Zwitterionic poly (sulfobetaine methacrylate) / kappa carrageenan double-network hydrogel

Abstract

The invention relates to a zwitterionic poly (sulfobetaine methacrylate) / kappa carrageenan double-network hydrogel and a preparation method thereof. The invention aims to design a hydrogel wound dressing capable of promoting wound healing. The hydrogel wound dressing is formed by physically interweaving a zwitterionic polysulfobetaine methacrylate hydrogel network and a kappa carrageenan hydrogel network. Through verification, the double-net hydrogel has good antifouling property, no cytotoxicity and remarkable promotion of wound healing, more importantly, the hydrogel also has good mechanical property, and has important significance as a hydrogel wound dressing which is easy to synthesize, high in wound healing promotion efficiency and good in biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to an amphoteric ion polymethacrylic acid sulfobetaine / kappa-carrageenan double-network hydrogel (SBMA / kappa-carrageenan double-network hydrogel), a preparation method thereof, and an application thereof. Background Art

[0002] The skin, as the largest organ of the human body, is a protective barrier against the external environment, and it also plays a crucial role in aspects such as excretion, body temperature regulation, and external stimuli. However, the exposed skin is prone to damage, including abrasions, burns, and cuts, which not only damage its barrier function and physiological processes but also cause persistent pain. In addition, the damaged skin is easily infected by external bacteria, leading to further deterioration and possibly serious consequences such as amputation or even death. Therefore, there is an urgent need for timely and effective treatment of damaged skin.

[0003] Currently, the mainstream wound dressings mainly consist of gauze and sponges. These traditional wound dressings are difficult to maintain a moist wound environment and often cause scabbing and scarring during the healing process. In addition, they are easily adhered to the newly formed tissue and are prone to cause secondary damage to the skin when changing the dressing. As an ideal wound dressing material, hydrogel materials have good application prospects in wound dressings. A hydrogel is a three-dimensional network of hydrophilic polymer chains that can absorb and retain a large amount of water, and its structure is similar to the extracellular matrix, which can maintain a moist wound environment and promote wound healing. Despite these advantages, however, hydrogel dressings are prone to biological flocculation when applied to wounds, leading to bacterial infection. If antibiotics are introduced, it may lead to the emergence of super bacteria. Therefore, adding an antifouling material to the hydrogel wound dressing seems to be a more feasible option.

[0004] Zwitterionic polymer materials have attracted attention in pollution prevention applications due to their unique properties, where each structural unit carries a pair of opposite charges. Currently, polysulfobetaine, polycarboxybetaine, and polyphosphatidylcholine are the most common zwitterions. Compared with the commonly used polyethylene glycol with antifouling properties, the zwitterionic group has a lower binding energy with water and is more easily hydrated. The difference in hydration free energy between the zwitterionic group (-261~-238 kJ / mol) and polyethylene glycol (-180 kJ / mol) makes the zwitterionic polymer more likely to bind with water molecules through ionic solvation and form a tightly bound water molecule layer on its surface. This phenomenon generates an energy barrier that prevents the adhesion of protein molecules and cells, thus producing excellent anti-bioadhesion ability. Among zwitterions, polysulfobetaine has the effect of promoting wound healing. However, the mechanical properties of polysulfobetaine hydrogels are poor, and it is reported that its shear modulus is only about 1 kPa, which may limit its application in wound dressings. To address this limitation, the double-network theory proposed by Gong in 2003 provides a promising method for significantly improving the mechanical strength and toughness of hydrogels. By combining two independent asymmetric polymer networks, the first rigid and brittle network is intertwined with the second soft and tough network, resulting in remarkable mechanical properties. At the same time, in our research, we observed that SBMA hydrogels also exhibited limited swelling properties, presumably making it difficult for them to absorb a large amount of wound exudate. Solving these drawbacks is the key to optimizing the application of SBMA-based hydrogels in wound dressings.

[0005] Carrageenan is a natural polysaccharide containing sulfate groups extracted from seaweed and has attracted much attention due to its rich content and non-toxicity. Kappa-carrageenan is a widely used variant of carrageenan and is known for its anti-inflammatory, antioxidant, and anti-tumor activities. In addition, it also has excellent gel-forming ability, liquid absorption ability, and retention ability. Kappa-carrageenan hydrogels can be easily formed through a simple heating and cooling process.

[0006] Considering these good properties, kappa-carrageenan was introduced during the formation of SBMA hydrogels by free radical polymerization. This method ensures the uniform dispersion of SBMA and kappa-carrageenan in the whole system during the heating process, thus forming SBMA and kappa-carrageenan networks. Subsequently, the cooling process enables the formation of the kappa-carrageenan hydrogel network. The networks formed by SBMA and kappa-carrageenan are intertwined and penetrated with each other to form a double-network structure. Summary of the Invention

[0007] Based on the above technical background, the object of the present invention is to provide a hydrogel dressing with good antibacterial properties. To achieve this technical object, the present invention provides the following technical solutions:

[0008] In the first aspect of the present invention, a zwitterionic poly(sulfobetaine methacrylate) / κ-carrageenan double-network hydrogel (SBMA / κ-carrageenan double-network hydrogel) is provided. The networks formed by SBMA and κ-carrageenan are intertwined and permeated with each other to form a double-network structure.

