDs were monitored. The results showed that after carbon dot treatment, E. coli and Staphylococcus aureus released a large amount of nucleic acids ( Figure 2D). The above results indicate that the positively charged CC-CDs have a high density of amine groups on their surface, which can selectively destroy bacterial cell walls through electrostatic interaction, leading to bacterial inactivation and the outflow of nucleic acids ( Figure 2 E).

[0053] At present, the design of antibacterial materials often only focuses on antibacterial properties, but bacteria often grow together with cells in the body, and how to balance antibacterial and cell activity has received less attention. In the present invention, human corneal epithelial cells (HCECs) and Staphylococcus aureus were labeled with CM-FDA probe (green) and CM-Dil probe (red), respectively. Subsequently, CM-FDA-labeled HCECs and CM-Dil-labeled Staphylococcus aureus were co-cultured with 100μg / ml CC-CDs. After 24 hours, gentamicin (60μ / ml) was used to eliminate extracellular bacteria. Figure 2 F and Figure 2 As shown in Figure G, compared to the blank group, most bacteria in the CC-CDs-treated group were in a dead state, with only a very small number of cells internalized and showing red fluorescence. Notably, no significant decrease in cell number was detected during this process, indicating that CC-CDs have excellent antibacterial and cell survival-promoting abilities.

[0054] Test Example 3. Characterization of burst pressure, adhesion, and other physical properties of CHB@CC

[0055] It is worth noting that the adhesive used in the present invention is based on an imine cross-linking (PIC) reaction triggered by strong light. Specifically, under light of 365 nm wavelength, the proto-nitrosobenzyl functional groups in the HA-NB precursor undergo photochemical conversion to proto-nitrosobenzaldehyde groups. These active aldehyde groups rapidly undergo imine cross-linking reactions with amines in the prepolymer solution or on the tissue surface, thereby producing strong direct adhesion and endowing the tissue with the potential for sealing. At the same time, the effect of the adhesive component (i.e., HA-NB) on the mechanical properties and optical transparency of the hydrogel was first explored. Figure 3 As shown in Figure A, with increasing angular frequency, all samples exhibited gel-like behavior characterized by a storage modulus (G') exceeding the loss modulus (G"). As the HA-NB content increased, the G' and G" values ​​of the CHB hydrogels gradually increased, indicating that the addition of HA-NB helped improve the mechanical toughness and viscoelastic behavior of the hydrogel structure. Tensile measurements showed that the ultimate stretch and elongation of CHB were closely related to the HA-NB content ( Figure 3B-3D). A reasonable explanation is that under ultraviolet irradiation, some of the generated benzaldehyde groups form dynamic covalent bonds with the amino groups in collagen, thereby enhancing the mechanical stability (from 38±3KPa to 76±4KPa) and resilience (from 20±0.6% to 62±5%) of the hydrogel network. In addition, although the addition of HA-NB gave the CHB hydrogel a slight yellow color, according to the transmittance analysis, it had little effect on its transmittance in the visible spectrum. In contrast, a more obvious effect was observed in the ultraviolet (UV) range ( Figure 3 E-3H). When the HA-NB concentration was 1%, the transmittance of the CHB hydrogel at a wavelength of 550 nanometers was 91 ± 2%, while the transmittance at a wavelength of 365 nanometers was only 42 ± 2.6 nanometers. These experiments demonstrate that the addition of HA-NB can enhance the UV resistance of the hydrogel matrix without affecting its overall optical clarity in the visible light region, which is particularly beneficial for the development of corneal substitutes.

