In-situ oxygen-generating hydrogel and preparation method and application thereof

Abstract

The application discloses in-situ oxygen-generating hydrogel and a preparation method and application thereof, and relates to the technical field of medical biomaterials.The hydrogel comprises phenylboronic acid modified quaternary ammonium chitosan, dopamine modified oxidized hyaluronic acid and composite nanoparticles.The application utilizes manganese dioxide to induce decomposition of endogenous active oxygen into oxygen, effectively improves oxidative stress and hypoxia, and introduces manganese dioxide nanoparticles into the antibacterial injection hydrogel, so that various requirements, namely antibacterial effect, active oxygen consumption and oxygen generation, can be met.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, specifically to an in-situ oxygen-generating hydrogel, its preparation method, and its applications. Background Technology

[0002] There are approximately 463 million people with diabetes worldwide, and this number is projected to increase to 579 million by 2030. Diabetes is a complex metabolic disease that adversely affects multiple organs in the body. Chronic diabetes is accompanied by long-term problems such as hyperglycemia, high inflammation, and bacterial infection, threatening the health of diabetic patients.

[0003] Normal skin wound regeneration primarily involves overlapping stages of inflammation, proliferation, and remodeling; however, the healing process of diabetic wounds is often delayed. The cascade of abnormal inflammatory responses interferes with the behavior of functional cells, including keratinocytes, fibroblasts, and endothelial cells, and can even cause cell damage and apoptosis. Simultaneously, inflammatory cells recruited at the wound site produce large amounts of reactive oxygen species, exacerbating oxidative stress and leading to various detrimental effects, including reduced collagen deposition and angiogenesis, inappropriate degradation of the extracellular matrix and growth factors, and delayed re-epithelialization. Hyperglycemia can cause vasoconstriction, inhibiting angiogenesis, blocking oxygen supply, and hindering the healing process. Furthermore, hypoxia has been shown to inhibit wound healing by blocking fibroblast proliferation, capillary angiogenesis, collagen production, and increasing the risk of infection. Theoretically, oxygen supply to diabetic wounds is beneficial for wound healing.

[0004] Hydrogels have attracted increasing attention due to their porous structure and are considered a potential clinical strategy for normal wound healing. Traditional hydrogel dressings suffer from weak tissue adhesion, are not adaptable to wound shapes, have insufficient functionality, and fail to achieve the desired therapeutic effect on irregular wounds. In recent years, injectable self-healing hydrogels have garnered significant attention due to their advantages such as strong shape adaptability, good tissue adhesion, ease of functionalization, and drug loading. Therefore, injectable self-healing hydrogels have potential application value as wound dressings in the future.

[0005] Biocompatible hydrogels can help optimize biological and molecular events involved in wound healing, including cell migration, proliferation, and differentiation, thereby accelerating the healing process. However, vascular lesions and damage caused by multidrug-resistant bacterial infections limit the application of hydrogels in diabetic wound healing.

[0006] To achieve the above objectives, those skilled in the art have used sodium alginate as the polymer backbone, and after simple modification with dopamine, mixed it with calcium peroxide / polymer oxygen-generating particles to prepare a physicochemically double-crosslinked injectable oxygen-generating hydrogel in one step. When used under physiological conditions, the polymer on the surface of the oxygen-generating particles in this injectable oxygen-generating hydrogel exhibits hydrophobic properties, which can prevent water from contacting the oxygen-generating particles, thereby prolonging the oxygen-generating time of the calcium peroxide oxygen-generating particles. However, excessive oxidative stress is generated after oxygen production, which cannot be effectively eliminated, thus hindering tissue repair. Therefore, the development of in-situ oxygen-generating hydrogels is particularly important for addressing the high oxidative stress and hypoxic microenvironment characteristics of diabetic wounds. Summary of the Invention

[0007] To address the shortcomings of the aforementioned background technologies, and specifically targeting the high oxidative stress and hypoxic microenvironment characteristics of diabetic wounds, existing technologies, due to the prolonged oxygen production time of calcium peroxide oxygen-producing particles, generate excessive oxidative stress after oxygen production, which cannot be effectively eliminated, thus hindering tissue repair. This invention provides an in-situ oxygen-generating hydrogel, its preparation method, and its applications. This hydrogel utilizes manganese dioxide to induce the decomposition of endogenous reactive oxygen species into oxygen, effectively improving oxidative stress and hypoxia. Introducing manganese dioxide nanoparticles into an antibacterial injectable hydrogel can meet multiple needs, namely antibacterial effects, reactive oxygen species depletion, and oxygen generation.

[0008] To achieve the above objectives, the first aspect of the present invention provides an in-situ oxygen-generating hydrogel, the hydrogel comprising phenylboronic acid-modified quaternized chitosan, dopamine-modified oxidized hyaluronic acid, and composite nanoparticles.

[0009] The composite nanoparticles include manganese dioxide nanoparticles, a chitosan coating on the surface of the manganese dioxide nanoparticles, and arginine adhered to the chitosan coating.

[0010] Preferably, the mass ratio of the phenylboronic acid-modified quaternized chitosan, the dopamine-modified oxidized hyaluronic acid, and the composite nanoparticles is (2-4):(6-10):(0.25-1).

[0011] Preferably, the hydrogel is prepared by forming Schiff base bonds and phenylboronic acid ester structures from phenylboronic acid-modified quaternized chitosan, dopamine-modified oxidized hyaluronic acid, and composite nanoparticles.

