A hydrogel with antibacterial, antioxidant and proangiogenic functions and a preparation method and application thereof

Abstract

This invention provides a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions, its preparation method, and its applications, belonging to the field of biomedical technology. This invention uses 4-carboxyphenylboronic acid to modify quaternary ammonium salt chitosan to prepare modified quaternary ammonium salt chitosan HACC-PBA; then, POSS-modified polyethylene glycol grafted with dihydrocaffeic acid is prepared by esterification reaction of POSS-PEG-OH and dihydrocaffeic acid; next, H2S donor molecules are encapsulated by the amphiphilic molecule PEG-PCL, and H2S-loaded nanoparticles are obtained by solvent displacement; finally, through the phenylboronic ester reaction between tannic acid, modified quaternary ammonium salt chitosan, and POSS-modified polyethylene glycol grafted with dihydrocaffeic acid, and simultaneously doping different concentrations of nanoparticles into the gel prepolymer solution, the hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions is obtained. The hydrogel of this invention exhibits excellent antibacterial, antioxidant, and angiogenesis-promoting properties, and can effectively promote the rapid healing of diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions, its preparation method and application. Background Technology

[0002] Hydrogels are three-dimensional hydrophilic network materials formed by physical or chemical cross-linking of natural or synthetic polymers. Their water content can reach up to 99%, giving them excellent softness, swelling properties, and biocompatibility. Modern research on this material began in the 1960s, initially applied to contact lenses. It has evolved from traditional static gels into smart gels capable of responding to external stimuli such as pH and temperature, and has further developed advanced functions such as high strength and self-healing. With its unique solid-liquid dual-phase properties, hydrogels have become one of the core materials in the biomedical field, widely used as wound dressings, drug delivery carriers, tissue engineering scaffolds, and repair materials such as artificial cartilage. They also play an important role in flexible electronic sensing, environmental engineering, and cosmetics.

[0003] Quaternary ammonium chitosan (HACC), a high-performance cationic biopolymer, has overcome the limitation of natural chitosan's solubility only in acids through chemical modification, achieving excellent water solubility in a full range of pH environments (especially neutral physiological environments). In the field of biomaterials, its high-density, strongly positively charged structure allows it to actively capture and disrupt bacterial cell membranes through electrostatic interactions, exhibiting extremely strong broad-spectrum antibacterial and antiviral activity. Currently, this material is widely used in the production of high-performance antibacterial dressings, artificial skin, and tissue-engineered scaffolds. The functionalized biopolymer caffeic acid chitosan, formed by covalently grafting natural polyphenol caffeic acid onto the chitosan backbone, cleverly solves the problem of caffeic acid's easy oxidation and degradation, while simultaneously endowing chitosan with powerful antioxidant activity.

[0004] Tannic acid (TA), a natural polyphenolic compound extracted from plants, relies on its extremely high-density phenolic hydroxyl structure in its molecular chain to interact extensively and strongly with proteins, polysaccharides, and metal ions through hydrogen bonds, coordination bonds, and hydrophobic interactions. In the biomedical field, it not only possesses natural antioxidant, broad-spectrum antibacterial, and anti-inflammatory activities, but also serves as a universal surface modification platform or cross-linking agent, greatly expanding the composite dimensions of natural products and synthetic materials. Patent CN118240245A discloses a linked network hydrogel made by soaking a chitosan-polyvinyl alcohol dual-network hydrogel in an aqueous tannic acid solution. This hydrogel possesses antibacterial, antioxidant, and angiogenesis-promoting functions, but lacks an intelligent drug release mechanism that actively responds to changes in the microenvironment, and drug release is mostly passive diffusion, making it difficult to achieve on-demand drug delivery during peak infection periods.

[0005] POSS-PEG is a typical inorganic-organic hybrid biomaterial composed of polyhedral oligomeric silsesquioxane (POSS) with a highly symmetrical cage-like structure and hydrophilic polyethylene glycol (PEG) through chemical bonding. The POSS cage-like structure provides robust physical support, and the active groups at the ends of the PEG chains can crosslink linear polymers into a three-dimensional network. Compared to traditional small-molecule crosslinking agents, POSS-PEG, as a crosslinking center, can significantly improve the mechanical toughness and dimensional stability of hydrogels or biological scaffolds, effectively preventing excessive swelling or premature degradation of materials under physiological conditions. Studies have shown that introducing POSS into hydrogel networks can increase multiple crosslinking sites, effectively improving the mechanical properties of hydrogels and endowing materials with tunable degradation rates. Patent CN119306951A discloses a multifunctional hydrogel prepared based on PEG-modified POSS nanoparticles. The introduction of nanoparticles significantly improves the material's mechanical strength, thermal insulation, and fluorescence properties, but it lacks the ability to regulate bioactivity against wound-specific pathological factors (such as bacteria and ROS), and cannot directly address infection and oxidative stress issues.

