A self-healing hydrogel and preparation method thereof

Abstract

The present invention relates to a self-healing hydrogel and a preparation method thereof, and belongs to the technical field of biomedical hydrogels. A self-healing hydrogel, wherein the hydrogel is formed by cross-linking two matrices of polyacrylamide and dopamine hydrochloride modified hyaluronic acid through hydrogen bonding and conjugation of polyphenol compounds to form a hydrogel. The present invention provides a method for preparing a polyphenol injectable self-healing hydrogel that promotes wound healing under mild conditions and a simple preparation method. The wound adhesive hydrogel material provided by the present invention has antibacterial properties. When Escherichia coli and Staphylococcus aureus are used as pathogenic bacteria, the hydrogel exhibits excellent antibacterial ability. At the same time, it also has antioxidant and antibacterial properties, can inhibit wound infection, remove free radicals, and limit inflammatory reactions on the wound.

Description

Technical Field

[0001] The invention relates to a self-healing hydrogel and a preparation method thereof, and belongs to the technical field of biomedical hydrogels. Background Art

[0002] Hydrogel is a macromolecular polymer material with a three-dimensional network structure. It is a new type of wound dressing material that can quickly stop bleeding, promote tissue repair, and reduce the formation of scars. Compared with traditional dressings, hydrogel has excellent air permeability, which can not only ensure the moisture of the wound but also absorb the wound exudate. The moist and open environment makes it easy for the wound tissue to be invaded by bacteria, so it is very necessary to give the hydrogel excellent antibacterial ability. Injectable hydrogel refers to a hydrogel with a certain fluidity that can be applied by injection. Injectable hydrogel with tissue adhesion can fit wounds in different states to achieve better therapeutic effects.

[0003] As one of the main components of connective tissues such as the intercellular matrix, vitreous body, and synovial fluid of the human body, hyaluronic acid has many advantages such as good biocompatibility and biodegradability. In addition, hyaluronic acid also has the functions of regulating the permeability of blood vessel walls, regulating the diffusion and movement of proteins, water and electrolytes, and promoting wound healing. However, hyaluronic acid has poor tissue adhesion and high brittleness. When used as a dressing, it is easy to cause drug loss and wound infection. The use of catechol modification can improve the tissue adhesion and toughness of hyaluronic acid hydrogels. Summary of the invention

[0004] The present invention aims to provide a polyphenol injectable self-healing hydrogel for promoting wound healing, and a preparation method and application thereof.

[0005] A self-healing hydrogel, wherein the hydrogel is formed by using polyacrylamide and hyaluronic acid modified by dopamine hydrochloride as gel matrix, and polyphenol compounds cross-link the two matrices through hydrogen bonding and conjugation to form the hydrogel.

[0006] The self-healing hydrogel of the present invention is injectable and can be injected through a syringe so as to better fit irregular wound surfaces in all directions to achieve better healing effects.

[0007] The self-healing hydrogel of the present invention may contain water. During the preparation process, polyacrylamide, modified dopamine hydrochloride and polyphenol compounds are uniformly mixed in water.

[0008] Preferably, the polyphenol compound is tannic acid or gallic acid.

[0009] Preferably, the mass ratio of the polyacrylamide, the hyaluronic acid modified with dopamine hydrochloride, and the polyphenol compound is 1:1-5:0.001-0.01.

[0010] Preferably, the dopamine hydrochloride modified hyaluronic acid is prepared by the following method: dissolving hyaluronic acid in a morpholineethanesulfonic acid buffer solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, after complete dissolution, performing a carboxyl activation reaction under stirring, the reaction temperature is 20-35° C., and the reaction time is 20-40 min; after the reaction is completed, adding dopamine hydrochloride to the above solution, performing a grafting reaction under a nitrogen protection atmosphere, the reaction temperature is 20-35° C., the reaction time is 12-72 h, dialyzing, and freeze-drying to obtain dopamine hydrochloride modified hyaluronic acid.

[0011] The mass ratio of hyaluronic acid: dopamine hydrochloride: 1-ethyl-(3-dimethylaminopropyl) carbodiimide: N-hydroxysuccinimide is 2:2:1:2; and the molecular weight of hyaluronic acid is 90,000 to 400,000.

[0012] Preferably, the polyacrylamide is prepared according to the following method: acrylamide is dissolved in water at a ratio of 1 g: 10 to 50 mL, and methylene bisacrylamide and ammonium persulfate are added to react for 15 to 20 minutes; after the reaction, unreacted acrylamide, catalyst and by-products are removed by dialysis, and the dialyzed solution is freeze-dried and sealed for storage, wherein the mass ratio of methylene bisacrylamide, ammonium persulfate and acrylamide is 0.001:0.01:1.

[0013] Furthermore, the molecular weight cut-off of dialysis is 3500-14000.