[0009] The present invention designs and obtains a polymer material with good mechanical properties and antibacterial properties. As a zwitterionic polymer, poly(sulfobetaine methacrylate) is more likely to combine with water molecules through ionic solvation and form a tightly bound water molecule layer on its surface. This phenomenon generates an energy barrier that prevents the adhesion of protein molecules and cells, thereby producing excellent anti-bioadhesion and antibacterial capabilities.

[0010] The present invention designs and prepares an SBMA / κ-carrageenan double-network hydrogel. Since κ-carrageenan has excellent gel-forming ability, liquid absorption ability, and retention ability, it can significantly improve the mechanical properties of SBMA. The introduction of κ-carrageenan has the least impact on the anti-pollution ability of the hydrogel and successfully prevents the adsorption of bacteria, proteins, and cells. In addition, the hydrogel exhibits good biocompatibility and proves its ability to effectively promote wound healing secondly.

[0011] Specifically, the SBMA / κ-carrageenan double-network hydrogel of the present invention has a three-dimensional porous network structure with a pore size greater than or equal to 50 μm and less than or equal to 80 μm.

[0012] Preferably, the SBMA / κ-carrageenan double-network hydrogel of the present invention is prepared by the following preparation method: adding a cross-linking agent to an SBMA solution to prepare an SBMA precursor solution, continuing to add a κ-carrageenan solution to the hydrogel precursor solution, maintaining at 45-55 °C (preferred temperature is 50 °C), adding an initiator, heating, and standing to obtain the SBMA / κ-carrageenan double-network hydrogel. Optionally, finally, it is soaked in a phosphate buffer solution to remove unreacted cross-linking agent and initiator.

[0013] More preferably, the cross-linking agent is N,N'-methylenebisacrylamide.

[0014] Also more preferably, the initiator contains ammonium persulfate and sodium metabisulfite.

[0015] Also more preferably, the SBMA solution is a 40% (w / v) SBMA aqueous solution.

[0016] Also more preferably, the κ-carrageenan solution is a 1% (w / v) κ-carrageenan aqueous solution.

[0017] Also more preferably, the dosage of N,N'-methylenebisacrylamide as the cross-linking agent relative to the amount of SBMA is 2%, 4%, 6%, 8%, preferably 4%.

[0018] More preferably, the concentrations of ammonium persulfate and sodium metabisulfite in the initiator are 40% (w / v) and 15% (w / v), respectively, and the dosage of the initiator is 10 μl per 1 g of SBMA.

[0019] More preferably, the heating conditions are 60 - 80°C for 15 - 45 min, and the preferred heating conditions are 70°C for 0.5 h.

[0020] More preferably, the standing is at room temperature for 24 hours.

[0021] Preferably, in the SBMA / κ-carrageenan double-network hydrogel of the present invention, the weight ratio of SBMA to κ-carrageenan is 0.2 - 0.6:0.01 - 0.07, preferably 0.24 - 0.56:0.014 - 0.06.

[0022] In addition, the present invention also provides a method for preparing the SBMA / κ-carrageenan double-network hydrogel, and the preparation method is as follows: Dissolve a certain mass of SBMA in deionized water, add an N,N'-methylenebisacrylamide cross-linking agent to the solution, and obtain the SBMA precursor solution after dissolution. Dissolve a certain amount of κ-carrageenan in deionized water. After mixing the SBMA precursor solution and the κ-carrageenan solution evenly, add an initiator composed of ammonium persulfate and sodium metabisulfite. After mixing evenly, transfer it to a mold, and react completely under heating conditions to form the SBMA / κ-carrageenan double-network hydrogel. Subsequently, use a phosphate buffer solution to remove the residual impurities in the hydrogel.

[0023] The hydrogel also has good antibacterial properties. Applying it to an animal model can effectively reduce the bacterial survival rate on the wound surface, promote the proliferation of fibroblasts, promote angiogenesis, and reduce the inflammatory response. Based on the good self-healing and antibacterial properties of the hydrogel, the above hydrogel can be used as an antibacterial product or as a wound dressing, especially suitable for use as a wound dressing at joints, limbs, and oral cavities with high mobility. Therefore, the present invention provides the application of the SBMA / κ-carrageenan double-network hydrogel as an antibacterial product or a wound dressing. Specifically, the present invention provides the application of the SBMA / κ-carrageenan double-network hydrogel of the present invention in the preparation of dressings.

[0024] Preferably, the dressing is used for wounds, preferably for the damaged parts of the skin or soft tissues caused by trauma or carbuncle.

[0025] The beneficial effects of the technical solution of the present invention are:

[0026] The present invention provides an SBMA / κ-carrageenan double-network hydrogel wound dressing, a preparation method thereof and an application. In the present invention, SBMA, N,N'-methylenebisacrylamide, κ-carrageenan, ammonium persulfate and sodium metabisulfite are uniformly mixed and then polymerized by heating initiation to form an SBMA / κ-carrageenan double-network hydrogel wound dressing. It is verified that the hydrogel prepared in the present invention has good compressive properties and successfully prevents the adsorption of bacteria, proteins and cells. The material can simultaneously absorb wound exudate, stimulate the proliferation of fibroblasts in the wound, promote collagen deposition, stimulate capillary regeneration, and thus promote wound tissue regeneration. The prepared double-skeleton gel material can also avoid bacterial growth by preventing bacterial adhesion. The prepared material has good biocompatibility and has the effect of promoting skin tissue healing, and has good practical application value. In addition, the material can return to its original state under external force and can meet the needs of protecting wounds with some special wound shapes by injecting the material. Based on these findings, the hydrogel wound dressing has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0028] Figure 1 A is the stress-strain curve of the hydrogels in Examples 1 to 4 of the present invention; Figure 1 B is a comparison of the compressive strengths of the hydrogels in Examples 1 to 4 of the present invention.

