A composite dynamic cross-linking self-healing hydrogel as well as a preparation method and application thereof

Abstract

The application belongs to the technical field of polymer gels, and particularly relates to a composite dynamic crosslinking self-healing hydrogel and a preparation method and application thereof. The preparation method of the composite dynamic crosslinking self-healing hydrogel comprises the following steps: S1. 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added into an oxidized hyaluronic acid aqueous solution to perform an activation reaction at room temperature, 3-aminobenzene boronic acid is added after sufficient reaction, and the reaction is continuously performed at room temperature in the dark under acidic conditions, and the benzene boronic acid oxidized hyaluronic acid is obtained by dialysis and freeze-drying; S2. the benzene boronic acid oxidized hyaluronic acid, tannic acid and hydrazide hyaluronic acid in S1 are uniformly mixed, and the composite dynamic crosslinking self-healing hydrogel is obtained by being left to gel. When the above hydrogel is used as a dressing, the wound surface can be self-repaired and protected again even if the hydrogel is broken in motion; meanwhile, the hydrogel has glucose / ROS / pH responsiveness, and the release rate of tannic acid can be regulated at the molecular level, so that the healing of a diabetic infected wound is better promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of high molecular gel, more particularly, to a kind of composite dynamic crosslinking self-healing hydrogel and its preparation method and application. BACKGROUND

[0002] Diabetes is a group of chronic hyperglycemia characterized by multiple causes, a lifelong metabolic disease. According to the statistics of the World Health Organization, there are more than 100 complications of diabetes, which is the most complicated disease known so far, especially the difficult-to-heal skin wounds caused by diabetes, which seriously affects the daily life of patients. Normal wound healing generally goes through four specific stages: hemostasis, inflammation, proliferation and remodeling. Chronic inflammation is induced by the imbalance of pro-inflammatory factors, high oxidative stress and the presence of biological membranes in diabetic wounds, which prevents the wound repair from developing from the inflammation stage to the proliferation stage, making it difficult to heal. At present, antibiotics, anti-inflammatory agents and growth factors are commonly used to relieve chronic non-healing wounds in diabetes, but there are also some problems, such as high price, drug resistance and side effects.

[0003] Tannic acid (TA) is a natural polyphenol that can be extracted from a variety of plants (mainly oak and lacquer tree), and has a variety of biological activities, including bactericidal, anti-inflammatory, antioxidant and anticancer, etc., so it is widely used in different fields. However, due to the poor bioavailability and easy oxidation of TA, its practical application is limited. In the prior art, tannic acid is often added to hydrogel dressings to deliver it directly to the wound site for bactericidal and anti-inflammatory effects. For example, the prior art discloses a kind of amorphous silver-containing long-acting antibacterial hydrogel dressing with photo-crosslinking solidification and its preparation method, which uses bacterial cellulose as a macromolecular skeleton, combines crosslinking monomer, tannic acid and silver ion photo-curing to form a composite antibacterial hydrogel dressing. Although the hydrogel dressing has good mechanical properties and can adapt to different shapes of wounds, the dressing is easily damaged or detached when moving in the affected area, which makes it difficult to closely adhere to the wound and effectively and accurately deliver the drug to the wound site. SUMMARY

[0004] The present application aims to overcome the defect or deficiency that the existing hydrogel dressing is easily broken during movement, which makes it difficult to closely adhere to the wound. A preparation method of a composite dynamic crosslinking self-healing hydrogel is provided, which uses benzene boronic acid oxidized hyaluronic acid, tannic acid and hydrazide hyaluronic acid to interact and cooperate with each other, and uses two dynamic bonds, acylhydrazone bond and borate ester bond, to construct a composite dynamic crosslinking self-healing hydrogel. When the hydrogel is used as a dressing, it can restore crosslinking and gel protection of the wound even if it is broken during movement. It also has glucose / ROS / pH responsiveness, which intelligently controls the release rate of tannic acid at the molecular level and better promotes the healing of diabetic infected wounds.

[0005] Another object of the present application is the composite dynamic crosslinking self-healing hydrogel prepared by the above preparation method.

[0006] Still another object of the present application is the use of the above composite dynamic crosslinking self-healing hydrogel in the preparation of wound dressings.