[0006] Thiocarbamate-type H2S donors are a cutting-edge chemical biology tool in the field of gaseous signaling molecular biology. Their development hinges on designing molecular structures to mimic the physiological slow-release of endogenous hydrogen sulfide (H2S). H2S can activate VEGFR2 through hydrosulfide modification, triggering downstream signaling pathways and inducing endothelial cell proliferation, migration, and tubular formation. Secondly, it can activate sensitive H2S channels, leading to cell membrane hyperpolarization, thereby regulating vascular tone and promoting microvascular emergence. In the field of biomaterials, these molecules, due to their high specificity and spatiotemporal controllability, are widely integrated into hydrogel scaffolds, nanomicelles, and implant coatings to address the problem of insufficient angiogenesis in chronic wounds.

[0007] Although various hydrogels for wound repair have been developed in existing technologies, they generally suffer from problems such as limited functionality, demanding gelation conditions, and difficulty in balancing biocompatibility and mechanical properties. Furthermore, they cannot simultaneously achieve antibacterial, antioxidant, and angiogenesis-promoting effects, making them unsuitable for the repair needs of complex wounds and limiting their widespread application in the biomedical field.

[0008] Therefore, developing a hydrogel that can simultaneously achieve highly efficient antibacterial and antioxidant angiogenesis-promoting effects, as well as excellent mechanical properties and high biosafety, is of great practical significance. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the main objective of this invention is to provide a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions, as well as its preparation method. The hydrogel of this invention continuously releases H2S through a pH-responsive mechanism, combining the potent antioxidant properties of tannic acid with the broad-spectrum antibacterial properties of quaternary ammonium chitosan to achieve synergistic treatment of antibacterial, antioxidant, and angiogenesis-promoting effects. This effectively eliminates reactive oxygen species, inhibits infection, accelerates angiogenesis, and promotes rapid wound healing.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] A method for preparing a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions includes the following steps: (1) Synthesis of phenylboronic acid modified quaternary ammonium salt chitosan: 4-Carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were added to deionized water and stirred to obtain activated 4-carboxyphenylboronic acid. Subsequently, the activated 4-carboxyphenylboronic acid was added dropwise to an aqueous solution of quaternary ammonium chitosan and stirred to obtain the final product, namely phenylboronic acid modified quaternary ammonium chitosan (HACC-PBA). (2) Synthesis of POSS-PEG-DA: POSS-PEG-OH and dihydrocaffeic acid were reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine under catalysis to obtain POSS-modified polyethylene glycol-grafted dihydrocaffeic acid (POSS-PEG-DA). (3) Synthesis of PPNP nanoparticles: PPNPs nanoparticles were prepared by encapsulating H2S donors in polyethylene glycol-polycaprolactone micelles using a solvent displacement method. (4) Synthesis of hydrogels: PPNP nanoparticles were dispersed in a POSS-PEG-DA solution, and HACC-PBA and tannic acid (TA) were added sequentially to initiate a dynamic cross-linking reaction, resulting in a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions.

[0012] Furthermore, in step (1), the molar ratio of 4-carboxyphenylboronic acid (PBA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxysuccinimide (NHS) and quaternary ammonium chitosan (HACC) is 0.5-1.5:0.5-1.5:0.5-1.5:0.2-0.3.

[0013] Furthermore, in step (2), the molar ratio of POSS-PEG-OH, dihydrocaffeic acid (DA), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) is 0.5-1.5:5-10:13-18:13-18.

[0014] Furthermore, in step (3), the mass ratio of polyethylene glycol-polycaprolactone (PEG-PCL) to H2S donor (PTCM) is 2-10:1.

[0015] Furthermore, in step (3), the effective concentration of PPNPs nanoparticles is 0.1-50 mmol / L.

[0016] Furthermore, in step (4), the molar ratio of the PBA group in HACC-PBA to the DA in POSS-PEG-DA is 1-5:1.

[0017] Furthermore, in step (4), the temperature of the dynamic cross-linking reaction is 25-40℃, the reaction solution is PBS or deionized water, and the reaction time is 0-30 min.

[0018] Furthermore, in step (4), the solid content of the hydrogel is 5-15%.

[0019] This invention utilizes the dynamic reversible borate ester bonds formed by phenylboronic acid and polyphenol groups to construct a smart network with self-healing capabilities. It works synergistically with the bactericidal effect of quaternary ammonium chitosan, the free radical scavenging effect of tannic acid, and the controlled release of H2S from nanocarriers to activate angiogenesis signaling pathways, thereby achieving pH / ROS responsive regulation and multiple treatments of the wound microenvironment.

[0020] A second aspect of the present invention provides a hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions obtained by the above preparation method.

[0021] A third aspect of the present invention provides the application of the hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions in wound dressing materials, bone repair materials, antibacterial materials or nerve conduit materials.

