CN117815438B - Multifunctional hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention discloses a multifunctional hydrogel and a preparation method and application thereof, wherein the material comprises polyethyleneimine grafted and modified by phenylboronic acid groups, polydopamine nanospheres loaded with BNN6, chitosan functionalized by catechol groups and oxidized dextran. The multifunctional hydrogel has good antibacterial performance and oxidation resistance through reasonable matching of the components and the dosage, can eliminate bacterial infection, remove DPPH, OH free radicals and ABTS free radicals, has a photo-thermal effect, can promote angiogenesis, has multiple functions, is used for preparing wound healing dressing for diabetics, and has good application potential and prospect.

Description

A multifunctional hydrogel and its preparation method and application

Technical Field

The invention relates to a multifunctional hydrogel and a preparation method and application thereof, in particular to a hydrogel for preparing a dressing for healing diabetic wounds, belonging to the field of preparation of hydrogels for wound dressings.

Background technique

Impaired wound healing is common in patients with chronic diabetes. The main reasons for delayed wound healing in diabetic patients are as follows: (1) The hyperglycemic environment of diabetic wounds is prone to bacterial infection and biofilm localization, leading to severe inflammatory responses in the wounds; (2) Sustained hyperglycemia leads to the production of advanced glycation end products in the blood, resulting in excessive reactive oxygen species (ROS) in the wounds, inducing oxidative stress and further exacerbating the inflammatory response; (3) Vascular damage caused by diabetes interferes with the delivery of nutrients and oxygen, adversely affecting wound healing. Therefore, when considering wound healing in diabetic patients, a therapeutic intervention with effective antimicrobial properties, the ability to alleviate oxidative stress, and the ability to promote angiogenesis is desirable.

Hydrogels are widely considered as promising materials for promoting diabetic wound healing due to their ability to mitigate the above challenges by integrating bioactive molecules or materials. Cationic polymers, as alternatives to traditional antibiotics, have been incorporated into antimicrobial hydrogel networks and have shown their bactericidal effects without developing drug resistance. However, cationic polymers have limited selectivity and their ability to induce toxicity in mammalian cells. Therefore, the balance between biocompatibility and antimicrobial properties of cationic polymers has always been a dilemma.

Effective management of oxidative stress levels is another important issue in promoting the healing process of chronic wounds. Hydrogels containing catechol groups have been shown to have antioxidant capacity and are expected to manage diabetic wounds by reducing oxidative stress. Catechol-based hydrogels have been used to prepare antioxidant hydrogels and show enhanced chronic wound healing ability. In addition, hydrogels containing catechol groups generally have good tissue adhesion, which makes the hydrogels have long-term effectiveness. In addition, angiogenesis also contributes to wound healing. Recently, nitric oxide (NO) has been added to hydrogels as an active molecule and has shown the ability to heal diabetic wounds by promoting angiogenesis, stimulating granulation formation, and promoting collagen deposition. However, most wound dressings have a single function and cannot meet the clinical treatment requirements of the complex microenvironment of chronic diabetic wounds.

Therefore, it is necessary to prepare a multifunctional hydrogel that integrates antibacterial, antioxidant and angiogenic properties for the treatment of chronic diabetic wounds.

Summary of the invention

In view of the defects of the hydrogel in the prior art such as single function and slow healing of diabetic wounds, the first purpose of the present invention is to provide a multifunctional hydrogel (CDP-PB) which has good antibacterial, antioxidant and angiogenic properties and has high biocompatibility.

The second object of the present invention is to provide a method for preparing a multifunctional hydrogel, which is simple, has relatively mild reaction conditions, is low in cost, has great application prospects, and can be prepared on a large scale.

The third purpose of the present invention is to provide an application of a multifunctional hydrogel, which is used in the preparation of a diabetic wound healing dressing. The multifunctional hydrogel in the dressing has good in vitro antibacterial and antioxidant properties, can eliminate bacterial infection, effectively remove a variety of free radicals, and reduce oxidative stress; it also has a photothermal effect, can control the release of NO in near-infrared light, promote angiogenesis, and accelerate the healing of diabetic wounds.

In order to achieve the above technical objectives, the present invention provides a multifunctional hydrogel, which includes polyethyleneimine (PEI-PBA) grafted with phenylboronic acid groups, polydopamine nanospheres loaded with BNN6 (PDA@BNN6 NPs), chitosan functionalized with catechol groups (Chi-Ca) and oxidized dextran (Dex-CHO); wherein the mass ratio of polyethyleneimine grafted with phenylboronic acid groups, polydopamine nanospheres loaded with BNN6, chitosan functionalized with catechol groups and oxidized dextran is (10-100): (1-10): (60-600): (100-1000).