[0007] Still another object of the present application is to provide a diabetic infected wound dressing prepared from the above composite dynamic crosslinking self-healing hydrogel.

[0008] The above objects of the present application are achieved by the following technical solutions.

[0009] The present application protects a preparation method of a composite dynamic crosslinking self-healing hydrogel, comprising the following steps:

[0010] S1. In the aqueous solution of oxidized hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to perform an activation reaction at room temperature, after sufficient reaction, 3-aminobenzoic acid is added to continue the reaction under acidic conditions at room temperature in the dark, and then dialysis and freeze-drying are performed to obtain benzene boronic acid oxidized hyaluronic acid;

[0011] S2. The benzene boronic acid oxidized hyaluronic acid and hydrazide hyaluronic acid in S1 are mixed and uniformly mixed with tannic acid, and then gelled to obtain a composite dynamic crosslinking self-healing hydrogel.

[0012] The mass ratio of the benzene boronic acid oxidized hyaluronic acid to the hydrazide hyaluronic acid in S2 is <1.

[0013] The composite dynamic crosslinking self-healing hydrogel of the present application cooperates with tannic acid through two dynamic bonds of acylhydrazone bonds and borate ester bonds, wherein the acylhydrazone bond is formed by the amide group of hydrazide hyaluronic acid and the aldehyde group of benzene boronic acid oxidized hyaluronic acid, and the borate ester bond is formed by the boronic acid group of benzene boronic acid oxidized hyaluronic acid and the ortho-phenol group of tannic acid. The existence of the dynamic bonds endows the hydrogel with self-healing and injectable properties, so that the hydrogel can better fit the wound.

[0014] Moreover, the acylhydrazone bond is stable under normal physiological conditions, but it can be broken by the induction of low pH value of a diabetic wound, so that the crosslinking of the hydrogel is reduced, thereby releasing the tannic acid in the hydrogel faster. When the borate ester bond contacts a large amount of active oxygen at a diabetic wound site, oxidation will occur, further promoting the rapid release of tannic acid. When glucose exists, the ortho-hydroxyl group of the glucose molecule will compete with tannic acid to bind the benzene boronic acid group, so that more tannic acid is free in the hydrogel and released. Therefore, the dynamic bonds endow the hydrogel with the intelligent controlled release ability for diabetic wounds.

[0015] It is also necessary to point out that the mass ratio of benzene boronic acid oxidized hyaluronic acid to hydrazide hyaluronic acid in the composite dynamic crosslinking self-healing hydrogel is <1, because the benzene boronic acid oxidized hyaluronic acid has a larger degree of oxidation (i.e. more aldehyde groups on the molecular chain), and the hydrazide hyaluronic acid has a lower hydrazide branch rate (i.e. fewer hydrazide groups on the molecular chain). When the amount of benzene boronic acid oxidized hyaluronic acid is too large, that is, there are too many aldehyde groups and too few hydrazide groups, the amount of acylhydrazone bonds formed is significantly reduced, resulting in reduced crosslinking of the hydrogel and failure to form a gel.

[0016] Preferably, the molar ratio of the oxidized hyaluronic acid to 3-aminobenzene boronic acid in S1 is 1:(0.5-2). More preferably, the molar ratio of the oxidized hyaluronic acid, 3-aminobenzene boronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in S1 is 1:(0.5-2):2:2.

[0017] Specifically, the mass ratio of the benzene boronic acid oxidized hyaluronic acid to the hydrazide hyaluronic acid in S2 is (1-4):(6-9). Alternatively, the mass ratio of the benzene boronic acid oxidized hyaluronic acid to the hydrazide hyaluronic acid in S2 is (2.5-3.5):(6.5-7.5), for example, it can be 1:9, 2:8, 3:7, 4:6, 2.5:7.5 or 3.5:6.5.

[0018] Specifically, the specific operation in S2 is to first mix the benzene boronic acid oxidized hyaluronic acid in S1 with tannic acid uniformly, and then mix it with the hydrazide hyaluronic acid; or first mix the hydrazide hyaluronic acid with tannic acid uniformly, and then mix it with the benzene boronic acid oxidized hyaluronic acid in S1.