[0056] Importantly, hydrogels used for sutureless repair of severe fistulas need to have sufficient adhesion to adjacent tissues to withstand high intraocular pressure and avoid detachment from the eyeball. Here, key properties of effective bioadhesion, including shear adhesion strength and burst pressure, were investigated. The adhesion properties of the hydrogels were first tested using the lap shear method ( Figure 3 I). Figure 3 The illustration in Figure 1 shows that the hydrogel formed in situ on pig skin tissue can withstand water flow impact, bending and twisting. Figure 3 As can be seen from Figure 1, the shear adhesion strength of pure MA-Col@CC hydrogel is relatively low (18±1.73kPa). With the increase of HA-NB concentration and the introduction of aldehyde groups, the shear adhesion strength of the hydrogel is greatly improved. CHB@CC-2% has the best shear adhesion strength (53.2±1.4kPa). Figure 3 As shown in J, a simple device was constructed to test the burst pressure. Figure 3 It can be clearly observed in Figure 1 that the burst pressure of the hydrogel gradually increased with increasing HA-NB concentration. When the HA-NB concentration reached 2%, the adhesion strength reached 236±30 mmHg. This quantitative data emphasizes the robust adhesion properties of the hydrogel at this concentration, indicating its potential for effective tissue fixation in clinical applications. Interestingly, there was no significant difference in the burst pressure between the CHB@CC-1% group and the CHB@CC-2% group, both of which were sufficient for sealing corneal defects. These results indicate that the addition of HA-NB can improve the mechanics, adhesion strength, and burst pressure of the hydrogel. Considering the effects on mechanics and transmittance, the hydrogel containing 1% HA-NB was selected for the next experiment.

[0057] The expansion rate is a key indicator for judging the quality of tissue adhesives. A lower expansion rate is beneficial for tissue adhesion. In terms of corneal transplantation applications, adhesives with low expansion rates help maintain perfect alignment between the graft and the recipient cornea, thereby reducing postoperative corneal astigmatism. The results of the study showed that the expansion rate of CHB hydrogel was low (about 28%). After the addition of CC-CDs, the expansion rate was even lower (about 23%), which may be because the amino groups on the surface of CC-CDs formed new bonds with the aldehyde groups on the polymer chain, increasing the density of the hydrogel network ( Figure 3 K). In addition, as an excellent corneal substitute, its excellent degradation resistance can match the corneal regeneration rate and prevent premature corneal shedding. Here, the present invention tested the enzymatic degradation of CHB and CHB@CC hydrogels under the action of collagenase. Figure 3 As shown in Figure 1, the introduction of CC-CDs improved the hydrogel's resistance to enzymatic degradation, a trend mirroring the change in swelling rate. After 24 hours, the CHB@CC hydrogel degraded by only 40%, while the CHB hydrogel degraded by 60%. This trend is readily explained by the increased degree of cross-linking. As a functional component, the slow release of CC-CDs is crucial for repairing keratitis damage. Figure 3 The release curves in Figure 2 show that CHB@CC hydrogel can achieve slow release of CC-CDs, with only 40% released after 20 h.

[0058] In summary, the designed hydrogel not only exhibits excellent mechanical, optical, and adhesive properties, but also exhibits low swelling and degradation resistance, and can achieve slow release of carbon dots when loaded. The integration of these properties is expected to develop a class of corneal substitutes with universal applicability for repairing severe keratitis.

[0059] Experimental Example 4. In vitro cytocompatibility, antioxidant and anti-inflammatory properties of CHB@CC

[0060] Good biocompatibility is the basis for the in vivo application of CHB@CC hydrogel. In order to evaluate the biocompatibility of CHB@CC hydrogel, live / dead staining and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) detection were used. Figure 4 As shown in A, strong green fluorescence and weak red fluorescence were observed in human corneal epithelial cells (HCECs), which confirmed that the prepared hydrogel had good biocompatibility. Figure 4The quantitative data in B showed that the survival rate of cultured cells in the hydrogel group was greater than 95% compared with the control group, with no obvious cytotoxicity. It is worth noting that the survival rate of the CC group alone was 80±5%, which suggests that the one-time addition of carbon dots may be toxic. Cytoskeleton staining showed that the morphology of the CHB@CC hydrogel was more dispersed, indicating that it can maintain the cell structure and morphology and promote the migration and proliferation of corneal epithelial cells. Then, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect the ROS scavenging effect of different coatings using H2O2-induced intracellular oxidative stress in HCECs. Figure 4 As shown in Figure C, the control group and CHB-Gel group showed clear green fluorescence, indicating that HCECs produced ROS in the cells under the stimulation of H2O2. Compared with the CC-CDs group, the CHB@CC group showed almost no green fluorescence, indicating that the CDs slowly released from the hydrogel effectively alleviated the oxidative stress in the cells.