In the hydrogel of the technical solution of the present invention, on the one hand, the amino groups of Chi-Ca, PEI-PBA, and PDA@BNN6NPs and the aldehyde groups of Dex-CHO can be used to react with Schiff bases to generate Schiff base bonds; on the other hand, the phenylboronic acid group of PEI-PBA and the catechol group of Chi-Ca can be used to form phenylboronic acid ester bonds, and both Schiff base bonds and phenylboronic acid ester bonds are dynamic covalent bonds, which give the hydrogel good injectability, self-healing properties and tissue adhesion, and can be used for a long time in irregular wound sites. In addition, Chi-Ca gives the hydrogel antioxidant properties, enabling it to scavenge a variety of free radicals and reduce oxidative stress; PEI-PBA gives the hydrogel antibacterial properties, and under the action of near-infrared light, due to the presence of PDA@BNN6 NPs, the hydrogel can exert a photothermal effect and can controllably release high concentrations of NO, further enhancing the antibacterial properties of the hydrogel; and Dex-CHO can reduce the cytotoxicity induced by PEI-PBA by cross-linking with PEI-PBA. Therefore, mild photothermal action is beneficial to the formation of new blood vessels. After photothermal action, the BNN6 of PDA@BNN6 NPs will gradually desorb and slowly decompose to generate low concentrations of NO, which can further promote angiogenesis. In summary, the CDP-PB hydrogel provided by the present invention has multiple functions, and through the synergistic effect of various components, it can quickly promote angiogenesis and wound healing during application.

The inventors found that the dosage of each component in the CDP-PB hydrogel is also very important. For example, the dosage of Chi-Ca directly determines the antioxidant properties of the hydrogel. The higher the dosage of Chi-Ca, the stronger the antioxidant properties of the hydrogel. If the Chi-Ca content in the hydrogel is too low, the degree of hydrogel is too low, it is not easy to gel, and the antioxidant capacity is limited; if the Chi-Ca content in the hydrogel is too high, due to the interaction between molecular chains (including hydrogen bonds, π-π stacking, etc.), Chi-Ca will automatically gel through physical cross-linking during the process of dissolving in water. This gel has poor mechanical properties and single functions, and cannot meet the needs of drug treatment of diabetic wounds. The dosage of PEI-PBA is one of the important factors that determine the antibacterial properties of hydrogels. The higher the dosage of PEI-PBA, the stronger the antibacterial properties of hydrogels. However, PEI-PBA is alkaline. If the PEI-PBA content in the hydrogel is too high, the pH of the system will be too high, and gelation will not be smooth, and it will cause serious cytotoxicity problems. If the PEI-PBA content in the hydrogel is too low, it will affect the antibacterial ability of the hydrogel. The dosage of Dex-CHO determines the balance between the biocompatibility and antibacterial properties of PEI-PBA. Therefore, it is necessary to select a suitable dosage to take into account the biocompatibility and antibacterial properties of the hydrogel. The dosage of PDA@BNN6 NPs directly determines the photothermal effect of the hydrogel and the release of NO. Therefore, only by balancing the dosage of each component of the hydrogel can the various functions of the hydrogel be guaranteed while having good mechanical properties.

Further preferably, the mass ratio of the polyethyleneimine grafted with phenylboronic acid groups, the polydopamine nanospheres loaded with BNN6, the chitosan functionalized with catechol groups and the oxidized dextran is (10-25): (1-2): (60-150): (100-250). Within the further preferred range, the obtained hydrogel has better comprehensive properties.

As a preferred solution, the PEI-PBA is obtained by substitution reaction of polyethyleneimine (PEI) and 4-(bromomethyl)phenylboronic acid (BPBA).

As a preferred solution, the mass ratio of PEI to BPBA is (3-4): 1. Within the selected range, the grafting rate of BPBA on the PEI molecular chain can be ensured to be as high as possible, which is beneficial to increase the content of phenyl borate bonds in the gel to improve the cross-linking degree of the gel.

As a preferred solution, the conditions for the substitution reaction are: temperature of 70-80° C. and time of 20-24 h.

As a preferred solution, the molecular weight or degree of polymerization of the polyethyleneimine is 300 to 3000 Da.

As a preferred solution, the PEI-PBA product obtained by the substitution reaction needs to be subjected to ice ether precipitation and water impurity removal treatment.

As a preferred scheme, the PDA@BNN6 NPs are obtained by self-assembly π-π stacking of polydopamine nanospheres (PDA NPs) and N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN6) under light-proof conditions.

As a preferred solution, the polydopamine nanospheres (PDA NPs) are prepared by the following method: F127, dopamine hydrochloride (DA) and 1,3,5-trimethylbenzene (TMB) are mixed and added with ammonia water for water bath reaction, and then the 1,3,5-trimethylbenzene template is removed by ultrasonication to obtain the product.