[0019] It is more conducive to forming a homogeneous system, and further more conducive to forming a hydrogel, to first mix tannic acid with either of the benzene boronic acid oxidized hyaluronic acid and the hydrazide hyaluronic acid uniformly, and then mix the other.

[0020] Preferably, the mass fraction of the tannic acid relative to the composite dynamic crosslinking self-healing hydrogel is ≤1%.

[0021] When the amount of tannic acid added is relatively large (mass fraction >1%), although the antibacterial and bactericidal effects of the composite dynamic crosslinking self-healing hydrogel can be improved, it will also cause greater toxicity. Therefore, the preferred mass fraction of tannic acid is ≤1%.

[0022] Specifically, the oxidized hyaluronic acid is prepared by the following preparation method:

[0023] Sodium periodate is added to an aqueous solution of hyaluronic acid, and after sufficient reaction under light-proof conditions, dialysis and freeze-drying are performed to obtain the oxidized hyaluronic acid.

[0024] The hyaluronic acid has a molecular weight of 100-200 million, and the molar ratio of the hyaluronic acid repeating unit to sodium periodate is 1:1.

[0025] Specifically, the hydrazide-converted hyaluronic acid is prepared by the following preparation method:

[0026] In the aqueous solution of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to perform an activation reaction at room temperature, then azelaic acid dihydrazide is added, and after reaction, the hydrazide-converted hyaluronic acid is obtained by dialysis and freeze-drying under dark and acidic conditions.

[0027] The hyaluronic acid has a molecular weight of 100-200 million, and the dialysis refers to using a dialysis bag with a molecular weight of 8-14 kDa to dialyze in pure water for 5 days, and the water is changed every 12 hours to remove unreacted small molecular raw materials.

[0028] Preferably, the molar ratio of the hyaluronic acid repeating unit to azelaic acid dihydrazide is 1:(1-8).

[0029] Specifically, the molar ratio of the hyaluronic acid repeating unit, azelaic acid dihydrazide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1-8):2:2.

[0030] A composite dynamic cross-linking self-healing hydrogel prepared by the above preparation method is also within the protection scope of the present application.

[0031] The present application also protects the use of the above-mentioned composite dynamic cross-linking self-healing hydrogel in the preparation of wound dressings.

[0032] Preferably, the wound dressing in the above-mentioned use is a dressing for treating chronic wound healing of diabetes.

[0033] A wound dressing prepared from the above-mentioned composite dynamic cross-linking self-healing hydrogel is also within the protection scope of the present application.

[0034] The present application has the following beneficial effects:

[0035] The present application constructs a hydrogel by two dynamic bonds of acylhydrazone bond and boronate ester bond, has self-healing property and injectability, is beneficial to closely adhere to irregular wounds, and can restore cross-linking and gelation when the dressing is broken in motion, thereby protecting the wound.

[0036] The cross-linked acylhydrazone bond is stable under normal physiological conditions, but it can be broken by the induction of low pH value of diabetic wounds; the borate ester bond can be changed by the presence of glucose and oxidation in the presence of H2O2, so that the network structure of the hydrogel is changed, and the hydrogel has glucose / ROS / pH responsiveness, thereby intelligently controlling the speed of TA release at the molecular level. The acylhydrazone bond and the borate ester bond are of great significance for constructing a drug carrier system that can sensitively respond to changes in glucose / ROS / pH concentration in the physiological environment. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The synthesis route of the benzene boronate oxidized hyaluronic acid (OHA-APBA) and the hydrazide hyaluronic acid (HAAD) of the application is shown in the figure.

[0038] Figure 2 The structure of tannic acid (TA) is shown in the figure.

[0039] Figure 3 The schematic diagram of dynamic cross-linking of the composite dynamic cross-linking self-healing hydrogel of the application is shown in the figure.

[0040] Figure 4 The self-repairing display of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application is shown in the figure.

[0041] Figure 5 The glucose / ROS / pH responsive release of TA of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application is shown in the figure.

[0042] Figure 6 The intracellular reactive oxygen species scavenging effect of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application is shown in the figure.

[0043] Figure 7 The in vitro antibacterial experiment results of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application are shown in the figure.

