Injectable self-healing hydrogel for treating radiation-induced oral ulceration as well as preparation method and application of injectable self-healing hydrogel

Abstract

The invention relates to the field of biomedical materials, and discloses injectable self-healing hydrogel for treating radiation-induced oral ulceration as well as a preparation method and application of the injectable self-healing hydrogel. The hydrogel provided by the invention is OCC / CDots / EGCG composite hydrogel, the OCC basic hydrogel is used as a carrier, and Vc-PEI-CDots and EGCG are loaded through a process. The composite hydrogel has an injectable characteristic, has strong antibacterial and in-vitro oxidation resistance, and has a remarkable healing promoting function. Animal experiments show that the hydrogel can effectively promote RIOM wound healing, the expression is epithelial continuous recovery, cell apoptosis reduction, and up-regulation of expression of migration promoting protein VIM and key antioxidant enzyme GPX3 in tissues, and a novel and efficient biomedical material selection is provided for treatment of clinical radiation-induced oral ulcerative mucositis.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an injectable self-healing composite hydrogel for treating radiation-induced oral ulcers, its preparation method, and its application in the preparation of wound dressings. Background Technology

[0002] Radiation-induced oral mucositis (RIOM) is one of the most common and distressing complications following radiotherapy in patients with head and neck tumors. Its pathological process is complex, involving multiple factors such as inflammatory response, oxidative stress, bacterial infection, and impaired tissue regeneration. Persistent accumulation of reactive oxygen species (ROS) damages cellular function, hinders macrophage polarization towards the reparative M2 phenotype, and inhibits epithelial cell migration, leading to prolonged wound healing. Currently, there is no specific treatment for this condition; conventional methods such as mouthwashes, analgesics, and nutritional support only provide symptomatic relief and cannot actively regulate the wound microenvironment or promote functional tissue regeneration.

[0003] Guiding tissue regeneration and creating a moist healing environment are effective strategies for promoting wound repair. Hydrogels, due to their high water content and three-dimensional network structure, are considered ideal wound dressings. However, traditional hydrogels have limited functionality and cannot simultaneously meet the multiple needs in the treatment of radiation-induced oral ulcers, including antibacterial, antioxidant, anti-inflammatory, cell migration-promoting, and adaptation to the complex anatomy of the oral cavity (such as injectability, self-healing, and strong adhesion).

[0004] Konjac glucomannan (KGM) is a natural polysaccharide whose mannose units can be recognized by macrophage mannose receptors, exhibiting immunomodulatory potential. Oxidized KGM (OKGM) can be infused with aldehyde groups, facilitating the construction of composite hydrogels via dynamic Schiff base reactions. Carbon dots (CDs) are a class of carbon-based nanomaterials with particle sizes less than 10 nm, possessing advantages such as good biocompatibility, ease of modification, and fluorescence properties. Studies have shown that specific carbon dots exhibit potential exceeding that of traditional materials in the biomedical field. For example, carbon dots (Vc-PEI-CDots) synthesized from ascorbic acid and polyethyleneimine (PEI) via a microwave-assisted method are rich in carboxyl and amine functional groups and carry a positive charge. They can not only achieve broad-spectrum antibacterial effects by disrupting bacterial cell membranes through electrostatic interactions, but more importantly, they have been shown to activate the transforming growth factor-β (TGF-β) / p38 mitogen-activated protein kinase (p38 MAPK) / Snail signaling pathway, thereby effectively inducing epithelial-mesenchymal transition (EMT) in epithelial cells. EMT is a key step in wound healing involving epithelial migration and reepithelialization, characterized by significantly enhanced cell migration capacity. This provides a novel approach for actively promoting epithelial coverage of RIOM using carbon dots.

