Photothermal response and nitric oxide release synergistic hydrogel and preparation method thereof

Abstract

This invention discloses a photothermal responsive and nitric oxide release synergistic hydrogel and its preparation method; belonging to the field of biomedical materials technology, the hydrogel uses N-isopropylacrylamide and acrylic acid copolymer as a backbone, and is loaded with polydopamine@polypyrrole, cerium dioxide, glucose oxidase, and L-arginine. Polydopamine@polypyrrole generates a local photothermal effect under near-infrared light irradiation, achieving physical antibacterial effects; cerium dioxide catalyzes the slow release of nitric oxide from L-arginine, achieving chemical antibacterial effects; glucose oxidase consumes glucose at the wound site, lowering local blood glucose and providing hydrogen peroxide substrate for nitric oxide generation. The synergistic effect of these three components endows the hydrogel with multiple functions, including photothermal antibacterial properties, controlled nitric oxide release, and microenvironment responsiveness. The hydrogel of this invention also exhibits good mechanical properties, tissue adhesion, and self-healing ability. Animal experiments show that this hydrogel can significantly promote the healing of chronic diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a photothermal responsive and nitric oxide release synergistic hydrogel and its preparation method. Background Technology

[0002] The healing process of chronic diabetic wounds is often slow and prone to complications such as bacterial infection, oxidative stress damage, impaired angiogenesis, and persistent damage from hyperglycemia. A hyperglycemic environment damages vascular endothelium and nerve tissue, reducing local blood circulation and nutrient delivery to the wound. Simultaneously, elevated oxidative stress levels induced by hyperglycemia weaken the body's immunity, significantly increasing the risk of infection and tissue damage. Nitric oxide (NO), as a key signaling molecule regulating wound healing, plays an important role in promoting angiogenesis, anti-inflammation, and antibacterial activity. However, in the pathological state of diabetes, the pathways for endogenous NO generation and release are significantly hindered. Therefore, developing a wound dressing capable of achieving exogenous, controllable NO release has become a research hotspot for promoting diabetic wound healing.

[0003] Current research has attempted to achieve local NO delivery by introducing NO donors into carrier materials such as hydrogels. However, single NO release systems often face challenges such as uncontrollable release behavior, short release cycles, and a lack of active response to microenvironments like high glycemia and acidity in wounds. Furthermore, for the common bacterial infections in diabetic wounds, single NO antibacterial mechanisms often have limited effectiveness and struggle to address challenges such as drug-resistant bacterial infections and biofilm formation.

[0004] In recent years, photothermal therapy (PTT) has shown promise in antibacterial treatment due to its advantages such as non-invasiveness, strong spatiotemporal controllability, and low likelihood of inducing drug resistance. Photothermal materials can convert light energy into heat energy under near-infrared light irradiation, killing bacteria through localized thermal effects. However, simple photothermal antibacterial treatment requires relatively high temperatures to completely kill bacteria, which may cause thermal damage to surrounding healthy tissues.

[0005] Therefore, how to construct a dressing material that can respond to the microenvironment of diabetic wounds, synergistically integrate photothermal antibacterial and NO-controlled release functions, thereby efficiently and safely promoting the healing of chronic diabetic wounds, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of existing diabetic chronic wound dressings in terms of synergistic antibacterial properties, controlled release of nitric oxide, and response to the wound microenvironment, and to provide a photothermal responsive and nitric oxide release synergistic hydrogel and its preparation method.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: A method for preparing a photothermal responsive and nitric oxide-releasing synergistic hydrogel includes the following steps: Step 1: Add N-isopropylacrylamide, hyaluronic acid, photothermal nanoparticles, nitric oxide donor, nanozyme, acrylic acid and glucose oxidase to deionized water, and dissolve by magnetic stirring to form a mixed solution. Step 2: Add crosslinking agent, catalyst and accelerator to the mixed solution, stir and mix well to obtain prepolymer solution; Step 3: Pour the prepolymer solution into the mold, defoam it by ultrasonication, and then carry out the polymerization reaction under constant temperature conditions. Demold and wash the polymerized gel to obtain the hydrogel.

[0008] Furthermore, the nanozyme is cerium dioxide, and the nitric oxide donor is L-arginine.

