CN117122729A - Preparation method and application of tissue-adhesive hydrogel - Google Patents

Abstract

The invention relates to a preparation method of tissue-adhesive hydrogel and its application. It belongs to the field of biomedicine technology and solves technical problems such as poor biocompatibility of tissue-adhesive hydrogel, complex preparation process, and difficulty in preservation. , the solution is: cross-link gelatin and casein through glutamine transferase to form a hydrogel; apply a layer of chitosan solution on the isolated pig skin, and then cover the hydrogel to finally make the hydrogel Glue and detached pig skin stick together. The tissue-adhesive hydrogel prepared by the invention has good biocompatibility and degradability, and has properties such as transparency, water retention, anti-freeze, drug loading, and local adhesion to tissues. This kind of natural hydrogel with excellent performance has more practical significance and can be used in tissue engineering scaffolds, skin dressings, biosensors, etc.

Description

Preparation method and application of tissue-adhesive hydrogel

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to a preparation method and application of tissue-adhesive hydrogel.

Background technique

In daily life, the skin will always be damaged to varying degrees (such as burns, cuts, surgical wounds, etc.). In order to heal the wounds quickly, various dressings have been developed. Traditional gauze patches and band-aids are weak when facing joint wounds, because these dressings cannot fit the wound well and can easily fall off when the joint moves.

Hydrogels have attracted widespread attention from scientists in recent years due to their unique and controllable physical and chemical properties, and have been widely studied and applied in the field of wound repair. The tissue-adhesive hydrogel can adhere firmly to the wound and has a certain degree of flexibility. This hydrogel will not fall off when the joints at the wound move, thereby effectively protecting the wound and preventing the wound from being infected by the external environment. In recent years, scientists have developed various strategies to prepare tissue-adhesive hydrogels, such as:

(1) Introducing polymers that can produce adhesion into the hydrogel system: polyphenol compounds, polymers grafted with N-hydroxysuccinimide, polymers rich in hydrogen bond donors or acceptors The surface groups of hydrogels prepared from other substances can interact with active groups on the tissue surface to cause adhesion;

(2) Preparation of bionic structures: Inspired by various adhesion phenomena in nature, researchers simulated the nanostructures on gecko foot hairs, beetle structures, etc. to prepare hydrogels to make them have good adhesion;

(3) Preparing a biomimetic mineralized transition layer to achieve strong interfacial adhesion, ultrasound-mediated adhesion, solvent exchange strategies, etc.

Although hydrogels with tissue adhesion are currently being continuously researched, they usually face the following problems: poor biocompatibility, complex preparation processes, and difficulty in preservation.

Contents of the invention

The main purpose of the present invention is to overcome the deficiencies in the prior art and solve technical problems such as poor biocompatibility of tissue-adhesive hydrogels, complex preparation processes, and difficulty in preservation. The present invention provides a chitosan-mediated Preparation method and application of pure natural tissue adhesive hydrogel.

The design concept of the present invention is: cross-link gelatin and casein through glutamine transferase to form a hydrogel; apply a layer of chitosan solution on pig skin, and then cover the hydrogel to finally make the hydrogel Glue and detached pig skin stick together. When used as a wound dressing, the hydrogel can cover the wound well and prevent the wound from being infected by the external environment, thereby promoting wound healing.

The present invention is realized through the following technical solutions:

A preparation method of tissue-adhesive hydrogel, including the following steps:

S1. Raw material preparation:

Prepare mixed solution A: Dissolve gelatin particles in deionized water, stir evenly at 50°C, and prepare mixed solution A for later use;

Prepare 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 prepare a mixed solution B is reserved for later use;

Prepare mixed solution C: Dissolve glutamine transferase powder in deionized water, stir evenly at room temperature, and prepare mixed solution C for later use;

S2. First, mix the mixed solution A and the mixed solution B prepared in step S1 at a volume ratio of 2:1 to prepare a mixed solution D; secondly, add glycerol to the mixed solution D, and the volume ratio of glycerin to the mixed solution D is 1 :10, and stir until the solution is evenly mixed at 50°C to prepare mixed solution E; again, add mixed solution C prepared in step S1 to mixed solution E, and the volume ratio of mixed solution C to mixed solution D is 1 :5, stir at room temperature until uniform; finally, defoam the mixed solution ultrasonically and place it in a plastic mold, and let it stand for 3 hours at a temperature of 40°C to prepare a gelatin/casein water tissue-adhesive hydrogel.