[0005] However, radiation-induced persistent oxidative stress is one of the core pathological factors leading to the persistent nature of oral ulcers. Excessive ROS induced by radiation damages intracellular macromolecules, induces mitochondrial dysfunction, and maintains a pro-inflammatory microenvironment, directly hindering the normal healing process. Therefore, effectively scavenging ROS and alleviating oxidative stress are key to treating RIOM (Related Inflammatory Occurrence of Occurrence). Epigallocatechin gallate (EGCG), as the most effective active ingredient in tea polyphenols, is a recognized potent natural antioxidant. Its phenolic hydroxyl group can efficiently neutralize free radicals and interrupt free radical chain reactions by providing hydrogen atoms. Studies have shown that EGCG can not only directly scavenge various free radicals such as DPPH and ABTS, but also enhance the overall antioxidant capacity of cells by upregulating endogenous cellular antioxidant defense systems (such as the glutathione metabolic pathway).

[0006] Therefore, combining carbon dots with migration-promoting functions with EGCG, which has excellent antioxidant capabilities, and integrating them into a hydrogel delivery system with injectable, self-healing, and tissue adhesion properties can achieve functional complementarity and synergistic effects. This design can simultaneously intervene in the complex pathological process of RIOM at multiple key levels, including broad-spectrum antibacterial activity, efficient ROS removal, regulation of the wound microenvironment, and provision of a physical barrier, thus promising to achieve efficient healing. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide an injectable self-healing hydrogel for treating radiation-induced oral ulcers. This hydrogel integrates excellent injectability, self-healing properties, broad-spectrum antibacterial activity, high-efficiency antioxidant activity, and significant cell migration-promoting ability, effectively addressing the complex pathological microenvironment of radiation-induced oral ulcers and accelerating wound healing.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The present invention provides a method for preparing an injectable self-healing composite hydrogel in a first aspect, comprising the following steps: Step 1, Preparation of Vc-PEI-CDots Ascorbic acid and polyethyleneimine were dissolved in deionized water and stirred until completely dissolved. The mixture was then heated until the solution turned dark brown. After cooling, the solution was centrifuged, purified, and concentrated for later use. Step 2, Preparation of OKGM Konjac glucomannan was dissolved in deionized water, sodium periodate was added, and the mixture was stirred in the dark to react. After the reaction was completed, ethylene glycol was added to quench the reaction. Stirring was continued, and the mixture was dialyzed and freeze-dried to obtain OKGM. Step 3, Preparation of OCC-based hydrogel The OKGM, carboxymethyl chitosan, and carboxymethyl cellulose obtained in step 2 are mixed to obtain the OCC-based hydrogel; Step 4, Loading CDots and EGCG Under light-protected conditions, the Vc-PEI-CDots solution and EGCG solution obtained in step 1 were added to the OCC basic hydrogel obtained in step 3, respectively, and mixed thoroughly. After static crosslinking, the injectable self-healing hydrogel OCC / CDots / EGCG was obtained.

[0010] Furthermore, in step 1, the ratio of ascorbic acid, polyethyleneimine, and deionized water is 1g:0.5g:20mL.

[0011] Furthermore, in step 1, the heating reaction refers to heating at 500W power for 4 minutes.

[0012] Furthermore, in step 1, centrifugation refers to centrifugation at 8000 rpm for 5 minutes.

[0013] Furthermore, in step 2, the ratio of konjac glucomannan, deionized water, sodium periodate, and ethylene glycol is 1g:100mL:0.7g:2mL.

[0014] Furthermore, in step 3, 3% w / v OKGM, 8% w / v carboxymethyl chitosan and 5% w / v carboxymethyl cellulose are mixed in a volume ratio.

[0015] Furthermore, in step 4, the loading concentration of CDots is 400-600 μg / mL.

[0016] Furthermore, in step 4, the loading concentration of EGCG is 500 μg / mL.

[0017] In a second aspect, the present invention provides an injectable self-healing composite hydrogel, which is prepared by the aforementioned preparation method.

[0018] In a third aspect, the present invention provides the use of the injectable self-healing hydrogel in the preparation of wound dressings.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1) Excellent physicochemical properties: Through dynamic Schiff base bonds and hydrogen bonds, a stable three-dimensional porous network structure is constructed, which has good injectability and rapid self-healing ability.

