3D printing bimetallic cross-linked hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a 3D printing bimetallic cross-linked hydrogel dressing, which is characterized in that bimetallic cross-linked nanoparticles composed of curcumin, tannic acid, copper ions and iron ions are loaded into SFMA to form a wound dressing with multiple biological activities, bacterial reproduction can be effectively inhibited, wound oxidative stress is reduced by regulating ROS (reactive oxygen species) level, and wound healing is promoted. The proliferation, migration and angiogenesis of skin cells are promoted, and the healing time of diabetic wounds is remarkably shortened. In animal experiments, the 3D-printed double-metal cross-linked hydrogel shows high tissue repair capacity, and healing of diabetic wounds can be accelerated. In addition, the hydrogel dressing has good biocompatibility and degradability, can realize accurate adaptation of wound surfaces, long-acting slow release of drugs and multi-mechanism synergistic anti-infection and healing promotion, and provides a novel functional biological material for treatment of diabetic wound surfaces.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a 3D-printed bimetallic cross-linked hydrogel dressing, its preparation method, and its application. Background Technology

[0002] The clinical treatment of diabetic wounds has long faced multiple challenges. For example, traditional wound dressings (such as gauze and ordinary hydrogels) are difficult to precisely fit irregular wound shapes, easily forming gaps that lead to exudate accumulation and increase the risk of infection. The drug release rate of most antibacterial dressings is uncontrollable; excessively high initial drug concentrations may irritate wound tissue, while a sudden drop in concentration later results in insufficient antibacterial effect, requiring frequent dressing changes to maintain efficacy. Frequent dressing changes not only damage newly formed granulation tissue and prolong the healing period but also significantly increase patient suffering and the workload of medical staff. While existing hydrogel scaffolds possess a certain degree of biocompatibility, they have limitations in balancing mechanical support performance and drug sustained-release effects: hydrogel scaffolds with only physical cross-linking lack sufficient mechanical strength and are prone to rupture during wound activity; while hydrogels with only chemical cross-linking offer superior mechanical properties, residual cross-linking agents may trigger wound inflammation, limiting biocompatibility. Meanwhile, for the repair needs of chronic wounds such as diabetic wounds, existing materials mostly focus only on antibacterial function and lack the ability to regulate the wound microenvironment, making it difficult to achieve the synergistic repair effect of "antibacterial-antioxidant-anti-inflammatory-tissue regeneration" at the same time.

[0003] Photopolymerized silk fibroin (SFMA), as a natural biomaterial, possesses excellent biocompatibility and biodegradability. Its degradation rate can be flexibly controlled through concentration adjustment, theoretically matching the wound repair needs of different healing stages. Bimetallic ions (such as iron and copper ions) play a clear role in wound repair: copper ions can activate antioxidant enzymes in wound tissue, inhibit the release of inflammatory factors, and possess broad-spectrum antibacterial activity. Iron ions, when combined with tannic acid, can endow hydrogel scaffolds with near-infrared photothermal therapy capabilities. Tannic acid, as a natural polyphenol, can form stable coordination bonds with metal ions, achieving long-term sustained release of bimetallic ions. Curcumin has potent anti-inflammatory, antioxidant, and antibacterial biofilm formation effects. Due to the highly conjugated β-diketone moiety in its chemical structure, curcumin can chelate various metal ions to form metal-curcumin complexes. Subsequently, the chemical properties of curcumin change, significantly enhancing its antioxidant properties and further improving the repair effect of infected wounds. Combining iron ions, copper ions, tannic acid, and curcumin into bimetallic cross-linked nanoparticles can effectively combine the effects of each component, resulting in a good therapeutic effect on diabetic wounds.

[0004] However, current technologies have not effectively combined SFMA with bimetallic cross-linked nanoparticles (such as iron ion-tannic acid-copper ion-curcumin), nor have they utilized the synergistic advantages of microfluidics and 3D printing. Microfluidics can disperse nanoparticles, avoiding uneven drug release caused by particle aggregation, and precisely construct biomimetic microstructures (adapting to keratinocyte infiltration and proliferation), simulating the skin's growth microenvironment; it can also help with NP loading and sustained release, synergistically regulating cell function, anti-infection, and angiogenesis; and it can customize mechanical properties and degradation rates to match the environment of diabetic wounds, improving clinical applicability. 3D printing technology, on the other hand, can customize and print hydrogel scaffolds that perfectly fit the wound morphology based on wound scanning data, solving the problem of poor adhesion of traditional dressings. Therefore, developing a 3D-printed hydrogel dressing based on SFMA, combined with bimetallic cross-linked nanoparticles and advanced preparation technology, for the treatment of chronic wounds such as diabetic wounds is of great significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a 3D printed bimetallic cross-linked hydrogel scaffold, which addresses the shortcomings of the prior art.

[0006] The technical problem that this invention also aims to solve is to provide a 3D-printed bimetallic cross-linked hydrogel scaffold prepared by the above-mentioned method.

[0007] The final technical problem to be solved by this invention is to provide the application of the above-mentioned 3D printed bimetallic cross-linked hydrogel scaffold in the treatment of chronic wounds.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0009] A method for preparing a 3D-printed bimetallic cross-linked hydrogel dressing includes the following steps:

[0010] Step 1: Add glycidyl methacrylate to the silk fibroin solution to carry out the first reaction, and obtain methacrylamide silk fibroin (SFMA). Dissolve it in water to obtain an aqueous solution of methacrylamide silk fibroin.