Further, in step S1, the concentration of gelatin in the prepared mixed solution A is 0.25g/mL.

Further, in step S1, the concentration of transglutaminase in the prepared mixed solution C is 6%-15%.

Further, in step S1, the concentration of transglutaminase in the prepared mixed solution C is 12%.

An application of tissue-adhesive hydrogel prepared by the above method, wherein: the gelatin/casein water tissue-adhesive hydrogel is used to prepare skin dressings, tissue engineering scaffolds or biosensors.

Application as described above involves the following steps:

First, prepare mixed solution F: morpholinoethanesulfonic acid, chitosan, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide powder according to Dissolve in deionized water in sequence and stir at room temperature for 12 hours to prepare mixed solution F; in mixed solution F: the concentration of morpholinoethanesulfonic acid is 1%, the concentration of chitosan is 1%~4%, 1- The concentration of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0-20mg/mL, and the concentration of N-hydroxysuccinimide powder is 0-20mg/mL;

Then, the mixed solution F is evenly applied to the surface of the isolated skin tissue and left for 2 minutes to form a semi-solidified or solidified solution F coating on the surface of the isolated skin tissue; then, the gelatin/casein water tissue adhesion The hydrogel is coated on the solution F coating, and the gelatin/casein water tissue-adhesive hydrogel is bonded to the ex vivo skin tissue through the solution F coating.

Further, in the prepared mixed solution F, the concentration of chitosan is 2%, and the concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 10 mg/mL, The concentration of N-hydroxysuccinimide is 20 mg/mL.

The beneficial effects of the present invention are: the gelatin/casein hydrogel prepared through the process steps provided by the present invention has excellent biocompatibility and degradability, and has the properties of water retention, antifreeze, drug loading, and local adhesion to tissues. Additional performance. This kind of natural hydrogel with excellent performance has more practical significance and can be used in tissue engineering scaffolds, skin dressings, biosensors, etc.

Description of the drawings

Figure 1 is a schematic diagram of the hydrogel shear test test;

Figure 2 is a comparison chart of the shear strength of G3C0 hydrogel, G2C1 hydrogel, G1C1 hydrogel, and G1C2 hydrogel prepared in Example 1;

Figure 3 is a comparison chart of the shear strength measured after adhesion of hydrogel and pig skin with chitosan solutions of different EDC/NHS concentrations in Example 2;

Figure 4 is a comparative diagram of the shear strength of the bond between hydrogels containing different glycerin concentrations and isolated pig skin in Example 3;

Figure 5 is a comparison chart of the water retention rate of the hydrogel prepared in Example 3;

Figure 6 is a comparison chart of the elastic modulus of the hydrogel prepared in Example 3;

Figure 7 is a comparative chart of the elongation at break of the hydrogel prepared in Example 3;

Figure 8 is a physical photo of the adhesion performance between the G2C1-10% hydrogel prepared in Example 3 and pig skin;

Figure 9 is a drug release chart of the hydrogel prepared in Example 4;

Figure 10 is a diagram showing the inhibitory effect of the hydrogel prepared in Example 4 on Staphylococcus aureus and Escherichia coli.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and examples.

The technical terms mentioned in the following description are represented by their corresponding abbreviations:

Gelatin (G), casein (C), transglutaminase (mTG), sodium carbonate (Na ₂ CO ₃ ), morpholinoethanesulfonic acid (MES), chitosan (CS), 1-ethyl- 3-(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), tetracycline hydrochloride (TH).

