A preparation method of a licorice acid / fish gelatin double-network composite hydrogel capable of promoting wound healing

Abstract

The application provides a preparation method of licorice acid / fish gelatin double-network composite hydrogel which can promote wound healing. The application uses licorice acid and fish gelatin as main raw materials, utilizes the characteristics that the licorice acid can self-assemble into nanofibers under certain conditions and is mixed with fish gelatin, and respectively forms a physical network in a staged cooling process, so as to form a double-network hydrogel with excellent performance. The obtained hydrogel has the characteristics of high mechanical strength, anti-freezing and moisture retention, high transparency, high adhesion, high biocompatibility and the like. And the obtained hydrogel is used for wound treatment, and has obvious promoting effect on wound healing. The licorice acid / fish gelatin double-network composite hydrogel has excellent mechanical performance, good biocompatibility, simple preparation method, mild reaction condition, easy scale production, green environmental protection, and has potential application prospect in the field of medical hydrogel.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing a glycyrrhizic acid / fish gelatin dual-network composite hydrogel that can promote wound healing. Background Technology

[0002] As the first line of defense for our immune system, the skin plays a vital role in maintaining and regulating body temperature, preventing water loss, and protecting and repairing itself. As the first line of defense, the skin is easily injured in daily life, resulting in acute full-thickness wounds of varying degrees. If these wounds are not treated promptly and effectively, they can easily lead to difficult-to-heal wounds, and in severe cases, even threaten life. Therefore, the development of biomaterials that promote wound healing is of great significance to human health management. In recent years, there has been extensive research on biomaterials that promote wound healing, and the types of materials are diverse. Among them, hydrogels are one of the most widely studied biomaterials. Hydrogels, as wound healing dressings, have the following advantages: a three-dimensional network structure, high water content, ability to absorb tissue exudate, ability to block microbial invasion, ability to create a low-oxygen environment, and good biocompatibility.

[0003] In recent years, the development and utilization of natural small molecules with certain biological functional activities that are both food and medicine have become a research hotspot. Among the many natural small molecules that are both food and medicine, those that can self-assemble into hydrogels under certain conditions have attracted widespread attention. These hydrogels have advantages such as simple preparation methods, strong bioactivity, tunable mechanical properties, and injectability, and have great application prospects in medical fields such as drug encapsulation and delivery, wound healing, and tissue engineering. Glycyrrhizic acid (GL), as an important active molecule in licorice, has a wide range of physiological activities, including antibacterial, anti-inflammatory, antiviral, and anticancer effects. In recent years, related studies have also reported that glycyrrhizic acid can self-assemble into hydrogels with a nanofiber network structure and has a positive healing effect on wounds. However, although pure glycyrrhizic acid hydrogels have great advantages for use as wound dressings, they still have some fatal defects that limit their further application in the biomedical field, such as poor storage stability, low mechanical strength, and the tendency for high self-assembly concentrations to induce cytotoxicity. Therefore, improving the properties of pure glycyrrhizic acid hydrogels remains a challenge.

[0004] Gelatin, a product of partial hydrolysis of collagen, possesses unique cell adhesion domains and matrix metalloproteinase reaction sequences, providing conditions for cell proliferation and diffusion. Currently, commonly used gelatin is mainly derived from mammals, often posing negative risks such as zoonotic disease risks, immune rejection reactions, and religious restrictions. Fish gelatin (FGel), derived from fish processing waste (fish skin, bones, scales, etc.), offers advantages such as low cost, high biocompatibility, lower immunogenicity, and fewer religious restrictions. Similarly, single-component fish gelatin hydrogels have poor mechanical strength. Some studies have shown that introducing the physical cross-linking network of fish gelatin into composite hydrogels can significantly enhance the mechanical strength and biocompatibility of fish gelatin hydrogels. In recent years, constructing hydrogels with dual-network structures has been considered one of the most effective methods to improve the mechanical strength of hydrogels. Therefore, inspired by the construction methods of dual-network structure hydrogels, this invention aims to solve the problem of how to utilize GL and FGel to construct a hydrogel with excellent mechanical strength and biological functional properties.

[0005] Based on the above considerations, this invention uses GL and FGel as the main raw materials, aiming to provide a GL / FGel dual-network composite hydrogel with excellent mechanical properties, good biocompatibility, and the ability to promote wound healing, as well as its preparation method and application. Summary of the Invention

[0006] This invention aims to overcome the shortcomings and deficiencies of current methods for constructing hydrogel wound dressings. It introduces a physical network, simplifies the preparation process, constructs a dual-network structure, and improves the mechanical strength, environmental stability, and biological activity of the hydrogel. It provides a GL / FGel dual-network composite hydrogel that can promote wound healing and its preparation method.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A composite hydrogel with a dual-network structure was prepared by a one-pot method using GL and FGel as raw materials.

[0009] Preferably, GL and FGel are further purified by dissolving, centrifuging, dialysis, and vacuum freeze-drying.