[0044] Figure 8 The in vitro anti-inflammatory experiment results of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application are shown in the figure.

[0045] Figure 9 The in vivo results of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application for promoting the healing of diabetic infected wounds are shown in the figure.

[0046] Figure 10 The in vivo results of the composite dynamic cross-linking self-healing hydrogel in Example 1 of the application for promoting the healing rate of diabetic infected wounds are shown in the figure.

[0047] Figure 11 The gel state of the composite dynamic cross-linking self-healing hydrogel in Examples 1-4 and Comparative Example 1 of the application is shown in the figure. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0050] (1) Oxidized hyaluronic acid (OHA)

[0051] The above-mentioned oxidized hyaluronic acid can be prepared by the following preparation method:

[0052] Completely dissolve 2g of hyaluronic acid in 100mL of ultrapure water, then add 1.064g of sodium periodate. Allow to react at room temperature in the dark for 24 hours. Terminate the reaction by adding 1.5mL of ethylene glycol. After completion of the reaction, transfer the product to an 8kDa to 14kDa dialysis bag and dialyze in pure water for 5 days, changing the water daily. After dialysis, freeze-dry to obtain oxidized hyaluronic acid (OHA).

[0053] (2) Hyaluronic acid hydrazide (HAAD)

[0054] The above hydrazide hyaluronic acid can be prepared by the following preparation method (eg Figure 1 shown):

[0055] Dissolve 1g of hyaluronic acid completely in 100mL of ultrapure water, then add 820mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 610mg of N-hydroxysuccinimide (NHS) for activation. After reacting at room temperature for 30 minutes, add 3.5g of azelaic acid dihydrazide, adjust the pH of the reaction system to 3-5, and react at room temperature in the dark for 24 hours. After completion of the reaction, transfer the product to an 8kDa-14kDa dialysis bag and dialyze against pure water for 5 days, changing the water every 12 hours. After dialysis, freeze-dry to obtain hydrazide-modified hyaluronic acid (HAAD).

[0056] Example 1

[0057] A method for preparing a composite dynamically cross-linked self-healing hydrogel comprises the following steps:

[0058] S1. Dissolve 1 g of hyaluronic acid in 100 mL of ultrapure water, then add 960 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 576 mg of N-hydroxysuccinimide for activation reaction, after 30 min of reaction at room temperature (25℃), add 687 mg of 3-aminophenylboronic acid (APBA), and adjust the pH of the reaction system to 3-5, continue to react for 24 h at room temperature in the dark; after the reaction is completed, the obtained product is transferred to a dialysis bag with a molecular weight of 8 kDa-14 kDa, dialyzed in pure water for 5 days, and the water is changed every 12 hours, and then freeze-dried to obtain phenylboronic acid hyaluronic acid (OHA-APBA);

[0059] S2. Mix the phenylboronic acid hyaluronic acid aqueous solution (2% by mass) in S1 with tannic acid (as shown in Figure 2 ), and then add a hydrazide hyaluronic acid aqueous solution (2% by mass) and mix uniformly, and then stand to form a gel, to obtain a composite dynamic crosslinking self-healing hydrogel (OAH@TA, as shown in Figure 3 ); wherein the mass ratio of phenylboronic acid hyaluronic acid and hydrazide hyaluronic acid is 3:7, and the mass fraction of tannic acid relative to the composite dynamic crosslinking self-healing hydrogel is 1%.

[0060] Example 2

[0061] A preparation method of a composite dynamic crosslinking self-healing hydrogel, which comprises substantially the same steps as in Example 1, except that the mass ratio of phenylboronic acid hyaluronic acid and hydrazide hyaluronic acid in step S2 is 2:8.

[0062] Example 3

[0063] A preparation method of a composite dynamic crosslinking self-healing hydrogel, which comprises substantially the same steps as in Example 1, except that the mass ratio of phenylboronic acid hyaluronic acid and hydrazide hyaluronic acid in step S2 is 4:6.

[0064] Example 4

[0065] A preparation method of a composite dynamic crosslinking self-healing hydrogel, which comprises substantially the same steps as in Example 1, except that the mass ratio of phenylboronic acid hyaluronic acid and hydrazide hyaluronic acid in step S2 is 1:9.