[0020] 2) Strong antibacterial and in vitro antioxidant capabilities: The hydrogel exhibits broad-spectrum and highly effective antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Simultaneously, it demonstrates scavenging rates of 87.69% and 99.28% for DPPH and ABTS free radicals, respectively, effectively removing excess ROS from RIOM wounds and alleviating oxidative stress.

[0021] 3) Significant healing-promoting function: In vitro experiments confirmed that this hydrogel, at a concentration of 400 μg / mL, can effectively induce EMT in human oral keratinocytes (HOK), significantly upregulate the expression of mesenchymal markers such as vimentin (VIM) and SNAI1 / 2, downregulate the expression of E-cadherin (E-Cad), and greatly enhance cell migration ability. CDots and EGCG showed a synergistic enhancing effect in promoting migration and in vivo antioxidant activity.

[0022] 4) Clear in vivo efficacy: Animal experiments have shown that this hydrogel can effectively promote the healing of RIOM wounds, manifested by the restoration of epithelial continuity, reduction of cell apoptosis, and upregulation of the expression of tissue-promoting migration protein VIM and key antioxidant enzyme GPX3.

[0023] 5) Good biocompatibility: CCK-8, apoptosis, colony formation and hemolysis experiments all proved that the hydrogel has good cell compatibility and blood compatibility, meeting the safety requirements of biomedical materials. Attached Figure Description

[0024] Figure 1 This diagram illustrates the synthesis and physicochemical properties of the OCC / CDots / EGCG hydrogel. A shows the synthetic mechanism via synergistic cross-linking through Schiff bases and hydrogen bonds. B shows successful gelation demonstrated by an inverted vial experiment. C shows a SEM image of the hydrogel. D shows the FT-IR spectrum of the hydrogel. E shows the injectability demonstration of the hydrogel using a syringe. F shows the swelling kinetics of the hydrogel over 12 hours. G shows the in vitro degradation curve of the hydrogel over 14 days. H shows its adhesion properties on porcine skin tissue. I shows the hydrogel's adaptability to simulated joint movements. J shows a macroscopic self-healing image after reconnection of severed hydrogel fragments. K shows the strain-scan rheological results. L shows the frequency-scan results of the hydrogel. M shows the results of step-strain cycling experiments. All data are from at least three independent experiments (n≥3).

[0025] Figure 2 Evaluation of the antibacterial, antioxidant, and blood compatibility properties of the OCC / CDots / EGCG hydrogel. A shows bacterial colony images on agar plates after co-culturing with different hydrogels. B shows the effect of the hydrogel on Staphylococcus aureus (…). S. aureus ), Escherichia coli ( E. coli ) and Pseudomonas aeruginosa ( P. aeruginosaThe quantitative antibacterial rate of the hydrogel is shown in Figure 1. C represents the scavenging ability against ABTS and DPPH free radicals. D shows the results of the hydrogel hemolytic activity test. E is a graph showing the hemolytic rate determination results of the hydrogel. All data are from at least three independent experiments (n≥3) and are expressed as mean ± standard deviation.

[0026] Figure 3 Biocompatibility assessment of the OCC / CDots / EGCG hydrogel. A: HOK cells subjected to 5 Gy ionizing radiation to construct a irradiated cell model. B: Cell viability results of unirradiated HOK cells (left) and irradiated HOK cells (right) treated with different concentrations of OCC / CDots / EGCG, as determined by CCK-8 assay. C: Flow cytometry apoptosis analysis results of unirradiated and irradiated HOK cells treated with different concentrations of OCC / CDots / EGCG. D: Representative images from the colony formation experiment. E: Quantitative assessment of colony number. All data are from at least three independent experiments (n≥3) and are expressed as mean ± standard deviation.