[0022] This invention, based on the dynamic reversible borate ester bond of phenylboronic acid-catechol, constructs a hydrogel system integrating H2S nanoparticles, quaternary ammonium chitosan, tannic acid, and POSS-PEG-DA. This system possesses excellent antibacterial, antioxidant, and angiogenesis-promoting advantages: The broad-spectrum antibacterial properties of quaternary ammonium chitosan rapidly kill pathogens, blocking infection sources and creating a sterile environment for healing; the highly efficient antioxidant capacity of tannic acid directly scavenges ROS to reduce tissue damage and relieves the inhibition of angiogenesis pathways by oxidative stress, restoring endothelial cell activity; combined with the pH-responsive mechanism of a slightly acidic environment, it releases H2S gaseous signaling molecules on demand, directly dilating blood vessels and improving microcirculation. Through the synergistic effect of these components, not only is precise targeted drug release at the lesion site achieved, but the vicious cycle of infection, oxidation, and ischemia in diabetic wounds is also improved, significantly enhancing the efficiency, quality, and durability of wound healing.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. The hydrogel provided by the present invention has the advantages of uniform pore structure, good mechanical properties and obvious response release effect, which not only facilitates nutrient transport and cell migration, but also can adapt to the deformation requirements of different wounds.

[0024] 2. Compared with traditional single-function wound healing materials, the hydrogel prepared in this invention integrates H2S donor-loaded nanoparticles, antibacterial quaternary ammonium salt chitosan, antioxidant tannic acid, and POSS-PEG-DA through dynamic and reversible borate ester bonds between phenylboronic acid and catechol. Chitosan's broad-spectrum antibacterial properties inhibit bacterial growth at the wound site, reducing the risk of infection and providing a good sterile environment for wound healing. Tannic acid efficiently scavenge reactive oxygen species, reducing oxidative stress damage and simultaneously relieving the inhibition of key signaling pathways for endothelial cell survival and angiogenesis by high oxidative stress, clearing obstacles for angiogenesis. The slightly acidic infected wound environment serves as a trigger signal, enabling pH-responsive on-demand release of H2S. H2S not only exerts a vasodilatory effect to improve local microcirculation but also forms a synergistic effect with TA, jointly promoting mature collagen deposition and fibroblast proliferation, accelerating the establishment of a functional vascular network. Through the synergistic and intelligent response mechanism of the above components, the stability and durability of the therapeutic effect are significantly improved.

[0025] 3. The components used in this invention (such as HACC-PBA, TA, etc.) all have good biocompatibility and no obvious cytotoxicity; the entire preparation process is simple, easy to operate, mild, and highly controllable, making it easy to promote and apply. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings: Figure 1This is a schematic diagram illustrating the synthesis of the quaternary ammonium salt phenylboronic acid chitosan (HACC-PBA) of the present invention; Figure 2 This is a schematic diagram illustrating the synthesis of the crosslinking agent (POSS-PEG-DA) of the present invention; Figure 3 This is a scanning electron microscope (SEM) image of the hydrogel from Example 1 of the present invention. Figure 4 The photoelectron spectroscopy spectrum of the hydrogel in Example 1 of this invention is shown. Figure 5 This is a diagram showing the H2S donor release of the hydrogel in Example 1 of the present invention at different pH and different H2O2 concentrations; Figure 6 A diagram illustrating the biocompatibility of the hydrogels used in the embodiments and comparative examples of this invention; Figure 7 The antioxidant detection graphs are for the hydrogels used in the embodiments and comparative examples of this invention. Figure 8 Antibacterial diagrams of hydrogels used in embodiments and comparative examples of the present invention; Among them, (a) bacterial plate test; (b) bacterial plate test statistics; (c) inhibition zone test; Figure 9 This is a rheological characterization diagram of the hydrogel provided in this invention; Figure 10 The diagram shows the angiogenesis-promoting effects of hydrogels in the embodiments and comparative examples of this invention under oxidative stress. Among them, (a) scratch test, (b) Transwell migration test, (c) tube formation test, (d) scratch healing rate statistics, (e) Transwell migration cell statistics, (f) vascular network common tube length statistics, and (j) vascular network number statistics. Figure 11 The images show the wounds of diabetic mice treated with hydrogel in the embodiments and comparative examples of this invention. Among them, (a) wound images at predetermined time points after different hydrogel treatments; (b) statistics of wound healing area at predetermined time points after different hydrogel treatments; (c) statistics of wound healing rate at predetermined time points after different hydrogel treatments; and (d) schematic diagram of changes in wound healing area after different hydrogel treatments. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Unless otherwise specified, all raw materials used in the following embodiments and comparative examples of this application are commercially available.

[0031] This invention provides a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions. It utilizes a framework of quaternary ammonium chitosan grafted with phenylboronic acid (HACC-PBA) and POSS-modified polyethylene glycol grafted with dihydrocaffeic acid (POSS-PEG-DA). A three-dimensional network is constructed through a dynamic esterification reaction between phenylboronic acid and catechol groups mediated by tannic acid (TA). Simultaneously, an H2S donor (PTCM) is encapsulated within a PEG-PCL amphiphilic molecule using a solvent displacement method to form nanoparticles (PPNPs), which are then uniformly doped into the hydrogel. The hydrogel prepared by this invention can gel in situ under physiological conditions and continuously release H2S gas, while also exhibiting excellent mechanical strength and self-healing ability. This material can effectively scavenge reactive oxygen species, inhibit bacterial growth, and promote angiogenesis.

