Method for preparing tissue-adhesive hydrogel and use thereof
A gelatin/casein hydrogel was prepared by cross-linking gelatin and casein with chitosan solution using glutamin transferase. This method solves the problems of biocompatibility and preservation difficulties of existing hydrogels, achieving excellent biocompatibility and adhesion, and is suitable for skin dressings and biosensors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- TAIYUAN UNIVERSITY OF TECHNOLOGY
- Filing Date
- 2023-08-31
- Publication Date
- 2026-04-24
AI Technical Summary
Existing tissue-adhesive hydrogels have poor biocompatibility, complex preparation processes, and are difficult to preserve, making them unsuitable for effectively protecting wounds and promoting healing.
Gelatin/casein hydrogels were prepared by cross-linking gelatin and casein with glutamine transferase and then coating with chitosan solution, enabling adhesion to isolated pig skin.
The prepared hydrogel has excellent biocompatibility and degradability, and possesses properties such as water retention, antifreeze, and drug loading, making it suitable for skin dressings and biosensors.
Smart Images
Figure CN117122729B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a tissue-adhesive hydrogel and its application. Background Technology
[0002] In daily life, the skin is often injured to varying degrees (such as burns, cuts, surgical wounds, etc.). In order to make wounds heal quickly, all kinds of dressings have been developed. Traditional gauze and adhesive bandages are not very effective when dealing with wounds on joints because these dressings cannot adhere well to the wound and are easy to fall off when the joint moves.
[0003] Hydrogels have attracted widespread attention from scientists in recent years due to their unique and tunable physicochemical properties, and have been extensively studied and applied in the field of wound repair. Hydrogels with tissue adhesion can firmly adhere to the wound site and possess a certain degree of flexibility. Even when joints move at the wound site, these hydrogels will not detach, thus effectively protecting the wound and preventing external infection. In recent years, scientists have developed various strategies to prepare tissue-adhesive hydrogels, such as:
[0004] (1) Introducing adhesive polymers into hydrogel systems: Hydrogels prepared with polyphenol compounds, polymers grafted with N-hydroxysuccinimide, polymers rich in hydrogen bond donors or acceptors, etc., can have their surface groups interact with the active groups on the tissue surface to produce adhesion.
[0005] (2) Preparation of biomimetic structures: Inspired by various adhesion phenomena in nature, researchers have simulated the nanostructures on gecko foot hairs and beetle structures to prepare hydrogels with good adhesion.
[0006] (3) Prepare biomimetic mineralization transition layers to achieve strong interfacial adhesion, ultrasound-mediated adhesion, solvent exchange strategies, etc.
[0007] Although hydrogels with tissue adhesion properties are constantly being developed, they usually face the following problems: poor biocompatibility, complex preparation process, and difficulty in preservation. Summary of the Invention
[0008] The main objective of this invention is to overcome the shortcomings of the prior art and solve the technical problems of poor biocompatibility, complex preparation process, and difficult preservation of tissue-adhesive hydrogels. This invention provides a method for preparing a chitosan-mediated pure natural tissue-adhesive hydrogel and its application.
[0009] The design concept of this invention is as follows: a hydrogel is formed by cross-linking gelatin and casein with glutamin transferase; a chitosan solution is applied to pigskin, and then the hydrogel is applied on top, ultimately causing the hydrogel to adhere to the detached pigskin. When used as a wound dressing, this hydrogel can effectively cover the wound, preventing external environmental infection and thus promoting wound healing.
[0010] This invention is achieved through the following technical solution:
[0011] A method for preparing a tissue-adhesive hydrogel includes the following steps:
[0012] S1. Raw material preparation:
[0013] Preparation of mixed solution A: Dissolve gelatin particles in deionized water and stir evenly at 50°C to obtain mixed solution A for later use;
[0014] Preparation of mixed solution B: First, dissolve sodium carbonate powder in deionized water, then add casein powder. The weight ratio of sodium carbonate powder to casein powder is 1:4. Stir evenly at 50°C to obtain mixed solution B for later use.
[0015] Preparation of mixed solution C: Dissolve glutaminase powder in deionized water and stir evenly at room temperature to obtain mixed solution C for later use;
[0016] S2. First, mix the mixed solution A and mixed solution B prepared in step S1 at a volume ratio of 2:1 to obtain mixed solution D. Second, add glycerol to mixed solution D at a volume ratio of 1:10 and stir at 50°C until the solution is uniformly mixed to obtain mixed solution E. Third, add the mixed solution C prepared in step S1 to mixed solution E at a volume ratio of 1:5 and stir at room temperature until uniform. Finally, after ultrasonically defoaming the mixed solution, place it in a plastic mold and let it stand at 40°C for 3 hours to obtain gelatin / casein water-based tissue adhesive hydrogel.