As a preferred solution, the conditions for the water bath reaction are: temperature of 20-25° C. and time of 1-2 h.

As a preferred solution, the ultrasonic removal of the template agent 1,3,5-trimethylbenzene uses a mixture of acetone and ethanol in a volume ratio of 1:2 as a solvent.

As a preferred solution, the BNN6 is prepared by the following method: N,N'-di-sec-butyl-p-phenylenediamine (BPA) and _NaNO2 are mixed under a nitrogen atmosphere, and hydrochloric acid is added to react in the dark to obtain the BNN6.

As a preferred solution, the mass ratio of PDA NPs to BNN6 is 1:(1-1.5).By controlling the mass ratio of PDA NPs to BNN6, BNN6 can be fully loaded in PDA NPs particles through π-π stacking.

As a preferred solution, the Chi-Ca is obtained by amidation reaction of chitosan (Chi) and 3,4-dihydroxyphenylpropionic acid (HCA). Due to the presence of _-NH2 groups in the chitosan structural unit, it is very easy to form salts with acids and dissolve, and the dissolved chitosan is in a gel state with strong adsorption capacity, so it is easy to react with the carboxyl groups on HCA for amidation reaction, thereby chemically modifying the catechol groups on HCA on the chitosan surface to obtain catechol-functionalized chitosan.

As a preferred solution, the amidation reaction uses EDC as a carboxyl activator, and the pH during the reaction is 4-6.

As a preferred solution, the mass ratio of Chi to HCA is 1:(1-1.5).

As a preferred solution, the Dex-CHO is obtained by oxidizing dextran (Dex) with sodium periodate.

As a preferred solution, the mass ratio of Dex to sodium periodate is 1:(1-2).

As a preferred solution, the oxidation conditions are: reacting in the dark at room temperature for 10 to 12 hours, and terminating the reaction with ethylene glycol.

As a preferred solution, the dextran is dialyzed using a cellulose membrane with a molecular weight cutoff of 3000 to 3500 Da after oxidation.

As a preferred solution, the oxidation degree of the Dex-CHO is 50-60%.

The present invention also provides a method for preparing a multifunctional hydrogel, which comprises dispersing polyethyleneimine grafted with phenylboronic acid groups, polydopamine nanospheres loaded with BNN6, chitosan functionalized with catechol groups, and oxidized dextran in water and mixing them to obtain the multifunctional hydrogel.

The present invention adopts an emulsion-induced interfacial anisotropic assembly method to prepare PDANPs nanospheres, and then uses BNN6 as a NO prodrug donor to load it into the mesoporous PDA NPs nanospheres through π-π stacking. In addition, chitosan (Chi), Dex and PEI are used as raw materials, and they are functionally modified respectively, and finally the modified raw materials are mixed and PDA@BNN6 NPs are added to prepare a multifunctional hydrogel. The preparation method of the present invention is simple, the reaction conditions are relatively mild, the cost is low, the application prospect is great, and large-scale preparation can be carried out.

The present invention also provides an application of a multifunctional hydrogel, which is used to prepare a diabetic wound healing dressing. Cell experiments on the dressing show that the CDP-PB hydrogel has good cell compatibility and can effectively remove a variety of free radicals produced in cells and alleviate oxidative damage. When used, the dressing has the effects of eliminating wound infection, reducing inflammation, alleviating oxidative damage, promoting angiogenesis, stimulating granulation formation and increasing collagen deposition, and can significantly promote the healing of diabetic wounds.

As a preferred solution, the dressing includes a multifunctional hydrogel and auxiliary materials.

The multifunctional hydrogel of the present invention can be applied to wounds of any shape by applying CDP-PB via a syringe.

As a preferred solution, the dressing is a pharmaceutically acceptable injection preparation; more preferably, it is a pharmaceutically acceptable local injection preparation.

As a preferred solution, the diabetic wound healing dressing has a photothermal effect, and has antibacterial, antioxidant and angiogenesis-promoting functions under the action of photothermal. Since the hydrogel of the present invention contains a suitable concentration of PDA@BNN6NPs, it gives the hydrogel a good photothermal effect. It is specifically manifested as follows: on the one hand, under the irradiation of near-infrared light, BNN6 is accelerated by heat to decompose and generate high concentrations of NO, and the synergistic photothermal effect of NO can enhance the antibacterial effect of PEI-PBA in the hydrogel; on the other hand, after near-infrared light irradiation, part of BNN6 will gradually desorb from PDA NPs, and slowly decompose to generate low concentrations of NO, which can effectively promote angiogenesis.

As a preferred solution, the conditions for the photothermal effect are: irradiation power density of 0.1-10W/ ^cm2 , irradiation time of 5-20min near-infrared laser. Within the selected range, the photothermal synergy has the best effect and can significantly improve the antibacterial properties of the hydrogel in the dressing.

