Multifunctional hydrogel for wound treatment and preparation method thereof

Abstract

The invention provides multifunctional hydrogel for wound treatment and a preparation method of the multifunctional hydrogel, and belongs to the technical field of polymer hydrogel and new biomedical materials. The preparation method comprises the following steps: mixing a functional polymer simultaneously containing an aldehyde group and a phenylboronic acid structure, a polymer containing an amino group, a polymer containing a diol structure and a small molecule compound simultaneously containing an aldehyde group and a catechol structure in a solvent, adding metal ions, and uniformly mixing to obtain the multifunctional hydrogel for wound treatment. The multifunctional hydrogel has the advantages of excellent toughness, ductility, injectability, controllable tissue adhesion, broad-spectrum antibacterial property, oxidation resistance and the like, and can effectively promote healing of full-thickness skin defect wounds. In addition, the preparation method of the multifunctional hydrogel is simple, the gel forming speed is high, the reaction condition is mild, the cost is low, and large-scale preparation and application are facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of high molecular hydrogel and biomedical new material technology, and particularly relates to a multifunctional hydrogel for wound treatment and a preparation method thereof. BACKGROUND

[0002] Skin is the largest organ of the human body and the first line of defense of the human body, which plays a key role in maintaining body homeostasis, blocking the invasion of pathogenic microorganisms from the outside world and regulating body temperature. However, the skin is directly exposed to the external environment and is prone to injury. At present, traumatic wounds caused by various traffic accidents, accidental injuries and natural disasters are increasingly becoming a heavy social and economic burden, and the treatment of wounds is still a serious challenge in clinical practice.

[0003] The normal wound healing process consists of four stages with some overlap and independence: hemostasis stage, inflammation stage, proliferation stage and remodeling stage. After trauma occurs, the body activates the coagulation system at the first time, in this stage, the blood vessels contract, the platelets are activated and aggregated, the fibrinogen is converted into fibrin under the action of thrombin, and then cross-linked to form a thrombus to adhere to the wound, achieving the purpose of hemostasis; in the inflammation stage, immune cells such as neutrophils and macrophages are recruited to the wound, their main role is to clean up pathogenic microorganisms and damaged tissues at the wound site, etc., while secreting various cytokines, which is conducive to the healing process entering the next stage; in the proliferation stage, various cells proliferate and migrate actively, the wound is gradually filled with granulation tissue, accompanied by re-epithelialization and angiogenesis; then the wound enters the remodeling stage, the wound surface continuously contracts, collagen remodels from type III to type I, and the wound gradually becomes a scar. As can be seen, skin wound healing is a very complex and dynamic process, if the normal healing pathway is hindered or destroyed, a chronic non-healing wound may be formed. Therefore, it is of great significance to develop a treatment strategy that can meet the needs of wound healing.

[0004] Medical dressings are a common wound treatment strategy in clinical practice, traditional dressings are mainly medical gauze, which mainly plays a role in protecting the wound and absorbing exudate, but the dried gauze after absorbing exudate is easy to adhere to the wound, causing pain during dressing change. In order to overcome this problem, gauze is combined with vaseline to make vaseline gauze, but this type of dressing still cannot meet the needs of wound treatment. In recent years, with the in-depth understanding of the mechanism of wound healing and the rapid development of biological materials, various new dressings, such as foam, hydrogel, film, nanofiber and hydrogel dressings, have been developed. Among them, hydrogel dressings have attracted widespread attention, the three-dimensional cross-linked network structure of hydrogel is similar to the extracellular matrix, which can promote cell proliferation and migration; at the same time, its high water content characteristics can provide a humid microenvironment; most hydrogels have good biocompatibility; in addition, the porous structure of hydrogel makes it exhibit unique advantages in drug delivery.

[0005] However, the conventional hydrogel-based dressings usually have the following problems: easy to fall off from the wound surface, leading to dressing failure; slow self-healing speed after the structure is damaged, leading to an increase in the number of dressing changes; poor mechanical properties unsuitable for skin tissue, unable to provide an effective barrier for the wound surface; poor antibacterial effect, leading to wound infection; unable to effectively regulate the immune microenvironment, affecting the healing process of the wound, etc. The current common hydrogel dressings on the market have relatively single functions and cannot meet the treatment needs of wounds. Therefore, it is an urgent technical problem to develop a new type of multifunctional hydrogel dressing. SUMMARY

[0006] In view of the above problems of the prior art, the present application provides a multifunctional hydrogel for wound treatment and a preparation method thereof. The multifunctional hydrogel has good self-healing and adhesion properties, suitable mechanical properties, excellent antibacterial properties, and good anti-inflammatory and antioxidant properties, and can effectively improve the wound healing microenvironment and promote wound healing.

[0007] To achieve the above-mentioned purposes, the specific technical solutions of the present application are as follows:

[0008] The present application provides a preparation method of a multifunctional hydrogel for wound treatment, comprising the following steps:

[0009] S1, oxidizing modification of a polymer containing an ortho-hydroxyl structure to obtain a functionalized polymer containing an aldehyde group;

[0010] S2, reacting the functionalized polymer containing an aldehyde group with an amino-containing phenylboronic acid compound to obtain a functionalized polymer containing both an aldehyde group and a phenylboronic acid structure;

[0011] S3, mixing the functionalized polymer containing both an aldehyde group and a phenylboronic acid structure, the amino-containing polymer, the diol-containing polymer, and the small molecule compound containing both an aldehyde group and an ortho-diphenol structure in a solvent, and adding metal ions to mix uniformly to obtain a multifunctional hydrogel for wound treatment.