[0017] Furthermore, in step S1, the concentration of gelatin in the prepared mixed solution A is 0.25 g / mL.
[0018] Furthermore, in step S1, the concentration of glutamine transferase in the prepared mixed solution C is 6%-15%.
[0019] Furthermore, in step S1, the concentration of glutamine transferase in the prepared mixed solution C is 12%.
[0020] An application of a tissue-adhesive hydrogel prepared by the method described above, wherein the gelatin / casein hydrogel is used to prepare skin dressings, tissue engineering scaffolds, or biosensors.
[0021] The application described above includes the following steps:
[0022] First, a mixed solution F was prepared: morpholine ethanesulfonic acid, chitosan, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide powder were dissolved in deionized water in sequence and stirred at room temperature for 12 hours to obtain mixed solution F; in mixed solution F: the concentration of morpholine ethanesulfonic acid was 1%, the concentration of chitosan was 1%~4%, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was 0-20 mg / mL, and the concentration of N-hydroxysuccinimide powder was 0-20 mg / mL;
[0023] Then, the mixed solution F is evenly applied to the surface of the isolated skin tissue and left to stand for 2 minutes to form a semi-solid or solidified solution F coating on the surface of the isolated skin tissue; then, the gelatin / casein aqueous tissue adhesive hydrogel is coated on the solution F coating, and the gelatin / casein aqueous tissue adhesive hydrogel adheres to the isolated skin tissue through the solution F coating.
[0024] Furthermore, in the prepared mixed solution F, the concentration of chitosan is 2%, the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 10 mg / mL, and the concentration of N-hydroxysuccinimide is 20 mg / mL.
[0025] The beneficial effects of this invention are as follows: the gelatin / casein hydrogel prepared by the process steps provided by this invention has excellent biocompatibility and degradability, and also possesses properties such as water retention, freeze resistance, drug loading, and local tissue adhesion. This high-performance natural hydrogel has significant practical value and can be applied to tissue engineering scaffolds, skin dressings, biosensors, and other fields. Attached Figure Description
[0026] Figure 1 This is a schematic diagram of a hydrogel shear test.
[0027] Figure 2 A comparison of the shear strength of G3C0 hydrogel, G2C1 hydrogel, G1C1 hydrogel, and G1C2 hydrogel prepared in Example 1;
[0028] Figure 3 This is a comparison of the shear strength measured after chitosan solutions with different EDC / NHS concentrations adhered to hydrogel and pigskin in Example 2.
[0029] Figure 4This is a comparison of the shear strength of the adhesion between hydrogels with different glycerol concentrations and isolated pigskin in Example 3;
[0030] Figure 5 This is a comparison chart of the water retention rates of the hydrogels prepared in Example 3;
[0031] Figure 6 A comparison chart of the elastic modulus of the hydrogels prepared in Example 3;
[0032] Figure 7 A comparison of the elongation at break of the hydrogel prepared in Example 3;
[0033] Figure 8 A photograph of the adhesion properties of the G2C1-10% hydrogel prepared in Example 3 to pigskin;
[0034] Figure 9 This is a drug release diagram of the hydrogel prepared in Example 4;
[0035] Figure 10 The image shows the inhibitory effect of the hydrogel prepared in Example 4 on Staphylococcus aureus and Escherichia coli. Detailed Implementation
[0036] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.
[0037] The technical terms mentioned in the following description are represented by their corresponding abbreviations:
[0038] Gelatin (G), casein (C), glutamine transferase (mTG), sodium carbonate (Na2CO3), morpholine ethanesulfonic acid (MES), chitosan (CS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), tetracycline hydrochloride (TH). Example 1
[0039] A method for preparing a tissue-adhesive hydrogel includes the following steps:
[0040] S1. Raw material preparation:
[0041] Preparation of mixed solution A: Dissolve 25g of gelatin particles in 100mL of deionized water and stir evenly at 50℃ to obtain mixed solution A for later use;
[0042] Preparation of mixed solution B: First, dissolve 2.5g of sodium carbonate powder in 100mL of deionized water, then add 10g of casein powder, stir evenly at 50℃ to obtain mixed solution B for later use;
[0043] Preparation of mixed solution C: Weigh 6.25g of glutamine transferase powder and dissolve it in 100mL of deionized water. Stir well at room temperature to obtain mixed solution C for later use.