[0066] Example 5

[0067] A preparation method of a composite dynamic crosslinking self-healing hydrogel, which comprises substantially the same steps as in Example 1, except that the mass fraction of tannic acid relative to the composite dynamic crosslinking self-healing hydrogel in step S2 is 0.3%.

[0068] Example 6

[0069] A method for preparing a composite dynamically cross-linked self-healing hydrogel comprises steps substantially the same as those in Example 1, except that in step S2, the mass fraction of tannic acid relative to the composite dynamically cross-linked self-healing hydrogel is 0.7%.

[0070] Comparative Example 1

[0071] A method for preparing a composite dynamically cross-linked self-healing hydrogel comprises steps substantially the same as those of Example 1, except that in step S2, the mass ratio of phenylborated oxidized hyaluronic acid to hydrazide hyaluronic acid is 1:1.

[0072] Performance Testing

[0073] (1) Self-repair performance test

[0074] The specific test method is as follows: the composite dynamic cross-linked self-healing hydrogel in Example 1 is made into a hydrogel sheet (length 25mm × width 10mm × height 1.5mm) and cut into two sections from the middle. The two sections are placed horizontally with the cut surfaces in contact with each other. After being placed at room temperature for 30 minutes, their self-healing ability is evaluated.

[0075] The test results are as follows Figure 4 As shown by Figure 4 It can be seen that the composite dynamic cross-linked self-healing hydrogel in Example 1 of the present invention can be closely attached to the surface of the finger skin. After being cut, it only needs to be kept in contact with the cross-section for 30 minutes at room temperature to achieve self-repair using reversible dynamic bonds. The repaired composite dynamic cross-linked self-healing hydrogel can firmly adhere to the surface of the bent finger joint. This fully demonstrates that the composite dynamic cross-linked self-healing hydrogel of the present invention can not only closely fit the irregular wound surface, but also can self-repair and cross-link into gel again when the dressing is broken during exercise to protect the wound surface. The self-healing performance of the composite dynamic cross-linked self-healing hydrogels of Examples 2 to 6 is basically the same as that of Example 1.

[0076] (2) Tannic acid release test

[0077] The specific test method is as follows: take 6 portions of the composite dynamic cross-linked self-healing hydrogel in Example 1 (100 μL each) and soak them in 6 different groups of 500 μL PBS, respectively, ① pH = 7.4, ② pH = 4.0, ③ pH = 7.4 + 1 mM hydrogen peroxide, ④ pH = 4.0 + 1 mM hydrogen peroxide, ⑤ pH = 7.4 + 50 mM glucose, ⑥ pH = 4.0 + 50 mM glucose, and then place the test tubes in a shaker at 37 ° C and 100 rpm, and take out all the PBS at specific times (6h, 12h, 24h, 36h, 48h and 72h) for analysis of TA concentration; then add 500 μL of fresh PBS into the test tube; finally, by measuring the absorbance of the solution at 278 nm and comparing it with the standard curve, the TA released into the medium is determined.

[0078] The test results are as follows Figure 5 As shown by Figure 5 Under physiological conditions, the TA release over 72 hours was approximately 46%, while at pH 4, the release was approximately 67%. This indicates that the cumulative release of tannic acid under acidic conditions was significantly greater than at physiological pH. This is likely due to the accelerated hydrolysis of the acylhydrazone bond in an acidic environment, leading to increased TA release. Furthermore, the addition of H₂O₂ and glucose to a pH 7.2 buffer also increased the cumulative TA release. This is because hydrogen peroxide specifically cleaves the C₂ bond of phenylboronic acid, releasing TA. The vicinal diol structure of glucose has a greater bonding constant with boronic acid than TA, leading to preferential binding of phenylboronic acid to glucose, leading to TA release. When low pH is combined with H₂O₂ or glucose, a synergistic effect occurs, resulting in 72-hour TA release greater than that observed when H₂O₂ or glucose are present at either pH 4 or pH 7.2. These results demonstrate that the OAH@TA hydrogel prepared in this invention exhibits glucose / ROS / pH-sensitive release properties.