[0032] This invention achieves synergistic effects of multiple functions by rationally proportioning and combining materials with different functions, such as POSS providing good mechanical properties, tannic acid providing antioxidant properties, quaternary ammonium salt chitosan enhancing antibacterial properties, PPNPs releasing H2S to promote angiogenesis, and phenylboronic acid participating in cross-linking reactions to construct network structures. This enables the hydrogel to play a role from multiple angles in the wound repair process, promoting rapid wound healing and tissue regeneration.

[0033] In the preferred embodiment, the selected materials, such as polyethylene glycol (PEG), TA, and quaternary ammonium chitosan, all have good biocompatibility, so that the prepared hydrogel will not cause obvious immune or toxic reactions when applied in vivo, thus ensuring the safety and reliability of use.

[0034] The sources of some of the raw materials used in the following examples and comparative examples are as follows: Allyl-PEG-OH, with a molecular weight of 2.5 kDa, was purchased from Beijing Jiankai Technology Co., Ltd. The quaternary ammonium chitosan was synthesized in the laboratory (reference: Injectable self-healingchitosan-based POSS-PEG hybrid hydrogel as wound dressing to promote diabeticwound healing [J]. Carbohydrate Polymers, 2023, 299: 120-198), and the average degree of substitution of the quaternary ammonium chitosan was 35%. The eight-arm PEG crosslinking agent precursor (POSS-PEG-OH) was a laboratory-synthesized product (reference: Injectable self-healing chitosan-based POSS-PEG hybrid hydrogel as wounddressing to promote diabetic wound healing [J]. Carbohydrate Polymers, 2023, 299: 120-198), and the average molecular weight of POSS-PEG-OH was 17 kDa; Tannic acid (TA) and PEG-PCL were both purchased from Titan Technology Co., Ltd. The H2S donor molecule (PTCM) was synthesized in the laboratory (Reference: Hydrogen Sulfide Donors Activated by Reactive Oxygen Species [J]. Angew Chem Int Ed. 2016 Oct24; 55(47):14638-14642).

[0035] Example 1: This embodiment provides a method for preparing a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions, comprising the following steps: (1) Synthesis of phenylboronic acid modified quaternary ammonium salt chitosan: 4-Carboxyphenylboronic acid (PBA), EDCI, and NHS were added to deionized water and stirred for 20 min to activate the carboxyl groups of 4-carboxyphenylboronic acid. Next, quaternary ammonium salt chitosan (HACC) was dispersed in water until completely dissolved. Then, the activated 4-carboxyphenylboronic acid was added dropwise to the chitosan solution, and the reaction was stirred at room temperature for 48 h. The reaction solution was dialyzed through a 14000 Da dialysis membrane for 7 days. The dialyzed solution was freeze-dried to obtain the final product, HACC-PBA, in which the molar ratio of PBA, EDCI, NHS, and HACC was 1:1:1:0.25.

[0036] (2) Synthesis of POSS-PEG-DA: POSS-PEG-OH, DA, EDCI, and DMAP were dissolved in dry dichloromethane and reacted at room temperature for 3 days. After the reaction was completed, the solvent was removed, and the crude product was dissolved in water and dialyzed using a 3500 Da cutoff dialysis membrane for 7 days. After dialysis, the dialysate was extracted with dichloromethane, the organic phase was collected, recrystallized with methyl tert-butyl ether, filtered, and dried to obtain POSS-PEG-DA. The molar ratio of POSS-PEG-OH, DA, EDCI, and DMAP was 1:8:16:16. (3) Synthesis of PPNP nanoparticles: PEG-PCL and H2S donor molecule PTCM were dissolved in N,N-dimethylformamide and gradually added dropwise to vigorously stirred ultrapure water. After the addition was complete, the mixture was dialyzed for 1 day using a 3500 Da retention dialysis membrane to remove unencapsulated drug. After dialysis, the PPNPs nanoparticles were obtained by freeze drying. The mass ratio of PEG-PCL to H2S donor molecule PTCM was 5:1, and the effective drug concentration of the PPNPs nanoparticles was 50 mmol / L. (4) Preparation of hydrogels: 44 mg of PPNPs nanoparticles were dissolved in an aqueous solution containing 12.6 mg of POSS-PEG-DA, followed by the sequential addition of 14.8 mg of HACC-PBA and 12.6 mg of TA. The mixture was then placed in a biochemical incubator at 37 °C for in-situ curing to form a gel for 20 min, resulting in a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions; the solid content of the hydrogel was 10%.

[0037] Comparative Example 1: The difference from Example 1 is that this comparative example does not contain PPNP nanoparticles.

[0038] In step (4), 14.8 mg HACC-PBA and 12.6 mg TA were added sequentially to an aqueous solution containing 12.6 mg POSS-PEG-DA, and the solution was in situ cured into a gel in a biochemical incubator at 37°C for 20 min to obtain a hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions, with a solid content of 10%.