[0012] The present application obtains a functional polymer containing aldehyde group by first oxidizing a polymer containing ortho-hydroxyl structure, then obtains a functional polymer containing aldehyde group and phenylboronic acid structure by reacting the functional polymer containing aldehyde group with an amino-containing phenylboronic acid compound, and finally obtains a multifunctional hydrogel for wound treatment by mixing the functional polymer containing aldehyde group and phenylboronic acid structure, an amino-containing polymer, a diol-containing polymer, a small molecule compound containing aldehyde group and ortho-phenol structure in a solvent, and adding metal ions. The multifunctional hydrogel contains a multi-network structure, in which a cross-linking network is formed between the functional polymer containing aldehyde group and phenylboronic acid structure and the amino-containing polymer through Schiff base; a cross-linking network is formed between the functional polymer containing aldehyde group and phenylboronic acid structure and the diol-containing polymer through borate ester bond, and a cross-linking network is formed between the metal ions and the small molecule compound containing aldehyde group and ortho-phenol structure through coordination; and the small molecule compound containing aldehyde group and ortho-phenol structure also exists as a "dynamic lasso" in the hydrogel, which is cross-linked with the functional polymer containing aldehyde group and phenylboronic acid structure through borate ester bond and with the amino-containing polymer through Schiff base bond, so as to further effectively improve the toughness and ductility of the hydrogel.

[0013] Further, in step S1, the polymer containing ortho-hydroxyl structure includes at least one of natural polysaccharides containing ortho-hydroxyl structure and derivatives thereof.

[0014] Further, the natural polysaccharides containing ortho-hydroxyl structure include but are not limited to at least one of dextran, hyaluronic acid, sodium alginate and cellulose.

[0015] Further, the specific operation of step S1 is as follows: the polymer containing ortho-hydroxyl structure is mixed with an oxidizing agent in water to perform oxidation reaction; after the reaction is completed, the reaction solution is purified and dried to obtain the functional polymer containing aldehyde group. The purification method includes but is not limited to dialysis method, and the drying method includes but is not limited to freeze-drying method.

[0016] Further, the mass ratio of the polymer containing ortho-hydroxyl structure to the oxidizing agent is 1: (0.3-1.2).

[0017] Further, the temperature of the oxidation reaction is 25-40 ℃, and the time is 2-8 h.

[0018] Further, the oxidizing agent includes but is not limited to sodium periodate.

[0019] Further, in step S2, the mass ratio of the functional polymer containing aldehyde group to the amino-containing phenylboronic acid compound is 1: (0.1-0.8).

[0020] Further, in step S2, the amino-containing phenylboronic acid compound includes but is not limited to 3-amino-phenylboronic acid.

[0021] Further, in step S2, the reaction temperature is 20-40℃, and the reaction time is 8-24 h.

[0022] Further, in step S2, the specific reaction process is as follows: the aldehyde group-containing functional polymer is dissolved in water, and then the amino-containing phenylboronic acid compound is added for reaction, and the reaction solution is purified and dried to obtain the functional polymer containing both aldehyde group and phenylboronic acid structure.

[0023] Further, in step S3, the final concentration of the functional polymer containing both aldehyde group and phenylboronic acid structure in the solvent is 1 wt%-6 wt%, the final concentration of the amino-containing polymer in the solvent is 0.5 wt%-4 wt%, the final concentration of the diol structure-containing polymer in the solvent is 1 wt%-6 wt%, and the concentration of the small molecule compound containing both aldehyde group and catechol structure in the solvent is 0.2 wt%-3 wt%, and the final concentration of the metal ion in the solvent is 0.2-3 mg / mL.

[0024] Further, the mass ratio of the functional polymer containing both aldehyde group and phenylboronic acid structure, the amino-containing polymer, and the diol structure-containing polymer is (1-12):(1-4):(1-12).

[0025] Further, in step S3, the amino-containing polymer includes but is not limited to at least one of chitosan and its derivatives, collagen and its derivatives, polylysine and its derivatives, and polyethyleneimine and its derivatives.

[0026] Further, in step S3, the diol structure-containing polymer includes but is not limited to at least one of hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives, polyvinyl alcohol and its derivatives, and sodium alginate and its derivatives.

[0027] Further, in step S3, the small molecule compound containing both aldehyde group and catechol structure includes but is not limited to 3,4-dihydroxybenzaldehyde.

[0028] Further, in step S3, the metal ion includes but is not limited to at least one of copper ion, silver ion, zinc ion, gold ion, iron ion, and gallium ion.

[0029] The application also provides a multifunctional hydrogel prepared by the method.

[0030] The application also provides the application of the multifunctional hydrogel in wound treatment, especially in acute wound treatment.

[0031] Compared with the prior art, the application has the advantages of:

[0032] 1、The multifunctional hydrogel provided by the application is prepared by first preparing a functional polymer containing an aldehyde group and a phenylboronic acid structure, and then allowing the functional polymer to interact with a biocompatible polymer containing an amino group and a polymer containing a diol structure. The functional polymer containing an aldehyde group and a phenylboronic acid structure simultaneously forms a crosslinked network with the polymer containing an amino group through a Schiff base, and forms a crosslinked network with the polymer containing a diol structure through a boronic ester bond. Meanwhile, metal ions form a crosslinked network with a small-molecule compound containing an aldehyde group and a catechol structure through coordination. The small-molecule compound containing an aldehyde group and a catechol structure simultaneously also exists in the hydrogel as a "dynamic lasso", which is crosslinked with the functional polymer containing an aldehyde group and a phenylboronic acid structure through a boronic ester bond, and is crosslinked with the polymer containing an amino group through a Schiff base bond, so as to further effectively improve the toughness and ductility of the hydrogel. The multifunctional hydrogel provided by the application does not need to use a crosslinking agent, can avoid the toxic effects caused by the crosslinking agent, and has the advantages of simple preparation process, fast gelation speed, mild reaction conditions, low cost and facilitation of industrial scale production.