[0009] Furthermore, the photothermal nanoparticles are polypyrrole coated with polydopamine.

[0010] Furthermore, the amounts of each component used in step 1 are as follows: N-isopropylacrylamide 0.1g, acrylic acid 1mL, hyaluronic acid 0.02g, photothermal nanoparticles 0.01g, glucose oxidase 0.04g, nanozyme 0.04g, and nitric oxide donor 0.02g.

[0011] Furthermore, in step 2, the crosslinking agent is 10 mg of N,N-methylenebisacrylamide, the catalyst is 10 mg of ammonium persulfate, and the accelerator is 13 μl of polyethylene glycol diacrylate.

[0012] A photothermal responsive and nitric oxide-releasing synergistic hydrogel prepared by the above method comprises: The hydrogel framework is formed by copolymerization of N-isopropylacrylamide and acrylic acid in the presence of a crosslinking agent; Photothermal nanoparticles, dispersed in a hydrogel matrix, are used to generate a photothermal effect under near-infrared light irradiation; Nitric oxide donors are dispersed in a hydrogel matrix; And nanozymes, used to catalyze the production of nitric oxide from nitric oxide donors.

[0013] Furthermore, the hydrogel exhibits an antibacterial rate of over 90% against both Staphylococcus aureus and Escherichia coli.

[0014] Furthermore, the hydrogel has a maximum tensile strength at break of 2700%, and maintains structural integrity after being cyclically stretched 5 times at a tensile strength of 400%.

[0015] Application of a photothermal responsive and nitric oxide release synergistic hydrogel in the preparation of drugs or dressings for treating chronic diabetic wounds.

[0016] The beneficial effects of this invention are as follows: The PDA@PPy hydrogel in this invention generates a photothermal effect under near-infrared light irradiation, locally heating to 40-45℃, directly disrupting the integrity of bacterial cell membranes. Simultaneously, L-arginine slowly releases NO under CeO2 catalysis, which can penetrate bacterial biofilms and damage bacterial DNA. The photothermal effect and NO release form a synergistic antibacterial mechanism of physical thermal damage + chemical destruction. Experiments show that the inhibition rate against Staphylococcus aureus and Escherichia coli both reach over 90%.

[0017] The hydrogel of this invention uses a NIPAM and AA copolymer as a backbone and exhibits differentiated swelling behavior under different pH conditions. It can respond to changes in the microenvironment of diabetic wounds and realize the on-demand release of functional materials.

[0018] The hydrogel of this invention has a maximum elongation of 2700%, exhibiting excellent fatigue resistance and self-healing ability; at the same time, through the interaction of dynamic hydrogen bonds and electrostatic interactions between components, it can heal itself after damage, extending the service life of the dressing.

[0019] In a diabetic mouse model of full-thickness skin defect infection, the wound closure rate reached 79.19% after 7 days of treatment with the hydrogel of this invention and 99.36% after 14 days, which was significantly higher than that of the control group. Attached Figure Description

[0020] Figure 1 The graphs show the water absorption and swelling curves of the hydrogels in the embodiments and comparative examples of this invention at pH=7.4.

[0021] Figure 2 This is a graph showing the water absorption and swelling curves of the hydrogel at different pH values ​​in an embodiment of the present invention.

[0022] Figure 3 These are scanning electron microscope images of embodiments and comparative examples of the present invention.

[0023] Figure 4 The image shows the mapping diagram of C, N, O, and Ce in the hydrogel of the example.

[0024] Figure 5 This is a scanning electron microscope image of CeO2.

[0025] Figure 6 for Figure 5 Average particle size distribution diagram.

[0026] Figure 7 This is a scanning electron microscope image of PDA@PPy nanoparticles.

[0027] Figure 8 for Figure 7 Average particle size distribution diagram.

[0028] Figure 9 The images show the tensile properties of the hydrogel under different treatments in this embodiment.

[0029] Figure 10 The tensile stress-strain curves of the hydrogels in the examples and comparative examples are shown.

[0030] Figure 11 The figures show the compressive stress-strain curves of the hydrogels in the examples and comparative examples.

[0031] Figure 12 This is a diagram illustrating the morphological changes of the hydrogel during compression and release in an example.

[0032] Figure 13 The stress-strain curve of the hydrogel under cyclic stretching is shown in the example.