[0010] Preferably, different ratios of glycerol / water solution are prepared, with volume ratios of 1:1, 1:3 and 1:5 respectively.

[0011] Preferably, the FGel is dissolved in the above glycerol / water solution, placed in a constant temperature water bath and heated to 60°C, and stirred with a magnetic stirrer at 200 rpm for 2.0 h, with a final concentration of 50-200 mg / mL.

[0012] Preferably, a certain amount of GL is added to the above FGel solution, placed in a constant temperature water bath and heated to 80°C, and stirred with a magnetic stirrer at 200 rpm for 0.5 h. The final addition amount is 5-25 mg / mL.

[0013] Preferably, the above GL / FGel mixture is placed in an ultrasonic cleaner and ultrasonically cleaned at a working frequency of 40KHz for 5 minutes to remove bubbles.

[0014] Preferably, the ultrasonically defoamed GL / FGel mixture is spread evenly in a hydrogel mold and first allowed to stand at room temperature (25°C) for 1-2 hours to induce GL self-assembly to form a nanofiber network. Then, it is transferred to a refrigerator at 4°C and allowed to stand for 12-24 hours to promote the formation of the FGel triple helix structure.

[0015] This invention provides a GL / FGel dual-network composite hydrogel that can promote wound healing and its preparation method, which can be obtained by using the preparation method described in the above technical solution.

[0016] This invention has the following advantages:

[0017] This invention proposes a method for preparing a GL / FGel dual-network composite hydrogel that promotes wound healing. It utilizes biocompatible FGel and highly bioactive GL as raw materials. The nanofiber network structure formed by the self-assembly of GL effectively incorporates the FGel network, jointly promoting the formation of the dual-network hydrogel. By optimizing the addition ratio of GL and FGel, a clever composite of the GL nanofiber network and the FGel network is achieved, resulting in a dual-network hydrogel with excellent properties. This method is simple to prepare; GL, FGel, and glycerol are mixed and reacted, and the hydrogel with the dual-network structure can be obtained through a one-pot method.

[0018] The GL / FGel dual-network composite hydrogel proposed in this invention possesses excellent biocompatibility, biofunctional activity, and biodegradability due to its constituent materials being derived from natural organisms, thus exhibiting a significant promoting effect on wound healing. Furthermore, the resulting hydrogel possesses superior mechanical strength, antifreeze and moisturizing properties, strong adhesion, and high transparency, making it a high-strength hydrogel wound dressing with promising applications. The preparation method of this invention is simple, low-cost, environmentally friendly, and exhibits significant functional activity, aligning with the goals of sustainable socio-economic development. The hydrogel obtained by this invention, when used in wound treatment, demonstrates a significant promoting effect on wound healing. This glycyrrhizic acid / fish gelatin dual-network composite hydrogel exhibits excellent mechanical properties, good biocompatibility, a simple preparation method, mild reaction conditions, ease of large-scale production, and is environmentally friendly, showing promising potential applications in the field of medical hydrogels. Attached Figure Description

[0019] Figure 1A schematic diagram illustrating the preparation method of GL / FGel dual-network composite hydrogel and its application in wound healing;

[0020] Figure 2 Real-world images showing the stretching, extension, and adhesion of GL / FGel high-transparency dual-network composite hydrogel to the skin;

[0021] Figure 3 Transmission electron microscope image of GL self-assembled fiber network;

[0022] Figure 4 This is a cryo-scanning electron microscope image of the GL / FGel dual-network composite hydrogel.

[0023] Figure 5 The diagram shows the mechanical tensile properties of the GL / FGel dual-network composite hydrogel.

[0024] Figure 6 The diagram shows the antifreeze properties of the GL / FGel dual-network composite hydrogel.

[0025] Figure 7 The graph shows the evaluation of the moisturizing performance of the GL / FGel dual-network composite hydrogel.

[0026] Figure 8 Figure 1 shows the in vitro biocompatibility evaluation of the GL / FGel dual-network composite hydrogel (Figure A is the hydrogel's blood compatibility evaluation; Figure B is the hydrogel's cytotoxicity evaluation; Figure C is the hydrogel's in vitro angiogenesis promotion ability evaluation).

[0027] Figure 9 The images show the wound healing effect after treatment with GL / FGel dual-network composite hydrogel (Figure A shows the wound healing process after hydrogel treatment; Figure B shows the healing rate of the wound after hydrogel treatment at different times). Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] This embodiment provides a method for preparing a GL / FGel dual-network composite hydrogel that can promote wound healing, including the following steps:

[0031] Weigh 1.5g of FGel and dissolve it in 10mL of a 1:1 glycerol / water mixture. Heat the mixture in a constant temperature water bath to 60℃ and stir magnetically at 200rpm for 2 hours. Then add 25.0mg of GL powder and heat the mixture in a constant temperature water bath to 80℃. Continue stirring magnetically at 200rpm for 0.5 hours. Finally, place the GL / FGel mixture in an ultrasonic cleaner and sonicate at 40kHz for 5 minutes to remove foam. After standing at room temperature (25℃) for 1 hour, place it at 4℃ for 12 hours to obtain a GL / FGel dual-network composite hydrogel, denoted as FGel / GL 1.