[0061] After bacterial infection, the immune system will produce inflammatory responses and oxidative stress. In order to simulate the anti-inflammatory and antioxidant effects of hydrogels in vitro, RAW 264.7 cells were selected as a cell model, and lipopolysaccharide (LPS) was stimulated to produce an inflammatory response. After immunofluorescence staining, the expression of various inflammatory factors in the cells was observed, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). The results showed that the cells in the CHB hydrogel group showed obvious fluorescence intensity, indicating that the inflammatory factors IL-1β, IL-6, and TNF-α were expressed in large quantities. However, in the CC group and CHB@CC group, the green fluorescence was significantly reduced, indicating that the expression of IL-1β, IL-6, and TNF-α was highly reduced, which demonstrated the anti-inflammatory effect by eliminating oxidative stress ( Figure 4 D). In addition, the fluorescence quantitative data showed that the expression level of the CHB@CC group was significantly lower than that of the CC group, indicating that the sustained-release anti-inflammatory effect of the gel-loaded CD was the best ( Figure 4 E-4G). In addition, Figure 4 Flow cytometry analysis in Figure 3 showed that CHB@CC hydrogels significantly upregulated the expression of CD206 and downregulated the expression of CD86. This suggests that CD-loaded hydrogels can promote the repolarization of M1 macrophages to the M2 phenotype, thereby alleviating inflammation.

[0062] Experimental Example 5. In Vivo Evaluation of Corneal Wound Healing CHB@CC:

[0063] The primary goal of designing CHB@CC hydrogels is to use them as corneal substitutes to accelerate wound healing in patients with severe IK. To further understand the potential of CHB@CC hydrogels to promote corneal wound healing, the present invention conducted in vivo studies using a New Zealand rabbit lamellar keratectomy corneal wound infection model.

[0064] Figure 5 A shows the detailed procedures for lamellar keratectomy, bacterial infection, and treatment. Specifically, a partial keratectomy corneal defect (3.5 mm in diameter and 250 μm in depth) was created in the rabbit cornea. A Staphylococcus aureus bacterial suspension was then injected into the corneal defect, followed by treatment with CHB-gel, CC-CDs, and CHB@CC hydrogel. All procedures were approved by the Animal Research Committee.

[0065] Rabbits were examined by slit lamp using conventional light and fluorescein staining at intervals of 1, 4, 7, 14, and 28 days. Figure 5 The slit lamp images on day 1 in B show that there is almost no bacterial infection in the CC@CC and CHB@CC groups compared with the control group, verifying the in vivo antibacterial effect of carbon dots. It is obvious that the CHB-Gel and CHB@CC groups can stably fill and fit the contours of the corneal defect, demonstrating their strong tissue adhesion properties. In addition, Figure 5 As shown in Figures C and 5D, the wounds treated with CHB@CC hydrogels were completely re-epithelialized within 4 days, while the epithelial defects in the control group persisted due to severe IK even after 28 days, indicating that CHB@CC hydrogels can accelerate wound re-epithelialization. Although the CHB-gel group achieved the filling of the corneal defect, the epithelialization process may be affected due to residual bacteria. The CC-CDs group alone also inhibited the epithelialization process because the rapid clearance mechanism of the ocular surface affected the antibacterial effect. AS-OCT images collected on day 28 showed that all hydrogel-treated groups had dense stromal tissue and newly formed corneal epithelium compared with the control group ( Figure 5 E). After 28 days of treatment, the CHB@CC-treated corneas displayed optical reflectivity and structural characteristics similar to native corneas, while corneas from the other treatment groups showed significant scarring in the pupil area. In summary, the CHB@CC hydrogel demonstrated excellent antimicrobial efficacy, robust tissue adhesion, and superior corneal wound healing in vivo. CHB@CC hydrogels promote rapid corneal reepithelialization and restore corneal structure, suggesting potential as a therapeutic approach for severe infectious keratitis.

