Preparation and application of multifunctional hydrogel based on reversible covalent bond crosslinking

Abstract

The invention discloses preparation and application of multifunctional hydrogel based on reversible covalent bond crosslinking, and belongs to the technical field of biomedical materials. The hydrogel is formed by crosslinking chlorogenic acid grafted oxidized hyaluronic acid and polylysine grafted with phenylboronic acid groups through imine bonds and phenylboronic acid ester bonds. Compared with the prior art, the hydrogel disclosed by the invention can respond to high active oxygen and low pH broken bonds in a chronic wound inflammation microenvironment, so that local delivery of drugs is realized, and excellent antibacterial, anti-inflammatory and chronic wound healing promoting effects can be obtained.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to the preparation and application of a multifunctional hydrogel based on reversible covalent crosslinking. Background Technology

[0002] Chronic, non-healing wounds, as a chronic inflammatory disease, have a high incidence rate worldwide. They not only cause immense pain and inconvenience to patients, leading to serious complications and even threatening lives, but also impose a significant economic burden on society. Their main pathological characteristic is that the wound healing process is stalled in the inflammatory phase, unable to enter the proliferative and remodeling stages. This creates a microenvironment with high levels of reactive oxygen species and inflammatory factors infiltrating impaired cell function, making the wound susceptible to bacterial infection. The resulting biofilm further exacerbates inflammation and oxidative stress, creating a vicious cycle that severely damages and hinders skin tissue regeneration.

[0003] Currently, traditional wound repair strategies mainly rely on skin grafts or synthetic skin. However, issues such as immune rejection, complex manufacturing processes, and high costs limit their application. Furthermore, these materials fail to adequately address key challenges such as bacterial infection control and functional skin regeneration. Clinically used antibiotics and silver-containing dressings struggle to completely penetrate and eliminate biofilms of mature bacteria, and issues such as bacterial resistance and cytotoxicity exist. Therefore, researching novel combination therapy strategies—enhancing antibacterial capabilities while improving the oxidative stress microenvironment—is crucial for promoting the healing of chronic, difficult-to-heal wounds.

[0004] Berberine (BBR), a natural alkaloid compound, not only exhibits significant inhibitory effects on various bacteria, fungi, and viruses, but also alleviates inflammatory responses by inhibiting the NF-κB signaling pathway. Catalase (CAT), while clearing hydrogen peroxide from wounds, can generate oxygen to alter the local hypoxic environment, thereby promoting wound repair. The combined application of these two agents can inhibit the growth of various bacteria and scavenge reactive oxygen species, synergistically improving the local oxidative stress environment, thus promoting tissue regeneration and accelerating wound healing, achieving safe and efficient chronic wound repair. However, how to achieve local delivery and sustained release of the drug, as well as improve its stability, remains a problem that urgently needs to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a method for preparing and applying multifunctional hydrogels based on reversible covalent crosslinking.

[0006] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a hydrogel formed by crosslinking chlorogenic acid-grafted oxidized hyaluronic acid and α-polylysine grafted with phenylboronic acid groups via imine bonds and phenylboronic ester bonds.

[0007] Optionally, the mass ratio of the chlorogenic acid-grafted oxidized hyaluronic acid to the α-polylysine grafted with phenylboronic acid groups is 1:1 to 1:5.

[0008] Optionally, the structural formula of the chlorogenic acid-grafted oxidized hyaluronic acid is: .

[0009] Optionally, the method for synthesizing chlorogenic acid-grafted hyaluronic acid (HA) includes the following steps: dissolving hyaluronic acid in pure water, adding sodium periodate solution dropwise after complete dissolution, stirring at 25°C in the dark for 4-8 h, adding ethylene glycol to the solution, continuing stirring to terminate the reaction, dialyzing with a dialysis bag (8000 kDa) for 1-4 days, and obtaining chlorogenic hyaluronic acid after lyophilization. Alternatively, dissolving chlorogenic hyaluronic acid in pure water, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine, stirring on ice for 1-5 h to activate the carboxyl group, adding chlorogenic acid to the solution, stirring for 12-48 h, dialyzing with a dialysis bag (3500 kDa) for 1-4 days, and obtaining chlorogenic acid-grafted hyaluronic acid after lyophilization.