[0029] Figure 2 A is the FTIR spectrum of the hydrogels in Examples 1 to 5 of the present invention. Figure 2 A are the SEM images of Examples 1(a), 2(b), 3(c), 4(d) in the examples of the present invention.

[0030] Figure 3 is the equilibrium water content of the hydrogels in Examples 1 to 4 of the present invention Figure 4 A is the swelling ratio of the hydrogels in Examples 1 to 4 of the present invention in water; Figure 4 B is the swelling ratio of the hydrogels in Examples 1 to 4 of the present invention in physiological saline.

[0031] Figure 5 A is the adsorption of bacteria by the hydrogels in Examples 1 to 5 of the present invention; Figure 5 B is the adsorption of proteins by the hydrogels in Examples 2, 3, 5 of the present invention; Figure 5C shows the cell adhesion on the surfaces of cell culture medium (a), Example 2 (c), Example 3 (d), and Example 5 (b).

[0032] Figure 6 A shows the hemolysis rate of the hydrogels of Example 2 and Example 3 of the present invention; Figure 6 B shows the cell viability of the hydrogels of Example 2 and Example 3 of the present invention.

[0033] Figure 7 A shows the wound healing photos of the positive control, Example 2, Example 3, and negative control at 12 days; Figure 7 B shows the wound healing rates of the positive control, Example 2, Example 3, and negative control; C: shows the H&E staining section photos of the wounds of the positive control (a), Example 2 (b), Example 3 (c), and negative control (d) at 6 days, magnification ratio 100 times; D: shows the H&E staining section photos of the wounds of the positive control (a), Example 2 (b), Example 3 (c), and negative control (d) at 12 days, magnification ratio 40 times Detailed Implementation Modes

[0034] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for describing specific implementation modes and are not intended to limit the exemplary implementation modes according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or their combinations.

[0036] As introduced in the background art, the weak mechanical properties of the existing zwitterionic poly(sulfobetaine methacrylate) (SBMA) antibacterial hydrogel matrix materials limit their development in the field of wound dressings. To solve the above technical problems, the present invention proposes an SBMA / κ-carrageenan double-network hydrogel.

[0037] In the first aspect of the present invention, an SBMA / κ-carrageenan double-network hydrogel is provided, in which the networks formed by SBMA and κ-carrageenan are intertwined and penetrated with each other to form a double-network structure.

[0038] It has been verified that the hydrogel prepared by the present invention has a three-dimensional porous network structure with a pore size greater than or equal to 50 μm and less than or equal to 80 μm.

[0039] The infrared spectrum of the SBMA / κ-carrageenan double-network hydrogel described in the first aspect of the present invention shows that in the SBMA / κ-carrageenan double-network structure sample of the hydrogel provided by the present invention, only the characteristic peaks of SBMA are visible. This may be because the absorption peaks of κ-carrageenan are relatively small and may be masked by the SBMA peaks. However, the peak value of SBMA has not changed, indicating that there is a physical mixture of the two components, which confirms that the SBMA / κ-carrageenan double-network structure is a chemical-physical double-network structure.

[0040] In the second aspect of the present invention, a preparation method of the SBMA / κ-carrageenan double-network hydrogel described in the first aspect is provided. The preparation method is as follows: A mixture composed of SBMA, N,N'-methylenebisacrylamide, κ-carrageenan, ammonium persulfate, and sodium metabisulfite is prepared, and the SBMA / κ-carrageenan double-network hydrogel is obtained by heating and initiating cross-linking. Phosphate buffered saline is used to soak and remove unreacted cross-linking agents and initiators.

[0041] Preferably, the cross-linking agent is N,N'-methylenebisacrylamide.

[0042] Preferably, the dosage of the cross-linking agent N,N'-methylenebisacrylamide is 2% - 8% of the amount of SBMA, and the preferred dosage is 4%.

[0043] Preferably, for the preparation method of the SBMA / κ-carrageenan double-network hydrogel, the temperature for dissolving and heating SBMA with stirring is 45 - 55°C, and the preferred temperature is 50°C.

[0044] Preferably, the initiator is a composition of ammonium persulfate and sodium bisulfite.

[0045] Preferably, the initiator is a composition of ammonium persulfate and sodium bisulfite, with concentrations of 40% (w / v) and 15% (w / v).

[0046] Preferably, for the composition of ammonium persulfate and sodium bisulfite as the initiator, with concentrations of 40% (w / v) and 15% (w / v), the dosage is 10 μl of the initiator added per 1 g of SBMA.

[0047] Preferably, the initiation conditions are 60 - 80°C and the time is 15 - 45 min. The preferred heating conditions are 70°C and the time is 0.5 h.