[0039] Comparative Example 2: The difference from Example 1 is that this comparative example does not contain PPNP nanoparticles and POSS-PEG-DA.

[0040] In step (4), 14.8 mg HACC-PBA and 25.2 mg TA were added to deionized water in sequence, and the mixture was cured in situ in a biochemical incubator at 37°C for 20 min to obtain a hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions, with a solid content of 10%.

[0041] Comparative Example 3: The difference from Example 1 is that this comparative example does not contain PPNP nanoparticles and TA.

[0042] In step (4), 14.8 mg of HACC-PBA was added to an aqueous solution containing 25.2 mg of POSS-PEG-DA, and the solution was in situ cured into a gel in a biochemical incubator at 37°C for 20 min to obtain a hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions, with a solid content of 10%.

[0043] Performance characterization: 1. Morphology analysis of hydrogels The morphology of the hydrogels prepared in the examples and comparative examples was observed using scanning electron microscopy (SEM). After freeze-drying and gold sputtering, the surface morphology of the prepared hydrogel samples was observed using SEM. The results are as follows: Figure 3 As shown.

[0044] Depend on Figure 3 It can be seen that the hydrogels prepared by this invention all have a porous sponge-like structure with uniform pore size, with an average pore size between 100 and 200 nm. They have a large porosity, which facilitates cell adhesion, migration, and nutrient exchange, creating an ideal microenvironment for cell and tissue regeneration.

[0045] 2. Photoelectron spectroscopy of hydrogels The valence state distribution of elements in the hydrogel prepared in Example 1 was characterized using a Thermo Escalab 250Xi instrument with X-ray photoelectron spectroscopy. Monochromatic aluminum X-rays were used as the excitation source (X-ray energy 1486.6 eV), the test spot area was 500 μm, and the instrument parameters were: X-ray tube voltage 15 kV, current 10 mA, and vacuum degree of the analysis chamber 2 × 10⁻⁶. -9 mbar.

[0046] After fully swelling the hydrogel from Example 1, the hydrogel was dried in an oven to obtain a thin sheet. The sheet was then cut into square pieces of 5mm × 5mm × 1mm (length × width × thickness) for testing. The results are as follows: Figure 4 As shown.

[0047] Depend on Figure 4 It can be seen that the main elements such as C, B, Si, O, and N in the hydrogel are all present in the broad spectrum. Among them, the BO bond absorption peak at 190 eV was observed in the fine spectrum of B, indicating the successful grafting of HACC-PBA; the Si-O bond absorption peak at 102 eV was observed in the fine spectrum of Si, indicating the presence of POSS in the hydrogel; the above chemical bonds indicate that the hydrogel network is successfully cross-linked through phenylboronic acid ester bonds.

[0048] 3. H2S release test of hydrogel Using PBS as the incubation solution, the hydrogel samples prepared in Example 1 were placed under different H2O2 concentrations (20, 100, 200, 500 μm) and different pH values ​​(4, 5, 6). The supernatant was collected, and the absorbance of the solution in the 300-700 nm wavelength range was measured using a UV-Vis spectrophotometer. The results are as follows: Figure 5 As shown.

[0049] Depend on Figure 5 It can be seen that the H2S release amount of the hydrogel sample varies under different pH and H2O2 concentrations. The release amount of the hydrogel in PBS buffer is approximately 42%, while at pH=4, the release amount is approximately 61%, and the release amount increases with increasing acidity, exhibiting excellent pH responsiveness. Furthermore, at an H2O2 concentration of 500 μM, the release amount is 50%, and the release amount also increases with increasing H2O2 concentration, demonstrating significant reactive oxygen species (ROS) responsiveness. These results indicate that the hydrogel of the present invention is cross-linked through phenylboronic acid ester bonds between HACC-PBA, POSS-PEG-DA, and TA. These chemical bonds break in response to changes in the pH and ROS levels of the microenvironment, thereby achieving pH and ROS responsiveness and releasing the drug.

[0050] 4. Biocompatibility testing of hydrogels The in vitro biocompatibility of hydrogels with HaCAT, L929, and HUVEC cells was determined according to the national standard (GB / T16886.5-2003 / ISO 10993-53199) using the extraction method. Hydrogel samples prepared in Comparative Examples 1-3 and Example 1 were sterilized and incubated in DMEM complete medium for 24 hours to prepare the extraction solution. HaCAT, L929, and HUVEC cells were digested and seeded into 96-well plates for pre-culture for 24 hours. Subsequently, the experimental groups were incubated with the above-mentioned serum-containing hydrogel extraction solution at 37°C and 5% CO2 for 24 hours (the control group used fresh complete medium). Following the manufacturer's instructions, CCK-8 solution was added to each well, and the absorbance (OD value) of the sample at 450 nm was measured using a microplate reader, and cell viability was calculated. HUVEC cells were stained with Calcein / PI detection working solution. After staining, cell morphology and distribution were observed and photographed using a fluorescence microscope. For blood compatibility, erythrocytes were collected from fresh blood of male C57BL / 6 mice. 800 μL of a 4% (v / v) erythrocyte solution was incubated with the test hydrogel sample (100 μL), PBS (negative control, 100 μL), and ddH2O (positive control, 100 μL) at 37°C for 2 h, centrifuged at 2000 rpm for 10 min, and the absorbance (OD value) of the samples at 540 nm was measured using a microplate reader. The hemolysis rate was calculated. Results are as follows: Figure 6 As shown.