[0010] The synthesis reaction formula is as follows: The method for synthesizing phenylboronic acid-grafted ε-poly-l-lysine (ε-PL) includes the following steps: dissolving 4-carboxyphenylboronic acid in an appropriate amount of dimethyl sulfoxide, adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stirring for 1-5 h to activate the carboxyl group, dissolving ε-poly-lysine in pure water, adding 4-carboxyphenylboronic acid solution dropwise after complete dissolution, stirring for 4-24 h, dialyzing with a dialysis bag (1000 kDa) for 1-4 days, and obtaining phenylboronic acid-grafted ε-poly-lysine after lyophilization.

[0011] The synthesis reaction formula is as follows: In a preferred embodiment of the present invention, the degree of oxidation of hyaluronic acid in the multifunctional hydrogel is 30%-70%, preferably 40%-50%; the grafting rate of chlorogenic acid-grafted hyaluronic acid is 5%-30%, preferably 10%-20%. As a preferred embodiment of the present invention, the grafting rate of ε-polylysine grafted with phenylboronic acid in the multifunctional hydrogel is 10%-50%, preferably 25%-35%.

[0012] As a preferred embodiment of the present invention, the mass ratio of chlorogenic acid-grafted oxidized hyaluronic acid to phenylboronic acid-grafted ε-polylysine in the multifunctional hydrogel is 1:1-1:5, preferably 1:2-1:4.

[0013] A second aspect of the present invention relates to a hydrogel composition comprising: Hydrogel matrix, including the hydrogels described above; And catalase loaded in the hydrogel matrix.

[0014] Optionally, it also includes berberine loaded in the hydrogel matrix.

[0015] Optionally, the concentration of berberine is from 1 mg / mL to 2.5 mg / mL, and the concentration of catalase is from 50 µg / mL to 200 µg / mL.

[0016] A third aspect of the present invention relates to the above-described hydrogel composition and the use of the above-described hydrogel in the preparation of a medicament or wound dressing for promoting chronic wound healing.

[0017] A fourth aspect of the present invention relates to a method for preparing a hydrogel, comprising the following steps: mixing chlorogenic acid-grafted oxidized hyaluronic acid with α-polylysine grafted with phenylboronic acid groups to form a hydrogel.

[0018] The beneficial effects of this invention are: The multifunctional hydrogel disclosed in this invention has strong mechanical properties (see Example 3), porous morphology (see Example 4), suitable tissue adhesion, good injectability and self-healing properties (see Example 5), low swelling and biodegradability (see Example 6), and the ability to release BBR in response to pH and ROS (see Example 7).

[0019] The multifunctional hydrogel disclosed in this invention can promote cell migration (see Example 8), regulate the pro-inflammatory phenotype of macrophages and alleviate oxidative stress (see Example 9), and inhibit the growth of various bacteria (see Example 10), thereby accelerating the healing of chronic wounds (see Example 11). In addition, the multifunctional hydrogel also has good in vivo safety (see Example 12).

[0020] The hydrogel network based on reversible covalent bonds of the present invention can not only adapt to irregular wound structures and withstand normal skin stretching by virtue of its in situ injectability and self-healing ability, but also respond to high reactive oxygen species and low pH bond breaking in the inflammatory microenvironment of chronic wounds through the responsiveness imparted by reversible covalent bonds, thereby achieving local drug delivery.

[0021] The innovation of this invention lies in the modification of natural polymer materials. Dynamic imine bonds are formed by the aldehyde groups of chlorogenic acid-grafted oxidized hyaluronic acid and the amino groups of 3-carboxyphenylboronic acid-grafted polylysine. Simultaneously, the phenolic hydroxyl groups of chlorogenic acid can form phenylboronic acid ester bonds with phenylboronic acid, ultimately preparing a reversible cross-linked hydrogel based on chlorogenic acid-grafted oxidized hyaluronic acid and phenylboronic acid-grafted ε-polylysine. This hydrogel possesses strong mechanical properties, suitable tissue adhesion, good injectability, self-healing properties, low swelling, and biodegradability. Furthermore, this multifunctional hydrogel enables the mild co-loading and local delivery of protein drugs and small molecule drugs, as well as responsive release within the wound inflammatory microenvironment. On the one hand, it inhibits the growth of various bacteria; on the other hand, it synergistically improves the local oxidative stress environment, thereby promoting tissue regeneration and accelerating wound healing. This provides a new combined delivery strategy and system for safe and efficient chronic wound repair. Attached Figure Description

[0022] The invention will now be further described with reference to the accompanying drawings.