[0011] Step 2: Mix tannic acid aqueous solution, iron ion aqueous solution, copper ion aqueous solution and curcumin solution to obtain the first mixture, and sonicate to obtain NP-Cur nanoparticles.

[0012] Step 3: Mix the methacrylamide silk fibroin aqueous solution prepared in Step 1, the photoinitiator, and the NP-Cur nanoparticles prepared in Step 2 evenly to obtain SF@NP-Cur mixture, introduce it into a 3D printing device for printing, and crosslink and cure it under ultraviolet light to obtain the final product.

[0013] In step 1, the solvent of the silk fibroin solution is a saturated lithium bromide aqueous solution; the concentration of the silk fibroin solution is 0.1 ~ 0.2 g / mL, preferably 0.2 g / mL; the volume ratio of the silk fibroin solution to the glycidyl methacrylate is 10 ~ 20:1, preferably 10:1.

[0014] Preferably, the preparation method of the silk fibroin is as follows: cut the silkworm cocoons into fragments, add them to a sodium carbonate aqueous solution of 3-5 g / L, boil and degumme for 30-60 min, drain and obtain pre-degummed silk, add it to a sodium carbonate aqueous solution of 3-5 g / L, boil and degumme for 30 min, drain and wash with pure water until the pH of the washing waste liquid is neutral, and dry at 60-80℃ to obtain silk fibroin; the amount of sodium carbonate aqueous solution used is based on submerging the silkworm cocoons.

[0015] Preferably, the silk fibroin solution is prepared by adding silk fibroin to a solvent and stirring at a constant temperature of 60-70°C until the silk fibroin is completely dissolved.

[0016] Preferably, in step 1, the addition of glycidyl methacrylate (GMA) to the silk fibroin solution is carried out under stirring and heating conditions; the stirring speed is 300-500 rpm; and the heating temperature is 60-70°C.

[0017] Preferably, in step 1, when adding glycidyl methacrylate to the silk fibroin solution, the glycidyl methacrylate is added slowly dropwise at a flow rate of 0.5 to 1 mL / min.

[0018] In step 1, the first reaction is carried out by stirring at 60-70°C and in the dark for 2-4 hours.

[0019] In step 1, the reaction solution obtained after the first reaction is dialyzed and freeze-dried to obtain the methacrylamide silk fibroin; the dialyz is aimed at removing lithium bromide and unreacted monomers.

[0020] Preferably, in step 1, the dialysis bag used has a molecular weight cutoff of 3500-7000 Da, preferably 3500 Da, the dialysis solution used is water, the dialysis duration is 48-72 hours, and the dialysis solution is changed every 2-4 hours during the process.

[0021] In step 1, the concentration of the methacrylamide silk fibroin aqueous solution is 0.1 ~ 0.2 g / mL, preferably 0.1 g / mL.

[0022] In step 2, the concentration of the curcumin solution is 20-40 mg / mL, and the solvent is preferably DMSO or ethanol; in the first mixture, the concentrations of tannic acid, iron ions, copper ions and curcumin are 0.24-0.48 mM, 18.50-73.98 mM, 31.33-94.00 mM and 0.27-0.54 mM, respectively; preferably, in the first mixture, the concentrations of tannic acid, iron ions, copper ions and curcumin are 0.24 mM, 18.5 mM, 31.33 mM and 0.54 mM, respectively.

[0023] In step 2, the ultrasonic power is 500-1000 W and the time is 5-15 min. Preferably, the ultrasonic power is 1000 W and the time is 5 min. After the ultrasonic treatment, the mixture is separated into solid and liquid, the solid is collected, washed and resuspended, and then freeze-dried to obtain the NP-Cur nanoparticles. Preferably, the solid-liquid separation is centrifugation at 9600 rpm for 10 min. The liquid used for washing and resuspending is water.

[0024] In step 3, the content of the NP-Cur nanoparticles and the photoinitiator in the SF@NP-Cur mixture is 2-4 mg / mL and 1-5 g / L, respectively; preferably, the content of the NP-Cur nanoparticles and the photoinitiator in the SF@NP-Cur mixture is 2 mg / mL and 5 g / L, respectively.

[0025] Preferably, in step 3, the method for printing a bimetallic cross-linked hydrogel scaffold using a 3D printing device is as follows: a matching three-dimensional structural model is constructed according to the wound morphology; the SF@NP-Cur mixture is loaded into an extrusion syringe; the SF@NP-Cur mixture is extruded through the needle of the syringe using a microfluidic injection pump; and then cross-linked and cured by ultraviolet light irradiation.

[0026] Preferably, in step 3, the wavelength of the ultraviolet light is 350 ~ 400 nm.

[0027] The present invention also claims protection for 3D printed bimetallic cross-linked hydrogel dressings prepared by the above-described preparation method.

[0028] The present invention also claims protection for the application of the above-described 3D-printed bimetallic cross-linked hydrogel dressing in the treatment of chronic wounds.

[0029] Figure 1 The diagram illustrates the preparation method of the 3D-printed bimetallic cross-linked hydrogel wound dressing of the present invention and its principle of application in wound treatment.

[0030] Beneficial effects:

[0031] (1) This invention uses 3D printing technology to customize and print hydrogel scaffolds, which can be perfectly adapted to the wound morphology, eliminating gaps between dressings and wounds from the root and reducing the risk of infection aggravation caused by exudate retention. At the same time, bimetallic cross-linked nanoparticles (NPs) achieve long-term sustained release of curcumin by means of the stable coordination between tannic acid and metal ions, maintaining a stable concentration of antibacterial, antioxidant, anti-inflammatory and repair-promoting effects, reducing the frequency of dressing changes. Compared with traditional dressings, this invention reduces patient pain, lightens the workload of medical staff, and avoids damage to newly formed wound tissue caused by frequent dressing changes.

