High-strength γ-polyglutamic acid / gelatin double network hydrogel and its preparation method and application

Abstract

The present invention provides a high-strength γ-polyglutamic acid / gelatin double-network hydrogel and its preparation method and application. γ-polyglutamic acid gelatin is used as a raw material, and γ-polyglutamic acid and ethylene glycol diglycidyl ether are covalently cross-linked to form a chemical cross-linked network. The product is then cooled, allowing the gelatin component in the system to form a physical cross-linked network at low temperatures, thereby forming a double-network hydrogel. The hydrogel obtained by the present invention is used for wound treatment and has a significant promoting effect on accelerating wound healing. In addition, the obtained hydrogel has super strong compressive properties, with a compressive strength of up to 40MPa, and is expected to be widely used in various medical environments.

Description

Technical Field

[0001] The invention belongs to the technical field of double network hydrogels, and in particular relates to a high-strength gamma-polyglutamic acid / gelatin double network hydrogel and a preparation method and application thereof. Background Art

[0002] Hydrogels are polymers with a high-water content, three-dimensional network structure. They are soft and skin-friendly, with internal hydrophilic groups that can absorb large amounts of water. Similar to the extracellular matrix, hydrogels allow biomolecules to diffuse and transfer through their porous network. Consequently, they have been used in a variety of biomedical fields, such as wound dressings, tissue engineering scaffolds, and drug delivery systems. Compared to traditional wound dressings, hydrogel wound dressings offer numerous advantages, such as absorbing wound exudate, maintaining moisture around the wound, preventing secondary damage during dressing changes, and being breathable and oxygen-permeable, effectively promoting wound healing.

[0003] γ-Polyglutamic acid (γ-PGA) is a polypeptide polymer formed by condensing L-glutamic acid and D-glutamic acid monomers through α-amino and γ-carboxyl groups in the form of amide bonds. γ-PGA has good biocompatibility and biodegradability, which is conducive to cell adhesion and proliferation. It is a biomaterial with great application prospects. Although γ-PGA has many advantages, its hydrogel still has the disadvantage of insufficient mechanical strength, which limits its application in fields such as wound dressings. Therefore, the preparation of γ-PGA-based hydrogel wound dressings with both good biocompatibility and mechanical properties remains a challenge.

[0004] Gelatin (GEL), a partially hydrolyzed product of collagen, has excellent biocompatibility and low immunogenicity, and is widely used in the biomedical field. Although single gelatin hydrogels have poor mechanical properties, due to their excellent low-temperature gelling properties, they are often introduced as a physical cross-linking network into composite hydrogels to enhance biocompatibility and mechanical properties.

[0005] Double-network hydrogels are considered an effective way to improve the mechanical properties of traditional single-network hydrogels. However, traditional double-network hydrogels usually require two chemical networks to form, and the synthesis steps are cumbersome. Summary of the Invention

[0006] The present invention proposes a high-strength γ-polyglutamic acid / gelatin double-network hydrogel, its preparation method and application. The introduction of a physical network simplifies the preparation process, significantly improves the mechanical properties of the hydrogel, and solves the current technical problems of γ-PGA hydrogel, such as insufficient mechanical strength at high water content and complicated synthesis.

[0007] The present invention provides a method for preparing a high-strength γ-polyglutamic acid / gelatin double-network hydrogel, comprising the following steps:

[0008] 1) dissolving γ-polyglutamic acid in deionized water to obtain a γ-polyglutamic acid solution;

[0009] 2) dissolving gelatin in the above-mentioned γ-polyglutamic acid solution, adding a cross-linking agent (ethylene glycol diglycidyl ether), adjusting the pH, and stirring to obtain a mixed solution;

[0010] 3) ultrasonically treating the mixed solution, removing bubbles in vacuo, and reacting the solution. The resulting hydrogel was allowed to stand and cooled to obtain a high-strength γ-polyglutamic acid / gelatin double-network hydrogel.