[0033] 2、The multifunctional hydrogel provided by the application is prepared based on a dynamic covalent bond, has good self-healing performance and injectability, and can meet the treatment needs of irregular wounds. In addition, the multifunctional hydrogel has controllable adhesion, can firmly adhere to skin tissue in a physiological environment, and can reduce the adhesion of the hydrogel to the skin tissue in a weak acid solution, which is conducive to reducing secondary damage that may be caused by changing dressings. More importantly, the multifunctional hydrogel has good toughness and ductility, can closely adhere to the skin, and is especially suitable for the treatment of wounds in active parts.

[0034] 3、The multifunctional hydrogel provided by the application has good biological activity, and natural polyphenols and metal ions together endow the hydrogel with good broad-spectrum antibacterial ability, which can effectively prevent wound infection and reduce the probability of the generation of drug-resistant bacteria. The hydrogel has good antioxidant performance and can eliminate excess free radicals. In addition, the multifunctional hydrogel can also inhibit excessive inflammation and improve the microenvironment of a wound.

[0035] 4、The multifunctional hydrogel provided by the application has good blood and cell compatibility, can effectively promote the healing of full-thickness skin defect wounds, and has a wide application prospect in wound treatment. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1NMR spectrum of OD-PBA in Example 1 of the present application;

[0037] Figure 2 Gelation of multifunctional hydrogel in Example 1 of the present application;

[0038] Figure 3 Self-healing property of multifunctional hydrogel in Example 1 of the present application;

[0039] Figure 4 Injectability of multifunctional hydrogel in Example 1 of the present application;

[0040] Figure 5 Adhesion property of multifunctional hydrogel in Example 1 of the present application;

[0041] Figure 6 Mechanical tensile property of multifunctional hydrogel in Example 1 of the present application;

[0042] Figure 7 Cytotoxicity result of multifunctional hydrogel in Example 1 of the present application;

[0043] Figure 8 Hemolysis test result of multifunctional hydrogel in Example 1 of the present application;

[0044] Figure 9 Antibacterial result of multifunctional hydrogel in Example 1 of the present application;

[0045] Figure 10 Anti-inflammatory result of multifunctional hydrogel in Example 1 of the present application;

[0046] Figure 11 Antioxidant result of multifunctional hydrogel in Example 1 of the present application;

[0047] Figure 12 Effect of multifunctional hydrogel in Example 1 of the present application in treating rat back wound;

[0048] Figure 13 H&E staining of wound tissue during treatment of wound by multifunctional hydrogel in Example 1 of the present application.

[0049] In the figures, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001. DETAILED DESCRIPTION

[0050] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0051] The present application provides a preparation method of a multifunctional hydrogel for wound treatment, comprising the following steps:

[0052] S1, oxidizing modification is performed on a polymer containing an ortho-hydroxyl structure to obtain an aldehyde group-containing functionalized polymer;

[0053] S2, the aldehyde group-containing functionalized polymer is reacted with an amino group-containing phenylboronic acid compound to obtain a functionalized polymer containing both an aldehyde group and a phenylboronic acid structure;

[0054] S3, the functionalized polymer containing both an aldehyde group and a phenylboronic acid structure, an amino group-containing polymer, a diol structure-containing polymer, and a small molecule compound containing both an aldehyde group and an ortho-phenol structure are mixed in a solvent, and a metal ion is added and uniformly mixed to obtain a multifunctional hydrogel for wound treatment.

[0055] In some examples, in step S1, the polymer containing an ortho-hydroxyl structure includes at least one of a natural polysaccharide containing an ortho-hydroxyl structure and a derivative thereof; and the natural polysaccharide containing an ortho-hydroxyl structure is selected from at least one of dextran, hyaluronic acid, sodium alginate, and cellulose.

[0056] In some examples, the specific operation of step S1 is as follows: the polymer containing an ortho-hydroxyl structure and sodium periodate are mixed in water at a mass ratio of 1: (0.3-1.2), and an oxidation reaction is performed at 25-40℃ for 2-8h; after the reaction is completed, the reaction solution is purified and dried to obtain the aldehyde group-containing functionalized polymer.

[0057] In some examples, in step S2, the mass ratio of the aldehyde group-containing functionalized polymer to the amino group-containing phenylboronic acid compound is 1: (0.1-0.8).

[0058] In some examples, in step S2, the reaction temperature is 20-40℃, and the reaction time is 8-24h.

[0059] In some examples, in step S3, the final concentration of the functionalized polymer containing aldehyde group and phenylboronic acid structure in the solvent is 1 wt% to 6 wt%, the final concentration of the polymer containing amino group in the solvent is 0.5 wt% to 4 wt%, the final concentration of the polymer containing diol structure in the solvent is 1 wt% to 6 wt%, the final concentration of the small molecule compound containing both aldehyde group and catechol structure in the solvent is 0.2 wt% to 3 wt%, and the final concentration of the metal ion in the solvent is 0.2 to 3 mg / mL.

[0060] In some examples, the mass ratio of the functionalized polymer containing aldehyde group and phenylboronic acid structure, the polymer containing amino group, and the polymer containing diol structure is (1-12):(1-4):(1-12).

[0061] In some examples, in step S3, the polymer containing amino group is selected from at least one of chitosan and its derivatives, collagen and its derivatives, polylysine and its derivatives, and polyethyleneimine and its derivatives; the polymer containing diol structure is selected from at least one of hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives, polyvinyl alcohol and its derivatives, and sodium alginate and its derivatives; the small molecule compound containing both aldehyde group and catechol structure is 3,4-dihydroxybenzaldehyde; and the metal ion is selected from at least one of copper ion, silver ion, zinc ion, gold ion, iron ion, and gallium ion.