[0051] Depend on Figure 6 As can be seen, after co-culturing for 24 hours, the survival rates of HaCAT cells, L929 cells, and HUVEC cells in the control group, comparative group, and example group were all above 90%, indicating that the hydrogel has no obvious cytotoxicity and good biocompatibility.

[0052] Depend on Figure 6 As can be seen from fluorescence microscopy imaging, the HUVEC endothelial cells in the control group, comparative group, and examples all exhibited high-density green fluorescence (live cells), with almost no red fluorescence (dead cells). Furthermore, the cell morphology remained intact in all groups, displaying a typical cobblestone-like arrangement, without shrinkage or detachment. These results indicate that the hydrogel material prepared in this invention has no significant negative impact on cell growth.

[0053] Depend on Figure 6 As can be seen from c, the supernatant of the red blood cell suspension in the comparative group and the example group was colorless and transparent, and the hemolysis rate was less than 5%, indicating that the hydrogel has good blood compatibility.

[0054] 5. Antioxidant performance test of hydrogel The hydrogel samples prepared in the examples and comparative examples were incubated in PBS buffer supplemented with H2O2. The supernatant was extracted and stained with DPPH, TMB, NBT, and indigo carmine (IC) staining agents to detect the antioxidant properties and scavenging effects on hydroxyl radicals, hydrogen peroxide, and superoxide anions. The results are as follows: Figure 7 As shown.

[0055] Depend on Figure 7 It can be seen that the hydrogels of both Example 1 and the comparative example have certain antioxidant effects, but the antioxidant capacity of the hydrogel of Example 1 is significantly better than that of the comparative example. This is mainly because the antioxidant TA in Example 1 has multiple phenolic hydroxyl groups, which gives it a strong hydrogen donation capacity and can effectively scavenge various free radicals. At the same time, the PPNPS nanoparticles added in Example 1 can respond to the reactive oxygen species (ROS) environment and release hydrogen sulfide. H2S, as an endogenous gaseous signaling molecule, has significant biological antioxidant function, which, together with TA, further enhances the hydrogel's ability to scavenge hydroxyl radicals, hydrogen peroxide, and superoxide anions.

[0056] Although Comparative Example 1 contains TA for antioxidant activity, its antioxidant performance is lower than that of Example 1 due to the lack of synergistic effect from PPNPS nanoparticles. Comparative Example 2 contains sufficient TA, but insufficient cross-linking between TA and HACC-PBA leads to the oxidation and consumption of some free TA during the test, thus reducing its antioxidant performance. Comparative Example 3 shows a significant decrease in antioxidant activity due to the lack of TA as an antioxidant component. These results demonstrate that the hydrogel of Example 1 achieves efficient scavenging of reactive oxygen species through the synergistic effect of multiple components, exhibiting excellent antioxidant activity.

[0057] 6. Antibacterial properties test of hydrogel Staphylococcus aureus and Escherichia coli were selected as model strains, and the bactericidal efficiency of the hydrogel samples prepared in the examples and comparative examples was quantitatively evaluated by plate count method. 200 μL of the hydrogel samples from the examples and comparative examples were injected into 48-well plates, and then 200 μL of bacterial suspension (10⁻⁶ g / L) was dropped onto the surface of the hydrogel. 6 CFU / mL). After 12 hours of contact culture (control group: 10). 6 CFU / mL bacterial suspension was serially diluted and spread onto agar plates. After incubation at 37°C for 24 hours, the number of colonies was counted and the bacterial survival rate was calculated. Using the inhibition zone assay, equal volumes of samples were placed on solid plates inoculated with bacteria, with an antibiotic control group included. The inhibitory activity of the material against surrounding pathogens was evaluated by observing and measuring the transparent inhibition zone formed around the samples. Results are as follows: Figure 8 As shown.

[0058] Depend on Figure 8As shown in a and 8b, Example 1 and Comparative Examples 1-2 all exhibited significant broad-spectrum antibacterial activity. Compared to the control group with dense colonies, the bacterial plates of the Example 1 and Comparative Examples 1-2 treatment groups showed almost no colony growth. Quantitative analysis showed that the killing rates of these three groups of materials against *S. aureus* and *E. coli* all exceeded 90%. Figure 8 As shown in c, the inhibition zone experiment results indicate that the hydrogels of Example 1 and Comparative Examples 1-2 formed clear and obvious inhibition zones on both bacterial plates. Their antibacterial effect against *S. aureus* was even close to that of the traditional antibiotic group, indicating that these three hydrogels possess excellent antibacterial component release capabilities and broad-spectrum inhibitory activity. However, Comparative Example 3, lacking TA, did not show a clear inhibition zone, indicating a lack of effective antibacterial activity. This result demonstrates that the antibacterial activity of the hydrogels mainly originates from the synergistic release of TA and HACC-PBA.