[0011] Furthermore, in step 1), the temperature of the deionized water is 40-60°C.

[0012] Furthermore, the mass ratio of γ-polyglutamic acid to gelatin is 0.1-10 g:1-10 g;

[0013] Preferably, the mass ratio of γ-polyglutamic acid to gelatin is 0.5-5.5 g:1-6 g.

[0014] Furthermore, in step 2), the mass of ethylene glycol diglycidyl ether is 30%-60% of the mass of γ-polyglutamic acid.

[0015] Furthermore, in step 2), the pH is adjusted in the range of 4-6.

[0016] Furthermore, in step 3), the ultrasonication time is 20 min.

[0017] Furthermore, in step 3), the reaction temperature is 40-60° C.; and the reaction time is 6-20 h.

[0018] Furthermore, in step 3), the reaction is placed in a reaction mold for reaction.

[0019] Furthermore, in step 3), the cooling condition is cooling at 4° C. for 2-12 hours.

[0020] The present invention also provides a high-strength γ-polyglutamic acid / gelatin double-network hydrogel prepared by any of the above preparation methods.

[0021] The present invention also proposes the use of the high-strength γ-polyglutamic acid / gelatin double network hydrogel prepared by any of the above preparation methods or any of the above high-strength γ-polyglutamic acid / gelatin double network hydrogels in the preparation of wound dressings for wound healing.

[0022] The present invention has the following advantages:

[0023] The present invention proposes a method for preparing a γ-polyglutamic acid / gelatin (γ-PGA / GEL) double-network hydrogel, which uses γ-polyglutamic acid and gelatin as raw materials, adds ethylene glycol diglycidyl ether as a cross-linking agent for γ-polyglutamic acid, and gelatin participates in the formation of the double-network hydrogel through physical network interaction. In addition, electrostatic interaction can also be generated between the network structure based on γ-polyglutamic acid and the network structure based on gelatin. By controlling the ratio between γ-polyglutamic acid and gelatin, a clever combination of chemically cross-linked network structure and physically cross-linked network structure is achieved, thereby obtaining a high-strength double-network hydrogel. The method is simple to operate. After mixing γ-polyglutamic acid, gelatin, and the cross-linking agent ethylene glycol diglycidyl ether and reacting them, a high-compressive-strength double-network hydrogel can be prepared through a "one-pot process."

[0024] The γ-PGA / GEL dual-network hydrogel proposed in this study is composed of natural polymer materials and exhibits excellent biocompatibility and biodegradability, as well as self-healing properties, promoting wound healing. Furthermore, the resulting hydrogel exhibits excellent compressive and tensile properties, particularly exceptional compressive strength of up to 40 MPa. This makes it a highly promising high-strength hydrogel wound dressing, expected to find widespread application in various medical settings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 is the compressive stress-strain curve of γ-PGA / GEL double network hydrogel;

[0027] Figure 2 is the tensile stress-strain curve of γ-PGA / GEL double network hydrogel;

[0028] Figure 3 This is a picture of the γ-PGA / GEL double network hydrogel self-healing sample;

[0029] Figure 4 The results of in vitro cell biocompatibility test of γ-PGA / GEL double network hydrogel are shown in Figure 2. Figure 4 (a) CCK-8 assay for the proliferation of L929 fibroblasts cultured in hydrogel extract for 1, 3, and 5 days; Figure 4 (b) Fluorescence staining of L929 fibroblasts cultured with hydrogel extract for 3 days; scale bar: 100 μm;

[0030] Figure 5 This is a diagram showing the wound healing effect after treatment with hydrogel dressing. Figure 5 (a) Photos of wounds in different groups on days 0, 4, 7, 10 and 14 of treatment. Figure 5 (b) Percentage of wound healing at different time points of treatment. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The embodiments of the present invention and the features in the embodiments can be combined with each other unless there is a conflict.