[0012] Preferably, the phenylboronic acid-modified quaternized chitosan is prepared according to the following steps:

[0013] Obtain quaternized chitosan;

[0014] Quaternized chitosan was dispersed in an aqueous solvent to obtain a quaternized chitosan solution;

[0015] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were dispersed in a quaternized chitosan solution, and then a methanol solution of 4-carboxyphenylboronic acid was added. The reaction was carried out for 12-24 hours, and after post-treatment, phenylboronic acid-modified quaternized chitosan was obtained.

[0016] Preferably, the dopamine-modified oxidized hyaluronic acid is prepared according to the following steps:

[0017] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride were dispersed in an aqueous solution of oxidized hyaluronic acid and reacted for 12-24 h under nitrogen protection. After post-treatment, dopamine-modified oxidized hyaluronic acid was obtained.

[0018] Preferably, the particle size of the composite nanoparticles is 105–165 nm.

[0019] Preferably, the method for preparing the composite nanoparticles includes:

[0020] Bovine serum albumin was dispersed in an aqueous solution of dopamine, and then potassium permanganate solution was added. After the reaction was completed, a manganese dioxide solution was obtained.

[0021] Chitosan solution was dispersed in manganese dioxide solution to obtain chitosan-manganese dioxide solution;

[0022] Arginine aqueous solution was dispersed in chitosan-manganese dioxide solution. After the reaction was completed, the composite nanoparticles were obtained by washing and drying.

[0023] A second aspect of this invention provides a method for preparing an in-situ oxygen-generating hydrogel, comprising the following steps:

[0024] Dopamine-modified oxidized hyaluronic acid was dispersed in deionized water to obtain a dopamine-modified oxidized hyaluronic acid solution; composite nanoparticles were dispersed in the dopamine-modified oxidized hyaluronic acid solution to obtain a composite precursor solution;

[0025] Benzylboronic acid-modified quaternized chitosan was dissolved and dispersed in deionized water to obtain a Benzylboronic acid-modified quaternized chitosan precursor solution.

[0026] A hydrogel with in-situ oxygen generation was obtained by mixing the phenylboronic acid-modified quaternized chitosan precursor solution and the composite precursor solution in a certain proportion.

[0027] Preferably, the volume ratio of the phenylboronic acid-modified quaternized chitosan precursor solution to the composite precursor solution is 1:1.5 to 3.

[0028] A third aspect of the present invention provides an application of an in-situ oxygen-generating hydrogel as a dressing for wounds damaged by high oxidative stress.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] This invention provides an in-situ oxygen-generating hydrogel, its preparation method, and its application. It consists of oxygen-generating composite nanoparticles and a biodegradable hydrogel. The oxygen-generating composite nanoparticles of this invention undergo biomineralization and polymerization between potassium permanganate, bovine serum albumin, and dopamine to generate manganese dioxide nanoparticles, which are negatively charged and have a particle size distribution of 105–120 nm. The manganese dioxide nanoparticles, chitosan, and arginine are assembled via an electrostatic self-assembly method. Positively charged chitosan is dispersed in an aqueous solution of negatively charged manganese dioxide nanoparticles. Through electrostatic self-assembly, chitosan-coated manganese dioxide nanoparticles are obtained. Then, arginine is loaded onto the manganese dioxide nanoparticles through chitosan adhesion, thus obtaining the oxygen-generating composite nanoparticles. The biodegradable hydrogel of this invention is prepared by grafting quaternary aminated chitosan with phenylboronic acid and dopamine-modified oxidized hyaluronic acid through the formation of Schiff base bonds and phenylboronic acid ester structures. The simple double cross-linking structure of Schiff base bonds and phenylboronic acid esters not only enhances the physical properties of the hydrogel, giving it self-healing capabilities, but also strengthens its biological efficacy. The introduced oxygen-generating composite nanoparticles react with aldehyde groups to form Schiff bases, further introducing the nanoparticles into the hydrogel. The composite nanoparticles are then released through hydrogel degradation. The manganese dioxide in the released composite nanoparticles consumes hydrogen peroxide to produce oxygen, and the arginine loaded in the nanoparticles generates nitric oxide under the action of nitric oxide synthase and oxygen. This effectively dilates blood vessels, increases blood flow, and promotes collagen synthesis.

[0031] This invention provides an in-situ oxygen-generating hydrogel that can catalyze the production of oxygen from hydrogen peroxide in a high-oxidative-stress microenvironment, promoting angiogenesis. Furthermore, under the action of oxygen and nitric oxide synthase, it catalyzes the production of nitric oxide from arginine, facilitating vasodilation and increasing blood flow. Simultaneously, the oxygen-generating hydrogel of this invention exhibits good biocompatibility, promoting cell proliferation and migration in vitro, and demonstrating good antibacterial effects against *Escherichia coli* (Gram-negative), *Staphylococcus aureus* (Gram-positive), and methicillin-resistant *Staphylococcus aureus* (drug-resistant bacteria). It can be applied to tissue repair of chronically infected wounds and shows promising application prospects in engineered tissue construction.

[0032] This invention provides a method for preparing an in-situ oxygen-generating hydrogel. Based on the Schiff base interaction between composite nanoparticles and the hydrogel, oxygen-generating composite nanoparticles are loaded into the hydrogel, simultaneously achieving antibacterial, oxidative stress relief, and slow oxygen release functions. This improves upon the problems of excessively rapid oxygen release and limited functionality in existing oxygen-generating materials. Regarding the healing mechanism, the hydrogel can absorb wound exudate, maintain wound moisture, and provide sufficient oxygen supply for wound healing; it can protect the wound from bacterial infection and oxidative stress damage; the timely release of arginine from the composite nanoparticles can promote angiogenesis and wound healing. Through the synergistic effect between the hydrogel and the composite nanoparticles—antibacterial activity, improvement of oxidative stress, simultaneous oxygen release, and promotion of angiogenesis—rapid wound healing is achieved. Attached Figure Description

[0033] Figure 1 A schematic diagram illustrating the function of oxygen-producing hydrogels.