[0048] Furthermore, the preparation method of the SBMA / κ-carrageenan double-network hydrogel is as follows: Dissolve a certain mass of SBMA in deionized water to form a 40% (w / v) SBMA solution. Subsequently, add 4% of N,N'-methylenebisacrylamide crosslinking agent based on the amount of SBMA, and wait until all are dissolved for standby. Dissolve a certain amount of κ-carrageenan in deionized water at 90 °C to obtain a solution with a concentration of 1% (w / v). After preparation, transfer the solution to a water bath at 50 °C and maintain the required temperature. Mix the SBMA precursor solution and the κ-carrageenan solution at 50 °C. After mixing evenly, add an initiator composed of 40% (w / v) ammonium persulfate and 15% (w / v) sodium metabisulfite. After mixing evenly, transfer it to a mold and react at 70 °C for 30 minutes. Take out the mold and let it stand at room temperature for 24 hours to form the SBMA / κ-carrageenan double-network hydrogel. Subsequently, extract the hydrogel from the mold and soak it in phosphate buffer for three days, and regularly change the buffer every other day to remove the remaining unreacted crosslinking agent and initiator in the hydrogel.

[0049] In the third aspect of the present invention, there is provided the application of the SBMA / κ-carrageenan double-network hydrogel described in the first aspect as an antibacterial product or a wound dressing.

[0050] In the fourth aspect of the present invention, there is provided a gel for wound surfaces, and the gel includes the SBMA / κ-carrageenan double-network hydrogel described in the first aspect.

[0051] Preferably, the gel preparation is applied to the injuries of skin and soft tissues caused by trauma, carbuncle sores, etc.

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with specific examples.

[0053] Examples 1 to 5

[0054] (I) Preparation and characterization of the SBMA / κ-carrageenan double-network hydrogel wound dressing

[0055] 1. Preparation of the SBMA / κ-carrageenan double-network hydrogel

[0056] Prepare the SBMA hydrogel by free radical polymerization. Dissolve a certain mass of SBMA in deionized water to form a 40% (w / v) SBMA solution. Subsequently, add 4% of N,N'-methylenebisacrylamide crosslinking agent based on the amount of SBMA, and wait until all are dissolved for standby. Dissolve a certain amount of κ-carrageenan in deionized water at 90 °C to obtain a solution with a concentration of 1% (w / v). After preparation, transfer the solution to a water bath at 50 °C and maintain the required temperature for further treatment.

[0057] Mix different amounts of SBMA precursor solution with κ-carrageenan solution at the ratio specified in Table 1 at 50 °C. After mixing evenly, add an initiator composed of 40% (w / v) ammonium persulfate and 15% (w / v) sodium metabisulfite. The addition amount of the initiator is 1 μl per 1 g of SBMA. After mixing evenly, transfer it to a mold and react at 70 °C for 30 minutes. Take out the mold and let it stand at room temperature for 24 hours to form an SBMA / κ-carrageenan double-network hydrogel. Subsequently, extract the hydrogel from the mold and soak it in phosphate buffer for three days, replacing the buffer regularly every other day to remove the remaining unreacted cross-linking agents and initiators in the hydrogel.

[0058] Table 1 Composition ratio of hydrogel samples with different ratios of SBMA / κ-carrageenan

[0059]

[0060]

[0061] 2. Characterization of materials

[0062] The mechanical strength of the hydrogel was evaluated using a micro-in-situ mechanical test system. The freeze-dried hydrogel samples were analyzed by Fourier transform infrared spectroscopy (FT-IR) to study the interactions between the components of the double-network hydrogel. The structure of the samples was observed by scanning electron microscopy. The equilibrium water content of the hydrogel samples was calculated using the drying loss method. The swelling ratio of the hydrogel was calculated using the change in water absorption weight.

[0063] (2) In vitro experimental study To study the effect of the SBMA / κ-carrageenan double-network hydrogel wound dressing on biological properties

[0064] 1. Bacterial adhesion test

[0065] Immerse the hydrogel sheet in a bacterial solution containing Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) with a bacterial density of 1x10 5 CFU / mL. Place the sample in a shaker and incubate at 37 °C and 120 rpm for 2 hours. Take out the hydrogel sample and wash the surface with phosphate buffer. Subsequently, place each hydrogel in a 24-well plate and add an appropriate amount of phosphate buffer to each well. Then, ultrasonically clean the 24-well plate to promote the adhesion of bacteria to fall off from the hydrogel sample into the solution. Dilute the bacterial suspension appropriately, take 100 μL and spread it evenly on Luria-Bertani medium. Incubate at 37 °C for 12 h, observe the colonies on the solid medium, and determine the number of bacteria adhering to the hydrogel sample.

[0066] 2. Protein adsorption test

[0067] Carefully arrange hydrogel discs (6 mm in diameter and 1 mm in thickness) in a 48-well plate and thoroughly soak them in 500 mL of phosphate buffer solution. The negative control uses Example 5, and the experimental groups use Example 2 and Example 3. Under dark conditions, supplement 200 mL of 2.5 mg / mL BSA-FITC (bovine serum albumin-fluorescein isothiocyanate) to each well. Place the 48-well plate in a carbon dioxide incubator and incubate for 30 minutes. Subsequently, take out the hydrogel samples from the wells, wash them clean with phosphate buffer solution, and transfer them to a new 48-well plate. Observe and photograph the hydrogels using an inverted fluorescence microscope.