[0079] (3) Intracellular reactive oxygen species clearance experiment

[0080] The specific test method is as follows: RAW264.7 cells (1×10 4 The cells were seeded in a 24-well cell culture plate and incubated for 24 hours. The cells were then treated with H2O2 (100 μM) for 1 hour and treated with DMEM (10% FBS) medium containing PBS, TA (10 mg / L), OAH, and OAH@TA (20 μL / mL) hydrogel for 2 hours. After incubation, the cells were treated with serum-free medium containing DCFH-DA (1 mL, 1 μL / mL) and incubated at 37°C in the dark for 30 minutes. Fluorescence was observed under an inverted fluorescence microscope.

[0081] The experimental results are as follows Figure 6 As shown by Figure 6It can be seen that after PBS and OAH treatment, the intracellular ROS level is significantly, a large area of fluorescence is observed in the figure; while after TA and OAH@TA treatment, the fluorescence area is significantly reduced, that is, tannic acid can scavenge intracellular active oxygen, and OAH@TA formed by the interaction of benzene boronic acid oxidized hyaluronic acid and hydrazide hyaluronic acid with tannic acid (TA) can fully guarantee the antioxidant activity of tannic acid, so that OAH@TA can well scavenge intracellular active oxygen.

[0082] (4) Bacterial activity detection experiment

[0083] The specific test method is: the colony counting method is used to detect the antibacterial activity of OAH and OAH@TA on Escherichia coli and Staphylococcus aureus. OAH hydrogel precursor solution (200 μL) and OAH@TA hydrogel precursor solution (200 μL) are sterilized by 0.22 μm filter, and then gelled in 48-well cell culture plate, and stored at 4℃ for standby. Then 10 μL of Escherichia coli or Staphylococcus aureus suspension (1.0×10 6 CFU mL -1 ) is respectively smeared on the surface of OAH and OAH@TA hydrogel, and 10 μL of bacterial suspension (200 μL PBS without hydrogel) is used as a control group. The inoculated hydrogel is incubated at 37℃ in a certain humidity environment for 24 h. Then, 1 mL of PBS is added to each well to resuspend the surviving bacteria, and 10 μL of bacterial suspension is spread on Luria-Bertani agar plate, and the number of colonies on Luria-Bertani agar plate is counted after 12 h of Escherichia coli culture and 24 h of Staphylococcus aureus culture, respectively.

[0084] The test results are shown in Figure 7 It can be seen from Figure 7 that OAH@TA can effectively inhibit the proliferation of Staphylococcus aureus and Escherichia coli, thereby inhibiting the formation of colonies and achieving the effect of antibacterial; while OAH only has a certain inhibitory effect on Escherichia coli and has no inhibitory effect on Staphylococcus aureus.

[0085] (5) In vitro anti-inflammatory experiment

[0086] The specific test method is: the change of the expression of inflammatory factors (IL-1β and iNOS) is detected by qPCR to evaluate the anti-inflammatory activity of the hydrogel. RAW264.7 cells (1×10 5Cells were seeded in 6-well cell culture plates and incubated for 24 hours. The cells were then pretreated with PBS, TA (10 mg / L), OAH, and OAH@TA hydrogel extracts (20 μL / mL) for 2 hours and then treated with 1 μg / mL LPS for 12 hours. Total RNA was extracted and its concentration was determined. The cDNA was synthesized by reverse transcription and then subjected to qPCR using the kit.

[0087] The test results are as follows Figure 8 As shown by Figure 8 It was found that the expression of IL-1β and iNOS in the TA and OAH@TA groups were significantly decreased compared with the PBS control group, while the expression of iNOS in the OAH group was not statistically different from that in the control group, while IL-1β was slightly decreased. This shows that the OAH@TA hydrogel material has good in vitro anti-inflammatory activity.

[0088] (6) Infected wound healing effect in diabetic mice

[0089] The specific testing method is to establish a diabetic mouse model by intraperitoneally injecting 1% streptozotocin (STZ) at 50 mg / kg in C57 mice. Successful modeling is indicated by a blood glucose level exceeding 16.7 mmol / L for three consecutive days. A full-thickness skin wound with a diameter of 6 mm was created on the back of the mice. Staphylococcus aureus was inoculated into the wound, and 500 μL of PBS, OAH, and OAH@TA hydrogel were injected into the wound. A control group was injected with PBS alone. Wound healing was recorded at 3, 6, 9, and 12 days to determine the extent and progress of wound repair.