Compared with the prior art, the technical solution of the present invention brings the following beneficial technical effects:

1) The CDP-PB multifunctional hydrogel prepared by the present invention has good antibacterial properties and can eliminate bacterial infections; has good antioxidant properties and can remove a variety of free radicals, especially DPPH, OH and ABTS free radicals; and has angiogenesis-promoting properties, and the hydrogel has high biocompatibility.

2) The present invention prepares CDP-PB hydrogel with excellent antibacterial properties through a reasonable formula ratio of PEI-PBA, PDA@BNN6 NPs, Chi-Ca and Dex-CHO, wherein PDA@BNN6 NPs have a photothermal effect (PTT), and the hydrogel containing catechol has the function of scavenging DPPH, OH and ABTS free radicals, which can reduce oxidative stress. In addition, under the synergistic effect of several substances, CDP-PB hydrogel treatment can increase cell migration and proliferation under oxidative stress conditions. CDP-PB hydrogel also has the effects of eliminating wound infection, promoting angiogenesis, stimulating granulation formation and increasing collagen deposition. These characteristics show the potential of CDP-PB hydrogel in clinical applications.

3) The preparation method of the multifunctional CDP-PB hydrogel provided by the present invention is simple, the reaction conditions are relatively mild, the cost is low, the application prospect is broad, and large-scale preparation can be carried out.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a TEM image of PDA NPs and PDA@BNN6 NPs prepared in Example 1 of the present invention, wherein Figure 1(a) and Figure 1(b) are TEM images of PDA NPs at different magnifications, and Figure 1(c) and Figure 1(d) are TEM images of PDA@BNN6NPs at different magnifications.

Figure 2 is a physical picture and SEM picture of the hydrogels prepared in Example 1 and Comparative Examples 1 to 5 of the present invention, wherein Figure 2(a) is a physical picture of the hydrogels prepared in Example 1 and Comparative Examples 1 to 5; Figure 2(b) is a SEM picture of the hydrogels prepared in Example 1 and Comparative Examples 1 to 5.

FIG3 is a graph showing the photothermal performance test results of the CDP-PB hydrogel prepared in Example 1 of the present invention.

Figure 4 is a graph showing the antibacterial test results of the hydrogels of different groups of the present invention; wherein (1) is the control group (PBS group), (2) is the CD group, (3) is the CDP group, (4) is the CDP-P group, (5) is the CDP-P+N group, and (6) is the CDP-PB+N group.

FIG. 5 is a graph showing the test results of DPPH removal by hydrogels of different types and concentrations prepared in Example 1 and Comparative Examples 1 to 5 of the present invention.

FIG6 is a graph showing the test results of ABTS removal by hydrogels of different types and concentrations prepared in Example 1 of the present invention and Comparative Examples 1 to 5.

FIG. 7 is a graph showing the detection results of removing OH radicals by hydrogels of different types and concentrations prepared in Example 1 of the present invention and Comparative Examples 1 to 5.

Figure 8 is a comparison of the wound healing effects of different groups of mice in the present invention; (1) is PBS, (2) is CD, (3) is CDP, (4) is CDP-P, (5) is CDP-P+N, and (6) is CDP-PB+N.

Figure 9 is a statistical graph of wound healing rates of different groups of mice wound healing effects of the present invention; wherein (1) is PBS, (2) is CD, (3) is CDP, (4) is CDP-P, (5) is CDP-P+N, and (6) is CDP-PB+N.

Detailed ways

The following examples are only specific descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of implementation of the present invention. For ordinary technicians in the technical field, any improvements made without departing from the present invention should be deemed to be within the scope of protection of the present invention.

Example 1

1) Preparation of PDA@BNN6 NPs:

PDA NPs nanoparticles were prepared by emulsion-induced interfacial anisotropic assembly method. F127, 0.9g DA, and 0.96mL TMB were added to 30mL water and 30mL ethanol, respectively, and ultrasonically dispersed until completely dissolved. Then, 2.25mL ammonia water was added dropwise while stirring, and the mixture was reacted in a water bath at 25°C for 2h. After centrifugation at 11000r/min for 15min, the supernatant was discarded and the solid was collected. Washed with ethanol and deionized water three times respectively. The template was removed by ultrasonication for 30min with 30ml acetone/ethanol mixture (1/2, v/v), centrifuged, and repeated three times. Finally, PDA NPs nanospheres were obtained by vacuum drying at 60°C.