[0068] 3. Cell adhesion test

[0069] Place the hydrogels of Example 5, Example 2, and Example 3 (6 mm in diameter and 1 mm in thickness) in separate wells of a 48-well plate and soak them in 500 mL of phosphate buffer solution for standby. Prepare an L929 cell suspension with a cell density maintained at 1×10 5 cells / ml. Discard the phosphate buffer solution and add 1 ml of the cell suspension. Then place the plate in a carbon dioxide incubator at a temperature of 37 °C and a carbon dioxide concentration of 5%, and incubate for 24 hours. Take out the culture medium and discard it, and wash the surface of the hydrogel 5 times with phosphate buffer solution. Subsequently, transfer the hydrogel samples to a new 48-well plate and wash them 5 times again with phosphate buffer solution. Then observe and photograph the samples using an inverted fluorescence microscope.

[0070] 4. In vitro hemolysis experiment

[0071] The hemolysis rate is determined by measuring the content of free hemoglobin in plasma through photometric colorimetry. Citrate-mixed rabbit whole blood is diluted 1:1 with 0.9% NaCl normal saline. The hydrogels of Example 2 and Example 3 are used as the experimental groups. The hydrogel samples are cut into three different sizes (10 mg, 30 mg, and 50 mg). Each sample is soaked in 10 ml of normal saline and incubated at 37 °C for 30 minutes. Then add 0.2 mL of the diluted blood and incubate for 60 minutes. Add 0.2 mL of the diluted blood to 10 mL of normal saline to form a negative control group (hemolysis rate 0%), and add 0.2 mL of the diluted blood to 10 mL of 0.1% Na 2 CO 3 solution to form a positive control group (hemolysis rate 100%). The control groups are incubated at 37 °C for 60 minutes. Subsequently, centrifuge the sample groups and control groups at 2000 rpm for 10 minutes, and measure the absorbance at 540 nm using a multifunctional enzyme marker.

[0072] The hemolysis rate calculation formula is as follows:

[0073]

[0074] 5. Cytotoxicity

[0075] In this study, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to evaluate the biocompatibility of the materials. Taking Example 2 and Example 3 as the experimental groups. After ultraviolet sterilization, hydrogel samples of different masses were immersed in the corresponding volumes of complete medium to obtain extracts of 10 mg / ml, 30 mg / ml, and 50 mg / ml. L929 mouse fibroblasts were cultured normally. When the cell density reached 80% and the cell growth was stable, cell counting was performed. In a 96-well plate, 1×10 4 cells were added to each well, and at the same time, the hydrogel extract was added. The control group was complete culture medium, and the liquid volume in each well was 100 μL. After incubation for 20 hours, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for about 4 hours. Then, the liquid in the wells was removed, and the cells were washed with phosphate buffer. 150 μL of DMSO was added to each well, and the plate was shaken for 30 minutes to measure the absorbance. There were 5 replicates in each group, and the cell survival rate was calculated.

[0076] The formula for calculating the cell survival rate is as follows:

[0077]

[0078] (III) In vivo experiments to detect the wound healing effect of SBMA / κ-carrageenan double-network hydrogel wound dressings on infected skin wounds

[0079] Sixteen 6-week-old SD rats with a body weight of 230 ± 20 g (Zhejiang Mukang Biotechnology Co., Ltd.) were selected and randomly divided into a positive control group, a negative control group, an Example 2 group, and an Example 3 group. All animal experiments were carried out in accordance with the guidelines for the use of experimental animals. A circular back wound model with a diameter of 10 mm was used for the wound. The hydrogel samples of Example 2 and Example 3 were used to cover the wounds of the rats as the experimental groups, a commercially available hydrogel wound dressing was used as the positive control, and gauze was used as the negative control. The dressings were changed every 2 days, and the wounds were measured and photographed on days 0, 3, 6, 9, and 12. The formula for calculating the wound healing rate is as follows:

[0080]

[0081] In the formula, S is the initial area, and Sn is the unhealed area of the rat wound on the nth day.

[0082] One rat was sacrificed on the 6th day and the 12th day respectively. Some of the wounds were stained with hematoxylin-eosin. Histological evaluation was performed using light microscopy, and appearance photos were taken.

[0083] II. Experimental results:

[0084] (I) Characterization of the materials

[0085] 1. Mechanical properties

[0086] In medical hydrogel dressings, compressive strain is more important than compressive strength. We may pay attention not to squeeze the wound, but poor mechanical properties may cause the hydrogel dressing to rupture due to stretching caused by human movement. In Figure 1 A, the SBMA hydrogel of Example 1 showed brittleness, with a compressive strain of only about 35%, and was vulnerable to damage. Due to its "rigid and brittle" network structure, the SBMA hydrogel had a certain compressive capacity, and the compressive strength could reach 45.51 ± 4.42 kPa. Therefore, we introduced κ-carrageenan, hoping that it would form a double-network structure with SBMA, where SBMA served as the main network to support the overall strength of the hydrogel. When an external force was applied, the main network resisted the external force first. If the main network broke due to excessive external force, the introduced κ-carrageenan hydrogel network had enhanced toughness, which could prevent instantaneous fracture and could also bear part of the external force, thereby improving the overall mechanical properties of the hydrogel. The experimental results showed that the addition of κ-carrageenan did achieve the expected purpose, greatly enhancing the toughness of the hydrogel. The higher the content of κ-carrageenan, the better the mechanical properties of the hydrogel. For the hydrogel of Example 2, its low compressive strength might be due to the relatively small amount of κ-carrageenan introduced, failing to form a suitable double-network structure. However, the compressive strain of the hydrogel of Example 2 still exceeded that of the SBMA hydrogel, reaching about 45%. With the increase in the content of κ-carrageenan and the appropriate ratio of the two substances, a good double-network structure was formed, and the compressive strength and compressive strain increased significantly. The compressive strain of the hydrogel of Example 3 reached 70%, and the compressive strength was 51.60 ± 2.00 kPa, both higher than those of the SBMA hydrogel. It should be noted that the compressive strain of the hydrogel of Example 4 could even reach 90%, and the compressive strength was 80.58 ± 1.25 kPa. Generally speaking, the modified hydrogel met the requirements of wound dressings.