[0023] Figure 1 : A schematic diagram of the biological mechanism verified by experiments in the embodiments of this application; Figure 2 : 1 According to the H NMR results, A is chlorogenic acid-grafted oxidized hyaluronic acid, and B is phenylboronic acid-grafted ε-polylysine. Figure 3 Rheological results of BBR / CAT@Gel and Gel: A is the frequency scan result of BBR / CAT@Gel and Gel; B is the viscosity change result of BBR / CAT@Gel and Gel under different shear rates; C is the storage modulus (G′) and loss modulus (G″) change result of Gel under different strain conditions; D is the G′ and G″ change result of BBR / CAT@Gel under different strain conditions. Figure 4 Representative SEM images of freeze-dried BBR / CAT@Gel; Figure 5The results of characterization of the adhesion, injectability and self-healing properties of gel are as follows: A is a picture of gel adhering to different materials and fingers at different angles; B is a schematic diagram of the overlap shear test; C is the overlap shear strength results of gels with different proportions; D is the injectability results of gel; E is the self-healing process of gel. Figure 6 Results of in vitro swelling and degradation of BBR / CAT@Gel: A is the swelling curve of BBR / CAT@Gel in different media, and B is the degradation curve of BBR / CAT@Gel in different media. Figure 7 : In vitro release curve of BBR; Figure 8 BBR / CAT@Gel and the cell migration promotion ability of Gel. A is the bright field image of different groups at different time points, and B is the statistical results of cell migration rate of different groups at different time points. Figure 9 Anti-inflammatory capabilities of BBR / CAT@Gel and Gel; A represents CD86 levels in RAW 264.7 cells after different treatment groups. + Horizontal flow cytometry analysis, B represents CD86 in RAW 264.7 cells after different treatment groups. + Statistical analysis at different levels, C represents the DCFH-DA levels in RAW 264.7 cells after different treatment groups. + Horizontal flow cytometry analysis, D represents DCFH-DA in RAW 264.7 cells after different treatment groups. + Statistical analysis at the level; Figure 10 Antibacterial activity of BBR / CAT@Gel hydrogel: A shows representative images of viable bacteria (Staphylococcus aureus and Escherichia coli) on plates after different treatment groups, and B shows the colony count of viable bacteria (Staphylococcus aureus and Escherichia coli) after different treatment groups. Figure 11 The ability of BBR / CAT@Gel and Gel to accelerate wound healing: A shows wound images and corresponding imagej area statistics for different groups on days 0, 1, 4, 7, 12 and 14; B shows the wound healing rate for different groups on days 4, 7, 12 and 14; C shows the H&E staining results of wound tissue sections on day 14. Figure 12 In vivo safety evaluation of BBR / CAT@Gel and Gel: A represents the organ index statistics of each group on day 14, and B represents the serum ALT, AST and UREA levels of each group on day 14. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Synthesis of hydrogel carrier materials This embodiment includes the synthesis of chlorogenic acid-grafted oxidized hyaluronic acid and phenylboronic acid-grafted ε-polylysine.

[0026] 500 mg of hyaluronic acid (800 kDa~1.0 MDa, H293501-25 g, Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in 40 mL of pure water. After complete dissolution, 2 mL of 10% sodium periodate solution was added dropwise. The reaction was carried out at 25°C in the dark with stirring for 6 h. 1 mL of ethylene glycol was added to the solution, and stirring was continued for 1 h to terminate the reaction. Dialysis was performed for 2 days using a dialysis bag (8000 kDa). Oxidized hyaluronic acid was obtained after lyophilization.