Add 1.17 ml of BPA to 9 ml of anhydrous ethanol, and inject enough nitrogen into the solution to remove the air in the solution. Under a nitrogen atmosphere, inject 10 ml of NaNO ₂ (6M) aqueous solution into the above solution while stirring. After stirring for 30 minutes, slowly add 10 ml of HCl aqueous solution (6M) with a syringe. The color of the reaction solution changes from orange to red, and a beige precipitate appears. After stirring for another 4 hours, collect the solid product by centrifugation and wash it three times with deionized water and 50% (v/v) ethanol respectively. Finally, the product BNN6 is vacuum freeze-dried overnight under light-proof conditions and stored at 4°C under light-proof protection.

Disperse 10 mg of PDA NPs nanospheres in 10 ml of deionized water, and dissolve 10 mg of BNN6 in 5 ml of ethanol. Slowly drip the BNN6 solution into the PDA NPs nanosphere dispersion, stir for 12 hours in the dark, and then let it stand for 2 hours to allow it to fully self-assemble. After centrifugation at 11000 r/min for 15 minutes, discard the supernatant and wash with deionized water three times to obtain PDA@BNN6NPs nanospheres. Finally, add 15 ml of deionized water to re-disperse and store at low temperature away from light.

2) Preparation of Chi-Ca:

3g chitosan was added to 300ml deionized water, and then 1M hydrochloric acid was added to completely dissolve the chitosan to obtain a light yellow transparent liquid. The pH of the solution was adjusted to 5.4 with NaOH (1M), and 3.6g HCA was added to obtain a coffee-colored transparent liquid. 7.5g EDC was dissolved in 300ml 50% (v/v) ethanol aqueous solution, and then gradually added to the above chitosan mixture to make it gradually turbid. The pH was adjusted to 4.6 with HCl (1M), and vigorously stirred for 2h. The mixture was dialyzed in 100mM NaCl solution (pH=3~3.5) for 48h with a cellulose membrane (MWCO=6000~8000Da), and the dialyzate was changed every 12h, and then dialyzed in deionized water (pH=5) for 12h, and finally freeze-vacuum dried to obtain Chi-Ca.

3) Preparation of Dex-CHO:

Dissolve 5g of dextran (Dex) in 50ml of deionized water and 5g of NaIO ₄ in 30ml of deionized water. Then slowly add the NaIO ₄ solution to the dextran solution and stir for 12h at room temperature in the dark. At the end of the reaction, terminate the reaction with 10ml of ethylene glycol. The product is dialyzed extensively with water for 3d using a cellulose membrane (MWCO=3500Da) to remove excess reactants. Finally, purified Dex-CHO is obtained by freeze drying and stored at 4°C. The degree of oxidation of Dex-CHO was determined to be 55.6% by hydroxylamine hydrochloride titration.

4) Preparation of PEI-PBA:

Dissolve 3.6g PEI and 1.08g BPBA in 30ml methanol, stir under reflux in an oil bath at 70℃ for 24h, cool to room temperature, precipitate in ice ether three times, and vacuum dry at 60℃ to obtain a crude product. Add the crude product to deionized water and filter to remove insoluble impurities. Finally, freeze-dry to obtain PEI-PBA, which is stored at 4℃.

5) Preparation of multifunctional hydrogel (CDP-PB):

Chi-Ca was dissolved in deionized water to obtain Chi-Ca solution, and then PDA@BNN6 NPs were added to the Chi-Ca solution and stirred to obtain Chi-Ca/PDA@BNN6 NPs solution. Dex-CHO and PEI-PBA were dissolved in deionized water to obtain Dex-CHO solution and PEI-PBA solution. Chi-Ca/PDA@BNN6 NPs solution, Dex-CHO solution and PEI-PBA solution were mixed in equal volume ratios and stirred vigorously to obtain hydrogel CDP-PB. The concentration of Chi-Ca in the hydrogel was 15 mg/ml, the concentration of PDA@BNN6 NPs was 0.2 mg/ml, the concentration of Dex-CHO was 25 mg/ml, and the concentration of PEI-PBA was 2.5 mg/ml.

As shown in Figures 1(a) and 1(b), the PDANPs prepared in this example have a mesoporous spherical structure; as shown in Figures 1(c) and 1(d), the PDA@BNN6 NPs have a uniform nanosphere structure.

Example 2

The difference between this embodiment and embodiment 1 is that the concentration of PEI-PBA in the hydrogel is changed to 5 mg/ml, and the other conditions and steps are the same. The microscopic morphology and physical image of the prepared hydrogel are consistent with those of embodiment 1.

Example 3

The only difference between this embodiment and embodiment 1 is that the concentration of PDA@BNN6 NPs in the hydrogel is changed to 0.1 mg/ml, and the other conditions, steps and conditions are the same. The microscopic morphology and physical image of the prepared hydrogel are consistent with those of embodiment 1, but the antibacterial effect of the prepared hydrogel under the photothermal action is lower than that of embodiment 1 due to the reduced concentration of PDA@BNN6 NPs.