[0087] 2. Fourier transform infrared spectroscopy

[0088] Figure 2 A shows the infrared spectra of each hydrogel sample. In the spectrum of the SBMA hydrogel, the absorption peaks at 1041 cm -1 and 1187 cm -1 mainly corresponded to the symmetric and asymmetric stretching vibrations of the -S=O group in SBMA, and the absorption peak at 1481 cm -1 represented the absorption of the C-N group in -N + (CH 3 ) 2 . In addition, the peaks at 1724 cm -1 and 1654 cm -1 belonged to the stretching vibrations of C=O and C=C in SBMA respectively. In the spectrum of the κ-carrageenan hydrogel, at 3378 cm-1 There is a broad peak at [x], indicating the presence of -OH groups. At 1227 cm -1 and 925 cm -1 The peaks at [x] are characteristic of sulfate groups and 3,6-anhydro-d-galactose in κ-carrageenan, which are typical features of κ-carrageenan.

[0089] However, in the SBMA / κ-carrageenan double-network structure sample, only the characteristic peaks of SBMA are visible, probably because the absorption peaks of κ-carrageenan are smaller in size and are masked by the SBMA peaks. However, the peak value of SBMA does not change, indicating the physical mixing of the two components, confirming that the SBMA / κ-carrageenan double-network structure is a chemo-physical double-network structure.

[0090] 3. Microscopic morphology

[0091] The microstructure of the hydrogel is affected by the ratio of SBMA to κ-carrageenan. As Figure 2 shown in B, the pore sizes of each hydrogel are different and are all less than 100 μm. Studies have shown that the pore size of wound dressings should be around 20 - 125 μm, which is beneficial for fibroblast migration and promotes wound repair. Therefore, the pore sizes of these hydrogels meet the requirements of wound dressings. However, the pore distribution is uniform, which is beneficial for gas exchange on the wound surface and is beneficial for wound healing. The surface of the SBMA hydrogel presents a relatively smooth and flat appearance. As the content of κ-carrageenan increases, the surface transforms into a flaky structure. This change is due to the smaller molecular weight and shorter molecular chain of κ-carrageenan compared to SBMA, resulting in an increase in porosity and more uniform pore sizes, concentrated in the range of 50 - 80 μm. The change in structure may lead to changes in the mechanical properties of the hydrogel. The introduction of flexible κ-carrageenan and the enhancement of the toughness of the hydrogel enable it to maintain integrity under external forces. The increased space inside the hydrogel allows displacement and prevents easy rupture. On the contrary, the SBMA hydrogel is mainly composed of a solid structure with fewer pores, has strong compressive resistance, but poor overall toughness and is easy to break. It should be noted that compared with other structural hydrogels, the pore distribution and size of the hydrogel in Example 3 are more uniform. When the ratio of κ-carrageenan exceeds SBMA, the layered structure increases, resulting in local collapse and a rough surface.

[0092] 4. Equilibrium water content

[0093] Compared with traditional wound dressings, hydrogels have obvious advantages because they create a favorable wound environment similar to the cytoplasmic matrix. This environment promotes cell growth, accelerates wound healing, and prevents scab formation. To further understand the properties of hydrogels, it is necessary to evaluate their water content. The experimental results show that the water content of the hydrogel in Example 1 is the lowest, only 70.53 ± 0.96%. On the contrary, the water content increases with the increase of κ-carrageenan content. Among them, the water content of the hydrogel in Example 2 is 76.65 ± 0.99%, the water content of the hydrogel in Example 3 is 83.48 ± 0.90%, and the water content of the hydrogel in Example 4 is the highest, 88.56 ± 0.31%. It is worth noting that the water contents of the three hydrogel groups are significantly higher than that of the SBMA hydrogel group in Example 1. This phenomenon can be attributed to the larger pores in the hydrogel molecular network caused by the double network structure, and water molecules can penetrate the hydrogel network more easily, which is consistent with the observed micro-morphology of each hydrogel sample.

[0094] 5. Swelling properties

[0095] As a wound dressing, the hydrogel must have appropriate liquid absorption capacity to effectively absorb wound exudate, minimize the risk of bacterial colonization, and maintain a moist environment conducive to wound healing.

