Dual-network callus hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of wound dressing and hydrogel preparation, in particular to double-network callus hydrogel and a preparation method thereof. The preparation method of the dual-network callus hydrogel comprises the following steps: dissolving chitosan in a solvent to obtain a chitosan solution; the method comprises the following steps: adding acrolein into a chitosan solution for reaction, washing, and freeze-drying to obtain a solid A; dissolving the solid A in a solvent, adding epigallocatechin gallate for reaction, and then washing and freeze-drying to obtain a solid B; the solid B is dissolved in a solvent, cysteine is added for a reaction, and a chitosan hydrogel solution is obtained; and mixing the chitosan hydrogel solution with a polyvinyl alcohol solution to obtain the dual-network callus hydrogel. The double-network callus hydrogel prepared by the method disclosed by the invention has anti-oxidation, antibacterial and anti-inflammatory functions.

Description

Technical Field

[0001] The invention relates to the technical field of wound dressing and hydrogel preparation, and in particular to a double-network wound healing hydrogel and a preparation method thereof. Background Art

[0002] The skin is an important defense system of the human body and plays a vital role in protecting the body from the invasion of external pathogenic microorganisms and environmental stimuli. Skin damage can trigger a series of physiological responses, including accelerated metabolism, decreased body temperature, loss of water and protein, and imbalance of the endocrine and immune systems. The skin wound healing process includes four main stages: hemostasis, inflammation, proliferation, and tissue remodeling. Skin damage is often associated with increased reactive oxygen species (ROS), impaired tissue oxygenation, and bacterial infection, which may hinder the rapid healing of wounds. Specifically, neutrophils quickly enter the injured area after injury and release ROS and proteases to destroy bacteria. In addition, they also release inflammatory cytokines, which enhance the inflammatory response. The overexpression of inflammatory factors leads to the continuous release of free radicals, reactive oxygen species, and proteases. Therefore, over-activation of the immune response and persistent infection usually lead to slow wound healing. Conventional treatments for infected wounds include wound dressings, antibiotics, and oxygen therapy. However, due to the complex microenvironment of the wound site, single treatment is not effective. Therefore, there is an urgent need to develop a variety of new multifunctional wound dressings. Summary of the invention

[0003] Based on the above content, the present invention provides a double network callus hydrogel and a preparation method thereof. The double network callus hydrogel has anti-oxidation, antibacterial and anti-inflammatory functions.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a method for preparing a double network wound healing hydrogel, comprising the following steps:

[0006] Step 1, dissolving chitosan in a solvent to obtain a chitosan solution; adding acrolein to the chitosan solution for reaction, washing and freeze-drying to obtain a solid A;

[0007] Step 2, dissolving the solid A in a solvent, adding epigallocatechin gallate to react, and then washing and freeze-drying to obtain a solid B;

[0008] Step 3, dissolving the solid B in a solvent, and adding cysteine ​​to react to obtain a chitosan hydrogel solution;

[0009] Step 4: mixing the chitosan hydrogel solution with the polyvinyl alcohol solution to obtain the double network wound healing hydrogel.

[0010] The second technical solution of the present invention is a double network wound healing hydrogel prepared according to the above-mentioned preparation method.

[0011] The third technical solution of the present invention is a wound dressing, the raw materials of which include the above-mentioned double-network wound healing hydrogel.

[0012] The present invention discloses the following technical effects:

[0013] The invention utilizes highly active acrolein to increase the loading amount of epigallocatechin gallate in chitosan hydrogel, thereby making the chitosan hydrogel have excellent antioxidant, antibacterial and anti-inflammatory functions. The mechanical properties of the hydrogel are improved by secondary cross-linking with polyvinyl alcohol.

[0014] The double-network wound healing hydrogel prepared by the method of the invention has the functions of anti-oxidation, anti-bacteria and anti-inflammatory. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is the appearance of the hydrogel prepared in Examples 1-4.

[0017] Figure 2 The ROS scavenging ability of the hydrogels prepared in Examples 1-4.

[0018] Figure 3 The antibacterial ability of the hydrogels prepared in Examples 1-4.

[0019] Figure 4 The effect of the hydrogel prepared in Examples 1-4 on in vitro cell migration.

[0020] Figure 5 The anti-inflammatory function of the hydrogel prepared in Examples 1-4.