[0034] Figure 2 This is a scanning electron microscope image of the oxygen-producing hydrogel.

[0035] Figure 3 This indicates the oxygen-generating effect of the oxygen-generating hydrogel.

[0036] Figure 4 This indicates the biocompatibility effect of the oxygen-generating hydrogel; among which, Figure 4 (A) Cell viability assay; Figure 4 (B) Cell live / dead staining results.

[0037] Figure 5 This indicates the antibacterial effect of the oxygen-producing hydrogel.

[0038] Figure 6 This indicates the therapeutic effect of oxygen-producing hydrogel on diabetic wounds infected with MRSA; among which, Figure 6 (A) shows wound images on days 0, 3, 7, and 14 and the antibacterial effects of different treatment groups; Figure 6 (B) Indicates the wound healing rate. Detailed Implementation

[0039] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0040] This invention provides an in-situ oxygen-generating hydrogel, the hydrogel comprising phenylboronic acid-modified quaternized chitosan, dopamine-modified oxidized hyaluronic acid, and composite nanoparticles; the composite nanoparticles comprising manganese dioxide nanoparticles, a chitosan coating on the surface of the manganese dioxide nanoparticles, and arginine adhered to the chitosan coating.

[0041] The composite nanoparticles used in this invention are used to catalyze the production of oxygen from hydrogen peroxide. The chitosan coating on the surface of the manganese dioxide nanoparticles in the composite nanoparticles is used for electrostatic assembly of arginine, and the arginine adhering to the chitosan coating is used to generate nitric oxide.

[0042] According to the present invention, the hydrogel is prepared by forming Schiff base bonds and phenylboronic acid ester structures by comprising phenylboronic acid-modified quaternized chitosan, dopamine-modified oxidized hyaluronic acid, and composite nanoparticles.

[0043] In one embodiment, an in-situ oxygen-generating hydrogel is obtained by mixing a phenylboronic acid-modified quaternized chitosan solution, a dopamine-modified oxidized hyaluronic acid solution, and composite nanoparticles. During the mixing reaction, the amino and aldehyde groups form Schiff base bonds, and the phenylboronic acid and catechol form a phenylboronic ester structure. The hydrogel contains dispersed composite nanoparticles, in which manganese dioxide can generate oxygen in the presence of hydrogen peroxide.

[0044] The hydrogel provided by this invention is composed of composite nanoparticles and a biodegradable hydrogel. The composite nanoparticles are synthesized from chitosan, arginine, and manganese dioxide nanoparticles. The hydrogel is formed by Schiff base and borate ester bonds between phenylboronic acid-modified quaternized chitosan and dopamine-modified oxidized hyaluronic acid. Introducing the composite nanoparticles into the hydrogel can catalyze the decomposition of hydrogen peroxide to produce oxygen, increasing tissue oxygenation and improving local oxidative stress and hypoxic microenvironment, thereby enhancing the therapeutic efficacy of hypoxia-related diseases.

[0045] According to the present invention, the mass ratio of the phenylboronic acid modified quaternized chitosan, the dopamine modified oxidized hyaluronic acid and the composite nanoparticles is (2-4):(6-10):(0.25-1).

[0046] According to the present invention, the phenylboronic acid-modified quaternized chitosan is prepared according to the following steps:

[0047] Obtain quaternized chitosan;

[0048] Quaternized chitosan was dispersed in an aqueous solvent to obtain a quaternized chitosan solution;

[0049] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were dispersed in a quaternized chitosan solution, and then a methanol solution of 4-carboxyphenylboronic acid was added. The reaction was carried out for 12-24 hours, and after post-treatment, phenylboronic acid-modified quaternized chitosan was obtained.

[0050] In one embodiment, chitosan was suspended in deionized water and dissolved in glacial acetic acid. After stirring at 55°C for 0.5–1 h, glycidyltrimethylammonium chloride was added and reacted at 55°C for 15–24 h. Then, the mixture was dialyzed with deionized water and freeze-dried to obtain quaternized chitosan. Quaternized chitosan was dissolved in deionized water and 4-carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 12–24 h, and then dialyzed in water and freeze-dried to obtain phenylboronic acid-modified quaternized chitosan.

[0051] The quaternized chitosan has a mass concentration of 0.5 wt%; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 4-carboxyphenylboronic acid is 1:1:(1~1.5).

[0052] According to the present invention, the dopamine-modified oxidized hyaluronic acid is prepared according to the following steps:

[0053] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride were dispersed in an aqueous solution of oxidized hyaluronic acid and reacted for 12-24 h under nitrogen protection. After post-treatment, dopamine-modified oxidized hyaluronic acid was obtained.

[0054] In one embodiment, hyaluronic acid was dissolved in deionized water, sodium periodate solution was added, and the reaction was carried out in the dark for 2–4 hours. Excess ethylene glycol was added to terminate the reaction for 1–2 hours. The mixture was then dialyzed in water and freeze-dried to obtain oxidized hyaluronic acid. Alternatively, oxidized hyaluronic acid was completely dissolved in deionized water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and dopamine hydrochloride were added. The reaction was carried out at 25°C for 12–24 hours, with the pH maintained at approximately 5.5, and nitrogen gas continuously purged. The mixture was then dialyzed in acidic water and freeze-dried to obtain dopamine-modified oxidized hyaluronic acid. The mass concentration of oxidized hyaluronic acid was 1 wt%; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and dopamine hydrochloride was 1:1:(1–2).