[0096] However, the swelling ability of the hydrogel should not be too large, otherwise covering the wound may cause pressure on the wound and hinder wound recovery. Therefore, it is necessary to characterize the swelling ability of the hydrogel. As can be seen from Figure 4 the data of A, the swelling ability of the SBMA hydrogel in Example 1 is poor, reaching the swelling equilibrium only within 1 hour, and the swelling rate is only 257.99 ± 31.47%. This is mainly because the molecular network structure of the simple SBMA hydrogel is compact, restricting the entry of water molecules into the hydrogel network. The swelling abilities of the double-network hydrogels in Examples 2 and 3 with different SBMA / κ-carrageenan ratios are significantly enhanced, reaching the swelling equilibrium in 6 hours, and the swelling rates are 617.25 ± 2.84% and 930.31 ± 1.34% respectively. The hydrogel in Example 4 has the highest swelling rate, reaching the swelling equilibrium within 4 hours, and the swelling rate is 1615.14 ± 14.14%. The incorporation of κ-carrageenan makes the network structure looser and the pores larger, thus increasing the water absorption rate and enhancing the swelling ability. It is worth noting that Example 4 shows the strongest swelling ability, which may be excessive for a wound dressing, may compress the wound, and hinder healing. Examples 2 and 3 show more appropriate swelling abilities and are more suitable for use as wound dressings.

[0097] 6. Influence of normal saline on the swelling properties of hydrogels

[0098] Compared with traditional materials, the SBMA hydrogel of Example 1 exhibits an "anti-polyelectrolyte effect" and has a higher swelling capacity in saline solution compared to being soaked in deionized water.

[0099] Generally, the swelling capacity of ordinary hydrogel materials weakens in physiological saline and is not suitable for highly exudative wounds. However, introducing SBMA into the double-network structure effectively counteracts this effect, making this hydrogel more advantageous as a wound dressing.

[0100] The experimental results show that the swelling rate of SBMA in physiological saline is significantly higher, reaching 401.97 ± 0.57%. In addition, with the increase in the proportion of κ-carrageenan, the saline solution has a significant effect on the swelling capacity of the hydrogel. Among them, the swelling amount of Example 2 decreased slightly (511.86 ± 0.72%), Example 3 decreased by 1 / 3 (607.50 ± 6.88%), and Example 4 decreased most significantly, only being 1 / 4 of the original swelling rate (467.30 ± 7.52%). Therefore, adding SBMA to the hydrogel effectively maintains the swelling capacity of the hydrogel and improves the absorption of wound exudate ( Figure 4 B).

[0101] (II) In vitro experiments

[0102] 1. Bacterial adhesion test

[0103] Wounds are prone to bacterial infection, and the use of antibiotics may lead to the emergence of drug-resistant superbugs. Therefore, using zwitterionic hydrogels with anti-fouling properties to prevent bacterial adsorption and achieve effective wound protection may be a more favorable choice.

[0104] The excellent water-binding ability of SBMA forms a hydrated layer on the surface of the hydrogel, effectively resisting bacterial adsorption. The experimental results show that SBMA does exhibit obvious resistance to bacterial adsorption, and only about 300 - 400 colonies were observed on the culture medium. With the decrease in the content of SBMA, the antibacterial adsorption effect weakens. Although the SBMA hydrogel of Example 1 lacks inherent bactericidal properties, it still shows low bacterial attachment, reducing the risk of wound infection and colonization. In cases where antibacterial properties are required, antibacterial agents can be incorporated into the hydrogel to achieve the desired effect. Given that the antibacterial adsorption effect of the hydrogel of Example 4 is not obvious, we will no longer conduct further research on this type of hydrogel ( Figure 5 A).

[0105] 2. Protein adsorption test

[0106] The protein adsorption test is an important indicator to measure the anti-fouling performance of the hydrogel. It has a dual role: one is to prevent wound bioflocculation and reduce bacterial infection; the other is to resist the adsorption of heterogeneous proteins and avoid specific reactions caused by wound inflammation.

[0107] Under an inverted fluorescence microscope, it can be seen that the hydrogel of Example 5 has an adsorption effect on proteins and exhibits obvious fluorescence, while the hydrogels of Example 2 and Example 3 do not show fluorescence. This observation indicates that SBMA has an anti-protein adsorption effect, and the incorporation of κ-carrageenan does not change this property( Figure 5 B).

[0108] 3. Cell adhesion assay

[0109] In this study, fibroblasts, which play a key role in the wound healing process, were used as the research object to explore the adsorption effect of the hydrogel on cells. The results showed that there was obvious cell adhesion on the surface of the hydrogel of Example 5, while no cell adhesion was observed on the surfaces of the hydrogels of Example 2 and Example 3. This indicates that the hydrogel effectively prevents cell adsorption during the wound healing process. The hydrogel wound dressing can resist the adhesion of newly formed tissues and prevent secondary wound damage that may occur during dressing change( Figure 5 C).

[0110] 4. In vitro hemolysis experiment

[0111] The hemolysis of materials is a key consideration for their biological safety. As Figure 6 shown in A, the hemolysis rates of the two hydrogels of Example 2 and Example 3 are both lower than 5%, demonstrating good safety( Figure 6 A). Therefore, these hydrogels meet the requirements of biomedical materials.

[0112] 5. Cytotoxicity

[0113] The wound healing process requires the regeneration of granulation tissue and the accumulation of matrix components such as collagen. Therefore, an ideal wound dressing should be non-toxic to wound cells and promote their growth. Fibroblasts play a crucial role in wound healing, so L929 fibroblasts are suitable for evaluating the biocompatibility of the hydrogel.