[0027] Figure 4 To evaluate the migration-promoting effect of OCC / CDots / EGCG hydrogels, hydrogels loaded with CDots and EGCG showed the best migration-promoting effect at a concentration of 400 µg / mL. A represents EMT-related marker genes in unirradiated and irradiated HOK cells treated with different concentrations of OCC / CDots / EGCG. SNAI1 , SNAI2 , VIM , CDH1 , CDH2 The relative RNA expression levels of OCC / CDots / EGCG were shown in Figure 1. B represents Western blot analysis of E-cadherin (E-Cad) and vimentin (VIM) protein levels in unirradiated and irradiated HOK cells treated with different concentrations of OCC / CDots / EGCG. C shows representative images from the Transwell migration assay. D shows quantitative analysis of cell migration rate. E shows representative images from the scratch assay. F shows quantitative assessment of scratch closure rate. All data were from at least three independent experiments (n≥3) and are expressed as mean ± standard deviation.

[0028] Figure 5To evaluate the migration-promoting effect of OCC / CDots / EGCG hydrogels, the co-loaded hydrogels exhibited stronger migration-promoting capabilities compared to hydrogels loaded with CDots or EGCG alone. A shows the relative RNA expression levels of EMT-related marker genes in unirradiated and irradiated HOK cells treated with Control, OCC / CDots, OCC / EGCG, or OCC / CDots / EGCG. B shows E-Cad and VIM protein levels analyzed by Western blot. C shows representative images from the Transwell migration assay. D shows the quantitative analysis of cell migration rate. E shows representative images from the scratch assay. F shows the quantitative assessment of scratch closure rate. All data are from at least three independent experiments (n≥3) and are expressed as mean ± standard deviation.

[0029] Figure 6 To evaluate the antioxidant effect of the OCC / CDots / EGCG hydrogel, the OCC / CDots / EGCG hydrogel showed a significant antioxidant effect. A represents antioxidant-related genes in unirradiated and irradiated HOK cells treated with Control, OCC / CDots, or OCC / CDots / EGCG. SLC7A11 , GCLC , GCLM , ANPEP A) Relative RNA expression levels. B) Intracellular ROS levels assessed by flow cytometry using DCFH-DA staining. C) Top 10 KEGG pathway enrichment analysis of differentially expressed genes, with glutathione metabolism pathway (marked) showing significant enrichment. D) Expression heatmap of glutathione metabolism between the OCC / CDots / EGCG group and the control group in irradiated HOK cells; glutathione peroxidase 3 (GPX3) was marked as a representative upregulated gene. E) Western blot analysis of GPX3 protein expression. All data were from at least three independent experiments (n≥3) and are presented as mean ± standard deviation.

[0030] Figure 7 This study evaluates the therapeutic effect of OCC / EGCG / CDots hydrogel on a mouse RIOM model. A shows a schematic diagram of the establishment of the RIOM mouse model and the administration of OCC / EGCG / CDots. B shows a representative photograph of the mouse tongue stained with toluidine blue, indicating the degree of mucosal lesions. C shows a representative histological image of the tongue tissue after H&E staining. D shows the quantitative analysis of tongue epidermal thickness based on H&E staining images. E shows representative immunohistochemical (IHC) staining images of GPX3 and VIM tongue tissue. F shows the quantitative analysis of AOD values ​​from immunohistochemical staining. All data are from at least three independent experiments (n≥3) and are expressed as mean ± standard deviation. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the following embodiments.

[0032] Example 1: Preparation of OCC / CDots / EGCG hydrogel Synthesis and purification of Vc-PEI-CDots: 1.0 g ascorbic acid (Vc) and 0.5 g polyethyleneimine (PEI) were dissolved in 20 mL of deionized water and magnetically stirred until completely dissolved. The solution was placed in a microwave reactor and heated at 500 W for 4 minutes, until the solution changed from colorless to dark brown, yielding the crude CDots product. After cooling to room temperature, the product solution was centrifuged at 8000 rpm for 5 minutes to remove aggregated particles. The supernatant was collected and purified by dialyzing in deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da. Finally, the solution was concentrated using a vacuum rotary evaporator and redissolved in deionized water for later use.