[0055] According to the present invention, the particle size of the composite nanoparticles is 105-165 nm.

[0056] According to the present invention, the method for preparing the composite nanoparticles includes:

[0057] Bovine serum albumin was dispersed in an aqueous solution of dopamine, and then potassium permanganate solution was added. After the reaction was completed, a manganese dioxide solution was obtained.

[0058] Chitosan solution was dispersed in manganese dioxide solution to obtain chitosan-manganese dioxide solution;

[0059] Arginine aqueous solution was dispersed in chitosan-manganese dioxide solution. After the reaction was completed, the composite nanoparticles were obtained by washing and drying.

[0060] In one embodiment, the composite nanoparticles are prepared by dissolving dopamine hydrochloride and bovine serum albumin in deionized water, adding potassium permanganate solution and reacting at room temperature for 2–4 hours, then adding chitosan solution and reacting for 0.5–1 hour to obtain positively charged nanoparticles; subsequently, arginine is added and sonicated for 15–30 minutes, followed by centrifugation, washing, and freeze-drying to collect the composite nanoparticles. The mass ratio of potassium permanganate, dopamine hydrochloride, bovine serum albumin, chitosan, and arginine is (0.2–1):1:2:(0.00125–0.01):(2–10).

[0061] Specifically, 100 mg of dopamine hydrochloride and 200 mg of bovine serum albumin were dissolved in 100 mL of water and added at 5-minute intervals. 30 mg of potassium permanganate was dissolved in 2 mL of deionized water and added dropwise to the mixture. The mixture was stirred at room temperature for 2 hours. Purified manganese dioxide nanoparticles were obtained by dialysis, washing, and freeze-drying.

[0062] Chitosan solution was pumped into manganese dioxide solution at a rate of 0.5 mL / min while stirring for 30 min to obtain positively charged nanoparticles. Chitosan-manganese dioxide nanoparticles were obtained after washing, centrifugation, and purification.

[0063] Dissolve 200 mg of arginine in water, add it to a chitosan-manganese dioxide solution, and sonicate for 15 min. Centrifuge at 13900 × g for 10 min, wash, and freeze-dry to collect the composite nanoparticles (arginine-chitosan-manganese dioxide nanoparticles).

[0064] The manganese dioxide nanoparticles are negatively charged and have a particle size of 78–190 nm, more preferably 105–120 nm. The chitosan-manganese dioxide nanoparticles are positively charged and have a particle size of 68–190 nm, more preferably 91–121 nm. The composite nanoparticles are negatively charged and have a particle size of 91–190 nm, more preferably 105–165 nm.

[0065] The chitosan has a viscosity of 100–200 mPa·s; the composite nanoparticles contain 4% manganese ions; and the composite nanoparticles have an arginine loading of 59.6 mg / g.

[0066] This invention provides a method for preparing an in-situ oxygen-generating hydrogel, comprising the following steps:

[0067] Dopamine-modified oxidized hyaluronic acid was dispersed in deionized water to obtain a dopamine-modified oxidized hyaluronic acid solution; composite nanoparticles were dispersed in the dopamine-modified oxidized hyaluronic acid solution to obtain a composite precursor solution;

[0068] Benzylboronic acid-modified quaternized chitosan was dissolved and dispersed in deionized water to obtain a Benzylboronic acid-modified quaternized chitosan precursor solution.

[0069] A hydrogel with in-situ oxygen generation was obtained by mixing the phenylboronic acid-modified quaternized chitosan precursor solution and the composite precursor solution in a certain proportion.

[0070] According to the present invention, the volume ratio of the phenylboronic acid-modified quaternized chitosan precursor solution to the composite precursor solution is 1:1.5 to 3. A preferred volume ratio is 1:2.

[0071] In one embodiment, a method for preparing an in-situ oxygen-generating hydrogel is as follows:

[0072] (1) Dissolve 1 g of quaternized chitosan in deionized water, and then add 332.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 246.6 mg of N-hydroxysuccinimide and 4-carboxyphenylboronic acid in sequence. Then react the mixed solution at room temperature for 12-24 h, and obtain phenylboronic acid modified quaternized chitosan after dialyzing and freeze drying.

[0073] (2) Dissolve 1g of oxidized hyaluronic acid in 100mL of deionized water, then add 0.474g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.285g of N-hydroxysuccinimide, and 0.388g of dopamine hydrochloride sequentially. React at 25℃ under a nitrogen atmosphere for 24h, maintaining the pH at 5.5. After dialyzing and freeze-drying, dopamine-modified oxidized hyaluronic acid is obtained.

[0074] (3) The composite nanoparticles were dispersed in a dopamine-modified oxidized hyaluronic acid solution at a concentration of 1 mg / mL to form an 8 wt% composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution.

[0075] (4) Dissolve phenylboronic acid-modified quaternary ammonium chitosan in deionized water and heat to dissolve, forming a 3wt% phenylboronic acid-modified quaternary ammonium chitosan precursor solution; then mix it with the composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution from step (3) at a volume ratio of 1:2. After mixing, maintain the pH at 7.5–8.5 to obtain an oxygen-generating hydrogel. The hydrogel is prepared by phenylboronic acid-modified quaternary ammonium chitosan and dopamine-modified oxidized hyaluronic acid through the formation of Schiff base bonds and phenylboronic acid ester structures. The amino group of arginine in the introduced composite nanoparticles reacts with the aldehyde group to form a Schiff base, further introducing the composite nanoparticles into the hydrogel, and releasing the composite nanoparticles through the degradation of the hydrogel.