[0032] Example 1 A method for preparing a double-network γ-polyglutamic acid hydrogel comprises the following steps:

[0033] Weigh 2.5g of γ-PGA powder, dissolve it in 44ml of deionized water, stir it magnetically and dissolve it completely, then add 4g of gelatin, stir it magnetically and dissolve it completely, add 1.5g of ethylene glycol diglycidyl ether, and adjust the pH to 5 to allow γ-PGA and ethylene glycol diglycidyl ether to undergo a cross-linking reaction, then pour the mixed solution into a mold, place it in a constant temperature and humidity chamber at 50℃ and react for 8h. After it forms a gel, take it out and then cool the hydrogel at 4℃ for 2h to obtain a high-strength γ-PGA / GEL double network hydrogel, recorded as γ-PGA / GEL-4.

[0034] Example 2 Preparation method of double-network gamma-polyglutamic acid hydrogel

[0035] Same as Example 1, except that 1.5 g of γ-PGA powder, 5 g of gelatin, and 0.9 g of ethylene glycol diglycidyl ether were used. This was designated as γ-PGA / GEL-5.

[0036] Example 3 Preparation method of double-network gamma-polyglutamic acid hydrogel

[0037] Same as Example 1, except that 3.5 g of γ-PGA powder, 3 g of gelatin, and 2.1 g of ethylene glycol diglycidyl ether were used. This was designated as γ-PGA / GEL-3.

[0038] Example 4 Preparation method of double-network gamma-polyglutamic acid hydrogel

[0039] Same as Example 1, except that 4.5 g of γ-PGA powder, 2 g of gelatin, and 2.7 g of ethylene glycol diglycidyl ether were used. This was designated as γ-PGA / GEL-2.

[0040] Example 5 Preparation method of double-network gamma-polyglutamic acid hydrogel

[0041] Same as Example 1, except that 5.5 g of γ-PGA powder, 1 g of gelatin, and 3.3 g of ethylene glycol diglycidyl ether were used. This was designated as γ-PGA / GEL-1.

[0042] Comparative Example 1 A preparation method of gamma-polyglutamic acid hydrogel

[0043] The same method as Example 1 was used, except that 6.5 g of γ-PGA powder, 0 g of gelatin, and 3.9 g of ethylene glycol diglycidyl ether were used. A single-network γ-polyglutamic acid hydrogel was obtained, designated as γ-PGA.

[0044] Comparative Example 2 A preparation method of gelatin hydrogel

[0045] Weigh 6.5 g of gelatin powder and dissolve it in 44 ml of deionized water. Stir magnetically until completely dissolved. Pour the solution into a mold and cool the hydrogel at 4°C for 2 hours to obtain a single-network GEL hydrogel (denoted as GEL).

[0046] Test Example 1 Example 1 The hydrogel obtained was used for testing cell biocompatibility

[0047] (1) L929 fibroblasts were cultured at a rate of 1×10 4 The cells were seeded at a density of 100 μg / mL on a 96-well plate and cultured overnight in an incubator at 37°C, 5% CO2. Before co-culturing with fibroblasts, the hydrogel was purified by repeated immersion in PBS and 75% ethanol to remove unreacted monomers and other residues. The hydrogel was then immersed in 15 mL of complete culture medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C for 24 hours, and the supernatant was collected to obtain the extract. The extract was mixed with DMEM containing 10% FBS to prepare a test culture medium, which was added to a 96-well plate at 100 μL / well and cultured in a 37°C, 5% CO2 incubator for 1 day, 3 days, and 5 days, respectively. Cells cultured normally in DMEM containing 10% FBS were set as the control group. After culturing for 1, 3, and 5 days, 10 μL of CCK8 (CA1210, Solarbio, China) was added to 100 μL of culture medium per well of cells and cultured at 37°C for 4 hours. The cells were analyzed using a multifunctional microplate reader (VICTOR Nivo TM , PerkinElmer) detected the OD value at a wavelength of 450nm and calculated the cell viability. Figure 4 (a).