[0044] S2. First, mix solutions A and B prepared in step S1 at volume ratios of 3:0, 2:1, 1:1, and 1:2, respectively. Stir the mixtures at 50°C until homogeneous to obtain four sets of mixed solutions D, each with a total volume of 24 mL. Second, add 2.4 mL of glycerol to each of the four sets of mixed solutions D and stir at 50°C until homogeneous to obtain four sets of mixed solutions E. Third, add 4.8 mL of mixed solution C prepared in step S1 to each of the four sets of mixed solutions E and stir at room temperature until homogeneous. Finally, after ultrasonic defoaming, place the four sets of mixed solutions in a plastic mold and let them stand at 40°C for 3 hours to obtain gelatin / casein aqueous tissue adhesive hydrogels. Based on the different ratios of gelatin (G) to casein (C), the obtained gelatin / casein aqueous tissue adhesive hydrogels are named G3C0 hydrogel, G2C1 hydrogel, G1C1 hydrogel, and G1C2 hydrogel, respectively.
[0045] An application of the tissue-adhesive hydrogel prepared by the method described in Example 1, wherein the gelatin / casein hydrogel is used to prepare skin dressings, tissue engineering scaffolds, or biosensors.
[0046] The application described above includes the following steps:
[0047] First, a mixed solution F was prepared: 0.976 g MES, 0.2 g CS, 700 μL of 1 mol / L NaOH solution, 100 mg EDC, and 100 mg NHS powder were sequentially dissolved in 10 mL of deionized water and stirred at room temperature for 12 hours to obtain mixed solution F; the concentration of CS in the prepared mixed solution F was 2%.
[0048] Then, the mixed solution F is evenly applied to the surface of the detached pigskin and left to stand for 2 minutes to form a semi-solid or solidified solution F coating on the surface of the detached pigskin; then, the gelatin / casein aqueous tissue adhesive hydrogel is coated on the solution F coating, and the gelatin / casein aqueous tissue adhesive hydrogel adheres to the detached pigskin through the solution F coating.
[0049] like Figure 1 As shown, a shear test was performed on the hydrogel prepared in Example 1. The test results of the shear test are as follows. Figure 2As shown, the shear strength of hydrogels with different gelatin-casein ratios varies, with the highest shear strength (15.37 ± 1.63 kPa) observed at a gelatin-casein ratio of 2:1. This reflects that the gelatin / casein aqueous tissue adhesive hydrogel exhibits the strongest adhesion and best adhesion to detached pig skin at a gelatin-casein ratio of 2:1. Example 2
[0050] A method for preparing a tissue-adhesive hydrogel includes the following steps:
[0051] S1. Raw material preparation:
[0052] Preparation of mixed solution A: Dissolve 25g of gelatin particles in 100mL of deionized water and stir evenly at 50℃ to obtain mixed solution A for later use;
[0053] Preparation of mixed solution B: First, dissolve 2.5g of sodium carbonate powder in 100mL of deionized water, then add 10g of casein powder, stir evenly at 50℃ to obtain mixed solution B for later use;
[0054] Preparation of mixed solution C: Weigh 6.25g of glutamine transferase powder and dissolve it in 100mL of deionized water. Stir well at room temperature to obtain mixed solution C for later use.
[0055] S2. First, mix 16 mL of mixed solution A and 8 mL of mixed solution B prepared in step S1, and stir at 50°C until the solution is homogeneous to obtain mixed solution D, with a total volume of 24 mL. Second, add 2.4 mL of glycerol to mixed solution D and stir at 50°C until the solution is homogeneous to obtain mixed solution E. Third, add 4.8 mL of mixed solution C prepared in step S1 to mixed solution E and stir at room temperature until homogeneous. Finally, after ultrasonically defoaming the mixed solution, place it in a plastic mold and let it stand at 40°C for 3 hours to obtain gelatin / casein water-based tissue adhesive hydrogel.
[0056] An application of the tissue-adhesive hydrogel prepared by the method described in Example 2, wherein the gelatin / casein hydrogel is used to prepare skin dressings, tissue engineering scaffolds, or biosensors.
[0057] The application described above includes the following steps:
[0058] First, a mixed solution F was prepared: 0.976 g MES, 0.2 g CS, 700 μL of 1 mol / L NaOH solution, EDC powder, and NHS powder were sequentially dissolved in 10 mL of deionized water. In this Example 2, four sets of experiments were set up according to the mass-volume ratio of EDC and NHS, with EDC and NHS concentrations of 0 and 0 mg / mL, 10 and 10 mg / mL, 10 and 20 mg / mL, and 20 and 20 mg / mL, respectively. The mixture was stirred at room temperature for 12 hours to obtain mixed solution F. In the prepared mixed solution F, the concentration of CS was 2%.