[0021] Figure 6 It is a schematic diagram of the double network structure of the double network wound healing hydrogel of the present invention. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The "room temperature" described in the present invention, unless otherwise specified, refers to 20-30°C.

[0028] Natural polymers and plant extracts have gained considerable attention as promising wound dressing ingredients, mainly due to their biocompatibility, biodegradability, and multiple bioactive properties. Chitosan (CS) is an ideal building block for the development of advanced bioactive hydrogel materials, with desirable properties such as biocompatibility, biodegradability, and antimicrobial activity. However, they have limitations in specific applications due to their poor mechanical properties. Therefore, hydrogels made by combining natural and synthetic polymers can have higher performance, such as high elasticity, gel strength, water absorption capacity, and biocompatibility. Among synthetic polymers, polyvinyl alcohol (PVA) has been widely used in various fields due to its advantages such as nontoxicity, biocompatibility, thermal stability, high hydrophilicity, excellent mechanical properties, and easy processing. In addition, PVA can be easily converted into hydrogels using physical cross-linking methods such as freeze-thaw without the need for chemical cross-linking agents. Epigallocatechin gallate (EGCG), a natural polyphenol, is the main bioactive compound in green tea and the most abundant substance among catechins. In recent years, EGCG has attracted much attention due to its multiple pharmacological effects, including antioxidant, antibacterial, anti-inflammatory, hypoglycemic, and angiogenesis-inducing effects. It has also begun to be used in the treatment of skin wounds as a component of wound healing agents.

[0029] The first aspect of the present invention provides a method for preparing a double network wound healing hydrogel, comprising the following steps:

[0030] Step 1, dissolving chitosan in a solvent to obtain a chitosan solution; adding acrolein to the chitosan solution for reaction, washing and freeze-drying to obtain a solid A;

[0031] Step 2, dissolving the solid A in a solvent, adding epigallocatechin gallate to react, and then washing and freeze-drying to obtain a solid B;

[0032] Step 3, dissolving the solid B in a solvent, and adding cysteine ​​to react to obtain a chitosan hydrogel solution;

[0033] Step 4: mixing the chitosan hydrogel solution with the polyvinyl alcohol solution to obtain the double network wound healing hydrogel.

[0034] In some embodiments of the present invention, in step 1 to step 3, the solvent is an aqueous solution of glacial acetic acid with a volume concentration of 0-5%.

[0035] Chitosan is a translucent amorphous solid that is insoluble in water and general organic solvents, but soluble in dilute acid solutions. If glacial acetic acid is replaced with other solvents such as water or alkaline solutions, the chitosan solid will agglomerate and become insoluble, thus affecting subsequent reactions.

[0036] In the present invention, adjusting the order of adding acrolein, epigallocatechin gallate and cysteine ​​will affect the network structure and functional properties of the prepared hydrogel.

[0037] In some embodiments of the present invention, the mass concentration of the chitosan in the chitosan solution is 0.5% to 3%.

[0038] In some embodiments of the present invention, the mass molar ratio of the chitosan to the acrolein is 1 g:0-2 mmol, and the added amount of acrolein is not zero.

[0039] The reason why the ratio of chitosan to acrolein is limited to the above parameters is that excessive addition of acrolein will lead to excessive gel strength (the hardness of the gel will be very large), which is not practical; too little acrolein will not have the effect of a cross-linking agent and will reduce the epigallocatechin gallate loading of chitosan.

[0040] In some embodiments of the present invention, the mass ratio of the chitosan to the epigallocatechin gallate is 1:0-0.5, and the added amount of epigallocatechin gallate is not zero.

[0041] The reasons why the mass ratio of chitosan to epigallocatechin gallate is limited to the above parameter range are: 1. Epigallocatechin gallate, as a functional component of hydrogel, has anti-inflammatory, antibacterial and antioxidant effects, so its content cannot be 0; 2. There is an upper limit on the amount of epigallocatechin gallate loaded on chitosan macromolecules. Even if an excessive amount of epigallocatechin gallate is added, the functional properties of the hydrogel will not be improved, and the unreacted epigallocatechin gallate will be removed during the preparation process; therefore, considering the comprehensive cost and efficacy, the mass ratio of chitosan to epigallocatechin gallate is limited to the above parameter range.