[0033] Preparation of OKGM: 1 g of KGM was dissolved in 100 mL of deionized water and mechanically stirred until fully swollen. 0.7 g of sodium periodate (NaIO4) was added to the solution, and the reaction mixture was stirred continuously for 5 hours in the dark. Then, 2 mL of ethylene glycol was added to quench the reaction, and stirring was continued for 2 hours. The reaction mixture was transferred to a dialysis bag and dialyzed for 72 hours to remove small molecule impurities. After dialysis, the solution was centrifuged, and the supernatant was freeze-dried to obtain OKGM powder, which was stored at 4°C.

[0034] Construction of composite hydrogels: OKGM (3% w / v), carboxymethyl chitosan (CMCS, 8% w / v), and carboxymethyl cellulose (CMC, 5% w / v) solutions were mixed at a certain volume ratio to form an OCC-based hydrogel. Under light-protected conditions, the Vc-PEI-CDots solution and EGCG solution prepared in step 1 were added to the OCC-based hydrogel, vortexed until homogeneous, and allowed to stand at room temperature for crosslinking, thus obtaining OCC / CDots, OCC / EGCG, and OCC / CDots / EGCG composite hydrogels. The preferred loading concentration of CDots was 400-600 μg / mL, and the preferred loading concentration of EGCG was 500 μg / mL.

[0035] Example 2: Physicochemical property characterization of hydrogels (1) Experimental methods Microstructure: The microstructure of the lyophilized hydrogel samples was observed using scanning electron microscopy (SEM, TESCAN MIRA LMS). All samples were sputter-coated with gold to improve conductivity before observation.

[0036] Chemical structure: Fourier transform infrared spectroscopy (FT-IR, Thermo Fisher Scientific Nicolet iN10) was used to determine the structure at 4000-400 cm⁻¹. -1 The sample is scanned within a wavelength range to analyze changes in its functional groups and chemical bonds.

[0037] Rheological properties: Tested using a rheometer (Haake Mars40). The linear viscoelastic region of the material was determined by strain scanning (strain range 0.1%-150%, fixed frequency 1 Hz); the frequency dependence of the modulus was evaluated by frequency scanning (frequency range 0.1-150 rad / s, fixed strain 0.1%); and the self-healing properties of the hydrogel were evaluated by step strain cycling test (alternating between 1% small strain and 150% large strain, each strain lasting 100 seconds).

[0038] Swelling properties: Weigh the initial mass of the lyophilized hydrogel sample, immerse it in excess artificial saliva, and incubate with shaking at 37°C. Remove the sample at a predetermined time point, blot the surface liquid with filter paper, weigh it, and calculate the swelling ratio of the hydrogel.

[0039] In vitro degradation performance: Weigh the initial mass of the lyophilized hydrogel sample and immerse it in artificial saliva containing salivary amylase. Incubate for 16 days at 37°C and 100 rpm in a shaker. Replace with fresh artificial saliva every 2 days. Remove the sample at the predetermined time point, rinse with PBS, lyophilize, and weigh.

[0040] Adhesion and self-healing properties: The hydrogel was adhered to the surface of pigskin or human joint models, and its adhesion stability was qualitatively observed by performing actions such as flipping and bending. Macroscopic experiments: The hydrogel was cut into two segments, with the cut surfaces in close contact. After standing at room temperature, it was observed whether they re-fused into one piece to qualitatively evaluate the self-healing ability.