[0062] Example 1

[0063] A multifunctional hydrogel for wound treatment is prepared by the following method:

[0064] (1) Preparation of functionalized dextran containing aldehyde group (OD)

[0065] 1 g of dextran was dissolved in 25 mL of ultrapure water, and 0.6 g of sodium periodate was dissolved in 5 mL of ultrapure water. The sodium periodate aqueous solution was added dropwise to the dextran solution in a dark environment, and the reaction was stirred at room temperature for 6 h in the dark. Then 1 mL of ethylene glycol was added to terminate the reaction, and the reaction solution was loaded into a 3500 Da dialysis bag and dialyzed in pure water for 3 days. After freeze-drying, the functionalized dextran containing aldehyde group was obtained.

[0066] (2) Preparation of functionalized dextran containing both aldehyde group and phenylboronic acid structure (OD-PBA)

[0067] Take 1 g of functionalized dextran containing aldehyde group dissolved in 20 mL of ultrapure water, then add 0.4 g of 3-aminobenzoic acid, stir at room temperature for 12 h. After the reaction is completed, the reaction solution is loaded into a 3500 Da dialysis bag, dialyzed in pure water for 3 days, freeze-dried to obtain functionalized dextran containing aldehyde group and phenylboronic acid structure.

[0068] (3) Preparation of multifunctional hydrogel

[0069] Dissolve the functionalized dextran containing aldehyde group and phenylboronic acid structure, carboxymethyl chitosan and polyvinyl alcohol in ultrapure water to prepare precursor solutions with concentrations of 10 wt%, 5 wt% and 10 wt% respectively, mix equal volumes of the three to obtain a basic hydrogel. Add 3,4-dihydroxybenzaldehyde to the basic hydrogel to obtain a functionalized hydrogel containing 3,4-dihydroxybenzaldehyde. Add gallium ions to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde to obtain a multifunctional hydrogel containing 3,4-dihydroxybenzaldehyde and gallium ions.

[0070] Example 2

[0071] A multifunctional hydrogel for wound treatment is prepared as follows:

[0072] (1) Preparation of aldehyde-containing functionalized hyaluronic acid (OHA)

[0073] Take 1 g of hyaluronic acid and dissolve it in 25 mL of ultrapure water, then take 0.6 g of sodium periodate and dissolve it in 5 mL of ultrapure water, add the sodium periodate solution dropwise to the hyaluronic acid solution in the dark, stir at room temperature for 3 h. Then add 1 mL of ethylene glycol to terminate the reaction, load the reaction solution into a 3500 Da dialysis bag, dialyze in pure water for 3 days, freeze-dry to obtain functionalized hyaluronic acid containing aldehyde group.

[0074] (2) Preparation of functionalized hyaluronic acid containing aldehyde group and phenylboronic acid structure (OHA-PBA)

[0075] Take 1 g of functionalized hyaluronic acid containing aldehyde group and dissolve it in 20 mL of ultrapure water, then add 0.2 g of 3-aminobenzoic acid, stir at room temperature for 12 h. After the reaction is completed, the reaction solution is loaded into a 3500 Da dialysis bag, dialyzed in pure water for 3 days, freeze-dried to obtain functionalized hyaluronic acid containing aldehyde group and phenylboronic acid structure.

[0076] (3) Preparation of multifunctional hydrogel

[0077] The functionalized hyaluronic acid containing both aldehyde group and phenylboronic acid structure, carboxymethyl chitosan and polyvinyl alcohol are dissolved in ultrapure water to prepare precursor solutions with concentrations of 6 wt%, 5 wt% and 10 wt% respectively, and then the three solutions are mixed in equal volume to obtain a basic hydrogel. 3,4-Dihydroxybenzaldehyde is added to the basic hydrogel to obtain a functionalized hydrogel containing 3,4-dihydroxybenzaldehyde with a final concentration of 1 wt%. Gallium ions are added to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde to obtain a multifunctional hydrogel containing both 3,4-dihydroxybenzaldehyde and gallium ions with a final concentration of 1 mg / mL.

[0078] Example 3

[0079] A multifunctional hydrogel for wound treatment is prepared as follows:

[0080] (1) Preparation of functionalized dextran (OD) containing aldehyde group

[0081] 1 g of dextran is weighed and dissolved in 25 mL of ultrapure water, and 0.6 g of sodium periodate is weighed and dissolved in 5 mL of ultrapure water. The sodium periodate aqueous solution is added dropwise to the dextran solution in a dark environment, and the reaction is stirred at room temperature for 6 h in the dark. Then 1 mL of ethylene glycol is added to terminate the reaction, and the reaction solution is placed in a 3500 Da dialysis bag and dialyzed in pure water for 3 days. After freeze-drying, functionalized dextran containing aldehyde group is obtained.

[0082] (2) Preparation of functionalized dextran (OD-PBA) containing both aldehyde group and phenylboronic acid structure

[0083] 1 g of functionalized dextran containing aldehyde group is weighed and dissolved in 20 mL of ultrapure water, and then 0.4 g of 3-aminophenylboronic acid is added. The reaction is stirred at room temperature for 12 h. After the reaction is completed, the reaction solution is placed in a 3500 Da dialysis bag and dialyzed in pure water for 3 days. After freeze-drying, functionalized dextran containing both aldehyde group and phenylboronic acid structure is obtained.