[0042] In some embodiments of the present invention, in step 2, the mass concentration of epigallocatechin gallate in the solvent is 0-0.5%.

[0043] In some embodiments of the present invention, the mass ratio of the chitosan to the cysteine ​​is 1:0-0.1, and the added amount of cysteine ​​is not zero.

[0044] In the present invention, cysteine ​​plays a role in eliminating the trace amount of acrolein remaining in the system, and because cysteine ​​is very active, adding a small amount of cysteine ​​can achieve a 100% elimination effect; at the same time, because cysteine ​​has a certain pungent odor, it cannot be added in excess.

[0045] In some embodiments of the present invention, in step 3, the mass concentration of cysteine ​​in the solvent is 0-0.1%.

[0046] In some embodiments of the present invention, the volume ratio of the chitosan hydrogel solution to the polyvinyl alcohol solution is 0.4-2.5:1; and the mass concentration of the polyvinyl alcohol solution is 0-10%.

[0047] In some embodiments of the present invention, in step 1 to step 3, the reaction is independently carried out at room temperature for 4-12 hours.

[0048] In some embodiments of the present invention, stirring is performed when chitosan is dissolved in a solvent to prepare a chitosan solution, and the stirring time is 1-4 hours.

[0049] In some embodiments of the present invention, in step 1 and step 2, ethyl acetate is used for washing, and the washing is performed 3-5 times until the color of the ethyl acetate phase is close to transparent. After the washing is completed, the step of removing the residual ethyl acetate by rotary evaporation is also included.

[0050] The present invention does not impose any particular limitation on the parameters such as temperature, pressure, and time of freeze drying in step 1 to step 2, and the parameters well known to those skilled in the art may be used; for example, the freeze drying time may be 12-48 hours.

[0051] In some embodiments of the present invention, in step 4, after the mixing is completed, the mixing step further includes stirring at room temperature for 12 hours, freezing the mixed solution at -20°C, and then thawing at room temperature for 4 hours, and repeating the freezing and thawing steps 0-5 times.

[0052] In the present invention, polyvinyl alcohol can be made to form a gel by freeze-thaw method.

[0053] The second aspect of the present invention provides a double-network wound healing hydrogel prepared according to the above preparation method.

[0054] The third aspect of the present invention provides a wound dressing, the raw materials of which include the above-mentioned double-network wound healing hydrogel.

[0055] Unless otherwise specified, the raw materials and reagents used in the examples of the present invention can be obtained through commercial sources.

[0056] The glacial acetic acid solution used in the embodiment of the present invention is prepared by adding glacial acetic acid into deionized water; the volume concentration of the glacial acetic acid solution is 1%.

[0057] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0058] Example 1

[0059] Dissolve 1g of chitosan in 100mL of glacial acetic acid solution and stir at room temperature for 12h to obtain a chitosan solution. Wash the chitosan solution repeatedly with ethyl acetate for 3-5 times until the color of the ethyl acetate phase is close to transparent; then remove the residual ethyl acetate in the chitosan solution by rotary evaporation; freeze-dry to obtain a solid. Dissolve the solid in 100mL of glacial acetic acid solution to obtain a chitosan hydrogel solution. Mix the chitosan hydrogel solution with a polyvinyl alcohol solution with a mass concentration of 5% in a volume ratio of 1:1, stir at room temperature for 4-12 hours, freeze the mixed solution at -20°C, and then thaw at room temperature for 4 hours. Repeat the freezing-thawing step 3 times to obtain a chitosan / PVA hydrogel.

[0060] Example 2

[0061] 1g chitosan was dissolved in 100mL glacial acetic acid solution and stirred at room temperature for 12h to obtain a chitosan solution. 0.2g EGCG was added to the chitosan solution and the reaction was continued for 12h; the solution obtained by the reaction was repeatedly washed with ethyl acetate for 3-5 times until the color of the ethyl acetate phase was close to transparent; then the residual ethyl acetate was removed by rotary evaporation; and the solid was freeze-dried to obtain a solid. The above solid was dissolved in 100mL glacial acetic acid solution to obtain a chitosan hydrogel solution. The chitosan hydrogel solution was mixed with a polyvinyl alcohol solution with a mass concentration of 5% in a volume ratio of 1:1, stirred at room temperature for 4-12 hours, and the mixed solution was frozen at -20°C, and then thawed at room temperature for 4 hours. The freezing-thawing step was repeated 3 times to obtain a chitosan-EGCG / PVA hydrogel.