[0027] 300 mg of oxidized hyaluronic acid was dissolved in 20 mL of pure water. EDC and DMAP were added and stirred on ice for 1 h to activate the carboxyl group. 177 mg of chlorogenic acid was added to the solution and stirred for 24 h. Dialysis was performed for 2 days using a dialysis bag (3500 kDa). After freeze-drying, chlorogenic acid-grafted oxidized hyaluronic acid was obtained.

[0028] 166 mg of 4-carboxyphenylboronic acid was dissolved in an appropriate amount of DMSO, and EDC and NHS were added. The mixture was stirred for 1 h to activate the carboxyl group. 128 mg of ε-polylysine (MV 2000-5000, P192512-25 g, Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in 10 mL of pure water. After complete dissolution, 4-carboxyphenylboronic acid solution was added dropwise and the mixture was stirred for 12 h. The mixture was dialyzed for 2 days using a dialysis bag (1000 kDa) and then lyophilized to obtain phenylboronic acid-grafted ε-polylysine.

[0029] pass 1 1H NMR results indicate that ε-polylysine grafted with phenylboronic acid (PDO) Figure 2 A) and chlorogenic acid-grafted oxidized hyaluronic acid (A) Figure 2 B) was successfully prepared. Based on the relative ratios of characteristic peaks, the grafting rate of phenylboronic acid-grafted ε-polylysine was 33.6%, and the grafting rate of chlorogenic acid-grafted oxidized hyaluronic acid was 14.0%. The degree of oxidation of oxidized hyaluronic acid was determined to be 44.3% using hydroxylamine hydrochloride titration.

[0030] Example 2: Preparation of BBR / CAT@Gel hydrogel Catalase (CAT) was dissolved in pure water, and chlorogenic acid-grafted oxidized hyaluronic acid was also dissolved to obtain a 100 mg / mL chlorogenic acid-grafted oxidized hyaluronic acid solution containing 0.01% CAT. Berberine (BBR) was uniformly dispersed in pure water, and phenylboronic acid-grafted ε-polylysine was also dissolved to obtain a 300 mg / mL phenylboronic acid-grafted ε-polylysine solution containing 0.2% BBR. Equal volumes of the two solutions were mixed to obtain a multifunctional hydrogel (BBR / CAT@Gel). The hydrogel without CAT and BBR is designated as a Gel hydrogel.

[0031] Example 3: Rheological characterization of BBR / CAT@Gel hydrogel Storage modulus (G′) and loss modulus (G″) of Gel and BBR / CAT@Gel hydrogels were measured using a rheometer at 37 °C. Frequency scanning was performed in the linear viscoelastic region at a constant strain from 1–100 Hz. G′ revealed the elastic properties of the hydrogel, while G″ revealed its viscous properties. Within the 1–100 Hz range, G′ was consistently higher than G″ for all groups, with no crossover marks. Figure 3 A) indicates that the hydrogel can form stably and has good viscoelasticity. The G′ of the BBR / CAT@Gel group is higher than that of the Gel group, reflecting that the addition of BBR and CAT increases the mechanical properties of the hydrogel.

[0032] The hydrogels of Gel and BBR / CAT@Gel were tested using a rheometer at 25°C in the range of 0.1–100 s⁻¹. -1 Viscosity changes within the shear rate range. With increasing shear rate, BBR / CAT@Gel and gel hydrogels exhibit shear-thinning properties, resulting in decreased viscosity. Figure 3 (B) This indicates that the hydrogel has injectable properties due to the physical and dynamic covalent interactions within it.

[0033] The changes in G′ and G″ of Gel and BBR / CAT@Gel hydrogels under different strain conditions were detected using a rheometer at 37℃. Under high strain conditions, G″ is greater than G′, indicating that the hydrogel network structure is damaged, losing its integrity and mechanical stability. Under low strain conditions, G′ is greater than G″, indicating that the hydrogel network structure can be restored. Figure 3 (C and D).