[0032] (2) The components of the bimetallic cross-linked nanoparticles in the hydrogel scaffold can exert synergistic effects: Cu 2+ It can activate antioxidant enzymes in wounds, inhibit the release of inflammatory factors, and possess broad-spectrum antibacterial activity, showing good antibacterial effects against Escherichia coli and Staphylococcus aureus; Fe 3+ When combined with tannic acid, it can aid in wound healing and synergize with Cu. 2+ This process achieves slow release of curcumin; curcumin possesses potent anti-inflammatory and antioxidant properties and inhibits bacterial biofilm formation; tannic acid stabilizes the bimetallic ions through coordination bonds, ensuring stable curcumin release. Simultaneously, after ultrasonic treatment, the nanoparticles exhibit uniform particle size (400 nm ~ 500 nm) and even dispersion. The addition of SFMA matrix does not alter SFMA's excellent biocompatibility and degradability. Animal experiments show that the hydrogel dressing exhibits no significant inflammatory response and demonstrates synergistic repair through multiple mechanisms, significantly accelerating the healing of diabetic wounds. It simultaneously regulates the wound microenvironment, synergistically achieving "antibacterial-antioxidant-anti-inflammatory-angiogenesis-tissue regeneration," accelerating the healing of chronic wounds such as diabetic wounds. Attached Figure Description

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0034] Figure 1 This diagram illustrates the preparation method of the 3D-printed bimetallic cross-linked hydrogel wound dressing of the present invention and its principle of application in wound treatment; wherein, Figure 1 a represents the preparation process of bimetallic nanoparticles. Figure 1 b represents the preparation process of the 3D-printed bimetallic cross-linked hydrogel. Figure 1 c is a schematic diagram of a diabetic rat wound treated with a 3D-printed bimetallic cross-linked hydrogel wound dressing. Figure 1 d represents the mechanism by which 3D-printed bimetallic cross-linked hydrogels are used to treat diabetic wounds.

[0035] Figure 2 The antibacterial properties of the 3D-printed bimetallic cross-linked hydrogel wound dressing in Example 2 and the different Fe...3+ Temperature changes of 3D-printed bimetallic cross-linked hydrogels after near-infrared irradiation at varying concentrations; among which, Figure 2 a represents the difference in Fe under the same near-infrared power irradiation. 3+ Temperature changes in the concentration of 3D-printed bimetallic cross-linked hydrogels Figure 2 b is 0.3% Fe 3+ Temperature changes of 3D-printed bimetallic cross-linked hydrogels of different concentrations under near-infrared power irradiation. Figure 2 c represents the temperature stability of the 3D-printed bimetallic cross-linked hydrogel under near-infrared lamp irradiation. Figure 2 Image d represents the plate coating experiment results for the Control group, SFMA@NP1 group, SFMA@NP2 group, and SFMA@NP2+NIR group. Figure 2 e is a statistical graph showing the survival rate of Staphylococcus aureus (S. aureus). Figure 2 f represents the bacterial morphology images under a scanning electron microscope after the Control group, SFMA@NP1 group, SFMA@NP2 group, and SFMA@NP2+NIR group were cultured on the bacterial wall. Figure 2 g represents Escherichia coli E. coli Survival rate statistics chart, Figure 2 h represents different Cu 2+ Images of the experimental results of plate coating of 3D printed bimetallic cross-linked hydrogels at different concentrations.

[0036] Figure 3 This figure shows the experimental results of the biocompatibility, cell migration promotion ability, and antioxidant capacity of the 3D-printed bimetallic cross-linked hydrogel wound dressing in Example 3; where, Figure 3 a and Figure 3 b shows the growth images of 3T3 cells and HUVEC cells after treatment with 3D-printed bimetallic cross-linked hydrogel wound dressings. Figure 3 Image c shows the cell growth of HUVEC cells after treatment with a 3D-printed bimetallic cross-linked hydrogel wound dressing following high-glucose induction. Figure 3 Figure d shows the results of 3T3 cell proliferation detection after different hydrogel treatments. Figure 3 e represents a statistical graph and image of blood compatibility. Figure 3 f is a statistical graph showing the number of live and dead cells in HUVEC cells after treatment with 3D-printed bimetallic cross-linked hydrogel wound dressings following high-glucose induction. Figure 3 Image g shows an experimental image of an anti-oxidation dressing made from a 3D-printed bimetallic cross-linked hydrogel. Figure 3 h represents the statistical analysis of antioxidant test results. Figure 3 i is the image from the scratch test. Figure 3 j represents the statistical results of the scratch test. Figure 3 k represents the experimental image at 24 hours after the angiogenesis experiment. Figure 3 l is a statistical graph of the blood vessel length detection results from the angiogenesis experiment.