[0041] (2) Experimental results Figure 1A schematic diagram of the synthesis and physicochemical characterization of the OCC / CDots / EGCG hydrogel are presented. As shown in the figure, the hydrogel achieves stable linkages through synergistic crosslinking of Schiff base bonds and hydrogen bonds. SEM images reveal a stable three-dimensional porous network structure with uniform pore size distribution. Furthermore, FT-IR spectroscopy analysis shows a redshift of the C=N characteristic peak and a significant enhancement of the CO stretching vibration peak, confirming that the introduction of EGCG and CDots enhances dynamic covalent crosslinking and hydrogen bond interactions. Rheological tests show that the hydrogel exhibits typical solid-like elastic behavior, with a storage modulus higher than the loss modulus and rapid modulus recovery, demonstrating excellent self-healing properties and structural stability. Swelling experiments show a moderate swelling rate, and in vitro degradation curves show that the material can be controlled to degrade to 13.96% of its initial mass within 14 days, meeting the requirements of wound dressings for maintaining a moist environment and timely degradation. Adhesion tests confirmed that the hydrogel exhibits good adhesion to tissue surfaces under moist conditions and can adapt to dynamic mechanical deformation. This is mainly attributed to the multiple non-covalent interactions between the catechol groups in EGCG and the proteins / polysaccharides on the tissue surface. In summary, this hydrogel possesses suitable injectability, a stable three-dimensional network, controllable swelling and degradation characteristics, good tissue adhesion, and self-healing capabilities, providing a solid physicochemical basis for its dressing application in the dynamically moist oral environment.

[0042] Example 3: Biological performance testing of hydrogels (1) Experimental methods ① Antibacterial performance test: Selected S. aureus , E. coli and P. aeruginosa All test strains were derived from Beijing Baocang Biotechnology Co., Ltd., China. The sterilized hydrogel (100 mg) was mixed with bacterial suspension (50 μL, 10... 6 The co-culture solution (CFU / mL) was co-cultured in PBS for 24 hours. Subsequently, the co-culture solution was serially diluted, spread on LB agar plates, and colony forming units (CFU) were counted after incubation.

[0043] ② Antioxidant performance test: DPPH free radical scavenging: The hydrogel was added to the DPPH working solution (absorbance 0.7±0.02), and the reaction was carried out in the dark for 30 minutes. The absorbance at 517 nm was measured. The calculation formula is as follows:

[0044] Where A1 and A2 are the absorbances of the DPPH working solution before and after the reaction, respectively.

[0045] ABTS radical scavenging: The method is the same as the DPPH method, measuring the change in absorbance at 734 nm and calculating the scavenging rate.

[0046] B1 and B2 are the absorbances of the ABTS working solution before and after the reaction, respectively.

[0047] ③ Blood compatibility test: Human blood was collected and centrifuged to obtain red blood cells, which were then resuspended in PBS. A sterile hydrogel was incubated with the red blood cell suspension for 2 hours, with PBS and deionized water used as negative and positive controls, respectively. After centrifugation, the supernatant was collected, and the absorbance was measured at 545 nm. The hemolysis rate was calculated using the following formula:

[0048] Where A in the formula X A represents the absorbance of the sample group. N Absorbance of the negative group, A P The absorbance is for the positive group.

[0049] ④ Cell compatibility test: Cell proliferation (CCK-8): HOK cells (including the unirradiated group and the model group irradiated with 5 Gy X-rays) were seeded in 96-well plates and co-cultured with hydrogel extracts of different concentrations for 1, 3, and 7 days. CCK-8 reagent was added at each time point, and the absorbance at 450 nm was measured after incubation.

[0050] Apoptosis: HOK cells were co-cultured with hydrogels of different concentrations for 48 hours, and then stained with Annexin V-FITC / PI apoptosis detection kit. The apoptosis rate was detected by flow cytometry.

[0051] Colony formation: HOK cells were seeded at low density in 12-well plates and co-cultured with hydrogels of different concentrations for 14 days. After fixation, crystal violet staining was used to count the cell colonies formed.

[0052] ⑤ Assessment of migration-promoting ability and EMT-induced effect: Scratch assay: Scratches were created in 6-well plates filled with cells, the medium was replaced with serum-free medium, and hydrogel was added. Photos were taken at 0 hours and 24 hours, and the scratch closure rate was calculated.

[0053] Transwell migration assay: Hydrogel-treated cells were seeded in the upper chamber of a Transwell chamber, while the lower chamber was filled with culture medium containing 10% FBS. After 24 hours of culture, cells that had crossed the membrane were fixed and stained, and then counted and statistically analyzed.

[0054] EMT-related gene expression (qRT-PCR): Total RNA was extracted from cells treated with hydrogels, reverse transcribed into cDNA, and the mRNA expression levels of genes such as SNAI1, SNAI2, VIM, CDH1, and CDH2 were detected using the SYBR Green assay.