[0076] In step (1), the mass concentration of quaternized chitosan is 0.5 wt%; the degree of quaternization of chitosan is 35%; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 4-carboxyphenylboronic acid is 1:1:(1~1.5); 4-carboxyphenylboronic acid needs to be dissolved in methanol; the modification rate of 4-carboxyphenylboronic acid is 23.1%.

[0077] In step (2), the mass concentration of oxidized hyaluronic acid is 1 wt%; the degree of oxidation of hyaluronic acid is 34%; the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride is 1:1:(1-2); and the dopamine grafting rate is 25.2%.

[0078] In step (4), the concentration range of phenylboronic acid modified quaternized chitosan is 2-4 wt%; the concentration range of dopamine modified oxidized hyaluronic acid is 6-10 wt%; and the concentration of composite nanoparticles in the hydrogel is 0.25-1 mg / mL.

[0079] This invention provides the application of an in-situ oxygen-generating hydrogel as a dressing for wounds damaged by high oxidative stress. Preferably, the in-situ oxygen-generating hydrogel is used as a wound repair gel for diabetic wounds infected with methicillin-resistant Staphylococcus aureus.

[0080] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0081] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods; and the reagents and materials used, unless otherwise specified, are all commercially available.

[0082] Example 1

[0083] Preparation of manganese dioxide oxygen-generating nanoparticles

[0084] Manganese dioxide nanoparticles were synthesized in the presence of bovine serum albumin via polymerization and biomineralization initiated by potassium permanganate. Specific steps:

[0085] Dissolve 30 mg of potassium permanganate in 2 mL of deionized water, and dissolve 100 mg of dopamine in 100 mL of water. Add 200 mg of bovine serum albumin at 5-minute intervals. Add the dissolved potassium permanganate solution dropwise to the mixture, and stir the mixture at room temperature for 2 hours. Dialyze the mixture using a dialysis bag (molecular weight 3500 Da) to remove unreacted small molecules. Freeze-dry the purified manganese dioxide nanoparticles, with a particle size of 78–190 nm, more preferably 105–140 nm, and these manganese dioxide nanoparticles are negatively charged.

[0086] Example 2

[0087] Preparation of chitosan-manganese dioxide oxygen-generating nanoparticles

[0088] Chitosan-manganese dioxide nanoparticles were prepared by electrostatic self-assembly, with the following specific steps:

[0089] 30 mg of potassium permanganate was dissolved in 2 mL of deionized water, and 100 mg of dopamine was dissolved in 100 mL of water. 200 mg of bovine serum albumin was added at 5-minute intervals. The dissolved potassium permanganate solution was added dropwise to the mixture, and the mixture was stirred at room temperature for 2 hours. A 1 mg / mL chitosan solution was pumped into a manganese dioxide solution at a rate of 0.5 mL / min, while stirring for 30 minutes to obtain positively charged nanoparticles. After washing, centrifugation, and purification, chitosan-manganese dioxide nanoparticles with a particle size of 68–190 nm, more preferably 91–141 nm, were obtained.

[0090] Example 3

[0091] Preparation of composite nanoparticles (arginine-chitosan-manganese dioxide oxygen-generating nanoparticles)

[0092] Arginine-chitosan-manganese dioxide nanoparticles were prepared by electrostatic self-assembly, with the following specific steps:

[0093] Dissolve 30 mg of potassium permanganate in 2 mL of deionized water, and dissolve 100 mg of dopamine in 100 mL of water. Add 200 mg of bovine serum albumin at 5-minute intervals. Add the dissolved potassium permanganate solution dropwise to the mixture and stir at room temperature for 2 hours. Pump a 1 mg / mL chitosan solution into the manganese dioxide solution at a rate of 0.5 mL / min while stirring for 30 minutes to obtain positively charged nanoparticles.

[0094] Dissolve 5 mg / mL arginine in water and add it to a chitosan-manganese dioxide solution, then sonicate for 15 min. Centrifuge at 13900×g for 10 min, wash three times with deionized water, and freeze-dry to collect arginine-chitosan-manganese dioxide nanoparticles. The arginine-chitosan-manganese dioxide nanoparticles are negatively charged and have a particle size of 91–190 nm, more preferably 105–165 nm.

[0095] Example 4

[0096] A method for preparing an in-situ oxygen-generating hydrogel includes the following steps:

[0097] (1) Preparation of quaternary ammonium chitosan modified with phenylboronic acid

[0098] Weigh 1.5 g of quaternized chitosan and dissolve it in 270 mL of deionized water. Stir at room temperature until the quaternized chitosan is completely dissolved. Add 997.8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 739.8 mg of N-hydroxysuccinimide to the above quaternized chitosan solution and continue stirring. Dissolve 1.545 g of 4-carboxyphenylboronic acid in 30 mL of methanol solution and slowly add it dropwise to the above mixed solution. Stir at room temperature for 24 h (pH 5.5). Transfer the mixture to a dialysis bag (MWCO: 8-14 kDa) and dialyze with deionized water at room temperature for 3 days to remove unreacted small molecules. Freeze-dry the dialysate to obtain phenylboronic acid-modified quaternized chitosan.

[0099] (2) Preparation of dopamine-modified oxidized hyaluronic acid

[0100] 2.0 g of oxidized hyaluronic acid was dissolved in 200 mL of deionized water and stirred at room temperature until a clear solution was obtained. 0.948 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.57 g of N-hydroxysuccinimide, and 0.776 g of dopamine hydrochloride were added to the oxidized hyaluronic acid solution, and the reaction was carried out under nitrogen protection for 12 h, maintaining the pH at 5.5. The mixture was then dialyzed with acidic deionized water for 3 days to remove unreacted byproducts. The purified product was freeze-dried to obtain dopamine-modified oxidized hyaluronic acid.