[0037] Figure 4 This is a diagram showing the results of a wound experiment on diabetic rats using the 3D-printed bimetallic cross-linked hydrogel wound dressing from Example 4; where... Figure 4 A describes the process of establishing a diabetic rat model, wound creation, and treatment. Figure 4 b shows the temperature changes of the back wounds of mice in each group after treatment with 3D-printed bimetallic cross-linked hydrogel wound dressings under near-infrared light irradiation. Figure 4 Image c shows a rat wound treated with a 3D-printed bimetallic cross-linked hydrogel wound dressing. Figure 4 d is a statistical graph showing wound healing in rats from different treatment groups. Figure 4 e shows the H&E and Masson images of the rat wound. Figure 4 f represents the statistical results of temperature changes in the back of rats after irradiation with near-infrared light. Figure 4 g is a statistical graph of the quantitative detection results of rat wound width. Figure 4 h represents the statistical results of quantitative collagen detection after Masson staining of rat wounds; the "ns" marker in the figure represents the differences between the test group and the control group after SPSS statistical analysis. p >0.05 indicates that the difference is not statistically significant. An asterisk (*) indicates that the difference between the experimental and control groups was statistically significant after SPSS analysis. p <0.05 indicates a statistically significant difference. The "**" symbol signifies that the difference between the experimental and control groups has been statistically analyzed using SPSS. p <0.01 indicates a highly significant difference; "***" marks indicate that the difference between the experimental group and the control group was statistically significant after SPSS analysis. p <0.001 indicates a higher significance level for the difference. Detailed Implementation

[0038] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0039] Example 1: Preparation of 3D-printed bimetallic cross-linked hydrogel wound dressing

[0040] (1) Synthesis of SFMA

[0041] Take dried silkworm cocoons, cut them into pieces, add 5 g / L sodium carbonate aqueous solution, degumm in a 98℃ constant temperature water bath for 30 min, drain and obtain pre-degummed silk, add 5 g / L sodium carbonate aqueous solution, boil and degumm in for 30 min, drain and wash with pure water until the pH of the washing waste liquid is neutral, place in a 60℃ vacuum drying oven and dry for 4 h to obtain silk fibroin.

[0042] Weigh 10 g of the dried silk fibroin and add it to 50 mL of saturated lithium bromide aqueous solution. Stir in a constant temperature water bath at 60 °C for 4 h until the silk fibroin is completely dissolved to obtain a silk fibroin solution.

[0043] 50 mL of the above silk fibroin solution was slowly added dropwise (flow rate 0.5 mL / min) to it while stirring at 60 °C. After the addition was complete, the solution was reacted in a constant temperature water bath at 60 °C for 4 h in the dark. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in deionized water for 72 h. During this period, the deionized water was changed every 2 h to remove lithium bromide and unreacted monomers and small molecule impurities. Finally, the dialyzed product was freeze-dried to obtain white, fluffy SFMA.

[0044] (2) Synthesis of bimetallic nanoparticles

[0045] To verify the functionality of the 3D-printed bimetallic crosslinked hydrogel, two types of bimetallic nanoparticles were designed: one containing curcumin and Cu. 2+ Fe 3+ Bimetallic nanoparticles containing tannins (NP2); another type contains only Cu. 2+ Fe 3+ The preparation methods for two types of bimetallic nanoparticles (NP1) are as follows: tannin-free bimetallic nanoparticles and tannic acid-free bimetallic nanoparticles (NP1).

[0046] Nanoparticles were prepared by coordination precipitation, and different concentration gradients of Fe³⁺ and Cu²⁺ were used during the preparation process to explore the optimal photothermal effect concentration and antibacterial concentration.

[0047] Preparation of different Fe 3+NP2 nanoparticles: 0.41 g of tannic acid (TA) was dissolved in 100 mL of deionized water and magnetically stirred at room temperature for 30 min until completely dissolved, yielding a 2.4 mM TA aqueous solution. Ferric chloride aqueous solutions with concentrations of 0.03 g / mL, 0.06 g / mL, and 0.12 g / mL were prepared. Curcumin (Cur) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 40 mg / mL curcumin solution. 100 μL of the TA aqueous solution was mixed with 0.03 g / mL, 0.06 g / mL, and 0.12 g / mL ferric chloride aqueous solutions at a volume ratio of 1:1. 100 μL of 0.05 g / mL copper sulfate and 5 μL of curcumin solution were added, followed by 695 μL of pure water. The resulting mixture was sonicated at 1000 W for 5 min, allowing Cur to be loaded into the coordination system of tannic acid and metal ions through hydrophobic interactions and hydrogen bonding, forming NP-Cur. After the reaction was completed, the mixture was centrifuged at 9600 r / min and 4℃ for 10 min. The supernatant was discarded to remove unreacted TA, metal ions and Cur. The precipitate was collected and resuspended in deionized water. The centrifugation purification was repeated 3 times. Finally, the purified nanoparticles were resuspended in deionized water and freeze-dried to obtain nanoparticle NP-Cur powder (NP2-0.3%Fe, NP2-0.6%Fe, NP2-1.2%Fe).

[0048] Preparation of different Cu 2+ NP2 nanoparticles with varying concentrations: 0.41 g of tannic acid (TA) was dissolved in 100 mL of deionized water and magnetically stirred at room temperature for 30 min until completely dissolved, yielding a TA aqueous solution with a concentration of 2.4 mM. Copper sulfate aqueous solutions with concentrations of 0.05 g / mL, 0.1 g / mL, and 0.15 g / mL were prepared. 100 μL of the TA aqueous solution was mixed with the 0.05 g / mL, 0.1 g / mL, and 0.15 g / mL copper sulfate aqueous solutions at a volume ratio of 1:1. 100 μL of 0.03 g / mL ferric chloride aqueous solution and 5 μL of curcumin solution were added, followed by 695 μL of pure water. The resulting mixture was sonicated at 1000 W for 5 min, allowing Cur to be loaded into the coordination system through hydrophobic interactions and hydrogen bonds, forming NP-Cur. After the reaction was completed, the mixture was centrifuged at 9600 r / min and 4℃ for 10 min. The supernatant was discarded to remove unreacted TA, metal ions and Cur. The precipitate was collected and resuspended in deionized water. The centrifugation purification was repeated 3 times. Finally, the purified nanoparticles were resuspended in deionized water and freeze-dried to obtain nanoparticle NP-Cur powder (NP2-0.5%Cu (also known as NP2-0.3%Fe), NP2-1.0%Cu, NP2-1.5%Cu).