[0084] (3) Preparation of multifunctional hydrogel

[0085] The functionalized dextran containing both aldehyde group and phenylboronic acid structure, carboxymethyl chitosan and dextran are dissolved in ultrapure water to prepare precursor solutions with concentrations of 10 wt%, 5 wt% and 6 wt% respectively, and then the three solutions are mixed in equal volume to obtain a basic hydrogel. 3,4-Dihydroxybenzaldehyde is added to the basic hydrogel to obtain a functionalized hydrogel containing 3,4-dihydroxybenzaldehyde with a final concentration of 1 wt%. Copper ions are added to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde to obtain a multifunctional hydrogel containing both 3,4-dihydroxybenzaldehyde and copper ions with a final concentration of 1 mg / mL.

[0086] Example 4

[0087] A multifunctional hydrogel for wound treatment is prepared as follows:

[0088] (1) Preparation of functionalized dextran containing aldehyde group (OD)

[0089] Weigh 1 g of dextran and dissolve it in 25 mL of ultrapure water. Weigh 0.6 g of sodium periodate and dissolve it in 5 mL of ultrapure water. Add the sodium periodate solution dropwise to the dextran solution in a dark environment. Stir the mixture at 30°C for 4 hours in the dark. Then add 1 mL of ethylene glycol to terminate the reaction. Place the reaction solution in a 3500 Da dialysis bag and dialyze it in pure water for 3 days. Freeze-dry the dialyzed solution to obtain functionalized dextran containing aldehyde groups.

[0090] (2) Preparation of functionalized dextran containing both aldehyde group and phenylboronic acid structure (OD-PBA)

[0091] Weigh 1 g of functionalized dextran containing aldehyde groups and dissolve it in 20 mL of ultrapure water. Then add 0.3 g of 3-aminophenylboronic acid and stir the mixture at room temperature for 16 hours. After the reaction is completed, place the reaction solution in a 3500 Da dialysis bag and dialyze it in pure water for 3 days. Freeze-dry the dialyzed solution to obtain functionalized dextran containing both aldehyde group and phenylboronic acid structure.

[0092] (3) Preparation of multifunctional hydrogel

[0093] Dissolve the functionalized dextran containing both aldehyde group and phenylboronic acid structure, chitosan quaternary ammonium salt, and polyvinyl alcohol in ultrapure water to prepare precursor solutions with concentrations of 10 wt%, 5 wt%, and 10 wt%, respectively. Mix equal volumes of the three solutions to obtain a basic hydrogel. Add 3,4-dihydroxybenzaldehyde to the basic hydrogel to obtain a functionalized hydrogel containing 3,4-dihydroxybenzaldehyde with a final concentration of 2 wt%. Add silver ions to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde to obtain a multifunctional hydrogel containing both 3,4-dihydroxybenzaldehyde and silver ions with a final concentration of 1 mg / mL.

[0094] Example 5

[0095] A multifunctional hydrogel for wound treatment is prepared as follows:

[0096] (1) Preparation of functionalized dextran containing aldehyde group (OD)

[0097] Take 1 g of dextran and dissolve it in 25 mL of ultrapure water, then take 0.6 g of sodium periodate and dissolve it in 5 mL of ultrapure water, add the sodium periodate aqueous solution dropwise to the dextran solution in the dark environment, stir the reaction at room temperature for 8 h. Then add 1 mL of ethylene glycol to terminate the reaction, load the reaction solution into a 3500 Da dialysis bag, dialyze in pure water for 3 days, freeze-dry to obtain functionalized dextran containing aldehyde groups.

[0098] (2) Preparation of functionalized dextran (OD-PBA) containing both aldehyde groups and phenylboronic acid structures

[0099] Take 1 g of functionalized dextran containing aldehyde groups and dissolve it in 20 mL of ultrapure water, then add 0.5 g of 3-aminophenylboronic acid, stir the reaction at room temperature for 12 h. After the reaction is completed, load the reaction solution into a 3500 Da dialysis bag, dialyze in pure water for 3 days, freeze-dry to obtain functionalized dextran containing both aldehyde groups and phenylboronic acid structures.

[0100] (3) Preparation of multifunctional hydrogel

[0101] Dissolve the functionalized dextran containing both aldehyde groups and phenylboronic acid structures, gelatin and polyvinyl alcohol in ultrapure water to prepare precursor solutions with concentrations of 10 wt%, 8 wt% and 10 wt% respectively, mix equal volumes of the three to obtain a basic hydrogel. Add 3,4-dihydroxybenzaldehyde to the basic hydrogel to obtain a functionalized hydrogel containing 3,4-dihydroxybenzaldehyde. Add gallium ions to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde to obtain a multifunctional hydrogel containing both 3,4-dihydroxybenzaldehyde and gallium ions.

[0102] Example 6

[0103] A multifunctional hydrogel for wound treatment is prepared as follows:

[0104] (1) Preparation of functionalized dextran (OD) containing aldehyde groups

[0105] Take 1 g of dextran and dissolve it in 25 mL of ultrapure water, then take 0.6 g of sodium periodate and dissolve it in 5 mL of ultrapure water, add the sodium periodate aqueous solution dropwise to the dextran solution in the dark environment, stir the reaction at room temperature for 8 h. Then add 1 mL of ethylene glycol to terminate the reaction, load the reaction solution into a 3500 Da dialysis bag, dialyze in pure water for 3 days, freeze-dry to obtain functionalized dextran containing aldehyde groups.

[0106] (2) Preparation of functionalized dextran (OD-PBA) containing both aldehyde groups and phenylboronic acid structures

[0107] 1 g of aldehyde-containing functionalized dextran was dissolved in 20 mL of ultrapure water, and then 0.4 g of 3-aminophenylboronic acid was added. The mixture was stirred at 35 °C for 12 h. After the reaction was completed, the reaction solution was placed in a 3500 Da dialysis bag and dialyzed in pure water for 3 days. The solution was then lyophilized to obtain functionalized dextran containing both aldehyde and phenylboronic acid structures.