[0048] (2) L929 fibroblasts were cultured at 5×10 4The cells were seeded at a density of 100 μg / well in a 24-well microplate and cultured at 37°C for 24 hours. They were then cultured with complete medium containing the hydrogel extract for 3 days. After 3 days of culture, the supernatant was removed, the cells were washed 3 times with buffer, and Calcein-AM stain and PI stain were added. After 15 minutes, the stained cells were observed using a fluorescence microscope (IX71, Olimpus) with excitation wavelengths of 488 and 546 nm, respectively. Live cells were stained green and dead cells were stained red. The test results are shown in the table. Figure 4 (b).

[0049] like Figure 4 As shown, Figure 4 (a) CCK-8 assay for the proliferation of L929 fibroblasts cultured in hydrogel extract for 1, 3, and 5 days. Figure 4 (b) Fluorescence staining of L929 fibroblasts after 3 days of culture with the hydrogel extract. Scale bar: 100 μm. The results demonstrate that the γ-PGA / GEL hydrogel promotes fibroblast proliferation and exhibits good biocompatibility.

[0050] Test Example 2 Example 1 The hydrogel obtained was used for wound healing research

[0051] Wound healing was evaluated in model SD rats. SD rats were randomly divided into three groups (n=3): control group, γ-PGA group and γ-PGA / GEL group. The rats were anesthetized and their backs were depilated, and a circular full-thickness skin defect model was constructed 1 cm below the armpit and on both sides of the spine, with a distance of 1 cm between the two wounds. The wounds of the control group, γ-PGA group and γ-PGA / GEL group were covered with gauze, γ-PGA hydrogel dressing and γ-PGA / GEL hydrogel dressing, respectively. The hydrogel group was covered with sterile gauze and fixed to the back of the rat with a bandage. The dressing was changed every two days. Wound photos were taken with a digital camera on the 0th, 4th, 7th, 10th and 14th days of treatment, and the wound area was measured and analyzed using ImageJ software. The test results are shown in Figure 5 .

[0052] like Figure 5 As shown, Figure 5 (a) is the photos of the wounds of different groups on the 2nd, 4th, 8th and 14th days of treatment. Figure 5 (b) The percentage of wound healing at different treatment time points. The results showed that the γ-PGA / GEL hydrogel group demonstrated a 98% healing rate on day 14 of treatment, showing the best healing effect compared to the gauze and γ-PGA groups, indicating that the γ-PGA / GEL hydrogel has a significant effect on wound healing.

[0053] Test Example 3 Compressive and tensile properties of hydrogels

[0054] In order to test the performance of the prepared γ-PGA / GEL double network hydrogel, the double network hydrogels of Examples 1-5 and the single network hydrogels of Comparative Examples 1-2 were subjected to the following analysis and tests.

[0055] Compression performance test: The hydrogel samples were tested using an instron 6959 universal strength tester. The compression sample was a cylindrical hydrogel with a diameter of 22 mm and a height of 18 mm. The compression rate was 10 mm / min. The results are shown in the table below. Figure 1 .

[0056] like Figure 1 Shown are compression test curves of γ-PGA, GEL, γ-PGA / GEL-1, γ-PGA / GEL-2, γ-PGA / GEL-3, γ-PGA / GEL-4 and γ-PGA / GEL-5 hydrogels (Note: the numbers in the above hydrogel names correspond to the mass of gelatin added in the examples). Figure 1 The compressive strength of the γ-PGA / GEL-4 double-network hydrogel was 40 MPa, which was 28 times that of pure γ-PGA hydrogel and 606 times that of pure GEL hydrogel. This indicates that the compressive strength of the γ-PGA / GEL double-network hydrogel was significantly improved compared to the single-network hydrogel. It can be seen that the γ-PGA / GEL double-network hydrogel has excellent compressive resistance. Among them, the hydrogel obtained with 2.5g of γ-PGA powder and 4g of gelatin in Example 1 had the highest strength.