[0032] See Figure 1 and Figure 2 , Figure 1 A schematic diagram illustrating the preparation method of GL / FGel dual-network composite hydrogel and its application in wound healing; Figure 2 Actual image of a GL / FGel high-transparency dual-network composite hydrogel exhibiting skin stretching, elongation, and adhesion.

[0033] Example 2

[0034] This embodiment provides a method for preparing a GL / FGel dual-network composite hydrogel that can promote wound healing, including the following steps:

[0035] Same as Example 1, except that 75 mg of GL powder was added, denoted as FGel / GL 3.

[0036] Example 3

[0037] This embodiment provides a method for preparing a GL / FGel dual-network composite hydrogel that can promote wound healing, including the following steps:

[0038] Same as Example 1, except that 125 mg of GL powder was added, denoted as FGel / GL 5.

[0039] Comparative Example 1

[0040] A method for preparing a GL hydrogel includes the following steps:

[0041] Same as Example 1, except that FGel powder is not added, denoted as GL.

[0042] Comparative Example 2

[0043] A method for preparing an FGel hydrogel includes the following steps:

[0044] Same as Example 1, except that GL is not added, denoted as FGel / GL 0.

[0045] Comparative Example 3

[0046] A method for preparing an FGel hydrogel includes the following steps:

[0047] Same as Example 1, except that the glycerol aqueous solution is replaced with pure water solvent and GL is not added, denoted as FGel-W.

[0048] Experimental Example 1

[0049] Transmission electron microscopy (TEM) was performed on the GL hydrogel obtained in Example 1, and the TEM image of the GL self-assembled fiber network is shown below. Figure 3 As shown.

[0050] Experimental Example 2

[0051] The cross-section of the GL / FGel dual-network composite hydrogel obtained in Example 2 was observed using cryo-scanning electron microscopy, and its cryo-scanning electron micrograph is shown below. Figure 4 As shown.

[0052] Experimental Example 3

[0053] The mechanical strength of the hydrogels obtained in Examples 1, 2, 3 and Comparative Example 2 was evaluated, and their tensile mechanical properties were obtained as shown in the figure. Figure 5 As shown.

[0054] The antifreeze properties of the hydrogels obtained in Example 2, Comparative Example 2, and Comparative Example 3 were evaluated, and their antifreeze properties are shown in the figure below. Figure 6 As shown.

[0055] The moisturizing properties of the hydrogels obtained in Examples 1, 2, 3, Comparative Example 2, and Comparative Example 3 were evaluated, and the evaluation graphs of their moisturizing properties are shown below. Figure 7 As shown.

[0056] Test Example 4

[0057] Biocompatibility was evaluated on the gel cross sections of the hydrogels obtained in Example 2 and Comparative Example 2. Specifically, evaluations included blood compatibility, cytotoxicity, and in vitro angiogenesis promotion. The resulting in vitro biocompatibility evaluation diagram is shown below. Figure 8 As shown.

[0058] Experimental Example 5

[0059] The hydrogels obtained in Example 2 and Comparative Example 2, as well as the commercially available Tegaderm... TM Wound dressings were used to evaluate acute full-thickness wound healing in mice. Observations and evaluations were conducted during the wound healing period (days 4, 8, 12, and 16), and the wound healing effect was obtained as shown in the following figures. Figure 9 As shown.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a glycyrrhizic acid / fish gelatin dual-network composite hydrogel that can promote wound healing, characterized in that, Includes the following steps: (1) Dissolve fish gelatin in a glycerol / water solution with a volume ratio of 1:1, 1:3 or 1:5, heat at 60°C and stir magnetically at 200 rpm for 2.0 h to prepare a fish gelatin solution with a concentration of 50-200 mg / mL. (2) Add glycyrrhizic acid powder to the fish gelatin solution obtained in step (1) to make the final concentration of glycyrrhizic acid 5-25 mg / mL, heat at 80°C and stir magnetically at 200 rpm for 0.5 h to obtain a mixed solution; (3) The mixed solution obtained in step (2) was ultrasonically treated at a frequency of 40 kHz for 5 min to remove air bubbles; (4) Pour the ultrasonically defoamed mixed solution into a mold and let it stand at 25°C for 1-2 hours. Then transfer it to 4°C and let it stand for 12-24 hours to obtain the glycyrrhizic acid / fish gelatin dual-network composite hydrogel.

2. The preparation method according to claim 1, characterized in that, The concentration of the fish gelatin solution mentioned in step (1) is 150 mg / mL.

3. The preparation method according to claim 1, characterized in that, The volume ratio of glycerol / water solution in step (1) is 1:

1.

4. The application of a glycyrrhizic acid / fish gelatin dual-network composite hydrogel prepared by any one of the methods described in claims 1-3 in the preparation of wound dressings.

Images