Comparative Examples 1 to 5

Comparative Examples 1 to 5 are the preparation of CD, CDP, CD-P, CDP-P and CD-PB hydrogels, respectively. The steps are basically the same as those in Example 1, except that the amounts of raw materials are different. Detailed information on the components of various hydrogels is shown in Table 1.

Table 1 Concentration of each component in the hydrogel (mg/ml)

The hydrogels obtained in Example 1 and Comparative Examples 1 to 5 were observed and tested by scanning electron microscope. The results are shown in Figure 2, where Figure 2(a) is a real picture of the hydrogel; Figure 2(b) is a SEM picture of the hydrogel. As shown in Figure 2(b), the particle sizes of CDP, CDP-P and CDP-PB are significantly smaller than those of the three hydrogel materials CD, CDP and CD-PB. This is because the three hydrogel materials CD, CDP and CD-PB are not doped with PEI-PBA, while CDP, CDP-P and CDP-PB are doped with PEI-PBA, which increases the crosslinking density of the hydrogel.

At the same time, it can be seen from Figure 2(b) that the CDP-PB hydrogel prepared in this embodiment has a uniform grid structure; this structure enables the hydrogel to have good adsorption capacity and moisturizing properties, while also having the characteristic of adaptive shape, which enables the hydrogel to better adapt to and meet actual needs in many applications.

The photothermal performance of the CDP-PB hydrogel prepared in Example 1 was tested:

500μl of hydrogel was placed in a 2ml glass bottle, and the temperature of the hydrogel was recorded every 30s with an infrared thermal imager (FLIR C2, USA) under 808nm laser irradiation. The relationship between laser power density and the photothermal properties of the material was studied, and the temperature rise of CDP-PB hydrogel within 10min under 808nm near-infrared laser irradiation with different power densities (0.3, 0.6, 0.9, 1.5, 2.1W/ ^cm2 ) was measured.

As shown in Figure 3, the photothermal effect of CDP-PB hydrogel is related to the light power density, and the photothermal effect increases with the increase of light power density. Among them, the temperature rise effect is better when the power density is 0.6-2.1W/ ^cm2 .

The antibacterial properties of the hydrogels obtained in Example 1 and Comparative Examples 1 to 5 were tested:

Methicillin-resistant Staphylococcus aureus (MRSA) was cultured in TSB medium and incubated at 37°C for 12 hours. After the bacterial cells grew to the logarithmic phase, the bacterial suspension was centrifuged (5000 rpm, 5 min), washed 3 times with sterile saline, and resuspended with sterile saline. The bacterial suspension was then diluted to 108 CFU/ml, i.e., its optical density (OD) at 600 nm was 0.1 as measured by an ELISA reader.

The antibacterial activity of the hydrogel was detected by the spot plate method. 50 μl of 108 CFU/ml bacterial suspension was added to the surface of 400 μl of 48-well plate hydrogel, and the hydrogel was irradiated or not treated with 808 nm near-infrared laser (0.6 W/cm ² ) for 5 min. The control group was the bacterial solution without irradiation treatment, and the test groups were CD, CDP, CDP-P, CDP-P+N, and CDP-PB+N. Among them, the CD, CDP and CDP-P groups were only incubated with the bacterial solution and hydrogel at 37°C for 4 hours, while the CDP-P+N and CDP-PB+N groups were irradiated with near-infrared light before the bacterial solution and hydrogel were incubated at 37°C for 4 hours. After incubation, an appropriate amount of sterile saline was added to the surface of the hydrogel to dilute the bacteria 10 times. The diluted bacterial suspension was transferred to a 96-well plate and diluted step by step with sterile saline. The diluted bacterial suspension was transferred to a plate covered with TSA and placed in a 37°C constant temperature incubator for 12 hours. The inhibition amount was calculated based on the number of colonies, and the formula is as follows:

Colony count (CFU/ml) = C/V×M(1)

Where C represents the average number of colonies grown on the plate at a given dilution, V represents the volume of diluent covering the plate (mL), and M represents the number of dilutions.

As shown in Figure 4, the CDP-PB+N treatment group has the best bactericidal effect, with a bactericidal rate of 99.9%. This indicates that the hydrogel contains PDA@BNN6 NPs, which is beneficial to the thermal decomposition and release of NO after irradiation with near-infrared light, thereby further improving the antibacterial properties of CDP-PB hydrogel.