[0014] Preferably, at room temperature, polyacrylamide and hyaluronic acid modified with dopamine hydrochloride are mixed, and then a polyphenol compound and a certain amount of water are added to react to obtain a self-healing hydrogel.

[0015] Furthermore, the concentration of the hyaluronic acid modified with dopamine hydrochloride is 0.03 to 0.15 g / mL, the concentration of the polyacrylamide is 0.03 to 0.15 g / mL, and the mass concentration of the polyphenol compound is 1 to 20 mg / mL.

[0016] The beneficial effects of the present invention are as follows: the present invention provides a method for preparing a polyphenol injectable self-healing hydrogel that promotes wound healing under mild conditions and a simple preparation method. The wound adhesive hydrogel material provided by the present invention has antibacterial properties. When Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are used as pathogenic bacteria, the hydrogel exhibits excellent antibacterial ability. It also has antioxidant and antibacterial properties, can inhibit wound infection, remove free radicals, and limit inflammatory reactions on the wound.

[0017] The wound adhesive hydrogel material provided by the present invention has high adhesion efficiency, can provide self-healing and repeatable adhesion. After purification, the hydrogel matrix has higher safety, is easy to make, and has high controllability. The wound adhesive polyphenol hydrogel material provided by the present invention has strong deformability and injectability, and can be injected by a syringe so as to better fit irregular wound surfaces in all directions to achieve better healing effects.

[0018] Naturally occurring and inexpensive tannic acid, as an effective gel adhesion unit, can grab polymer chains through hydrogen bonds and ionic bonds and cross-link polymer chains. Tannic acid-based hydrogels show diverse functions, adjustable mechanical properties, rapid self-healing, etc. In addition, tannic acid has a stronger conjugation effect due to its biphenyl structure, and as a cross-linking agent, it can give hydrogels higher adhesion. Tannic acid also has certain antioxidant and antibacterial properties, which can inhibit wound infection, scavenge free radicals, and limit the inflammatory response of the wound, thereby more effectively promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the synthesis of injectable self-healing polyphenol hydrogel for promoting wound healing according to an example of the present invention.

[0020] Figure 2 H 1 NMR spectra.

[0021] Figure 3 IR graph of the prepared polyphenol injectable self-healing hydrogel.

[0022] Figure 4 (a) and (b) are scanning electron micrographs of the prepared polyphenol injectable self-healing hydrogel.

[0023] Figure 5 The rheological properties of the polyphenol hydrogels prepared in Examples 3 to 5 are shown below.

[0024] Figure 6 Figure 3 shows the tissue adhesion and injectability of the prepared polyphenol injectable self-healing hydrogel.

[0025] Figure 7 This is a diagram showing the antibacterial effect of the prepared polyphenol injectable self-healing hydrogel.

[0026] Figure 8 This is a diagram showing the effect of the prepared polyphenol injectable self-healing hydrogel in promoting wound healing in rats.

[0027] Fig. 9 (a) and (b) are the cell survival and viability data of Example 3 and Comparative Example. DETAILED DESCRIPTION

[0028] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0029] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0030] One of the specific implementations:

[0031] A polyphenol injectable self-healing hydrogel for promoting wound healing and a preparation method thereof, comprising the following steps:

[0032] Step 1, taking hyaluronic acid or its sodium salt and a modifier, and reacting to obtain a hyaluronic acid derivative, wherein the hyaluronic acid derivative is catechol-modified hyaluronic acid;

[0033] Step 2, preparing polyacrylamide by taking acrylamide, methylene bisacrylamide and ammonium persulfate;

[0034] Step 3, taking the hyaluronic acid derivative and polyacrylamide, adding a polyphenol compound, adding a certain amount of water, and reacting to obtain a self-healing hydrogel.

[0035] According to the self-healing hydrogel and preparation method thereof of some examples of the present invention, the modifier is dopamine hydrochloride.

[0036] According to the self-healing hydrogel and preparation method thereof of some examples of the present invention, step (1) is specifically as follows: dissolving hyaluronic acid (HA) in a morpholineethanesulfonic acid buffer solution (0.05M, pH=5.5), adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and after complete dissolution, performing an activation carboxyl reaction under stirring, the reaction temperature is 20-35°C, and the reaction time is 20-40min. After the reaction is completed, dopamine hydrochloride (DA) is added to the above solution, and a grafting reaction is performed under a nitrogen protection atmosphere, the reaction temperature is 20-35°C, and the reaction time is 12-72h. After the reaction is completed, dialysis is performed to remove impurities. The dialyzed solution is freeze-dried and sealed to be stored in a dark place to obtain catechol-grafted hyaluronic acid.

[0037] According to the method for preparing the self-healing hydrogel of some examples of the present invention, in step (1), the mass ratio of hyaluronic acid: dopamine hydrochloride: 1-ethyl-(3-dimethylaminopropyl) carbodiimide: N-hydroxysuccinimide is 2:2:1:2.