[0049] Preparation of NP1 nanoparticles: 0.41 g of tannic acid (TA) was dissolved in 100 mL of deionized water and magnetically stirred at room temperature for 30 min until completely dissolved to obtain a 2.4 mM TA aqueous solution. 100 μL of the TA aqueous solution was mixed with 0.05 g / mL of copper sulfate aqueous solution and 0.03 g / mL of ferric chloride solution at a volume ratio of 1:1:1. The resulting mixture was sonicated at 1000 W for 5 min and NP1 was obtained. The mixture was centrifuged at 9600 r / min and 4℃ for 10 min. The supernatant was discarded to remove unreacted TA and metal ions. The precipitate was collected and resuspended in deionized water. The centrifugation purification was repeated 3 times. Finally, the purified nanoparticles were resuspended in deionized water and freeze-dried to obtain NP1 nanoparticle powder.

[0050] (3) 3D printed bimetallic cross-linked hydrogel wound scaffold

[0051] The SFMA obtained in step (1) was dissolved in water to prepare 6 SFMA solutions with a concentration of 0.1 g / mL, each 10 mL; 20 mg of the nanoparticles NP2 and NP1 obtained in step (2) were weighed and added to the 6 SFMA solutions respectively, and 0.05 g of LAP was added to each. The mixture was mixed evenly to prepare 6 SF@NP-Cur mixtures. 5 mL of the SF@NP-Cur mixture was loaded into an extrusion syringe, fixed to a microfluidic injection pump (flow rate of 1.2 mL / h), and extruded through a 21G (inner diameter 510 μm) flat-tipped needle; then 400 Six yellow-black SF@NP-Cur printing dressings (SF@NP2-0.3%Fe (also known as SF@NP2-0.5%Cu), SF@NP2-0.6%Fe, SF@NP2-1.2%Fe, SF@NP2-1.0%Cu, SF@NP2-1.5%Cu, and SF@NP1) were prepared by cross-linking and curing the structure under ultraviolet (UV) irradiation.

[0052] Example 2: Near-infrared photothermal effect and antibacterial effect of 3D-printed bimetallic cross-linked hydrogel wound dressing

[0053] (1) Different Fe prepared in Example 1 3+ Near-infrared photothermal effect experiment on hydrogel dressings with high concentrations of [specific components]:

[0054] Measurement of different Fe under near-infrared lamp irradiation at a power density of 2.5 W / cm² 3+ The temperature rise of 3D-printed bimetallic cross-linked hydrogel wound dressings with varying content is shown in the experimental results. Figure 2As shown in Figure a, the temperature rise of SF@NP2-0.3%Fe under near-infrared lamp irradiation best matches the temperature range of the treated wound. Therefore, SF@NP2-0.3%Fe was selected to conduct photothermal effect experiments on the excipient under near-infrared lamp irradiation with different power densities, and its temperature rise at 2.0 W / cm² was measured. 2 2.5 W / cm 2 3 W / cm 2 The temperature rise under near-infrared lamp irradiation at power density is shown in the experimental results. Figure 2 As shown in b, where 2.5 W / cm 2 The temperature of the dressing under near-infrared lamp irradiation at a power density that matches the temperature range of the treated wound is suitable; therefore, 2.5 W / cm² is selected. 2 The power density was used as the power density for the near-infrared lamp irradiation in subsequent experiments.

[0055] In addition, the different Fe values ​​under near-infrared lamp irradiation at a power density of 2.5 W / cm² were measured. 3+ The photothermal stability of a 3D-printed bimetallic cross-linked hydrogel wound dressing with a specific concentration was investigated. The experiment consisted of five cycles, with each cycle involving 150 seconds of irradiation followed by a 150-second rest period. The dressing temperature was measured and recorded every 30 seconds during the experiment. The experimental results are as follows: Figure 2 As shown in c, the SF@NP-Cur hydrogel scaffold maintained its photothermal performance during five on / off NIR irradiation cycles, indicating that it has excellent photothermal stability.

[0056] (2) Different Cu prepared in Example 1 2+ Antimicrobial activity of hydrogel dressings with high concentrations of [specific components] was evaluated.

[0057] The target bacterium in this experiment was Escherichia coli (Escherichia coli). E. coli ) and Staphylococcus aureus ( S.aureus ).

[0058] SF@NP2-0.5%Cu, SF@NP2-1.0%Cu, SF@NP2-1.5%Cu, and PBS buffer were co-cultured with 2 mL of target bacterial suspension for 24 hours. After the co-culture was completed, the co-culture system was subjected to 10... 5 Serial dilutions were performed. 20 μL of the diluted bacterial suspension was evenly spread onto LB agar plates. After 24 hours of incubation, colony forming units (CFUs) were quantitatively analyzed and images were captured using ImageJ software. The experimental results are shown below. Figure 2 As shown in h, different Cu 2+ The antibacterial effects of dressings with different Cu content were not significantly different, but those with excessive Cu content... 2+ This might lead to poor biocompatibility, so we will choose Cu in the future. 2+ A dressing with a content of 0.5% was used as the subsequent experimental group.