Example 1

A preparation method of tissue-adhesive hydrogel, including the following steps:

S1. Raw material preparation:

Prepare mixed solution A: Dissolve 25g gelatin particles in 100mL deionized water, stir evenly at 50°C, and prepare mixed solution A for later use;

Prepare mixed solution B: first dissolve 2.5g sodium carbonate powder in 100mL deionized water, then add 10g casein powder, stir evenly at 50°C, and prepare mixed solution B for later use;

Prepare mixed solution C: Weigh 6.25g of transglutaminase powder and dissolve it in 100 mL of deionized water, stir evenly at room temperature, and prepare mixed solution C for later use;

S2. First, mix the mixed solution A and the mixed solution B prepared in step S1 according to the volume ratio of 3:0, 2:1, 1:1, and 1:2 respectively. The mixed solutions in each ratio are stirred at a temperature of 50°C until the solution is Mix evenly to prepare four groups of mixed solutions D. The total volume of each group of mixed solutions D is 24 mL. Secondly, add 2.4 mL of glycerin to the four groups of mixed solutions D, and stir at a temperature of 50°C until the solutions are evenly mixed. , to prepare four groups of mixed solutions E; again, add 4.8 mL of the mixed solution C prepared in step S1 to the four groups of mixed solutions E, and stir at room temperature until uniform; finally, the four groups of mixed solutions are ultrasonically defoamed and placed in a plastic mold , and let it stand for 3 hours at a temperature of 40°C to prepare a gelatin/casein water tissue-adhesive hydrogel. According to the different ratios of gelatin (G) and casein (C), the prepared gelatin/casein water tissue adhesive hydrogels were named G3C0 hydrogel, G2C1 hydrogel, G1C1 hydrogel, and G1C2. Hydrogels.

An application of tissue-adhesive hydrogel prepared by the method described in Example 1, wherein: the gelatin/casein water tissue-adhesive hydrogel is used to prepare skin dressings, tissue engineering scaffolds or biological sensor.

Application as described above involves the following steps:

First, prepare mixed solution F: 0.976g MES, 0.2g CS, 700 μL NaOH solution with a concentration of 1 mol/L, 100 mg EDC, and 100 mg NHS powder were dissolved in 10 mL deionized water in order, and stirred at room temperature for 12 hours to prepare a mixed solution. Solution F; In the prepared mixed solution F, the concentration of CS is 2%;

Then, apply the mixed solution F evenly on the surface of the isolated pig skin and let it stand for 2 minutes to form a semi-solidified or solidified solution F coating on the surface of the isolated pig skin; then, the gelatin/casein water tissue adhesion The hydrogel is coated on the solution F coating, and the gelatin/casein water tissue-adhesive hydrogel is bonded to the isolated pig skin through the solution F coating.

As shown in Figure 1, the hydrogel prepared in Example 1 was subjected to a shear test. The test results of the shear test are shown in Figure 2. The shear strength of hydrogels with different gelatin to casein ratios is different. The shear strength is the highest when the gelatin and casein ratio is 2:1, which is 15.37±1.63kPa. This also reflects that when the ratio of gelatin to casein is 2:1, the gelatin/casein water tissue adhesive hydrogel has the greatest bonding force with isolated pig skin and has the best adhesion.

Example 2

A preparation method of tissue-adhesive hydrogel, including the following steps:

S1. Raw material preparation:

Prepare mixed solution A: Dissolve 25g gelatin particles in 100mL deionized water, stir evenly at 50°C, and prepare mixed solution A for later use;

Prepare mixed solution B: first dissolve 2.5g sodium carbonate powder in 100mL deionized water, then add 10g casein powder, stir evenly at 50°C, and prepare mixed solution B for later use;

Prepare mixed solution C: Weigh 6.25g of transglutaminase powder and dissolve it in 100 mL of deionized water, stir evenly at room temperature, and prepare mixed solution C for later use;