[0059] Then, the mixed solution F was evenly applied to the surface of the detached pigskin and left to stand for 2 minutes, forming a semi-solid or solidified coating of solution F on the surface of the detached pigskin. Next, a gelatin / casein aqueous tissue adhesive hydrogel was applied onto the solution F coating, and the gelatin / casein aqueous tissue adhesive hydrogel adhered to the detached pigskin through the solution F coating. In this Example 2, the groups were named 0 / 0, 10 / 10, 10 / 20, and 20 / 20 according to the concentrations of EDC and NHS, respectively.
[0060] Figure 3 The shear strength was measured after bonding the hydrogel and detached pigskin with chitosan solutions of different EDC / NHS concentrations (0 / 0, 10 / 10, 10 / 20, 20 / 20). The shear force between the hydrogel and detached pigskin was the largest, 37.88 ± 1.56 kPa, when the concentrations of EDC and NHS were 10 and 20 mg / mL, respectively, indicating the strongest bond. Example 3
[0061] A method for preparing a tissue-adhesive hydrogel includes the following steps:
[0062] S1. Raw material preparation:
[0063] Preparation of mixed solution A: Dissolve 25g of gelatin particles in 100mL of deionized water and stir evenly at 50℃ to obtain mixed solution A for later use;
[0064] Preparation of mixed solution B: First, dissolve 2.5g of sodium carbonate powder in 100mL of deionized water, then add 10g of casein powder, stir evenly at 50℃ to obtain mixed solution B for later use;
[0065] Preparation of mixed solution C: Weigh 6.25g of glutamine transferase powder and dissolve it in 100mL of deionized water. Stir well at room temperature to obtain mixed solution C for later use.
[0066] S2. First, mix 16 mL of mixed solution A and 8 mL of mixed solution B prepared in step S1, and stir at 50°C until the solution is homogeneous to obtain mixed solution D, with a total volume of 24 mL. Second, set up three comparative experiments, adding 0 mL, 2.4 mL, and 4.8 mL of glycerol respectively, and stirring at 50°C until the solution is homogeneous to obtain three mixed solutions E. Third, add 4.8 mL of mixed solution C prepared in step S1 to each mixed solution E, and stir at room temperature until homogeneous. Finally, after ultrasonically defoaming the mixed solutions, place them in plastic molds and let them stand at 40°C for 3 hours to obtain three groups of gelatin / casein aqueous tissue adhesive hydrogels. According to the different amounts of glycerol added in the gelatin / casein aqueous tissue adhesive hydrogel system, the gelatin / casein aqueous tissue adhesive hydrogels are divided into three groups: G2C1, G2C1-10%, and G2C1-20%.
[0067] An application of the tissue-adhesive hydrogel prepared by the method described in Example 3, wherein the gelatin / casein hydrogel is used to prepare skin dressings, tissue engineering scaffolds, or biosensors.
[0068] The application described above includes the following steps:
[0069] First, a mixed solution F was prepared: 0.976 g MES, 0.2 g CS, 700 μL of 1 mol / L NaOH solution, 100 mg EDC, and 200 mg NHS powder were dissolved sequentially in 10 mL of deionized water and stirred at room temperature for 12 hours to obtain mixed solution F; the concentration of CS in the prepared mixed solution F was 2%.
[0070] Then, the mixed solution F is evenly applied to the surface of the detached pigskin and left to stand for 2 minutes to form a semi-solid or solidified solution F coating on the surface of the detached pigskin; then, the gelatin / casein aqueous tissue adhesive hydrogel is coated on the solution F coating, and the gelatin / casein aqueous tissue adhesive hydrogel adheres to the detached pigskin through the solution F coating.
[0071] Figure 4 This refers to the shear strength of the adhesion between hydrogels containing different concentrations of glycerol and pigskin. The addition of glycerol reduces the adhesive properties of the hydrogel, but significantly improves its water retention. For example... Figure 5 As shown, the water retention capacity of the hydrogel increases with increasing glycerol concentration. The tensile properties of the hydrogel after being placed in environments of -20℃ or 37℃ for 72 hours were tested, and the results are as follows. Figure 6 , Figure 7As shown, the addition of glycerol can effectively protect the mechanical properties of the hydrogel, with minimal changes in its elastic modulus and elongation at break, thus preventing it from becoming hard due to water loss or low-temperature environments during practical applications.