[0062] Example 3

[0063] Dissolve 1g chitosan in 100mL glacial acetic acid solution and stir at room temperature for 12h to obtain a chitosan solution. Add 1.5mmol acrolein to the chitosan solution and stir at room temperature for 12h. Wash the resulting solution repeatedly with ethyl acetate for 3-5 times until the ethyl acetate phase is nearly transparent; then remove the residual ethyl acetate by rotary evaporation; freeze-dry to obtain solid A. Dissolve the above solid A in 100mL glacial acetic acid solution, add 0.2g EGCG, and continue stirring for 12h; wash the resulting solution repeatedly with ethyl acetate for 3-5 times until the ethyl acetate phase is nearly transparent; then remove the residual ethyl acetate by rotary evaporation; freeze-dry to obtain solid B. Dissolve the above solid B in 100mL glacial acetic acid solution to obtain a chitosan hydrogel solution. The chitosan hydrogel solution was mixed with a polyvinyl alcohol solution with a mass concentration of 5% in a volume ratio of 1:1. After stirring at room temperature for 4-12 hours, the mixed solution was frozen at -20°C and then thawed at room temperature for 4 hours. The freezing-thawing step was repeated 3 times to obtain chitosan-ACR-EGCG / PVA hydrogel.

[0064] Example 4

[0065] 1.0g chitosan was dissolved in 100mL glacial acetic acid solution and stirred at room temperature for 12h to obtain a chitosan solution. 1.5mmol acrolein was added to the chitosan solution and stirred at room temperature for 12h. The solution obtained by the reaction was repeatedly washed with ethyl acetate for 3-5 times until the color of the ethyl acetate phase was close to transparent; then the residual ethyl acetate was removed by rotary evaporation; and solid A was obtained by freeze drying. The above solid A was dissolved in 100mL glacial acetic acid solution, 0.2g EGCG was added, and the reaction was continued by stirring for 12h. The solution obtained by the reaction was repeatedly washed with ethyl acetate for 3-5 times until the color of the ethyl acetate phase was close to transparent; then the residual ethyl acetate was removed by rotary evaporation; and solid B was obtained by freeze drying. The above solid B was dissolved in 100mL glacial acetic acid solution, 0.01g cysteine ​​(Cys) was added, and the reaction was stirred for 12 hours to obtain a chitosan hydrogel solution. The chitosan hydrogel solution was mixed with a 5% polyvinyl alcohol solution in a volume ratio of 1:1. After stirring at room temperature for 4-12 hours, the mixed solution was frozen at -20°C and then thawed at room temperature for 4 hours. The freezing-thawing step was repeated 3 times to obtain chitosan-ACR-EGCG-Cys / PVA hydrogel.

[0066] Figure 1 This is the appearance of the hydrogel prepared in Example 1-4. Figure 1 It can be seen that the four hydrogels prepared in Examples 1-4 all have good gel properties.

[0067] Figure 2 is the ROS scavenging ability of the hydrogels prepared in Examples 1-4. Figure 2 It can be seen that the fluorescence intensity of the hydrogel in Example 4 is the weakest among the four hydrogels, close to that of the negative control group, showing good ROS scavenging ability. The experiment quantitatively analyzed the fluorescence intensity by flow cytometry detection. The negative control is the group not treated with lipopolysaccharide, and the positive control is the group treated with lipopolysaccharide only.

[0068] Figure 3 The antibacterial ability of the hydrogels prepared in Examples 1-4. Figure 3 It can be seen that the antibacterial effect of the hydrogel in Example 4 is the best among the four hydrogels. The experimental method of this experiment is to co-culture the hydrogel with Escherichia coli and Staphylococcus aureus, take the culture solution on the surface of the hydrogel for secondary culture, and judge the antibacterial ability of the hydrogel by comparing the number of surviving bacteria after the secondary culture. The control group is a group without hydrogel, in which Escherichia coli and Staphylococcus aureus are co-cultured with the culture medium.