S2. First, mix 16 mL of mixed solution A and 8 mL of mixed solution B prepared in step S1, and stir at a temperature of 50°C until the solutions are evenly mixed to prepare mixed solution D. The total volume of mixed solution D is 24 mL; secondly, add Add 2.4 mL of glycerin to mixed solution D, and stir at 50°C until the solution is evenly mixed to prepare mixed solution E; again, add 4.8 mL of mixed solution C prepared in step S1 to mixed solution E, and stir at room temperature until uniform. ; Finally, the mixed solution was placed in a plastic mold after ultrasonic defoaming, and left to stand at 40°C for 3 hours to prepare a gelatin/casein water tissue-adhesive hydrogel.

An application of tissue-adhesive hydrogel prepared by the method described in Example 2, wherein: the gelatin/casein water tissue-adhesive hydrogel is used to prepare skin dressings, tissue engineering scaffolds or biological sensor.

Application as described above involves the following steps:

First, prepare mixed solution F: 0.976g MES, 0.2g CS, 700 μL NaOH solution with a concentration of 1 mol/L, EDC powder, and NHS powder were dissolved in 10 mL deionized water in order. In Example 2, according to the Four sets of tests were set up for the mass volume ratio. The concentrations of EDC and NHS were: 0 and 0 mg/mL, 10 and 10 mg/mL, 10 and 20 mg/mL, 20 and 20 mg/mL. Stir at room temperature for 12 hours to prepare a mixed solution. F; In the prepared mixed solution F, the concentration of CS is 2%;

Then, apply the mixed solution F evenly on the surface of the isolated pig skin and let it stand for 2 minutes to form a semi-solidified or solidified solution F coating on the surface of the isolated pig skin; then, the gelatin/casein water tissue adhesion The hydrogel is coated on the solution F coating, and the gelatin/casein water tissue-adhesive hydrogel is bonded to the isolated pig skin through the solution F coating. In this Example 2, four groups were named 0/0, 10/10, 10/20, and 20/20 according to the concentrations of EDC and NHS respectively.

Figure 3 shows the shear strength measured after using chitosan solutions with different EDC/NHS concentrations (0/0, 10/10, 10/20, 20/20) to adhere hydrogel and isolated pig skin. When the concentrations of EDC and NHS were 10 and 20 mg/mL, the shear force between the hydrogel and the isolated pig skin was the largest at 37.88±1.56 kPa, and the bond was the tightest.

Example 3

A preparation method of tissue-adhesive hydrogel, including the following steps:

S1. Raw material preparation:

Prepare mixed solution A: Dissolve 25g gelatin particles in 100mL deionized water, stir evenly at 50°C, and prepare mixed solution A for later use;

Prepare mixed solution B: first dissolve 2.5g sodium carbonate powder in 100mL deionized water, then add 10g casein powder, stir evenly at 50°C, and prepare mixed solution B for later use;

Prepare mixed solution C: Weigh 6.25g of transglutaminase powder and dissolve it in 100 mL of deionized water, stir evenly at room temperature, and prepare mixed solution C for later use;

S2. First, mix 16 mL of mixed solution A and 8 mL of mixed solution B prepared in step S1, stir at a temperature of 50°C until the solutions are evenly mixed, and prepare mixed solution D. The total volume of mixed solution D is 24 mL; secondly, set Three groups of comparative tests were performed. In the comparative tests, 0mL, 2.4mL, and 4.8mL of glycerol were added respectively, and the solutions were stirred at 50°C until the solutions were evenly mixed to prepare three groups of mixed solutions E; again, add 0mL, 2.4mL, and 4.8mL of glycerin to each group of mixed solutions E. 4.8 mL of mixed solution C prepared in step S1 was stirred at room temperature until uniform; finally, the mixed solution was ultrasonically defoamed and placed in a plastic mold, and left to stand at 40°C for 3 hours to prepare three groups of gelatin/casein water Tissue-adhesive hydrogels. According to the different amounts of glycerol added in the gelatin/casein water tissue adhesive hydrogel system, the gelatin/casein water tissue adhesive hydrogel is divided into three groups: G2C1, G2C1-10%, and G2C1-20%. .