The hydrogels obtained in Example 1 and Comparative Examples 1 to 5 were tested for their antioxidant properties:

400 mg of the hydrogels obtained in Example 1 and Comparative Examples 1 to 5 were added to 4 ml of PBS (0.01 M, pH = 7.2 to 7.4) and dispersed into a homogenate using a tissue grinder to form 6 groups of hydrogel dispersions. The 6 groups of hydrogel dispersions were then diluted to 10 mg/ml, 5 mg/ml and 2.5 mg/ml with PBS (0.01 M, pH = 7.2 to 7.4), respectively.

First, the effects of different types and concentrations of hydrogels on removing 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) were evaluated. 500 μl of hydrogel dispersions of different types and concentrations were mixed with 500 μl of 200 μM DPPH. The concentrations of the hydrogel dispersions in the final test solution system were 5 mg/ml, 2.5 mg/ml, and 1.25 mg/ml, respectively. After incubation at 37°C for 10 minutes, the supernatant was collected by centrifugation and the absorbance at 517 nm was measured with an enzyme-labeled instrument. The results are shown in Figure 5.

Then, the scavenging effect of different types and concentrations of hydrogels on 2,2′-amino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was evaluated. Equal amounts of 7.4 mM ABTS and potassium persulfate (K ₂ S ₂ O ₈ , 2.6 mM) were mixed. After stirring for 12 h in a dark environment at room temperature, the mixed solution was diluted with PBS (0.01 M, pH=7.2-7.4). 500 μl of hydrogel dispersions of different types and concentrations were mixed with 500 μl of diluted ABTS, and the concentrations of the hydrogel dispersions in the final test solution system were 5 mg/ml, 2.5 mg/ml and 1.25 mg/ml, respectively. After incubation at room temperature for 10 min, the supernatant was collected by centrifugation, and the absorbance at 405 nm was measured using an enzyme marker. The results are shown in Figure 6.

Finally, the scavenging effect of different types and concentrations of hydrogels on hydroxyl radicals (·OH) was evaluated. 500 μl of hydrogen peroxide (100 μM) and 50 μl of horseradish peroxidase (1 mg/ml) were reacted to generate hydroxyl radicals (·OH), which were incubated with 500 μl of mixed hydrogel dispersions of different types and concentrations at room temperature for 10 minutes. 100 μl of (3,3',5,5'-tetramethylbenzidine (2.5 mg/ml) dissolved in dimethyl sulfoxide (DMSO) was added for color reaction, the supernatant was collected by centrifugation, and the absorbance at 650 nm was measured using an enzyme reader.

In the above experiment, PBS (0.01M, pH=7.2-7.4) was used instead of the hydrogel dispersion as a control experiment. The free radical scavenging rate was calculated as follows:

Clearance (%) = ((A _b -A _h ))/A _b × 100%

Where A _b is the absorbance of the control group, and _Ah is the absorbance of the hydrogel group.

The results of the scavenging effects of different types and concentrations of hydrogels on hydroxyl radicals (·OH) are shown in Figure 7.

As shown in Figures 5 to 7, the scavenging rate of hydrogels for DPPH, ·OH and ABTS is concentration-dependent. When the concentration of hydrogel homogenate is as high as 5 mg/ml, all hydrogels show a high level of antioxidant activity and can effectively scavenge DPPH, ·OH and ABTS free radicals. By comparison, when the concentration of hydrogel homogenate is lower than 5 mg/ml, the scavenging effect of hydrogels doped with PDANPs or PDA@BNN6 NPs is better than that of undoped hydrogels. This is mainly because the free radical scavenging effect in hydrogels is attributed to free catechol groups and catechol-boric acid complexes. In addition, PDA NPs or PDA@BNN6 NPs increase the content of free catechol groups and catechol-borate complexes in the hydrogel system, thereby enhancing the scavenging ability of nanoparticle-doped hydrogels for free radicals.

The hydrogels obtained in Example 1 and Comparative Examples 1 to 5 were tested for wound healing performance:

All animal experiments were completed in accordance with the experimental protocols and ethical rules at the Experimental Animal Center of Xiangya School of Medicine, Central South University (Changsha, China). Male BALB/c mice (6 weeks old) were acclimated for 2 weeks before administration. To establish streptozotocin (STZ)-induced diabetic mice, STZ was injected for 5 consecutive days. Specifically: the mice were fasted for 12 hours, then intraperitoneally injected with freshly prepared STZ (50 mg/kg), and fasted for 2 hours. After the completion of administration, blood was collected from the tail vein every 5 days, and blood glucose levels were measured using a glucometer. After 3 weeks, when the fasting glucose concentration of the mice was higher than 300 mg/l, the mice were considered to have diabetes.