[0108] (3) Preparation of multifunctional hydrogels

[0109] Functionalized dextran, carboxymethyl chitosan, and polyvinyl alcohol containing both aldehyde and phenylboronic acid groups were dissolved in ultrapure water to prepare precursor solutions with concentrations of 10 wt%, 6 wt%, and 8 wt%, respectively. These solutions were then mixed in equal volumes to obtain a basic hydrogel. 3,4-Dihydroxybenzaldehyde was added to the basic hydrogel to a final concentration of 1 wt% to obtain a functionalized hydrogel containing 3,4-dihydroxybenzaldehyde. Zinc ions were then added to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde to a final concentration of 1 mg / mL to obtain a multifunctional hydrogel containing both 3,4-dihydroxybenzaldehyde and zinc ions.

[0110] Taking the multifunctional hydrogel prepared in Example 1 as an example, tests were conducted. In the following tests and accompanying figures, unless otherwise specified, hydrogels 1-3 represent: Hydrogel 1: The basic hydrogel obtained by uniformly mixing equal volumes of solutions containing both aldehyde and phenylboronic acid functionalized dextran, carboxymethyl chitosan, and polyvinyl alcohol. Hydrogel 2: The functionalized hydrogel containing 3,4-dihydroxybenzaldehyde obtained by adding 3,4-dihydroxybenzaldehyde to the basic hydrogel. Hydrogel 3: The multifunctional hydrogel containing both 3,4-dihydroxybenzaldehyde and gallium ions obtained by adding gallium ions to the functionalized hydrogel containing 3,4-dihydroxybenzaldehyde. The specific test procedures and results are as follows:

[0111] 1. The structure of OD-PBA in Example 1 was characterized by nuclear magnetic resonance spectroscopy, and the results are shown in [Figure 1]. Figure 1 . Figure 1 The results showed that, compared with OD, the NMR spectrum of OD-PBA exhibited a phenyl proton peak at 7.3-7.7 ppm, which is a characteristic peak of 3-aminophenylboronic acid, proving that 3-aminophenylboronic acid was successfully grafted into the aldehyde-containing functionalized dextran molecule.

[0112] 2. The prepared hydrogel was characterized, and the results are shown in the figure. Figures 2-4 . Figure 2 The results show that hydrogels 1-3 are all structurally stable hydrogels. Figure 3The self-healing property of the hydrogel 3 is demonstrated. After two pieces of the hydrogel are contacted with each other for 30 seconds, one end of one piece of the hydrogel is picked up by a pair of tweezers. It is found that the two pieces of the hydrogel have been healed. The healed hydrogel can bear the weight of the two pieces of the hydrogel without breaking. Moreover, after the two pieces of the hydrogel are contacted with each other for 30 minutes, the healed hydrogel can bear a larger tensile force without breaking. It is indicated that the multifunctional hydrogel of the present application has not only a fast healing speed but also a high healing quality. Figure 4 The injectability of the hydrogel 3 is demonstrated. The experimental results show that the hydrogel 3 can be extruded by a syringe and maintained in a gel state. It is indicated that the multifunctional hydrogel of the present application has good injectability.

[0113] 3. The adhesion property of the prepared hydrogel is detected. The hydrogel 3 is adhered to the knuckles of a finger of an experimenter, and the finger is bent into different angles. Whether the hydrogel falls off from the knuckles is observed. Figure 5 As shown in A, the hydrogel 3 can be firmly adhered to the skin of a human body, and does not fall off even when the finger is bent into different angles. It is indicated that the multifunctional hydrogel of the present application has good skin adhesion. In addition, the adhesion property of the hydrogel 3 to the surfaces of different materials is tested. The hydrogel 3 is contacted with plastic, alloy, rubber and glass, respectively, Figure 5 B shows that the hydrogel 3 has good adhesion to different materials. In addition to this, the controllable adhesion of the hydrogel 3 is detected. The hydrogel 3 is adhered to pigskin, and a weakly acidic buffer solution is dropped on the hydrogel. After 20 minutes of action at room temperature, the hydrogel is picked up by a pair of tweezers again. Whether the hydrogel can be removed more easily is observed. Figure 5 As shown in C, the weakly acidic solution can reduce the adhesion of the hydrogel to the pigskin, so that the hydrogel can be removed more easily.

[0114] 4. The mechanical property of the prepared hydrogel is detected. The tensile property of the hydrogel is determined by an electronic universal testing machine. The results are shown in Figure 6 As shown in D, the tensile strength of the hydrogel 3 is about 60 kPa, and the elongation at break is more than 1500%. It is indicated that the multifunctional hydrogel of the present application has good toughness and ductility, and can be well combined with the skin in the actual application process. Meanwhile, it is suitable for joints and other easily movable parts.