Claims

1. An antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesive hydrogel, characterized by: The bio-based carbon dots are prepared from curcumin and carboxymethyl chitosan, mixed with double-bonded collagen and o-nitrobenzene functionalized hyaluronic acid, and chemically cross-linked under ultraviolet light to prepare a hydrogel; wherein the carboxymethyl chitosan and curcumin are prepared into carbon dots by a one-pot method; the double-bonded collagen is methacrylate collagen.

2. The antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel according to claim 1, characterized in that: The weight ratio of curcumin, carboxymethyl chitosan, double-bonded collagen, and o-nitrobenzene functionalized hyaluronic acid is: 1-3 parts of curcumin, 1-3 parts of carboxymethyl chitosan, 1-4 parts of double-bonded collagen, and 1-4 parts of o-nitrobenzene functionalized hyaluronic acid.

3. The antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel according to claim 1, characterized in that: The weight ratio of curcumin, carboxymethyl chitosan, double-bonded collagen, and o-nitrobenzene functionalized hyaluronic acid is: 1 part of curcumin, 2 parts of carboxymethyl chitosan, 2 parts of double-bonded collagen, and 2 parts of o-nitrobenzene functionalized hyaluronic acid.

4. The antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method of the bio-based carbon dots is: Curcumin and carboxymethyl chitosan were weighed, placed in a beaker, and stirred rapidly until evenly dispersed to form a dark yellow suspension; the mixture was transferred to a reactor lined with polytetrafluoroethylene and placed in an oven at 180°C for 8 hours; after the reaction, the mixture was cooled to room temperature; the reaction solution was centrifuged at 8000 r / min for 10 minutes to remove insoluble precipitates, and the supernatant was placed in a 500 Dalton dialysis bag and dialyzed for 3 days, with the water changed every day; the dialyzed liquid was transferred to a culture dish for freeze-drying, and the black powder obtained was the bio-based carbon dots CC-CDs.

5. The antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method of the double-bonded collagen is as follows: Use acetic acid solution to dissolve type I collagen at a concentration of 1% w / v; add methacrylic acid under magnetic stirring, and use NaOH to adjust the pH value to 7.4, and react for 4 hours; transfer the solution into an 8000-14000 Da dialysis bag to remove unreacted small molecules, freeze-dry, and store in the dark.

6. The antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel according to any one of claims 1 to 3, characterized in that: The preparation method of the o-nitrobenzene functionalized hyaluronic acid is: Hyaluronic acid and amino-nitrobenzene were dissolved in deionized water, and 1-hydroxybenzotriazole (HOBt) was added. The pH of the mixed solution was adjusted to 4.5, and 0.2 g of EDC hydrochloride was added. The mixture was reacted at room temperature for 48 hours. After the reaction was completed, the solution was transferred to an 8000-14000 Da dialysis bag to remove unreacted small molecules, and then freeze-dried and stored in the dark.

7. The method for preparing the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in-situ adhesion hydrogel according to any one of claims 1 to 6, characterized in that: It includes the following steps: a. Prepare a 0.05% photoinitiator solution with 0.1 M acetic acid; b. Dissolve the double-bonded collagen and o-nitrobenzene-functionalized hyaluronic acid using the initiator solution, maintaining the final concentration of the double-bonded collagen at 1%, and the final concentrations of the o-nitrobenzene-functionalized hyaluronic acid at 0.5%, 1%, and 2%, respectively; c. The above solution was mixed with 100 μg / ml of bio-based carbon dots, the pH was adjusted to 7, and the mixture was incubated at 37°C for 30 min. The mixture was then irradiated with UV light for 60 s to achieve chemical cross-linking. Finally, the antibacterial and anti-inflammatory bio-based carbon dots-photocross-linked in situ adhered hydrogel CHB@CC was obtained.

8. Use of the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in situ adhesion hydrogel according to any one of claims 1 to 6 in the preparation of a topical medicament for treating infectious keratitis.

9. The use according to claim 8, characterized in that: The medicine is an external medicine for eyes.

10. Use of the antibacterial and anti-inflammatory bio-based carbon dot-photocrosslinked in situ adhesion hydrogel according to any one of claims 1 to 6 in the preparation of a corneal substitute.

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