All surgeries were performed under sterile conditions. The diabetic mice were fixed, and the hair on the back of the mice was shaved with an electric shaver, and the shaved area was further depilated with a depilatory cream. The surgical area was disinfected with 75% alcohol. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (6 mg/ml), and a circular full skin wound with a diameter of 8 mm was made on the back of the mice. Then 50 μl of MRAS bacterial suspension (108 CFU/ml) was added to each wound and left to stand for 48 h. The diabetic mice were randomly divided into a control group (PBS), CD group, CDP group, CDP-P group, CDP-P+N group, and CDP-PB+N group. After the hydrogel dressing was added to the infected wound in the near-infrared group, the wound was irradiated with 808 nm near-infrared laser (0.6 W/cm ² ) for 5 min, and the thermal image of the wound was recorded with a thermal imager. The hydrogel was replaced every 2 days, for a total of 4 treatments. The wounds were photographed on days 0, 4, 6, 8, 10, and 14. Image J software was used to measure and analyze the wound area.

As shown in Figures 8 and 9, the wounds of the (1) PBS, (2) CD, (3) CDP, and (4) CDP-P treatment groups showed obvious suppuration on the 4th day, indicating the presence of severe bacterial infection. Among them, the wound healing of the PBS group was the slowest, with a healing rate of only 63.4±5.6% on the 14th day. After 14 days of treatment, the wound healing rates of the CD group, CDP group, CDP-P group, CDP-P+N group, and CDP-PB+N group were 71.8±8.6%, 77.1±5.7%, 85.5±4.2%, 89.0±3.3%, and 96.7±2.3%, respectively. The results showed that the CDP-PB+N treatment group showed excellent effects in wound healing.

In addition, on the 14th day, the in vivo antibacterial effect of each group of hydrogels was evaluated by smear method to detect whether there were residual bacteria in the wound tissue. The CDP-PB+N treatment group showed a strong antibacterial effect, with an antibacterial rate of 97.3%, which was significantly higher than that of other groups. This shows that the good antibacterial activity of CDP-PB hydrogel is the result of the positive charge of PEI-PBA cationic polymer, the release of a large amount of NO gas by PDA@BNN6 NPs nanospheres, and the PTT effect.

Claims (8)

1. A multifunctional hydrogel, characterized in that it comprises polyethyleneimine grafted with phenylboronic acid groups, polydopamine nanospheres loaded with BNN6, chitosan functionalized with catechol groups, and oxidized dextran; wherein the mass ratio of polyethyleneimine grafted with phenylboronic acid groups, polydopamine nanospheres loaded with BNN6, chitosan functionalized with catechol groups, and oxidized dextran is (10-100): (1-10): (60-600): (100-1000); The polyethyleneimine grafted with phenylboronic acid groups is obtained by a substitution reaction between polyethyleneimine and 4-(bromomethyl)phenylboronic acid; The mass ratio of the polyethyleneimine to 4-(bromomethyl)phenylboronic acid is (3-4):1; The catechol group-functionalized chitosan is obtained by amidation reaction of chitosan and 3,4-dihydroxyphenylpropionic acid; The mass ratio of the chitosan to 3,4-dihydroxyphenylpropionic acid is 1:(1-1.5). 2. A multifunctional hydrogel according to claim 1, characterized in that: The BNN6-loaded polydopamine nanospheres are obtained by self-assembly π-π stacking of polydopamine nanospheres and BNN6 under light-proof conditions; The polydopamine nanospheres are prepared by the following method: F127, dopamine hydrochloride and 1,3,5-trimethylbenzene are mixed and added with ammonia water for water bath reaction, and then the 1,3,5-trimethylbenzene template is removed by ultrasonication to obtain the product. 3. A multifunctional hydrogel according to claim 1 or 2, characterized in that: the amidation reaction uses EDC as a carboxyl activator, and the pH during the reaction is 4-6. 4. A multifunctional hydrogel according to claim 3, characterized in that: The oxidized dextran is obtained by oxidizing dextran with sodium periodate; The mass ratio of the dextran to sodium periodate is 1:(1-2); The oxidation condition is: reacting at room temperature in the dark for 10 to 12 hours. 5. A method for preparing a multifunctional hydrogel according to any one of claims 1 to 4, characterized in that polyethyleneimine grafted with phenylboronic acid groups, polydopamine nanospheres loaded with BNN6, chitosan functionalized with catechol groups and oxidized dextran are dispersed in water and mixed to obtain the hydrogel. 6. Use of the multifunctional hydrogel according to any one of claims 1 to 4, characterized in that it is used to prepare diabetic wound healing dressings. 7. The use of a multifunctional hydrogel according to claim 6, characterized in that the diabetic wound healing dressing has a photothermal effect, and has antibacterial, antioxidant and angiogenesis-promoting functions under the action of photothermal. 8. The use of a multifunctional hydrogel according to claim 7, characterized in that the conditions of the photothermal effect are: irradiation power density of 0.1-10 W/ ^cm2 , irradiation time of 5-20 min of near-infrared laser.