[0101] (3) Preparation of oxygen-generating hydrogel

[0102] Dopamine-modified oxidized hyaluronic acid was dissolved in deionized water at a concentration of 6 wt%, and composite nanoparticles were dispersed in the dopamine-modified oxidized hyaluronic acid solution at a concentration of 0.5 mg / mL to form a composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution. Phenylated boric acid-modified quaternized chitosan was dissolved in deionized water to prepare a 2 wt% precursor solution. The phenylboronic acid-modified quaternized chitosan precursor solution and the composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution were mixed evenly at a ratio of 1:2 to prepare the in-situ oxygen-generating hydrogel. (See [link to documentation]). Figure 1 As shown, from Figure 1 As can be seen, a dynamic hydrogel network is ultimately formed through the Schiff base reaction between amino and aldehyde groups and the borate ester structure between phenylboronic acid and catechol, while oxygen-generating composite nanoparticles are loaded, thereby forming an in-situ oxygen-generating hydrogel. The oxygen-generating nanoparticles can catalyze the production of oxygen from hydrogen peroxide in a high oxidative stress environment, and generate nitric oxide in the presence of nitric oxide synthase and oxygen. Figure 1 In Chinese: PBA-QCS represents phenylboronic acid grafted quaternized chitosan; DA-OHA represents dopamine-modified oxidized hyaluronic acid; ACMNPs represents oxygen-generating composite nanoparticles.

[0103] Example 5

[0104] Similar to Example 4, except that in step (3), dopamine-modified oxidized hyaluronic acid is dissolved in deionized water at a concentration of 8 wt%, and composite nanoparticles are dispersed in the dopamine-modified oxidized hyaluronic acid solution at a concentration of 0.5 mg / mL to form a composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution. Phenylated boric acid-modified quaternized chitosan is dissolved in deionized water to prepare a 3 wt% precursor solution. The phenylboronic acid-modified quaternized chitosan precursor solution and the composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution are mixed evenly at a ratio of 1:2 to prepare the in-situ oxygen-generating hydrogel.

[0105] Example 6

[0106] Similar to Example 4, except that in step (3), dopamine-modified oxidized hyaluronic acid is dissolved in deionized water at a concentration of 10 wt%, and composite nanoparticles are dispersed in the dopamine-modified oxidized hyaluronic acid solution at a concentration of 0.5 mg / mL to form a composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution. Phenylated boric acid-modified quaternized chitosan is dissolved in deionized water to prepare a 2.5 wt% precursor solution. The phenylboronic acid-modified quaternized chitosan precursor solution and the composite nanoparticle / dopamine-modified oxidized hyaluronic acid precursor solution are mixed evenly at a ratio of 1:2 to prepare the in-situ oxygen-generating hydrogel.

[0107] Comparative Example 1

[0108] A method for preparing a hydrogel is the same as in Example 4, except that no composite nanoparticles are added in step (3).

[0109] To illustrate the properties of the composite nanoparticles and hydrogels provided by the present invention, only the properties of the composite nanoparticles provided in Example 3, the oxygen-generating hydrogel provided in Example 4, and the hydrogel provided in Comparative Example 1 will be described.

[0110] To characterize the structure and properties of the oxygen-generating hydrogel provided in Example 4, samples of oxygen-generating hydrogels with different concentrations were first prepared. These samples were then immediately and rapidly frozen in liquid nitrogen and freeze-dried in a freeze dryer. The freeze-dried hydrogels were then adhered to conductive adhesive and vacuum-sputtered with gold. The surface morphology of the hydrogels was observed using a tungsten filament scanning electron microscope, and the distribution of each element in the hydrogels was analyzed by EDS. The pore size of the hydrogels was analyzed using ImageJ software (see [link to relevant documentation]). Figure 2 As shown. Figure 2 This is a scanning electron microscope (SEM) image of the oxygen-producing hydrogel. (From...) Figure 2 It can be seen that the oxygen-generating hydrogel has a uniform internal pore structure and the composite nanoparticles are evenly distributed.

[0111] For characterization of the oxygen generation performance of the composite nanoparticles (arginine-chitosan-manganese dioxide nanoparticles) provided in Example 3 and the hydrogel provided in Example 4, please refer to [link to relevant documentation]. Figure 3 As shown. Figure 3 This indicates the oxygen-generating effect of the oxygen-generating hydrogel provided in Example 4. Figure 3 In the table, PBS+ACM represents the oxygen release characteristics of oxygen-generating composite nanoparticles in PBS solution; PBS+PDM represents the oxygen release characteristics of oxygen-generating hydrogel in PBS solution; H2O2+ACM represents the oxygen release characteristics of oxygen-generating composite nanoparticles in PBS solution containing 10mM H2O2; and H2O2+PDM represents the oxygen release characteristics of oxygen-generating hydrogel in PBS solution containing 10mM H2O2. Figure 3 It was found that, under the same conditions, the composite nanoparticles produced almost no oxygen in PBS solution, but continuously produced oxygen for up to 6 hours in H2O2 solution. This is beneficial for improving oxidative stress in diabetic wounds and alleviating the hypoxic microenvironment of the wound, thus promoting wound healing. The oxygen release capacity of the hydrogel was monitored in PBS and H2O2, respectively. A 600 μL L PDM hydrogel sample was added to 20 mL of 10 mL H2O2 solution, and the probe of a dissolved oxygen meter was immediately immersed in it. The oxygen concentration was recorded at a predetermined time. As a control, 600 μL of the sample was added to 20 mL of PBS for oxygen concentration detection. Figure 3 It can be seen that the oxygen-generating hydrogel prepared in Example 4 can continuously release oxygen in H2O2 solution, and the hydrogel can alleviate the release of oxygen.