[0034] Example 4: Morphological and structural characterization of BBR / CAT@Gel hydrogel The morphology of the lyophilized BBR / CAT@Gel hydrogel was observed by SEM. The BBR / CAT@Gel hydrogel exhibited an interconnected porous structure with intact pore walls and a pore size of approximately 30 μm. Figure 4 ).

[0035] Example 5: Evaluation of the adhesion, injectability and self-healing properties of the gel hydrogel By observing the adhesion of the hydrogel to different materials and at different angles of the finger, the adhesive ability of the gel hydrogel was preliminarily evaluated. Figure 5 A). The bioadhesion properties of the hydrogel were then evaluated using an overlap shear test. Figure 5 B), as the ratio of phenylboronic acid-grafted ε-polylysine to chlorogenic acid-grafted oxidized hyaluronic acid gradually increases, the lap shear strength of the gel hydrogel also gradually increases, exhibiting an lap shear strength of 6.5 ± 3.8 kPa at a ratio of 3:1. Figure 5 C), enabling it to withstand deformation and stretching to conform to the wound site.

[0036] The injectability of the gel hydrogel was evaluated by injecting the hydrogel into water with a syringe and spelling out different letters. Figure 5 D).

[0037] By physically cutting two stained heart-shaped hydrogels into four pieces and then immediately contacting and splicing them together, the self-healing process was observed. Figure 5 E).

[0038] like Figure 4 The results show that the gel hydrogel has strong mechanical properties, suitable tissue adhesion, good injectability, and self-healing properties.

[0039] Example 6: Evaluation of the in vitro swelling and degradation capacity of BBR / CAT@Gel hydrogel Swelling experiments of the hydrogel were conducted in PBS solution at pH 7.4 (37℃) and PBS solution at pH 6.5 (containing 0.5 mM H2O2). The initial weight of the BBR / CAT@Gel hydrogel was recorded as W0. At different time points, the swollen hydrogel was removed, and after the surface moisture was absorbed with filter paper, the weight of the hydrogel was recorded as W1. The swelling rate (%) was calculated as (W1 - W0) × 100% / W0. The BBR / CAT@Gel hydrogel exhibited suitable swelling properties in different media, reaching swelling equilibrium after 24 h. Figure 6 A) indicates that BBR / CAT@Gel hydrogel can accelerate the absorption of wound fluid in a short time, which helps wound healing.

[0040] The degradation experiments of the BBR / CAT@Gel hydrogel were conducted in PBS solution at pH 7.4 (37℃) and PBS solution at pH 6.5 (containing 0.5 mM H2O2). The initial weight of the BBR / CAT@Gel hydrogel after complete swelling was recorded as W0. At different time points, the hydrogel was removed, and after the surface moisture was absorbed with filter paper, the weight of the remaining hydrogel was recorded as W1. The percentage of remaining mass (%) was calculated as (W0 - W1) × 100% / W0. The mass of the BBR / CAT@Gel hydrogel decreased to 18.2 ± 3.33% within six days. The mass loss of the hydrogel accelerated and further increased in PBS solution at pH 6.5 (containing 0.5 mM H2O2). Figure 6 (B), indicating that the BBR / CAT@Gel hydrogel has good degradation performance and pH and ROS sensitivity.

[0041] Example 7: Evaluation of BBR in vitro release capacity of BBR / CAT@Gel hydrogel The in vitro drug release of BBR / CAT@Gel hydrogel in PBS solution at pH 7.4 and PBS solution at pH 6.5 containing 0.5 mM H2O2 was tested using HPLC. In PBS solution at pH 7.4, BBR was rapidly released from the hydrogel within 24 hours, gradually reaching release equilibrium over four days. However, in PBS solution at pH 6.5 containing 0.5 mM H2O2, a higher BBR release rate was observed, reaching 78.57 ± 0.87% on the fourth day. Figure 7 This indicates that BBR / CAT@Gel hydrogel exhibits more efficient release behavior in high ROS and low pH environments, which is beneficial for the healing of chronic wounds.