An application of tissue-adhesive hydrogel prepared by the method described in Example 3, wherein: the gelatin/casein water tissue-adhesive hydrogel is used to prepare skin dressings, tissue engineering scaffolds or biological sensor.

Application as described above involves the following steps:

First, prepare mixed solution F: 0.976g MES, 0.2g CS, 700 μL NaOH solution with a concentration of 1 mol/L, 100 mg EDC, and 200 mg NHS powder were dissolved in 10 mL deionized water in order, and stirred at room temperature for 12 hours to prepare a mixed solution. Solution F; In the prepared mixed solution F, the concentration of CS is 2%;

Then, apply the mixed solution F evenly on the surface of the isolated pig skin and let it stand for 2 minutes to form a semi-solidified or solidified solution F coating on the surface of the isolated pig skin; then, the gelatin/casein water tissue adhesion The hydrogel is coated on the solution F coating, and the gelatin/casein water tissue-adhesive hydrogel is bonded to the isolated pig skin through the solution F coating.

Figure 4 shows the shear strength of the bond between hydrogel containing different glycerin concentrations and pig skin. The addition of glycerin reduces the adhesion performance of the hydrogel, but greatly improves the water retention performance of the hydrogel. As shown in Figure 5, as the glycerol concentration increases, the water retention performance of the hydrogel increases. The tensile properties of the hydrogel were tested after being placed in an environment of -20°C or 37°C for 72 hours. The results are shown in Figures 6 and 7. The addition of glycerin can well protect the mechanical properties of the hydrogel and its elasticity. The modulus and elongation at break have small changes, which prevents it from becoming hard due to water loss or low temperature environment in practical applications.

Figure 8 is a visual display of the adhesive properties of the G2C1-10% hydrogel prepared in Example 3. Figures a and b show that the hydrogel can adhere closely to pig skin, bending, twisting, and water flow. No shock will separate the two. Since the hydrogel and pig skin are further adhered to each other through 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 cause wound damage.

Example 4

On the basis of the above-mentioned Examples 1 to 3, this Example 4 further conducts a verification test on the drug loadability of the gelatin/casein water tissue-adhesive hydrogel, specifically: tetracycline hydrochloride as a broad-spectrum antibiotic It can be loaded into gelatin/casein water tissue-adhesive hydrogel to exert antibacterial function, including the following steps:

First, prepare four beakers, measure 100 mL of deionized water in each beaker, and weigh 0g, 0.01g, 0.03g, and 0.05g tetracycline hydrochloride as four sets of comparative examples and dissolve them in the corresponding beakers;

Secondly, prepare mixed solution A’: add 25g gelatin particles to each beaker, stir evenly at 50°C, and prepare mixed solution A’ for later use;

Prepare mixed solution B: first dissolve 2.5g sodium carbonate powder in 100mL deionized water, then add 10g casein powder, stir evenly at 50°C, and prepare mixed solution B for later use;

Prepare mixed solution C: Weigh 6.25g of transglutaminase powder and dissolve it in 100 mL of deionized water, stir evenly at room temperature, and prepare mixed solution C for later use;

Again, mix the 8 mL mixed solution B prepared in the previous step with the four groups of 16 mL mixed solutions A', then add 2.4 mL of glycerol to the four groups of comparative examples, stir at 50°C until the solutions are evenly mixed, and finally add 2.4 mL of glycerin to the four groups of comparative examples. Add 4.8 mL of solution C to the comparative example and stir at room temperature until uniform;

Finally, the mixed solutions in the four groups of comparative examples were placed in plastic molds after ultrasonic defoaming, and left to stand for 3 hours at a temperature of 40°C. According to the different concentrations of tetracycline hydrochloride (TH) in the hydrogel system, the water was The gel is divided into four groups: 0 TH, 0.01% TH, 0.03% TH, and 0.05% TH.