[0112] Biocompatibility characterization of the composite nanoparticles provided in Example 3, the oxygen-generating hydrogel provided in Example 4, and the hydrogel provided in the comparative example was performed by seeding L929 cells at a density of 10,000 cells / well in 48-well plates and allowing them to adhere and grow for 12 hours. Hydrogel discs with a diameter of 5 mm and a thickness of 1 mm were prepared, sterilized by soaking in 75% medical alcohol, and washed three times with PBS. The sterilized oxygen-generating hydrogel was added to the cells for co-incubation. The control groups included those without oxygen-generating hydrogel, the composite nanoparticles provided in Example 3, and the hydrogel provided in Comparative Example 1. After incubation for 1, 3, and 5 days, the hydrogel and culture medium were discarded, and the cells were washed once with PBS. MTT solution was added and the cells were cultured for 4 hours. DMSO was added, and the cells were incubated on a shaker for 10 minutes. The absorbance at 490 nm was measured using a multi-functional microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = (ODh - ODn) / ODn × 100%; see [link to relevant documentation]. Figure 4 As shown. Figure 4 (A) Cell viability assay; Figure 4 (B) Cell viability / deadness staining results. Wherein: ODh represents the absorbance values ​​after hydrogel incubation for 1, 3, and 5 days, and ODn represents the absorbance values ​​of the control group. Results are as follows... Figure 4 (A). Among them, Figure 4 This indicates the biocompatibility of the oxygen-generating hydrogel; Figure 4 In the table, Control: No treatment; ACM: Composite nanoparticles provided in Example 3; PD: Hydrogel provided in Comparative Example 1; PDM: Oxygen-generating hydrogel provided in Example 4. L929 cells were co-incubated with the cells without oxygen-generating hydrogel, the composite nanoparticles provided in Example 3, the hydrogel provided in Comparative Example 1, and the oxygen-generating hydrogel provided in Example 4, respectively. After 5 days of culture, Calcein-AM / PI detection working solution was added, and the cells were incubated at 37°C in the dark for 30 minutes. The staining effect was observed under an inverted fluorescence microscope. The results are as follows: Figure 4 (B) Co-culturing oxygen-producing hydrogel with L929 cells showed good cell compatibility and no cytotoxicity.

[0113] To characterize the antibacterial properties of the oxygen-producing hydrogel provided in Example 4, bacteria were incubated with the hydrogel for 4 hours, fixed overnight with 4% paraformaldehyde, and dehydrated using a gradient of alcohols (70%, 75%, 80%, 85%, 90%, 95%, and anhydrous ethanol), each for 10 minutes. The gel was then lyophilized and sputter-coated with gold. Bacterial morphology was observed using SEM. (See [link to SEM]). Figure 5 As shown. Figure 5To demonstrate the antibacterial effect of the oxygen-producing hydrogel, SEM was used to study the morphological changes of *E. coli*, *S. aureus*, and *MRSA* after incubation with the oxygen-producing hydrogel. Normal bacteria showed smooth surfaces with no obvious damage. However, when microorganisms adhered to the surface of the oxygen-producing hydrogel, their morphology was affected, the surface became rough, and the cell membranes were damaged and indented. This indicates that the oxygen-producing hydrogel has excellent antibacterial effects. The control group was used, with no oxygen-producing hydrogel added. Figure 5 In this context, E. coli refers to *Escherichia coli*, S. aureus to *Staphylococcus aureus*, and MRSA to methicillin-resistant *Staphylococcus aureus*.

[0114] The oxygen-generating hydrogel provided in Example 4 was used for wound healing testing in diabetic animals infected with MRSA. The wound healing effect of the oxygen-generating hydrogel in vivo was evaluated based on a mouse model of full-thickness diabetic wounds caused by MRSA infection. In short, a 7mm diameter circular wound was created in diabetic mice by removing full-thickness skin at the spine (2cm above the tail) using a special punch. The wound depth reached the subcutaneous layer, without damaging the fat and fascia in the paravertebral or abdominal muscles. 30 μL of MRSA bacterial solution was added to the wound. Four treatments were then applied: no treatment, 3M membrane treatment, hydrogel treatment, and oxygen-generating hydrogel treatment. The wound condition was photographed and recorded on days 0, 3, 7, and 14. (See [link to relevant documentation]). Figure 6 As shown.

[0115] Figure 6 This indicates the therapeutic effect of oxygen-producing hydrogel on diabetic wounds infected with MRSA; Figure 6 Control: No treatment performed; ACM: Composite nanoparticles provided in Example 3; PD: Hydrogel provided in Comparative Example 1; PDM: Oxygen-generating hydrogel provided in Example 4. Figure 6 (A) shows wound images on days 0, 3, 7, and 14 and the antibacterial effects of different treatment groups; Figure 6 (B) Indicates the wound healing rate.