[0038] According to the method for preparing the self-healing hydrogel of some examples of the present invention, the molecular weight of the hyaluronic acid in step (1) is 90,000-400,000.

[0039] According to the method for preparing the self-healing hydrogel of some examples of the present invention, step (2) is specifically as follows: dissolving acrylamide in water to obtain a solution with a mass fraction of 1g:10-50mL, adding methylene bisacrylamide and ammonium persulfate to react for 15-20 minutes. After the reaction is completed, dialyzing is performed to remove unreacted acrylamide, catalyst and by-products, and the dialyzed solution is freeze-dried and sealed for storage.

[0040] According to the method for preparing the self-healing hydrogel of some examples of the present invention, the dialysis supplies used in steps (1) and (2) are dialysis bags or dialysis tubes with a molecular weight cutoff (MWCO) of 3500-14000.

[0041] In step (2) of the method for preparing the self-healing hydrogel according to some examples of the present invention, the mass ratio of methylenebisacrylamide, ammonium persulfate and acrylamide is 0.001:0.01:1.

[0042] According to the method for preparing the self-healing hydrogel of some examples of the present invention, the polyphenol compound in step (3) is one of tannic acid, gallic acid and the like.

[0043] According to the method for preparing the self-healing hydrogel of some examples of the present invention, in step (3), the concentration of the hyaluronic acid derivative is 0.03-0.15 g / mL, and the concentration of the polyacrylamide is 0.03-0.15 g / mL.

[0044] According to the method for preparing the self-healing hydrogel of some examples of the present invention, the mass concentration of tannic acid in step (3) is 1 to 20 mg / mL.

[0045] The polyphenol injectable self-healing hydrogel for promoting wound healing is prepared by the method.

[0046] Example 1

[0047] Weigh 0.5g of hyaluronic acid (HA) and dissolve it in a morpholineethanesulfonic acid buffer solution (0.05M, pH=5.5), add 0.5g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 0.25g of N-hydroxysuccinimide (NHS), and react for 20 minutes under stirring after complete dissolution. After the reaction is completed, add 0.5g of dopamine hydrochloride (DA) to the above solution and react for 12 hours under a nitrogen protection atmosphere. After the reaction is completed, the product is dialyzed for 72 hours with a 8000-14000Da dialysis bag to remove impurities. The dialyzed solution is freeze-dried to obtain catechol-grafted hyaluronic acid and sealed for storage in the dark.

[0048] Example 2

[0049] Weigh 0.5g of acrylamide, add 150μL of 1% methylene bisacrylamide and 100μL of 2% ammonium persulfate, add deionized water to make 2mL, mix well and react for 30 minutes to obtain polyacrylamide. After the reaction is completed, the product is dialyzed with a 8000-14000Da dialysis bag for 48h to remove impurities. The dialyzed solution is freeze-dried to obtain polyacrylamide and sealed to avoid light for storage.

[0050] Example 3

[0051] Weigh 0.1 g of the hyaluronic acid derivative prepared in Example 1 and 0.1 g of the polyacrylamide prepared in Example 2, add 1 mg of tannic acid, and add the remaining amount of water to make 2 ml of hydrogel solution. After stirring evenly, let it stand to obtain a polyphenol injectable self-healing hydrogel that promotes wound healing.

[0052] Example 4

[0053] Weigh 1 g of the hyaluronic acid derivative prepared in Example 1 and 1 g of the polyacrylamide prepared in Example 2, add 30 mg of tannic acid, and add the remaining amount of water to make 20 ml of hydrogel solution. After stirring evenly, let it stand to obtain a polyphenol injectable self-healing hydrogel that promotes wound healing.

[0054] Example 5

[0055] Weigh 2 g of the hyaluronic acid derivative prepared in Example 1 and 1 g of acrylamide prepared in Example 2, add 50 mg of tannic acid, add the remainder of water to make 20 ml of a hydrogel solution, stir evenly and let stand to obtain a polyphenol injectable self-healing hydrogel that promotes wound healing.

[0056] Comparative Example 1

[0057] Weigh 0.1 g of acrylamide, add 30 μL of 1% methylene bisacrylamide and 100 μL of 1% ammonium persulfate, add deionized water to make 2 mL, weigh 0.1 g of the hyaluronic acid derivative prepared in Example 1, add it, mix well and react for 40 minutes to obtain polyacrylamide. Add 1 mg of tannic acid, stir well and let stand to obtain a polyphenol injectable self-healing hydrogel that promotes wound healing.

[0058] Performance Testing

[0059] The catechol-grafted hyaluronic acid synthesized in Example 1 was analyzed by nuclear magnetic resonance spectroscopy ( 1 H NMR) Figure 2 shown.