[0042] Example 8: Evaluation of the cell migration-promoting ability of BBR / CAT@Gel hydrogel Inoculate 1×10⁻⁶ cells into 6-well plates 6 Cells were suspended at 2 mL / ml and cultured in each well. After the cells were fully confluent and adhered to the well, horizontal and vertical scratches were made with a pipette tip. The cells were washed three times with culture medium and incubated with different drug solutions. Bright-field images were taken at different time points, and Imagej was used to analyze the area of ​​the incubated regions and calculate the migration rate. Compared with free CAT, BBR, and BBR / CAT, the cell migration rates of the Gel and BBR / CAT@Gel groups were significantly increased at 12, 24, and 48 h. Figure 8 (A and B) indicates that the hydrogel material can effectively promote cell migration, demonstrating that the BBR / CAT@Gel group has the ability to promote rapid wound healing.

[0043] Example 9: Evaluation of the anti-inflammatory ability of BBR / CAT@Gel hydrogel The ROS scavenging ability of BBR / CAT@Gel hydrogels was detected by flow cytometry. 1×10⁶ cells were seeded in 6-well plates. 6 Cells were suspended in RAW 264.7 cells / mL, 2 mL per well, and pre-cultured for approximately 3 h. After good cell adhesion, the culture medium was removed, and 1 μg / mL LPS solution was added to each well of the culture plate. The plates were then incubated for 6 h. The supernatant was discarded, and the cells were washed with PBS. 2 mL of complete culture medium containing hydrogel extract was added to each well, and the plates were incubated for 24 h. The supernatant was discarded, and the cells were washed with PBS. 5 μM DCFH-DA solution was added to each well, and the plates were incubated for 15 min. Cells were collected, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were washed with PBS and resuspended in PBS. Compared with the control group, the ROS level in the LPS group was significantly increased, demonstrating that macrophages tend to produce ROS under LPS stimulation. The BBR, BBR / CAT, Gel, CAT@Gel (CAT-only), BBR@Gel (BBR-only), and BBR / CAT@Gel groups significantly reduced intracellular ROS levels. Figure 9 The results (A and B in the figure) indicate that the hydrogel has a certain ROS resistance capacity. At the same time, the BBR / CAT@Gel group showed the most significant reduction, indicating that the components in the hydrogel can synergistically improve oxidative stress.

[0044] The inhibitory effect of BBR / CAT@Gel hydrogel on the pro-inflammatory phenotype of macrophages was investigated using flow cytometry. 5 × 10⁶ cells were seeded in 6-well plates. 5 Cells were suspended in RAW 264.7 cell suspension at 2 mL per well and pre-cultured for approximately 6 h. After good cell adhesion, the culture medium was removed, and 100 ng / mL LPS solution was added to each well of the culture plate. The plates were then incubated for 12 h. The supernatant was discarded, and 2 mL of complete culture medium containing hydrogel extract was added to each well. The plates were then incubated for 24 h. Cells were collected, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were washed with PBS. The cells were resuspended in 100 μL of CD86 dilution buffer, incubated at room temperature in the dark for 30 min, centrifuged at 600 g for 6 min, washed with PBS, and resuspended in PBS before loading onto the plate. Compared with the control group, the CD86 positivity rate in the LPS group was significantly increased, demonstrating that macrophages tend to polarize to the M1 phenotype under LPS stimulation. The CD86 positivity rate in the BBR / CAT@Gel group was 56.9%, nearly 1.6 times lower than that in the LPS group. The CD86 positivity rates in the CAT@Gel group and the BBR@Gel group were 72.3% and 66.5%, respectively. Figure 9 The C and D values ​​indicate that the components of BBR / CAT@Gel can synergistically inhibit the pro-inflammatory phenotype polarization of macrophages.

[0045] Example 10: Evaluation of the antibacterial activity of BBR / CAT@Gel hydrogel Dilute Staphylococcus aureus or Escherichia coli bacterial suspension in the logarithmic growth phase to 1×10⁻⁶. 6 CFU / ml was added to each well of a 96-well plate at a concentration of 100 μL, followed by 100 μL of hydrogel extract. The plates were incubated at 37°C for 12 h, and the OD value at 600 nm was measured using a microplate reader. Each solution was then diluted 10⁻⁶ times. 8 The concentration was increased by 10 times, and 50 μL was plated and incubated at 37°C for 24 h before colony counting. Compared with the control group, both Gel and BBR / CAT@Gel effectively inhibited the growth of Staphylococcus aureus and Escherichia coli. Figure 10 (A and B).