[0069] Figure 4The effect of the hydrogels prepared in Examples 1-4 on in vitro cell migration. Figure 4 It can be seen that the four hydrogels have a certain promoting effect on in vitro cell migration, among which the promoting effect of the hydrogel in Example 4 is the best. The experimental method of this experiment is the cell scratch method, in which a scratch is artificially made with a 1mL gun tip in a culture dish full of cells, and then the culture medium after 24 hours of co-culture with the hydrogel is added to the above culture dish, and the area of ​​the scratch is observed and compared after continuing to culture for 24 hours. The control group is a group without hydrogel, and fresh culture medium is added.

[0070] Figure 5 The anti-inflammatory function of the hydrogel prepared in Examples 1-4. Figure 5 It can be seen that different hydrogels have different effects on macrophage transformation. The CD206 expression (green fluorescence) of the hydrogel treatment group in Example 4 is high, proving that it has the best anti-inflammatory effect. The experimental method of this experiment is to first use lipopolysaccharide to induce macrophage polarization into M1 type, and then Figure 5 It can be seen that CD86 is highly expressed (red fluorescence). Next, the culture medium after co-culture with the hydrogel for 24 hours was added to the above M1 cells to compare the expression intensity of CD206 (green fluorescence) in different groups of hydrogels to determine the anti-inflammatory effect of the hydrogel. The control group is a group without hydrogel, and fresh culture medium is added. Figure 5 In the figure, blue fluorescence represents all cells; red fluorescence represents CD86 expression (red fluorescence and blue fluorescence overlap to show purple, i.e. Figure 5 The color of the first row); green fluorescence represents CD206 expression (green fluorescence and blue fluorescence overlap to show bright blue, i.e. Figure 5 The colors in the second row). Among them, the purple ratio in the picture is large, which means that CD86 is highly expressed, and CD86 represents M1 macrophages (M1 macrophages are a type of pro-inflammatory cell). The bright blue ratio in the picture is large, which means that CD206 is highly expressed, and CD206 represents M2 macrophages (M2 macrophages are a type of anti-inflammatory cell).

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a double network wound healing hydrogel, characterized in that: The following steps are involved: Step 1, dissolving chitosan in a solvent to obtain a chitosan solution; adding acrolein to the chitosan solution for reaction, washing and freeze-drying to obtain a solid A; Step 2, dissolving the solid A in a solvent, adding epigallocatechin gallate to react, and then washing and freeze-drying to obtain a solid B; Step 3, dissolving the solid B in a solvent, and adding cysteine ​​to react to obtain a chitosan hydrogel solution; Step 4: mixing the chitosan hydrogel solution with the polyvinyl alcohol solution to obtain the double network wound healing hydrogel.

2. The method for preparing the double network wound healing hydrogel according to claim 1, characterized in that: In step 1 to step 3, the solvent is a glacial acetic acid aqueous solution with a volume concentration of 0-5%.

3. The method for preparing the double network wound healing hydrogel according to claim 1, characterized in that: The mass concentration of the chitosan in the chitosan solution is 0.5% to 3%.

4. The method for preparing the double network wound healing hydrogel according to claim 1, characterized in that: The mass molar ratio of the chitosan to the acrolein is 1 g:0-2 mmol; the mass molar ratio of the chitosan to the epigallocatechin gallate is 1:0-0.

5.

5. The method for preparing the double network wound healing hydrogel according to claim 1, characterized in that: The mass ratio of the chitosan to the cysteine ​​is 1:0-0.

1.

6. The method for preparing the double network wound healing hydrogel according to claim 5, characterized in that: In step 3, the mass concentration of cysteine ​​in the solvent is 0-0.1%.

7. The method for preparing the double network wound healing hydrogel according to claim 6, characterized in that: The volume ratio of the chitosan hydrogel solution to the polyvinyl alcohol solution is 0.4-2.5:1; the mass concentration of the polyvinyl alcohol solution is 0-10%.

8. The method for preparing the double network wound healing hydrogel according to claim 1, characterized in that: In step 1 to step 3, the reaction is independently carried out at room temperature for 4-12 hours.

9. A double network wound healing hydrogel prepared according to the preparation method according to any one of claims 1 to 9.

10. A wound dressing, characterized in that: The raw materials include the double network wound healing hydrogel described in claim 9.

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