Four groups of hydrogels prepared in Example 4 were subjected to drug release tests, and the results are shown in Figure 9. The drug is released quickly and in large amounts within the first day, after which the drug begins to be released slowly. As the drug loading capacity increases, the drug release amount of the hydrogel also increases. After that, an inhibition zone experiment was conducted on the antibacterial effect of the hydrogel. The experimental results are shown in Figure 10. The loading of tetracycline hydrochloride gave the hydrogel a good bacterial effect and inhibited Staphylococcus aureus and Escherichia coli. Effect.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily imagined by those skilled in the technical field within the technical scope disclosed in the present invention are all should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (7)

1. A method for preparing a tissue-adhesive hydrogel, comprising the steps of:

s1, preparing raw materials:

preparing a mixed solution A: dissolving gelatin particles in deionized water, and uniformly stirring at 50 ℃ to prepare a mixed solution A for later use;

preparing a mixed solution B: firstly, dissolving sodium carbonate powder into deionized water, then adding casein powder, wherein the weight ratio of the sodium carbonate powder to the casein powder is 1:4, and uniformly stirring at 50 ℃ to prepare a mixed solution B for later use;

preparing a mixed solution C: dissolving the transglutaminase powder in deionized water, and uniformly stirring at room temperature to prepare a mixed solution C for later use;

s2, firstly, mixing the mixed solution A prepared in the step S1 and the mixed solution B according to the volume ratio of (1-3) (0-2) to prepare a mixed solution D; secondly, adding glycerol into the mixed solution D, wherein the volume ratio of the glycerol to the mixed solution D is 1:10, and stirring the mixed solution at 50 ℃ until the mixed solution is uniformly mixed to prepare a mixed solution E; thirdly, adding the mixed solution C prepared in the step S1 into the mixed solution E, wherein the volume ratio of the mixed solution C to the mixed solution D is 1:5, and stirring at room temperature until the mixed solution is uniform; finally, the mixed solution is placed in a plastic mould after ultrasonic bubble removal, and is kept stand for 3 hours at the temperature of 40 ℃ to prepare the gelatin/casein water tissue adhesive hydrogel.

2. A method of preparing a tissue-adhesive hydrogel according to claim 1, wherein: in the step S1, the concentration of gelatin in the prepared mixed solution A was 0.25g/mL.

3. A method of preparing a tissue-adhesive hydrogel according to claim 1, wherein: in the step S1, the concentration of the transglutaminase in the prepared mixed solution C is 6% -15%.

4. A method of preparing a tissue-adhesive hydrogel according to claim 3, wherein: in the step S1, the concentration of the transglutaminase in the prepared mixed solution C was 12%.

5. Use of a tissue-adhesive hydrogel obtainable by the method of claim 1, wherein: the gelatin/casein water tissue adhesive hydrogel is used for preparing skin dressing, tissue engineering scaffold or biosensor.

6. The use according to claim 5, characterized by the steps of:

first, a mixed solution F: sequentially dissolving morpholine ethanesulfonic acid, chitosan, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide powder in deionized water, and stirring at room temperature for 12 hours to obtain a mixed solution F; mixed solution F: the concentration of morpholinoethanesulfonic acid is 1%, the concentration of chitosan is 1% -4%, the concentration of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is 0-20mg/mL, and the concentration of N-hydroxysuccinimide powder is 0-20mg/mL;

then, uniformly coating the mixed solution F on the surface of the isolated skin tissue and standing for 2 minutes to form a semi-coagulated or coagulated solution F coating on the surface of the isolated skin tissue; the gelatin/casein water tissue-adhesive hydrogel is then coated on the solution F coating, through which the gelatin/casein water tissue-adhesive hydrogel adheres to the ex vivo skin tissue.

7. The use according to claim 6, characterized in that: in the prepared mixed solution F, the concentration of chitosan is 2%, the concentration of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is 10mg/mL, and the concentration of N-hydroxysuccinimide is 20mg/mL.