[0059] Taking into account both photothermal effect and antibacterial effect, SF@NP2-0.3%Fe (i.e., SF@NP2-0.5%Cu) is the optimal Fe. 3+ and Cu 2+ In subsequent examples, the concentration ratio of SF@NP2-0.3%Fe (also known as SF@NP2-0.5%Cu) will be referred to as SF@NP2.

[0060] Four groups of target bacterial cultures were prepared, with 2 mL in each group. One group served as a blank control, with PBS buffer added and co-cultured for 24 hours; another group was the SF@NP1 group, with SF@NP1 added and co-cultured for 24 hours; the remaining two groups were the SF@NP2 group and the SF@NP2+NIR group, with SF@NP2 added to each group and co-cultured for 24 hours. The SF@NP2+NIR group also received additional 2.5 W / cm² near-infrared light irradiation for 5 minutes during co-culture. After co-culture, the absorbance of each co-cultured bacterial culture was measured at 600 nm using a microplate reader, and the survival rate of each group relative to the blank control was calculated. The survival rates of Staphylococcus aureus and Escherichia coli are shown below. Figure 2 As shown in e and 2g, the near-infrared photothermal effect combined with the role of nanoparticles can achieve a better antibacterial effect. Meanwhile, the co-culture system was subjected to 10... 5 The bacterial suspension was serially diluted, with 20 μL of the diluted suspension evenly spread onto LB agar plates. Images of the plates were then captured using a camera. The results are shown below. Figure 2 As shown in Figure d, the SF@NP2+NIR group had fewer bacteria than the SF@NP2 group, indicating that the temperature rise of the hydrogel dressing after near-infrared light irradiation can achieve a better bactericidal effect. Bacterial morphology was also observed under a microscope.

[0061] The four co-culture systems were transferred to 6-well cell culture plates and cultured overnight using the bacterial wall-climbing culture method. After culture, the bacterial wall-climbing layers were sequentially dehydrated and fixed, and the bacterial surface morphology was then observed using scanning electron microscopy. The scanning electron micrographs are shown below. Figure 2 As shown in f, the influence of different samples on the structure was analyzed. Electron microscopy images showed that the bacteria in the Control group had intact morphology, while the bacteria in other groups showed shrinkage, lysis, etc., with the bacteria in the SF@NP2+NIR group showing the most severe morphological damage.

[0062] Example 3: Biocompatibility of 3D-printed bimetallic cross-linked hydrogel wound dressing, promoting cell migration and antioxidant capacity.

[0063] One SF@NP1 hydrogel and two SF@NP2 hydrogels were sterilized with ultraviolet light and then cultured overnight in complete culture medium. One SF@NP2 hydrogel was additionally irradiated with 2.5 W / cm² near-infrared light for 5 minutes during culture. A blank control group was also included, with an equal volume of PBS buffer added to the complete culture medium and cultured overnight. After culture, the culture medium from each group (Control, SF@NP1, SF@NP2, and SF@NP2+NIR) was used as the extraction solution for the following experiments.

[0064] (1) Biocompatibility assessment

[0065] 3T3 and HUVEC cells were seeded in 24-well plates, with each cell type divided into four groups. The extraction solutions described above were added to each of the four groups. At 24, 48, and 72 hours of culture, the cells were stained using the Calcein-AM / PI double staining method. Each group of cells was mixed with the Calcein-AM / PI staining solution and incubated for 30 minutes. After staining, the solution was removed, and the cells were gently washed twice with phosphate-buffered saline (PBS) to remove residual stain. Three fields of view were randomly selected for observation and photography using an Olympus CKx53 inverted fluorescence microscope.

[0066] Experimental results are as follows Figure 3 a and Figure 3 As shown in b, the results show that the proportion of live cells in the cells treated with the hydrogel dressing is basically the same as that in the untreated cells, indicating that the hydrogel excipients prepared in this invention have good biocompatibility.

[0067] (2) Assessment of the effect on 3T3 cell proliferation

[0068] Four groups of 3T3 cells were set up and co-cultured with the four extracts mentioned above for 24 h. After co-culture, the cells in each group were incubated with CCK-8 solution at 37℃ for 24, 48, and 72 h. The absorbance was then measured at 450 nm using a microplate reader to evaluate the effect of the hydrogel dressing on cell proliferation. The experimental results are as follows: Figure 3 As shown in d, the results indicate that the hydrogel dressing treatment did not affect the normal proliferation of cells.

[0069] (3) Evaluation of the protective effect of hydrogel excipients against cell damage under high sugar conditions

[0070] A high-glucose induction model of HUVEC cells was established: Five groups of HUVEC cells were set up. Four groups were co-cultured with the extracts of the four groups mentioned above for 48 h, and the remaining group was co-cultured with ordinary complete medium for 48 h as a positive control group. After the culture, HUVEC cells in each group were cultured in complete medium with a glucose concentration of 25 mmol / L, and the high-glucose medium was replaced daily to maintain stable induction for 48 hours. During this period, mannitol was added to the high-glucose medium of the control group to counteract the osmotic pressure effect of high glucose. After the culture of each group, the cells were stained with Calcein-AM / PI double staining method. Three fields of view were randomly selected for observation and photography using an Olympus CKx53 inverted fluorescence microscope, and the live / dead cell count was performed. The percentage of live cells was calculated as (number of live cells / total number of cells) × 100%, and the specific values ​​were obtained by ImageJ software.