[0057] Tensile properties and tensile toughness test: The hydrogel samples were tested using an instron 6959 universal strength tester. The tensile test sample size was dumbbell-shaped, the gauge length was set to 25 mm, and the tensile speed was 100 mm / min. The tensile strength is the maximum stress at which the sample breaks. The results are shown in Figure 2 .

[0058] like Figure 2 Shown are the tensile test curves of γ-PGA, GEL, γ-PGA / GEL-1, γ-PGA / GEL-2, γ-PGA / GEL-3, γ-PGA / GEL-4, and γ-PGA / GEL-5 hydrogels. As can be seen from the figure, the tensile strength of the γ-PGA / GEL-4 double network hydrogel is 0.27 MPa, which is 19 times that of pure γ-PGA hydrogel and 10 times that of pure GEL hydrogel, indicating that the tensile strength of the γ-PGA / GEL double network hydrogel has been significantly improved; at the same time, the tensile strain is 580%, which is 3.7 times that of pure γ-PGA hydrogel and 10.7 times that of pure GEL hydrogel. This shows that the γ-PGA / GEL double network hydrogel has good tensile properties and tensile toughness.

[0059] Self-healing performance test: γ-PGA / GEL double network hydrogel was cut into two sections, dyed blue and pink respectively, and then the two sections were tightly connected and sealed and stored at 60℃ for 2 hours, then taken out and cooled at 4℃ for 12 hours. Figure 3 .

[0060] like Figure 3 As shown, after the γ-PGA / GEL double network hydrogel was cut ( Figure 3 a), after self-healing treatment, it can be observed that the two segments of hydrogel can be reconnected together ( Figure 3 b) and has a certain strength, and successfully lifted a 200g weight without breaking ( Figure 3 c), showing certain self-healing properties.

[0061] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A high-strength γ-polyglutamic acid / gelatin double network hydrogel, wherein the compressive strength of the hydrogel is 40 Mpa. The preparation method of the hydrogel comprises the following steps: 1) dissolving γ-polyglutamic acid in deionized water to obtain a γ-polyglutamic acid solution; 2) dissolving gelatin in the above-mentioned γ-polyglutamic acid solution, adding a cross-linking agent, ethylene glycol diglycidyl ether, adjusting the pH, and stirring to obtain a mixed solution; wherein, The mass ratio of γ-polyglutamic acid to gelatin is 2.5g:4g; the mass of ethylene glycol diglycidyl ether is 1.5g; 3) ultrasonically treating the mixed solution, removing bubbles in vacuo, and reacting the resulting hydrogel, allowing it to stand and cool at 4° C. for 2-12 hours to obtain a high-strength γ-polyglutamic acid / gelatin double-network hydrogel; The test method for the compressive strength of the hydrogel includes: using an instron 6959 universal strength tester to test the hydrogel sample, the compression sample is a cylindrical hydrogel with a diameter of 22 mm and a height of 18 mm, and the compression rate is 10 mm / min.

2. The high-strength γ-polyglutamic acid / gelatin double network hydrogel according to claim 1, characterized in that: In step 1), the temperature of the deionized water is 40-60°C.

3. The high-strength γ-polyglutamic acid / gelatin double network hydrogel according to claim 1, characterized in that: In step 2), the pH is adjusted in the range of 4-6.

4. The high-strength γ-polyglutamic acid / gelatin double network hydrogel according to claim 1, characterized in that: In step 3), the ultrasonication time is 20 min.

5. The high-strength γ-polyglutamic acid / gelatin double network hydrogel according to claim 1, characterized in that: In step 3), the reaction temperature is 40-60° C. and the reaction time is 6-20 h.

6. Use of the high-strength γ-polyglutamic acid / gelatin double network hydrogel according to any one of claims 1 to 5 in the preparation of a wound dressing for wound healing.

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