[0114] The experimental results showed that both the hydrogels of Example 2 and Example 3 had good biocompatibility with L929 cells. The hydrogels of Example 2 and Example 3 could promote cell proliferation, and the cell survival rate exceeded 120%( Figure 6 B). Therefore, when these hydrogels are used as wound dressings, they can directly interact with the wound site and promote wound healing at the same time.

[0115] (III) In vivo experiments

[0116] Generally, in terms of the overall trend, the wound healing rates of the positive control group and the hydrogel group of Example 3 were the fastest, and the difference between the two groups was extremely small. The healing effect of the hydrogel group of Example 2 was the second best, and all hydrogel groups were superior to the negative control group. The hydrogel of the Example 2 group was relatively hard in material, which might affect wound healing. It is worth noting that at 12 days, the wound healing rates of the three hydrogel groups all exceeded 90%, the healing rate of the positive control group was 94.39 ± 1.07%, and the healing rate of the hydrogel group of Example 3 was 95.18 ± 1.67%( Figure 7 B). The results indicate that the double-network hydrogel dressing has good application prospects.

[0117] H&E staining was used to more effectively observe the wound healing process. At day 6( Figure 7 C), the H&E results showed that initial signs of wound healing appeared in all groups, with fibroblasts proliferating and differentiating into epidermal tissue. Obvious epidermal tissue formation was observed in the positive control group, Example 2, and Example 3, while it was not obvious in the negative control group. It is worth noting that the degree of healing in the positive control group was comparable to that of the Example 3 group, while the newly formed epidermal tissue in the Example 2 group was thinner. In addition, collagen deposition and granulation tissue were observed to increase rapidly. Granulation tissue plays a key role in wound healing, providing growth factors, ECM, and nutrients to support the growth of fibroblasts and blood vessels, ultimately promoting wound repair. At day 12( Figure 7 D), epidermal regeneration occurred in all groups, but the thickness of the regenerated epidermis varied. The wound healing effect of the Example 3 group was the best, with the thickest epidermal tissue, followed by the positive control group, and the negative control group had the slowest healing, with epidermal tissue still accumulating.

[0118] In summary, the SBMA / κ-carrageenan double-network hydrogel wound dressing successfully prepared in this example has good long-term antibacterial and effect of promoting the healing of infected skin.

[0119] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A zwitterionic poly(sulfobetaine methacrylate) (SBMA) / kappa-carrageenan (κ-carrageenan) double-network hydrogel, Characterized in that, The networks formed by SBMA and κ-carrageenan in the hydrogel are intertwined and permeated with each other to form a double-network structure.

2. The SBMA / κ-carrageenan double-network hydrogel according to claim 1, Characterized in that, The hydrogel has a three-dimensional porous network structure with a pore size greater than or equal to 50 μm and less than or equal to 80 μm.

3. The SBMA / κ-carrageenan double-network hydrogel according to claim 1 or 2, Characterized in that, It is prepared by the following preparation method: A cross-linking agent is added to the SBMA solution to prepare a SBMA precursor solution, and then the κ-carrageenan solution is added to the hydrogel precursor solution. While maintaining at 45-55 °C (preferably at 50 °C), an initiator is added, heated, and allowed to stand to obtain the SBMA / κ-carrageenan double-network hydrogel. Optionally, finally, it is soaked in a phosphate buffer solution to remove unreacted cross-linking agent and initiator.

4. The SBMA / κ-carrageenan double-network hydrogel according to claim 3, Characterized in that, The cross-linking agent is N,N'-methylenebisacrylamide, and / or, the initiator contains ammonium persulfate and sodium metabisulfite.

5. The SBMA / κ-carrageenan double-network hydrogel according to claim 3, Characterized in that, The SBMA solution is a 40% (w / v) aqueous SBMA solution, and / or, the κ-carrageenan solution is a 1% (w / v) aqueous κ-carrageenan solution.

6. The SBMA / κ-carrageenan double-network hydrogel according to claim 4, Characterized in that, The dosage of N,N'-methylenebisacrylamide as the cross-linking agent is 2%, 4%, 6%, 8% relative to the amount of SBMA, preferably 4%; and / or, the concentrations of ammonium persulfate and sodium metabisulfite in the initiator are 40% (w / v) and 15% (w / v) respectively, and the dosage of the initiator is 10 μl per 1 g of SBMA.

7. The SBMA / κ-carrageenan double-network hydrogel according to claim 3, Characterized in that, The heating conditions are 60-80 °C for 15-45 min, preferably the heating conditions are 70 °C for 0.5 h; and / or, the standing is at room temperature for 24 hours.

8. The SBMA / κ-carrageenan double-network hydrogel according to claim 1, Characterized in that, The weight ratio of SBMA to κ-carrageenan is 0.2-0.6:0.01-0.07, preferably 0.24-0.56:0.014-0.

06.

9. Use of the SBMA / κ-carrageenan double-network hydrogel according to any one of claims 1-8 in the preparation of a dressing.

10. The use according to claim 9, Characterized in that, The dressing is used for wounds, preferably for the damaged parts of the skin or soft tissues caused by trauma or carbuncles.

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