[0090] The test results are as follows Figure 9 and Figure 10 As shown in the figure, the wound area of ​​the control group, OAH group, and OAH@TA group gradually decreased with the extension of healing time. However, there was no statistical difference in the wound healing area between the control group and OAH group, while the wound area of ​​OAH@TA was significantly smaller than that of the control group and OAH group. This shows that OAH@TA hydrogel material can significantly accelerate the healing of infected wounds in diabetic mice.

[0091] Depend on Figure 11 It can be seen that when the mass ratio of phenylborated oxidized hyaluronic acid to hydrazide hyaluronic acid is ≥1.5, the mixture of phenylborated oxidized hyaluronic acid, tannic acid mixture and hydrazide hyaluronic acid is still in a solution state and cannot form a gel; when the mass ratio of phenylborated oxidized hyaluronic acid to hydrazide hyaluronic acid is 1, although the mixture of phenylborated oxidized hyaluronic acid, tannic acid mixture and hydrazide hyaluronic acid is a solution with a higher viscosity, it still cannot form a gel. Therefore, only phenylborated oxidized hyaluronic acid and hydrazide hyaluronic acid with a suitable ratio can form a composite dynamic cross-linked self-healing hydrogel together with tannic acid.

[0092] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A method for preparing a composite dynamically cross-linked self-healing hydrogel, characterized in that: The steps include: S1. Adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to an aqueous solution of oxidized hyaluronic acid at room temperature for activation reaction. After sufficient reaction, adding 3-aminophenylboronic acid and continuing the reaction at room temperature in the dark under acidic conditions. Dialysis and lyophilization are performed to obtain phenylboronic acid-oxidized hyaluronic acid. S2, mixing the phenylborated oxidized hyaluronic acid, tannic acid and hydrazide hyaluronic acid in S1, and allowing to stand to form a gel to obtain a composite dynamic cross-linked self-healing hydrogel; Wherein, the mass ratio of the phenylboronated oxidized hyaluronic acid to the hydrazide hyaluronic acid in S2 is less than 1; The oxidized hyaluronic acid described in S1 is prepared by the following preparation method: Sodium periodate is added to a hyaluronic acid aqueous solution, and after sufficient reaction under light-proof conditions, the solution is dialyzed and freeze-dried to obtain oxidized hyaluronic acid; The molar ratio of the hyaluronic acid repeating unit to sodium periodate is 1:1; The hydrazide hyaluronic acid described in S2 is prepared by the following preparation method: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added to an aqueous solution of hyaluronic acid at room temperature for activation reaction. After sufficient reaction, azelaic acid dihydrazide is added. The mixture is reacted at room temperature in the dark under acidic conditions, and then dialyzed and freeze-dried to obtain hydrazide hyaluronic acid. The molar ratio of the hyaluronic acid repeating unit to azelaic acid dihydrazide is 1:(1-8).

2. The preparation method according to claim 1, characterized in that The mass ratio of the phenylboronated oxidized hyaluronic acid to the hydrazide hyaluronic acid in S2 is (1-4):(6-9).

3. The preparation method according to claim 1, characterized in that The specific operations of S2 are: First, the phenylboronic acid-oxidized hyaluronic acid in S1 is mixed evenly with tannic acid, and then mixed with hydrazide-oxidized hyaluronic acid; Alternatively, hydrazide-modified hyaluronic acid and tannic acid are first mixed evenly, and then mixed with the phenylborated oxidized hyaluronic acid in S1.

4. The preparation method according to claim 1, characterized in that The mass fraction of the tannic acid relative to the composite dynamic cross-linked self-healing hydrogel is ≤1%.

5. A composite dynamic cross-linked self-healing hydrogel prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the composite dynamically cross-linked self-healing hydrogel according to claim 5 in preparing a wound dressing.

7. A wound dressing, characterized in that: The wound dressing is prepared from the composite dynamically cross-linked self-healing hydrogel according to claim 5.

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