[0116] from Figure 6It was observed that the oxygen-generating hydrogel group showed better wound healing than the other three groups. On day 3, due to the absence of antibacterial components in the control group and the composite nanoparticle group, a yellow bacterial film appeared on the wound surface, resulting in a significantly lower wound healing rate compared to the hydrogel and oxygen-generating hydrogel groups. After hydrogel treatment, on day 3, the control group had the lowest wound healing rate at 10.7%. Compared to the control group, treatment with hydrogel alone and composite nanoparticles significantly reduced wound area, with healing rates of 16.0% and 27.5%, respectively. Notably, the oxygen-generating hydrogel exhibited the best therapeutic effect, with a cure rate of 49.6%. On day 7, the wounds in all groups showed significant shrinkage. The hydrogel and composite nanoparticle groups maintained similar wound healing rates of 43.1% and 46.7%, respectively, significantly higher than the control group (33.8%). The oxygen-generating hydrogel group had the highest wound healing rate at 72.7%. On day 14, the skin defects in the oxygen-generating hydrogel group almost completely disappeared, while wound areas were still observed in the control group, the composite nanoparticle group, and the hydrogel group, with wound healing rates of 89.9%, 94.9%, and 96.2%, respectively. The in vivo antibacterial properties of the oxygen-generating hydrogel were further evaluated by culturing bacteria from the wound site on LB agar plates. The results showed that a large number of bacterial colonies were observed in the control group, while the oxygen-generating group had the fewest colonies, indicating that the oxygen-generating hydrogel has antibacterial activity in vivo, effectively inhibiting bacterial growth at the skin defects of MRSA-infected diabetic mice and promoting wound healing. In summary, the positive effects of hydrogel alone and composite nanoparticles alone on diabetic wound healing are limited. The combined effect of oxygen production from both showed the best therapeutic effect, suggesting a synergistic wound-healing effect between the oxygen release of the composite nanoparticles and the antibacterial properties of the hydrogel. This indicates that the oxygen-generating hydrogel prepared in Example 4 has a promoting effect on the repair of MRSA-infected diabetic wounds.

[0117] The oxygen-generating hydrogel provided by this invention, based on the synergistic effect between the in-situ oxygen-generating hydrogel and composite nanoparticles, exhibits antibacterial properties, improves oxidative stress, and releases oxygen, enabling rapid tissue angiogenesis, increasing wound healing rate, and achieving rapid wound healing. This in-situ oxygen-generating hydrogel can effectively improve oxidative stress and hypoxic microenvironment in diabetic wounds. It also possesses good biocompatibility, antibacterial properties, hemostatic function, and cell proliferation-promoting function. The oxygen-generating hydrogel formulation provided by this invention can be used for the treatment of wounds damaged by high oxidative stress.

[0118] This invention describes preferred embodiments and their effects. However, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to include both the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0119] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An oxygen-generating hydrogel in situ, characterized in that, The hydrogel comprises benzene boronic acid modified quaternary ammonium chitosan, dopamine modified oxidized hyaluronic acid and composite nanoparticles; The composite nanoparticles comprise manganese dioxide nanoparticles, a chitosan coating on the surface of the manganese dioxide nanoparticles and arginine adhered to the chitosan coating; The mass ratio of the benzene boronic acid modified quaternary ammonium chitosan, dopamine modified oxidized hyaluronic acid and composite nanoparticles is (2-4):(6-10):(0.25-1); The hydrogel is prepared by forming a Schiff base bond and a benzene boronic acid ester structure from the benzene boronic acid modified quaternary ammonium chitosan, dopamine modified oxidized hyaluronic acid and composite nanoparticles; The benzene boronic acid modified quaternary ammonium chitosan is prepared by the following steps: Obtaining quaternary ammonium chitosan; Dispersing the quaternary ammonium chitosan in an aqueous solvent to obtain a quaternary ammonium chitosan solution; Dispersing 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in the quaternary ammonium chitosan solution, then adding a methanol solution of 4-carboxyphenyl boronic acid, and reacting for 12-24 hours to obtain the benzene boronic acid modified quaternary ammonium chitosan after post-processing; The dopamine modified oxidized hyaluronic acid is prepared by the following steps: Dispersing 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride in an aqueous solution of oxidized hyaluronic acid, and reacting for 12-24 hours under the protection of nitrogen to obtain the dopamine modified oxidized hyaluronic acid after post-processing; The preparation method of the composite nanoparticles comprises: Dispersing bovine serum albumin in a dopamine aqueous solution, then adding a potassium permanganate solution, and obtaining a manganese dioxide solution after the reaction is completed; Dispersing a chitosan solution in the manganese dioxide solution to obtain a chitosan-manganese dioxide solution; Dispersing an arginine aqueous solution in the chitosan-manganese dioxide solution, and obtaining the composite nanoparticles after the reaction is completed, washing and drying.

2. The in situ oxygen-generating hydrogel of claim 1, wherein, The particle size of the composite nanoparticles is 105-165 nm.

3. A method for preparing the in-situ oxygen-generating hydrogel according to claim 1 or 2, characterized in that, The method comprises the following steps: Dispersing the dopamine modified oxidized hyaluronic acid in deionized water to obtain a dopamine modified oxidized hyaluronic acid solution; and dispersing the composite nanoparticles in the dopamine modified oxidized hyaluronic acid solution to obtain a composite precursor solution; Dispersing the benzene boronic acid modified quaternary ammonium chitosan in deionized water to obtain a benzene boronic acid modified quaternary ammonium chitosan precursor solution; Mixing the benzene boronic acid modified quaternary ammonium chitosan precursor solution and the composite precursor solution uniformly at a certain ratio to obtain the in-situ oxygen-producing hydrogel.

4. The method for preparing the in-situ oxygen-generating hydrogel according to claim 3, characterized in that, The volume ratio of the benzene boronic acid modified quaternary ammonium chitosan precursor solution to the composite precursor solution is 1:1.5-3.

5. The in-situ oxygen-producing hydrogel of claim 1 or 2 as a high oxidative stress injury wound dressing.

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