[0060] The structure of the injectable self-healing hydrogel prepared in Example 3 was characterized by Fourier transform infrared spectroscopy (FTIR). Figure 3 shown.

[0061] After the polyphenol injectable self-healing hydrogel was freeze-dried, its morphology was observed using a scanning electron microscope (ZEISSMERLIN Compact, Oxford x-max). The SEM electron microscope image is shown in the figure. Figure 4 shown.

[0062] The self-healing hydrogels prepared in Examples 3 to 5 were tested using a TA rheometer (AR2000ex). The test temperature was 25°C, the mold plate spacing was 1000 μm, and the storage modulus (G') and loss modulus (G") were obtained in the range of 0.1 to 100 rad / s under a constant strain of 1%. The results are shown in Table 1. Figure 5 .

[0063] Pigskin was used to test the adhesion of the self-healing hydrogel. The self-healing hydrogel was injected from a syringe and adhered to clean pigskin. The pigskin was also bent and twisted to simulate body movement to test whether the hydrogel had cracks. Furthermore, the gel was injected into the finger joints to simulate trauma at the joints. The joints were rotated to test whether the hydrogel could fully adhere to the human skin. The results are as follows: Figure 6 .

[0064] The hydrogels prepared in Example 3 and Comparative Example 1 were co-cultured with L929 cells for three days, and the co-cultured cells were stained with DAPI and PI dyes, and their states were observed under an inverted fluorescence microscope. Fig. 9 , the number of cells is not much different, but from the fluorescence staining image, it can be seen that the cells co-cultured in Example 3 present an irregular spindle shape, while the cells in the control group present a spherical shape, and their development is somewhat impaired.

Claims

1. A self-healing hydrogel, characterized in that: The hydrogel is a hydrogel formed by cross-linking the two matrices of polyacrylamide and dopamine hydrochloride modified hyaluronic acid as gel matrix, and the polyphenol compound is tannic acid. The self-healing hydrogel is prepared by the following method: at room temperature, polyacrylamide and hyaluronic acid modified by dopamine hydrochloride are mixed, a polyphenol compound is added, a certain amount of water is added, and the self-healing hydrogel is obtained by reaction, wherein the concentration of the hyaluronic acid modified by dopamine hydrochloride is 0.03-0.15 g / mL, the concentration of polyacrylamide is 0.03-0.15 g / mL, and the mass concentration of the polyphenol compound is 1-20 mg / mL.

2. The self-healing hydrogel according to claim 1, characterized in that: The mass ratio of the polyacrylamide, the hyaluronic acid modified by dopamine hydrochloride and the polyphenol compound is 1:1-5:0.001-0.

01.

3. The self-healing hydrogel according to claim 1, characterized in that: The dopamine hydrochloride modified hyaluronic acid is prepared by the following method: dissolving hyaluronic acid in a morpholineethanesulfonic acid buffer solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and after complete dissolution, performing a carboxyl activation reaction under stirring, the reaction temperature is 20-35° C., and the reaction time is 20-40 min; after the reaction is completed, adding dopamine hydrochloride to the above solution, performing a grafting reaction under a nitrogen protection atmosphere, the reaction temperature is 20-35° C., the reaction time is 12-72 h, dialyzing, and freeze-drying to obtain dopamine hydrochloride modified hyaluronic acid. The mass ratio of hyaluronic acid: dopamine hydrochloride: 1-ethyl-(3-dimethylaminopropyl) carbodiimide: N-hydroxysuccinimide is 2:2:1:2; and the molecular weight of hyaluronic acid is 90,000 to 400,000.

4. The self-healing hydrogel according to claim 1, characterized in that: The polyacrylamide is prepared according to the following method: acrylamide is dissolved in water at a ratio of 1 g: 10 to 50 mL, and methylene bisacrylamide and ammonium persulfate are added to react for 15 to 20 minutes; after the reaction, unreacted acrylamide, catalyst and by-products are removed by dialysis, and the dialyzed solution is freeze-dried and sealed for storage, wherein the mass ratio of methylene bisacrylamide, ammonium persulfate and acrylamide is 0.001:0.01:

1.

5. The self-healing hydrogel according to claim 4, characterized in that: The molecular weight cut-off for dialysis is 3500-14000.

6. The self-healing hydrogel according to claim 1, characterized in that: The obtained hydrogel is injectable.

7. The method for preparing the self-healing hydrogel according to claim 1, characterized in that: At room temperature, polyacrylamide and hyaluronic acid modified with dopamine hydrochloride are mixed, and then a polyphenol compound and a certain amount of water are added to react to obtain a self-healing hydrogel, wherein the concentration of the hyaluronic acid modified with dopamine hydrochloride is 0.03-0.15 g / mL, the concentration of polyacrylamide is 0.03-0.15 g / mL, and the mass concentration of the polyphenol compound is 1-20 mg / mL.

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