[0055] EMT-related protein expression (Western Blot): Total cellular protein was extracted, transferred to a membrane, blocked by SDS-PAGE electrophoresis, and then incubated with primary antibodies such as E-cadherin and Vimentin, as well as corresponding HRP-labeled secondary antibodies. Finally, protein bands were detected by ECL chemiluminescence method.

[0056] ⑥ Investigation into the antioxidant mechanism: Intracellular ROS level detection: The ROS level in HOK cells treated with hydrogel was detected by flow cytometry after co-incubation with the DCFH-DA fluorescent probe.

[0057] RNA sequencing (RNA-seq) and KEGG pathway analysis: Total RNA was extracted from irradiated HOK cells treated with OCC / CDots / EGCG and subjected to transcriptome sequencing. Differentially expressed genes were then subjected to KEGG pathway enrichment analysis.

[0058] Key protein validation (Western Blot): The expression level of GPX3, a key antioxidant protein screened in RNA-seq, was validated using the Western Blot method.

[0059] ⑦ In vivo animal experiments: Establishment of a radiation-induced oral ulcer model: Six-week-old female BALB / c mice were irradiated with 15 Gy X-rays to induce radiation-induced oral ulcers.

[0060] Hydrogel therapy: Starting on the 5th day after irradiation, OCC / CDots / EGCG hydrogel was injected into the oral ulcers of mice. The dressing was changed every morning and evening for 14 days.

[0061] Efficacy assessment: Mice were sacrificed on day 14 after irradiation, and tongue tissue was collected. Toluidine blue staining was used to initially observe the damage; hematoxylin and eosin (H&E) staining was used to observe tissue morphology, epithelial continuity, and inflammatory infiltration; TUNEL staining was used to detect cell apoptosis; and immunohistochemical (IHC) staining was used to detect the expression and localization of VIM and GPX3 proteins in the tissue.

[0062] (2) Experimental results Results of antibacterial properties, antioxidant properties, and blood compatibility tests are as follows: Figure 2 As shown. By Figure 2 It can be seen that this hydrogel is effective against... S. aureus , E. coli and P. aeruginosaAll hydrogels exhibited broad-spectrum and highly effective antibacterial activity, with inhibition rates exceeding 90%, primarily due to the synergistic antibacterial effects of CMCS, Vc-PEI-CDots, and EGCG through multiple mechanisms. Regarding antioxidant properties, the hydrogels achieved scavenging rates of 87.69% and 99.28% for DPPH and ABTS free radicals, respectively, demonstrating excellent free radical scavenging capabilities. This activity mainly derives from the potent antioxidant effect of EGCG. Blood compatibility experiments further showed that the hemolysis rate of each hydrogel group was less than 2%, meeting the safety standards for biomedical materials and proving the material's good blood compatibility. In summary, the OCC / CDots / EGCG hydrogel combines highly effective antibacterial and antioxidant properties with good blood safety, providing an important biological performance basis for its application in radiation-induced oral ulcer wounds.

[0063] Cell compatibility test results as follows Figure 3 As shown. By Figure 3 It was found that, compared with the control group, different concentrations of OCC / CDots / EGCG hydrogel treatment groups did not exhibit significant cytotoxicity against normal and 5 Gy radiation-damaged HOK cells. By establishing a radiation-damaged cell model to simulate the pathological microenvironment, comprehensive evaluation showed that the hydrogel did not significantly inhibit cell viability within the concentration range of 0-1000 μg / mL; apoptosis analysis further confirmed that treatment at 0-600 μg / mL did not increase the apoptosis rate; and colony formation assays also indicated that this concentration range did not affect the long-term proliferation capacity of cells. These results collectively demonstrate that the OCC / CDots / EGCG hydrogel possesses good in vitro biocompatibility, providing a reliable safety basis for its subsequent applications.