[0059] 7. Rheological property testing of hydrogels The hydrogel samples (20 mm in diameter, 2 mm thick) prepared in Example 1 and Comparative Examples 1-3 were placed on the parallel plate fixture of a rotational rheometer (25 mm in diameter plate, with a measurement gap of 1.5 mm). The test temperature was kept constant at 37 °C. Strain scanning experiments (0.01%~100%, frequency 1 Hz) were conducted, and the storage modulus (G') and loss modulus (G'') at each frequency point were recorded. Each sample was tested in triplicate, and the average value was used for data analysis. The results are as follows: Figure 9 As shown.

[0060] Depend on Figure 9 It can be seen that all hydrogel samples exhibit similar nonlinear rheological logic behavior across the entire frequency range: the storage modulus (G') is consistently higher than the corresponding loss modulus (G"), and both remain stable with increasing strain, without obvious yield points or structural damage, indicating that the prepared hydrogels are stable elastic solids with good mechanical stability. Furthermore, the addition of TA and POSS-PEG-DA increases the storage modulus of the hydrogels, indicating that the phenolic hydroxyl groups in TA form a dynamic phenylboronic acid ester crosslinking network with HACC-PBA, while the introduction of POSS-PEG-DA further constructs a multi-layer crosslinking network. The strength and rigidity of the hydrogel were enhanced. However, the storage moduli of Comparative Example 2 (without POSS-PEG-DA) and Comparative Example 3 (without TA) were significantly lower than those of Comparative Example 1 and Example 1, indicating that the mechanical properties of the cross-linked network formed by a single component (TA or POSS-PEG-DA) and HACC-PBA are limited. Only when all three components work together can a dense composite cross-linked structure be formed, enabling the hydrogel to exhibit excellent mechanical properties. These results demonstrate that the synergistic cross-linking of TA, POSS-PEG-DA, and HACC-PBA is crucial for obtaining hydrogels with high mechanical strength, and none of them can be omitted.

[0061] 8. Hydrogels promote angiogenesis under oxidative stress The effects of each hydrogel group on the migration function and angiogenesis ability of endothelial cells (HUVECs) were evaluated using cell scratch assay, Transwell migration assay and Matrigel tube formation assay.

[0062] Transwell migration assay: HUVEC cells were seeded in the upper chamber of a Transwell chamber (pore size: 8 μm), and the hydrogel sample to be tested and 600 µL of complete culture medium containing 300 µM H2O2 were added to the lower chamber. The control group was filled with fresh complete culture medium, and the PBS group was filled with complete culture medium containing 300 µM H2O2. After incubation for 24 h, the cells on the surface of the upper chamber were carefully wiped off, and the cells that migrated to the lower chamber were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and the number of migrated cells was counted.

[0063] Cell scratch assay: Scratches were created on HUVEC layers with a cell density of 90% using a 200µL pipette tip. Floating cells were then washed away with PBS buffer, and images of the scratches were taken immediately (0h). The hydrogel samples to be tested were placed in the upper chamber of a Transwell chamber, and 1mL of low-serum medium (containing 1% serum) with 300µM H2O2 was added to the lower chamber. The control group used fresh low-serum medium, and the PBS group used low-serum medium containing 300µM H2O2. After incubation for 24h, crystal violet staining was used for imaging, and the healing rate was calculated.

[0064] In vitro tube formation assay: HUVECs were cultured in 12-well plates. Once the cell confluence reached 80%, the hydrogel sample was placed in the upper chamber of a Transwell chamber, while the lower chamber was filled with complete culture medium containing 300 µM H2O2. The control group received fresh complete culture medium, and the PBS group received complete culture medium containing 300 µM H2O2. After 12 hours of incubation, HUVECs from different treatment groups were digested and seeded into 48-well culture plates pre-coated with Matrigel basement membrane matrix (60,000 cells per well). After another 6 hours of incubation, the number of vascular networks was observed and counted. Results are as follows: Figure 10 As shown.

[0065] Depend on Figure 10It was found that high-concentration hydrogen peroxide treatment significantly inhibited endothelial cell migration and tubular formation. However, all comparative examples 1-3 and the hydrogel of Example 1 reversed this inhibitory effect to some extent, exhibiting varying degrees of pro-angiogenic ability to promote vascular migration and tubular formation. Among them, the hydrogel of Example 1 showed significantly better pro-angiogenic ability than the other comparative examples. This is because it contains TA with antioxidant protective properties and H2S-responsive PPNP nanoparticles, which not only possess excellent antioxidant properties but also directly activate pro-angiogenic pathways within endothelial cells, thus jointly promoting vascular regeneration. Comparative examples 1-2 also showed significant pro-angiogenic ability, mainly due to the effective scavenging of reactive oxygen species (ROS) by the tannic acid (TA) it contains, reducing the damage of oxidative stress to HUVEC function and protecting the original vascular regeneration function of cells. The above results indicate that this hydrogel achieves functional complementarity through the antioxidant defense of TA and the active pro-angiogenic stimulation of H2S, exhibiting excellent pro-angiogenic ability under oxidative stress.