[0046] Example 11: Evaluation of the ability of BBR / CAT@Gel hydrogel to promote wound healing An 8 mm full-thickness skin wound was created on the dorsal side of male C57BL / 6 mice using a punch, and 10 μL of Staphylococcus aureus suspension (1×10⁻⁶) was injected. 7 CFU / mL was injected into the wound surface, and the wound was fixed with a silicone pad and covered with a 3M waterproof dressing. Optical images of the wound were recorded at 0, 1, 3, 7, 12, and 14 days to calculate the wound healing rate. At day 4, the wound healing rates of the CAT / @Gel, BBR@Gel, and BBR / CAT@Gel groups were significantly different from those of the Model group. At day 12, the wound healing rate of the Gel group was significantly different from that of the Model group. Meanwhile, the wound in the BBR / CAT@Gel group was essentially completely healed by day 14. Figure 11 A and B). Wound and surrounding tissue samples were collected on day 14 for H&E staining. Compared to the normal group, the model group had damaged epidermis with inflammatory infiltration, while the epidermis of the Gel, CAT / @Gel, and BBR@Gel groups was basically repaired. The BBR / CAT@Gel group showed complete epidermal repair and the appearance of hair follicle structures. Figure 11 C). The experimental results stated above prove that the hydrogel can effectively promote the healing of chronically infected wounds and restore the normal function of the skin.

[0047] Example 12: In vivo safety evaluation of BBR / CAT@Gel hydrogel Serum from mice in each group was collected on day 14 for safety evaluation, and organ indices were calculated from the heart, liver, spleen, and kidneys. There were no significant differences in organ indices among the groups, and no significant differences were found in ALT, AST, and UREA levels, indicating that the hydrogel has good in vivo safety. Figure 12 (A and B).

[0048] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0049] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A hydrogel, characterized in that, It is formed by cross-linking chlorogenic acid grafted with oxidized hyaluronic acid and α-polylysine grafted with phenylboronic acid groups through imine bonds and phenylboronic acid ester bonds.

2. The hydrogel according to claim 1, characterized in that, The mass ratio of the chlorogenic acid-grafted oxidized hyaluronic acid to the α-polylysine grafted with phenylboronic acid groups is 1:1 to 1:

5.

3. The hydrogel according to claim 1, characterized in that, The structural formula of the chlorogenic acid-grafted oxidized hyaluronic acid is as follows: 。 4. The hydrogel according to claim 1, characterized in that, In the chlorogenic acid-grafted oxidized hyaluronic acid, the degree of oxidation of hyaluronic acid is 30% to 70%, and the grafting rate of chlorogenic acid is 5% to 30%. In the α-polylysine grafted with phenylboronic acid groups, the grafting rate of phenylboronic acid groups is 10% to 50%.

5. The hydrogel according to claim 1, characterized in that, The grafting rate of phenylboronic acid to ε-polylysine was 33.6%, the grafting rate of chlorogenic acid to hyaluronic acid was 14.0%, and the degree of oxidation of hyaluronic acid was 44.3%.

6. A hydrogel composition, characterized in that, Include: The hydrogel matrix includes the hydrogel according to any one of claims 1 to 5; And catalase loaded in the hydrogel matrix.

7. The hydrogel composition according to claim 6, characterized in that, It also contains berberine loaded in the hydrogel matrix.

8. The hydrogel composition according to claim 6, characterized in that, The concentration of berberine is from 1 mg / mL to 2.5 mg / mL, and the concentration of catalase is from 50 µg / mL to 200 µg / mL.

9. The use of the hydrogel composition of claim 7 or 8, or the hydrogel of any one of claims 1 to 5, in the preparation of a medicament or wound dressing for promoting chronic wound healing.

10. A method for preparing a hydrogel, characterized in that, include: A hydrogel was prepared by mixing chlorogenic acid-grafted oxidized hyaluronic acid with α-polylysine grafted with phenylboronic acid groups.

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