[0072] Figure 8 This is a visual demonstration of the adhesive properties of the G2C1-10% hydrogel prepared in Example 3. Figures a and b show that the hydrogel can adhere tightly to the pigskin, and bending, twisting, and water flow impact will not cause them to separate. Since the hydrogel and pigskin are further adhered by the chitosan solution, local adhesion can be achieved (Figure c). When used as a wound dressing, it can avoid direct adhesion to the wound and damage to the wound. Example 4
[0073] Based on Examples 1 to 3 above, Example 4 further verifies the drug-carrying capacity of the gelatin / casein aqueous tissue adhesive hydrogel. Specifically, tetracycline hydrochloride, as a broad-spectrum antibiotic, can be loaded into the gelatin / casein aqueous tissue adhesive hydrogel to exert its antibacterial function, including the following steps:
[0074] First, prepare four beakers. Measure 100 mL of deionized water into each beaker, and weigh out 0 g, 0.01 g, 0.03 g, and 0.05 g of tetracycline hydrochloride as four comparative proportions and dissolve them in the corresponding beakers.
[0075] Next, prepare mixed solution A': Add 25g of gelatin particles to each beaker and stir evenly at 50℃ to obtain mixed solution A' for later use;
[0076] Preparation of mixed solution B: First, dissolve 2.5g of sodium carbonate powder in 100mL of deionized water, then add 10g of casein powder, stir evenly at 50℃ to obtain mixed solution B for later use;
[0077] Preparation of mixed solution C: Weigh 6.25g of glutamine transferase powder and dissolve it in 100mL of deionized water. Stir well at room temperature to obtain mixed solution C for later use.
[0078] Next, the 8 mL mixed solution B prepared in the previous step was mixed with the four groups of 16 mL mixed solutions A' respectively. Then, 2.4 mL of glycerol was added to each of the four comparative examples. The mixture was stirred at 50°C until the solution was homogeneous. Finally, 4.8 mL of solution C was added to the four comparative examples and stirred at room temperature until homogeneous.
[0079] Finally, the mixed solutions in the four comparative examples were ultrasonically defoamed and placed in plastic molds. They were then allowed to stand at 40°C for 3 hours. Based on the different concentrations of tetracycline hydrochloride (TH) in the hydrogel system, the hydrogels were divided into four groups: 0 TH, 0.01% TH, 0.03% TH, and 0.05% TH.
[0080] Drug release tests were performed on the four groups of hydrogels prepared in Example 4, and the results are as follows: Figure 9 As shown in the figure, the drug is released rapidly and in large quantities on the first day, after which the release becomes slower. The amount of drug released from the hydrogel increases with increasing drug loading. Subsequently, an inhibition zone experiment was conducted to assess the antibacterial effect of the hydrogel; the results are shown in the figure. Figure 10 As shown, the loading of tetracycline hydrochloride gives the hydrogel a good antibacterial effect, inhibiting both Staphylococcus aureus and Escherichia coli.
[0081] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for preparing a tissue-adhesive hydrogel, characterized in that, Includes the following steps: S1. Raw material preparation: Preparation of mixed solution A: Dissolve gelatin particles in deionized water and stir evenly at 50°C to obtain mixed solution A for later use; Preparation of mixed solution B: First, dissolve sodium carbonate powder in deionized water, then add casein powder. The weight ratio of sodium carbonate powder to casein powder is 1:
4. Stir evenly at 50°C to obtain mixed solution B for later use. Preparation of mixed solution C: Dissolve glutaminase powder in deionized water and stir evenly at room temperature to obtain mixed solution C for later use; S2. First, mix the mixed solution A and mixed solution B prepared in step S1 at a volume ratio of 2:1 to obtain mixed solution D. Second, add glycerol to mixed solution D at a volume ratio of 1:10 and stir at 50°C until the solution is uniformly mixed to obtain mixed solution E. Third, add the mixed solution C prepared in step S1 to mixed solution E at a volume ratio of 1:5 and stir at room temperature until uniform. Finally, after ultrasonically defoaming the mixed solution, place it in a plastic mold and let it stand at 40°C for 3 hours to obtain gelatin / casein tissue adhesive hydrogel.
2. The method for preparing a tissue-adhesive hydrogel as described in claim 1, characterized in that: In step S1, the concentration of gelatin in the prepared mixed solution A is 0.25 g / mL.
3. The method for preparing a tissue-adhesive hydrogel as described in claim 1, characterized in that: In step S1, the concentration of glutamine transferase in the prepared mixed solution C is 6%-15%.
4. The method for preparing a tissue-adhesive hydrogel as described in claim 3, characterized in that: In step S1, the concentration of glutamine transferase in the prepared mixed solution C is 12%.
5. An application of a tissue-adhesive hydrogel prepared by the method described in claim 1, characterized in that: The gelatin / casein aqueous tissue adhesive hydrogel is used to prepare skin dressings, tissue engineering scaffolds, or biosensors.