[0066] 9. Experiment on wound healing of diabetic mice using hydrogels: Eight-week-old male C57BL / 6 mice were selected, and a diabetic model was established by intraperitoneal injection of streptozotocin (STZ, 50 mg / kg, for 5 consecutive days) (fasting blood glucose ≥16.7 mmol / L was considered successful modeling). After hair removal and disinfection of the backs of all mice, a full-thickness skin defect was prepared using a 10 mm diameter skin biopsy puncture device, and 50 μm of Staphylococcus aureus (10 μL) was applied simultaneously. 8 A diabetic mouse model of wound infection was established 2 days after infection with CFU / mL. The mice were randomly divided into 5 groups: a blank control group (no treatment), comparative groups 1-3, and the example group (n=7 per group). Each group had its corresponding hydrogel (100μL) evenly applied to the wound and fixed with sterile dressings. The wounds were photographed on days 0, 4, 7, and 10 after treatment. The wound area was calculated using ImageJ software, and the wound healing rate was statistically analyzed. Results are as follows: Figure 11 As shown.

[0067] Depend on Figure 11 It was found that the wound treated with the hydrogel in the example showed a significantly higher healing rate on day 7 than the comparative groups, with no obvious signs of infection; by day 14, the wound was almost completely epithelialized, with a healing rate of over 95%; while the healing rates of comparative groups 1-3 were 78%, 65%, and 52%, respectively, and the blank control group was only 45%. These results indicate that the hydrogel of the present invention, through synergistic antibacterial, antioxidant, and angiogenesis-promoting effects, overcomes the inhibitory effects of high glucose and infection environments in diabetic mice on wound healing, significantly accelerating the healing process of chronic wounds in diabetic mice.

[0068] The above embodiments are merely preferred technical solutions of the present invention and should not be considered as limitations on the present invention. The scope of protection of the present invention should be limited to the technical solutions described in the claims, including equivalent substitutions of the technical features described in the claims. That is, equivalent substitutions and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel with antibacterial, antioxidant, and angiogenesis-promoting functions, characterized in that, Includes the following steps: (1) Synthesis of phenylboronic acid modified quaternary ammonium salt chitosan: 4-Carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added to deionized water and stirred to obtain activated 4-carboxyphenylboronic acid; then the activated 4-carboxyphenylboronic acid was added dropwise to an aqueous solution of quaternary ammonium chitosan HACC and stirred to obtain the final product, namely phenylboronic acid modified quaternary ammonium chitosan. (2) Synthesis of POSS-modified polyethylene glycol grafted with dihydrocaffeic acid: POSS-PEG-OH and dihydrocaffeic acid were reacted with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine under catalysis to obtain POSS-modified polyethylene glycol-grafted dihydrocaffeic acid. (3) Synthesis of PPNP nanoparticles: PPNPs nanoparticles were prepared by encapsulating H2S donors in polyethylene glycol-polycaprolactone micelles using a solvent displacement method. (4) Synthesis of hydrogels: PPNP nanoparticles were dispersed in a POSS-modified polyethylene glycol-grafted dihydrocaffeic acid solution, and then phenylboronic acid-modified quaternary ammonium salt chitosan and tannic acid were added sequentially to initiate a dynamic cross-linking reaction, resulting in a hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of 4-carboxyphenylboronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and quaternary ammonium chitosan is 0.5-1.5:0.5-1.5:0.5-1.5:0.2-0.

3.

3. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of POSS-PEG-OH, dihydrocaffeic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine is 0.5-1.5:5-10:13-18:13-18.

4. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of polyethylene glycol-polycaprolactone to H2S donor is 2-10:

1.

5. The preparation method according to claim 1, characterized in that, In step (3), the effective concentration of PPNPs nanoparticles is 0.1-50 mmol / L.

6. The preparation method according to claim 1, characterized in that, The molar ratio of phenylboronic acid groups in phenylboronic acid-modified quaternary ammonium salt chitosan to dihydrocaffeic acid groups in POSS-modified polyethylene glycol grafted dihydrocaffeic acid is 1-5:

1.

7. The preparation method according to claim 1, characterized in that, In step (4), the temperature of the dynamic cross-linking reaction is 25-40℃, the reaction solution is PBS or deionized water, and the reaction time is 0-30 min.

8. The preparation method according to claim 1, characterized in that, In step (4), the solid content of the hydrogel is 5-15%.

9. The hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions obtained by the preparation method according to any one of claims 1 to 8.

10. The application of the hydrogel with antibacterial, antioxidant and angiogenesis-promoting functions obtained by the preparation method according to any one of claims 1 to 8 in wound dressing materials, bone repair materials, antibacterial materials or nerve conduit materials.

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