[0115] 6、Biocompatibility test of the prepared hydrogel. L929 cells were used as test objects to evaluate the cell compatibility of the hydrogel. The cells were inoculated in a 96-well plate at a density of 2000 cells per well, and placed in a cell incubator for culture. The prepared hydrogel was soaked in complete culture medium in advance, and the hydrogel extract was obtained after 24 h of extraction at 37 ℃. The extract was then diluted to different concentrations with complete culture medium, filtered to remove bacteria, and used as a reserve. After 24 h of cell culture, the original culture medium was discarded, and different concentrations of culture medium containing hydrogel extract were added to each group, with a blank control group. The cells were then cultured in the cell incubator for another 24 h. After 24 h, the original culture medium was discarded, 100 μL of CCK-8 working solution was added, and the mixture was incubated at 37 ℃ for 1 h. The absorbance of each well at 450 nm was measured by a microplate reader. The results are shown in Figure 7 Fig. 2, which shows that hydrogels 1-3 did not exhibit toxic effects on L929 cells, and even promoted cell proliferation to some extent, indicating that the multifunctional hydrogel of the present application has good cell compatibility. In addition, the hemolysis experiment was used to test the blood compatibility of the hydrogel. Mouse anticoagulant whole blood was centrifuged at 2000 rpm for 10 minutes, and the supernatant was discarded. The red blood cell precipitate was repeatedly washed with normal saline until the supernatant was colorless. The red blood cells were then diluted to a 5% suspension with normal saline. The red blood cell suspension and the hydrogel were mixed, and a blank control group and a positive control group were set up. The mixture was incubated at 37 ℃ for 30 minutes, centrifuged at 2000 rpm for 10 minutes, and the hemolysis of each group was observed. The supernatant was measured for absorbance at 540 nm. The results are shown in Figure 8 Fig. 3, which shows that the positive control group (Triton X-100) exhibited obvious hemolysis, while the hydrogel 1-3 treatment groups did not exhibit hemolysis, indicating that the multifunctional hydrogel of the present application has good blood compatibility.

[0116] 6、Antibacterial performance test of the prepared hydrogel. Staphylococcus aureus and Escherichia coli were used as test objects to evaluate the antibacterial performance of the hydrogel. After activation and culture, the bacterial suspension was adjusted to a concentration of 10 6 CFU / mL with LB liquid medium, and 1 mL of the bacterial suspension was incubated with hydrogels 1-3, with a blank control group. The incubated bacterial suspension was diluted and spread on agar plates, which were inverted and incubated in a 37 ℃ incubator overnight. The colony growth on the agar plates of each group was observed and counted. The results are shown in Figure 9 Fig. 4, which shows that the blank control group had the most colonies, followed by the hydrogel 1 treatment group, the hydrogel 2 treatment group had fewer colonies than the hydrogel 1 treatment group, and the hydrogel 3 treatment group had very few colonies, indicating that the multifunctional hydrogel of the present application has good antibacterial effect.

[0117] 7. The anti-inflammatory properties of the prepared hydrogel were tested. First, the effect of the hydrogel extract on the polarization of RAW264.7 cells was evaluated. Cells were divided into groups of 2 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in a cell culture incubator. A hydrogel extract was prepared in advance, filtered, and sterilized for later use. After 24 h of cell culture, the original culture medium was discarded, and each group was added to culture medium containing the hydrogel extract. Cells were cultured for another 24 h, the culture medium was discarded, and cells were fixed with 4% paraformaldehyde. CD206 was then subjected to immunofluorescence staining, and finally, images were acquired and observed using a laser confocal microscope. Results are as follows: Figure 10 As shown in Figure A, compared with the blank control group, CD206 expression was significantly increased in cells treated with hydrogels 2 and 3, indicating that they can promote the polarization of M2 macrophages. In addition, an in vitro inflammation model was constructed using lipopolysaccharide stimulation of RAW264.7 cells to evaluate the effect of hydrogels on inflammation. Cells were cultured at 5 × 10⁻⁶ cells per cell line. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured in a cell culture incubator. Hydrogel extract was prepared in advance, filtered, and sterilized for later use. After 24 h of culture, the original culture medium was discarded. The hydrogel-treated group was treated with complete culture medium containing hydrogel extract and lipopolysaccharide (LPS), the positive control group was treated with complete culture medium containing LPS, and the blank control group was treated with only fresh complete culture medium. Cells were cultured for another 24 h. The concentration of nitric oxide in the supernatant of each group was further determined. The results are as follows: Figure 10 As shown in Figure B, the concentration of nitric oxide in the cell supernatant of the hydrogel 2 and hydrogel 3 treatment groups was significantly lower than that of the positive control group (LPS), indicating that the multifunctional hydrogel of the present invention has a good anti-inflammatory effect and can effectively regulate the inflammatory microenvironment of the wound.

[0118] 7. The antioxidant properties of the prepared hydrogel were tested. The in vitro antioxidant properties of the hydrogel were evaluated using a DPPH scavenging assay. First, DPPH was dissolved in anhydrous ethanol to prepare a 100 μM DPPH working solution, which was stored in the dark for later use. Then, the hydrogel and the DPPH working solution were co-incubated, and the absorbance at 570 nm was measured using the supernatant. The results are as follows: Figure 11 As shown, hydrogels 2 and 3 have a high DPPH scavenging rate, indicating that the multifunctional hydrogels of the present invention have good antioxidant properties, can effectively scavenge free radicals, and help alleviate oxidative stress in wounds.

[0119] 8. The prepared hydrogel is subjected to in vivo wound healing promotion effect detection. The wound healing promotion ability of the hydrogel is evaluated by a rat full-thickness skin defect model. SD rats are randomly divided into 5 groups: a PBS group (Control), a hydrogel 1 group, a hydrogel 2 group, a hydrogel 3 group and a positive drug group, 6 rats in each group. The rats are anesthetized by sodium pentobarbital, the back hair is shaved and sterilized with 75% alcohol, and a full-thickness skin defect wound is created on the back of the rat by using a skin puncher. The PBS group drops PBS on the wound surface; the hydrogel group covers different hydrogels on the wound surface; and the positive drug group applies a commercial dressing Hydrosorb gel to the wound surface. The wound healing is continuously observed and photographed during the entire treatment period. The results are shown in FIG. 8, and the wound healing speed of all hydrogel treatment groups is faster than that of the PBS treatment group. By the 10th day, the wound surface of the hydrogel 3 treatment group has been basically healed, and the healing speed is significantly better than that of the other treatment groups, while the wound surface of the PBS treatment group still has a large area that has not completed re-epithelialization. Moreover, the healing speed of the wound surface of the positive drug treatment group is also slower than that of the hydrogel 2 and hydrogel 3 treatment groups. Figure 12 For histological analysis, wound tissues are taken on the 7th day and the 14th day for H&E staining, and the results are shown in FIG. 9. The hydrogel 2 and hydrogel 3 significantly promote the vascular regeneration of the wound surface. In addition, compared with the PBS treatment group, the hydrogel 2 and hydrogel 3 treatment groups have skin appendages such as hair follicles on the wound surface, which proves that the multifunctional hydrogel of the application has good ability to promote wound healing. Figure 13