[0071] Results of observation under an inverted fluorescence microscope are as follows Figure 3 As shown in c, hydrogel dressings can effectively reduce cell damage induced by high glucose, and the percentage of viable cells is statistically shown in the following figures. Figure 3 As shown in f, treatment with SF@NP2+NIR can effectively reduce the damage to cells caused by a high-glucose environment.

[0072] (4) Blood compatibility assessment

[0073] To evaluate the blood compatibility of SF@NP2 hydrogel, we used SD rats for hemolytic efficacy testing. The experimental procedure included: 1) After anesthetizing SD rats with isoflurane, orbital venous blood was collected through a glass capillary tube and immediately transferred to an anticoagulant tube containing heparin sodium. 2) The anticoagulant blood was diluted to 5 mL with physiological saline and centrifuged at 1500 rpm for 5 minutes. 3) Damaged red blood cells were removed by centrifugation three times, and intact red blood cells were collected from the precipitate. 4) The intact red blood cells were resuspended in physiological saline to prepare a 5% suspension. 5) Five groups of red blood cell suspensions were prepared, 1 mL per group. Four groups of red blood cell suspensions were mixed with the above four extracts (500 μL), and the remaining group of red blood cell suspensions was mixed with 500 μL of sterile water as a positive control. All samples were incubated in a 37°C water bath for 4 hours, and the supernatant was separated by centrifugation at 1500 rpm for 5 minutes. The absorbance of the supernatant was measured using an ELISA reader at a wavelength of 570 nm. The blood compatibility of the SF@NP2 hydrogel was assessed by calculating the percentage of hemolysis in each group.

[0074] Experimental results are as follows Figure 3 As shown in e, the 3D-printed bimetallic cross-linked hydrogel wound dressing exhibits good blood compatibility.

[0075] (5) Assessment of the effect on the migration ability of human vascular endothelial cells (HUVECs)

[0076] The migration ability of hydrogel dressings on human vascular endothelial cells (HUVECs) was evaluated using a scratch assay to investigate the effect of hydrogel dressings on cell function. The experimental procedure was as follows: HUVECs were sputtered at a density of 1 × 10⁶ cells per well. 6 Cells were seeded at a density of [number] cells per well in 6-well cell culture plates. The four extraction solutions described above were added to each well, and the plates were incubated at 37°C with 5% CO2 for 24 hours. When cell confluence reached 80%–90%, horizontal scratches were made on the surface of the monolayer of cells using a sterile 200 μL pipette tip to establish an artificial wound model. The scratched areas were gently rinsed twice with PBS buffer to remove detached cells, and the four extraction solutions were re-added to each well. Microscopic images of the scratched areas were taken at 0 and 24 hours post-scratching. Quantitative analysis of the scratch images was performed using ImageJ software to calculate the cell migration rate of each group, thus characterizing the cell migration ability.

[0077] Figure 3 i represents images of HUVEC at 0 h and 24 h after treatment with the leachate from co-incubation of 3D-printed bimetallic cross-linked hydrogel wound dressing and cell culture medium. The dashed line represents the initial state of the cell scratch. Figure 3 j represents the cell migration rate test results, showing that the cell migration rate of the SF@NP2+NIR group was significantly improved compared with other groups after treatment with 3D printed bimetallic cross-linked hydrogel wound dressing.

[0078] (6) Assessment of in vitro angiogenesis capacity

[0079] In vitro angiogenesis capacity was assessed using a matrix gel angiogenesis assay. Matrix gel, melted at 4°C, was uniformly spread onto 96-well plates and incubated at 37°C for 30 minutes to solidify. Logarithmically growing HUVECs were introduced at a rate of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells onto the gel surface, and then the four extraction solutions described above were added. The cells were incubated at 37°C and 5% CO2 for 24 hours. The tubular structures formed by the cells were observed and photographed using an inverted optical microscope. The number of lumens and branching points were quantitatively analyzed using ImageJ software to characterize the in vitro angiogenesis capacity.

[0080] Figure 3 k is an image at 24 hours in the matrix gel angiogenesis experiment; Figure 3 The results of the matrix gel angiogenesis experiment were statistically analyzed, showing that the angiogenesis capacity of HUVECs in the SF@NP2+NIR group was significantly improved compared with other groups after treatment with 3D printed bimetallic cross-linked hydrogel wound dressing.

[0081] (7) In vitro reactive oxygen species scavenging experiment

[0082] First, 3T3 cells were seeded in 24-well plates and treated with the four sets of extraction solutions mentioned above for 24 hours. Then, DCFH-DA probe diluted 1000 times with DMEM was added, and the cells were incubated in the dark for 1 hour. Subsequently, the cells were washed three times with serum-free DMEM and then treated with 10 mM hydrogen peroxide for 2 hours. Finally, the fluorescence intensity of the cells was observed and measured using a fluorescence microscope.

[0083] Figure 3 g represents the in vitro reactive oxygen species scavenging experiment; Figure 3 h represents the test results of the in vitro reactive oxygen species scavenging experiment, showing that the cells treated with 3D-printed bimetallic cross-linked hydrogel wound dressings have significantly higher antioxidant capacity than other groups.

[0084] Example 4: Experiment on diabetic rat wounds with 3D-printed bimetallic cross-linked hydrogel wound dressing.