[0064] Pro-migration effects such as Figure 4 , Figure 5 As shown. By Figure 4 It can be seen that the hydrogel loaded with CDots and EGCG exhibited the best migration-promoting effect at a concentration of 400 µg / mL; Figure 5 It can be seen that, compared with hydrogels loaded with CDots or EGCG alone, hydrogels co-loaded with carbon dots and EGCG exhibit a stronger migration-promoting effect.

[0065] Antioxidant test results as follows Figure 6 As shown. By Figure 6 It is known that the OCC / CDots / EGCG hydrogel has a significant in vivo antioxidant effect.

[0066] Evaluation of the therapeutic effect of OCC / EGCG / CDots hydrogel on a mouse RIOM model, as follows: Figure 7 As shown. By Figure 7The results showed that by establishing a mouse model of localized RIOM induced by 15 Gy X-ray irradiation and administering local hydrogel treatment for 14 days, the hydrogel significantly promoted ulcer healing. Macroscopically, toluidine blue staining showed that the ulcer area of ​​the tongue mucosa in the treatment group was significantly smaller than that in the irradiated control group. Histological observation further confirmed that the epithelial continuity in the treatment group was better, the progressive tissue damage caused by radiation was effectively alleviated, and the epidermal thickness was also better maintained. Immunohistochemical results showed that the expression levels of vimentin (VIM) and glutathione peroxidase 3 (GPX3) in the tongue tissue of the treatment group were significantly higher than those in the irradiated control group, suggesting that the hydrogel can not only promote cell migration and epithelial repair by inducing epithelial-mesenchymal transition in vivo, but also activate the endogenous antioxidant defense system to effectively scavenge radiation-induced reactive oxygen species. In summary, the OCC / CDots / EGCG hydrogel significantly inhibited the pathological progression of RIOM and promoted ulcer healing and mucosal function recovery in vivo by synergistically exerting its migration-promoting and antioxidant effects.

[0067] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing an injectable self-healing hydrogel, characterized in that, Includes the following steps: Step 1, Preparation of Vc-PEI-CDots Ascorbic acid and polyethyleneimine were dissolved in deionized water and stirred until completely dissolved. The mixture was then heated until the solution turned dark brown. After cooling, the solution was centrifuged, purified, and concentrated for later use. Step 2, Preparation of OKGM Konjac glucomannan was dissolved in deionized water, sodium periodate was added, and the mixture was stirred in the dark to react. After the reaction was completed, ethylene glycol was added to quench the reaction. Stirring was continued, and the mixture was dialyzed and freeze-dried to obtain OKGM. Step 3, Preparation of OCC-based hydrogel The OKGM and carboxymethyl chitosan obtained in step 2 are mixed with carboxymethyl cellulose to obtain the OCC-based hydrogel; Step 4, Loading CDots and EGCG Under light-protected conditions, the Vc-PEI-CDots solution and EGCG solution obtained in step 1 were added to the OCC basic hydrogel obtained in step 3, respectively, and mixed thoroughly. After static crosslinking, the injectable self-healing hydrogel OCC / CDots / EGCG was obtained.

2. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 1, the ratio of ascorbic acid, polyethyleneimine, and deionized water is 1g:0.5g:20mL.

3. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 1, the heating reaction refers to heating at 500W power for 4 minutes.

4. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 1, centrifugation refers to centrifugation at 8000 rpm for 5 minutes.

5. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 2, the ratio of konjac glucomannan, deionized water, sodium periodate, and ethylene glycol is 1g:100mL:0.7g:2mL.

6. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 3, 3% w / v OKGM, 8% w / v carboxymethyl chitosan and 5% w / v carboxymethyl cellulose are mixed in a volume ratio.

7. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 4, the loading concentration of CDots is 400-600 μg / mL.

8. The method for preparing the injectable self-healing hydrogel according to claim 1, characterized in that, In step 4, the loading concentration of EGCG is 500 μg / mL.

9. An injectable self-healing hydrogel obtained by the preparation method according to any one of claims 1-8.

10. The use of the injectable self-healing hydrogel of claim 9 in the preparation of wound dressings.

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