[0120] In summary, the multifunctional hydrogel for wound treatment is obtained by mixing the functional polymer containing aldehyde group and phenylboronic acid structure, the polymer containing amino group, the polymer containing diol structure and the small molecule compound containing aldehyde group and catechol structure in a solvent, and adding metal ions to mix uniformly. The multifunctional hydrogel network is mainly constructed by dynamic covalent bonds, has excellent flexibility and ductility, can quickly self-heal, and has controllable tissue adhesion. The multifunctional hydrogel has good antibacterial effect, can prevent infection of the wound surface, can remove excess free radicals, promote the polarization of macrophages to M2 phenotype, inhibit the inflammatory response induced by lipopolysaccharide, reduce the secretion of pro-inflammatory factors, and promote the healing of full-thickness skin defect wounds. In addition, the multifunctional hydrogel provided by the application has high biological safety, the preparation method is simple and easy to scale up, has strong applicability, and can be applied to the fields of dressing development, drug delivery, tissue engineering, biosensing and beauty and skin care.

[0121] The above detailed description of the embodiments of the application, but the application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the application, the technical solutions of the application can be modified and changed in many ways, and these simple modifications all belong to the protection scope of the application.​

Claims

1. A method for preparing a multifunctional hydrogel for wound therapy, characterized by, The method comprises the following steps: S1, oxidizing a polymer containing an ortho-hydroxyl structure to obtain a functional polymer containing an aldehyde group; S2, reacting the functional polymer containing an aldehyde group with an amino-containing phenylboronic acid compound to obtain a functional polymer containing both an aldehyde group and a phenylboronic acid structure; S3, mixing the functional polymer containing both an aldehyde group and a phenylboronic acid structure, an amino-containing polymer, a glycol-containing polymer, a small molecule compound containing both an aldehyde group and an ortho-phenol structure, and adding metal ions in a solvent to obtain a multifunctional hydrogel for wound treatment.

2. The method for preparing a multifunctional hydrogel for wound therapy according to claim 1, characterized by, In step S1, the polymer containing an ortho-hydroxyl structure comprises at least one of natural polysaccharides containing an ortho-hydroxyl structure and derivatives thereof. In step S2, the amino-containing phenylboronic acid compound includes but is not limited to 3-amino phenylboronic acid. In step S3, the amino-containing polymer includes but is not limited to at least one of chitosan and derivatives thereof, collagen and derivatives thereof, polylysine and derivatives thereof, and polyethyleneimine and derivatives thereof; the glycol-containing polymer includes but is not limited to at least one of hyaluronic acid and derivatives thereof, dextran and derivatives thereof, cellulose and derivatives thereof, polyvinyl alcohol and derivatives thereof, and sodium alginate and derivatives thereof; the small molecule compound containing both an aldehyde group and an ortho-phenol structure includes but is not limited to 3,4-dihydroxybenzaldehyde; and the metal ion includes but is not limited to at least one of gold ion, silver ion, copper ion, iron ion, zinc ion, and gallium ion.

3. The method for preparing a multifunctional hydrogel for wound therapy according to claim 2, characterized in that, The natural polysaccharide containing an ortho-hydroxyl structure includes but is not limited to at least one of dextran, hyaluronic acid, sodium alginate, and cellulose.

4. The method for preparing a multifunctional hydrogel for wound therapy according to claim 1, characterized by, In step S1, the polymer containing an ortho-hydroxyl structure is mixed with an oxidizing agent in water to perform an oxidation reaction; after the reaction is completed, the reaction solution is purified and dried to obtain the functional polymer containing an aldehyde group.

5. The method for preparing a multifunctional hydrogel for wound therapy according to claim 4, characterized in that, The mass ratio of the polymer containing an ortho-hydroxyl structure to the oxidizing agent is 1: (0.3-1.2).

6. The method for preparing a multifunctional hydrogel for wound therapy according to claim 1, characterized by, In step S2, the mass ratio of the functional polymer containing an aldehyde group to the amino-containing phenylboronic acid compound is 1: (0.1-0.8).

7. The method for preparing a multifunctional hydrogel for wound therapy according to claim 1, characterized by, In step S3, the final concentration of the functional polymer containing both an aldehyde group and a phenylboronic acid structure in the solvent is 1 wt%-6 wt%, the final concentration of the amino-containing polymer in the solvent is 0.5 wt%-4 wt%, the final concentration of the glycol-containing polymer in the solvent is 1 wt%-6 wt%, the final concentration of the small molecule compound containing both an aldehyde group and an ortho-phenol structure in the solvent is 0.2 wt%-3 wt%, and the final concentration of the metal ion in the solvent is 0.2-3 mg / mL.

8. The method for preparing a multifunctional hydrogel for wound therapy according to claim 7, characterized in that, The mass ratio of the functional polymer containing both an aldehyde group and a phenylboronic acid structure to the amino-containing polymer to the glycol-containing polymer is (1-12):(1-4):(1-12).

9. The multifunctional hydrogel prepared by the method of any one of claims 1-8.

10. The multifunctional hydrogel of claim 9 for use in wound treatment.

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