[0085] A diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ, 55 mg / kg dissolved in 0.1 mol / L citrate buffer) for seven consecutive days. Blood glucose levels were measured via tail vein sampling on day seven after the last injection; animals with blood glucose ≥16.7 mmol / L were considered to have successfully established the diabetic model. Subsequently, the diabetic rats were anesthetized by isoflurane inhalation. After skin preparation on the back, a full-thickness skin defect was created using a 1 cm sterile punch. Rats were randomly divided into four groups (n=5 per group). Two groups had their wounds treated with SF@NP1 gel and SF@NP2 gel, respectively. One group also had its wound treated with SF@NP2 gel and irradiated with 2.5 W / cm² near-infrared light for 5 minutes (SF@NP2+NIR group). The wounds of the last group were rinsed three times with PBS buffer. Wound images were taken using a digital camera under the same lighting and distance conditions on days 0, 3, 6, 9, and 12 to record the healing process. Rats were sacrificed on day 12, and tissue samples containing an intact wound and 5 mm of surrounding normal skin were obtained. The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned before being stained with hematoxylin and eosin (H&E) and Masson's stain. Figure 4 a represents the process of establishing a diabetic rat model, creating a wound model, and the treatment process.

[0086] After 12 days of observation, the wound healing process in the SF@NP2 group and SF@NP2+NIR group was significantly faster than that in the Control and SF@NP1 groups. (Based on actual wound images...) Figure 4 c) It can be seen that in the SF@NP2+NIR group (G4), the wound shrinks rapidly within 3 to 12 days, the scab falls off, and epithelial regeneration occurs; the healing rate is quantitatively measured ( Figure 4 d) Confirmed that the healing rate of the SF@NP2+NIR group was close to 100% on day 12. Photothermal imaging and temperature change analysis ( Figure 4 b and Figure 4 f) indicates that under near-infrared irradiation, the SF@NP2 hydrogel can heat to approximately 58°C within 5 minutes. The photothermal effect not only directly eliminates bacteria from the wound surface but also improves local blood supply, providing metabolic support for repair. Histological staining results ( Figure 4 In (e), H&E staining showed that the SF@NP2+NIR group had more complete reepithelialization of the wound, with uniform thickness of new tissue and less inflammatory infiltration; Masson staining ( Figure 4 e) and collagen quantification ( Figure 4 h) showed that the collagen fibers in the SF@NP2+NIR group were densely arranged and the collagen content was significantly higher than that in other groups ( p <0.001 indicates excellent tissue remodeling effect. Quantitative analysis of wound width ( Figure 4 g) It was also confirmed that the SF@NP2+NIR group had more efficient wound contraction and the tissue morphology after repair was closer to that of normal skin.

[0087] This invention provides a concept and method for 3D printing bimetallic crosslinked hydrogels, their preparation, and applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a 3D printed bimetallic cross-linked hydrogel dressing, characterized in that, Comprising the following steps: Step 1, glycidyl methacrylate is added to the silk fibroin solution to perform a first reaction to obtain methacrylated silk fibroin, which is dissolved with water to obtain a methacrylated silk fibroin aqueous solution; Step 2, a first mixed solution is prepared by mixing a tannic acid aqueous solution, an iron ion aqueous solution, a copper ion aqueous solution and a curcumin solution, and then ultrasonic treatment is performed to obtain NP-Cur nanoparticles; Step 3, a light initiator, the methacrylated silk fibroin aqueous solution prepared in step 1 and the NP-Cur nanoparticles prepared in step 2 are uniformly mixed to obtain an SF@NP-Cur mixed solution, which is introduced into a 3D printing device for printing, and then ultraviolet light is irradiated for crosslinking and curing to obtain the product.

2. The production method according to claim 1, characterized by, In step 1, the solvent of the silk fibroin solution is saturated lithium bromide aqueous solution; the concentration of the silk fibroin solution is 0.1-0.2 g / mL; and the volume ratio of the silk fibroin solution to glycidyl methacrylate is 10-20:

1.

3. The production method according to claim 1, characterized by, In step 1, the first reaction is performed under the conditions of 60-70℃ and light shielding, and the reaction is stirred for 2-4 hours.

4. The method of claim 1, wherein, In step 1, the reaction solution obtained after the first reaction is dialyzed and freeze-dried to obtain the methacrylated silk fibroin.

5. The preparation method according to claim 1, characterized in that, In step 1, the concentration of the methacrylated silk fibroin aqueous solution is 0.1-0.2 g / mL.

6. The method of claim 1, wherein, In step 2, the concentration of the curcumin solution is 20-40 mg / mL; and in the first mixed solution, the concentrations of tannic acid, iron ions, copper ions and curcumin are 0.24-0.48 mM, 18.50-73.98 mM, 31.33-94.00 mM and 0.27-0.54 mM, respectively.

7. The preparation method according to claim 1, characterized in that, In step 2, the ultrasonic treatment is performed at a power of 500-1000 W for 5-15 min; after the ultrasonic treatment, the first mixed solution is subjected to solid-liquid separation, and the collected solid is washed, resuspended and freeze-dried to obtain the NP-Cur nanoparticles.

8. The method of claim 1, wherein, In step 3, in the SF@NP-Cur mixed solution, the content of the NP-Cur nanoparticles and the light initiator is 2-4 mg / mL and 1-5 g / L, respectively.

9. The 3D printed bimetallic crosslinked hydrogel dressing prepared by the preparation method of any one of claims 1-8.

10. The use of the 3D printed bimetallic crosslinked hydrogel dressing of claim 9 in the treatment of chronic wounds.

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