[0033] Figure 14 The stress-strain curve of the hydrogel under cyclic compression is shown in the example.

[0034] Figure 15 The illustration shows the application status of the hydrogel in the heart, kidney, lung, spleen, and liver.

[0035] Figure 16 This is a schematic diagram of adhesion and stretching.

[0036] Figure 17 Load-displacement curves for different substrates (metal, rubber, plastic, wood, skin) and the hydrogel of the example.

[0037] Figure 18 A bar chart comparing the shear strength of different substrates (metal, rubber, plastic, glass, wood, skin) with the hydrogel of the example.

[0038] Figure 19 The image shows a macroscopic view of the self-healing properties of the hydrogel in this example.

[0039] Figure 20 Photothermal images of the hydrogels of Comparative Examples 1, 2 and Examples within 0-5 minutes; Figure 21 The photothermal cycle curve is shown in the example diagram; Figure 22 Photothermal curves of the hydrogels in Comparative Examples 1, 2, and Examples; Figure 23 NO release kinetics curve; Figure 24 This is a diagram illustrating the reaction mechanism of NO formation catalyzed by CeO2 NPs.

[0040] Figure 25 The images show the antibacterial properties of the hydrogels in the examples and comparative examples.

[0041] Figure 26The diagram shows the antibacterial properties of the hydrogels against Escherichia coli in the examples and comparative cases.

[0042] Figure 27 The diagram shows the antibacterial properties of the hydrogels against Staphylococcus aureus in the examples and comparative examples.

[0043] Figure 28 Figure I shows the animal experiment test results of the hydrogels used in the examples and comparative examples.

[0044] Figure 29 Figure II shows the animal experiment test results of the hydrogels used in the examples and comparative examples.

[0045] Figure 30 Figure III shows the animal experiment test results of the hydrogels used in the examples and comparative examples. Detailed Implementation

[0046] 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. Example

[0047] Step S1: Add 0.1g of N-isopropylacrylamide, 20mg of hyaluronic acid (HA), 10mg of polydopamine@polypyrrole (PDA@Ppy), 40mg of glucose oxidase (Gox), 40mg of CeO2, 20mg of L-arginine (L-Arg), and 1ml of acrylic acid (AA) to 4ml of deionized water and stir magnetically to dissolve, forming a transparent mixed solution.

[0048] Step 2: Add 10 mg of N,N-methylenebisacrylamide, 10 mg of ammonium persulfate, and 13 μl of polyethylene glycol diacrylate (PEGDA with a molecular weight of 200) to the solution obtained in Step 1. Stir at room temperature for 2 hours to mix well and obtain a prepolymer solution. Step S3: Pour the prepolymer solution obtained in step S2 into a mold, sonicate to remove all air bubbles, and react in a 60℃ constant temperature oven for 2 hours. Demold the polymerized gel and wash it three times with deionized water at 5℃ to remove unreacted substances. The resulting hydrogel is HPGCL.

[0049] Comparative Example 1 Step 1: Add 0.1g of N-isopropylacrylamide, 20mg of hyaluronic acid (HA) and 1ml of acrylic acid (AA) to 4ml of deionized water, stir magnetically to dissolve, and form a transparent solution.

[0050] Step 2: Add crosslinking agent (i.e., 25 mg of N,N-methylenebisacrylamide), catalyst (i.e., 20 mg of ammonium persulfate) and accelerator (i.e., 13 μl of polyethylene glycol diacrylate) to the solution in Step 1 and stir at room temperature for 2 hours to obtain a prepolymer solution.

[0051] Step S3: Pour the prepolymer solution from step S2 into a mold, sonicate to remove all air bubbles, and react in a 60°C oven for 2 hours. Demold the polymerized gel and wash it three times with deionized water at 5°C to remove unreacted material. Hydrogel H is obtained.

[0052] Comparative Example 2 Step 1: Add 0.1g N-isopropylacrylamide, 20mg HA, 10mg polydopamine@polypyrrole (PDA@Ppy) and 1ml AA to 4ml deionized water, stir magnetically to dissolve, and form a transparent solution.

[0053] Step 2: Add 10 mg of N,N-methylenebisacrylamide as a crosslinking agent, 20 mg of ammonium persulfate as a catalyst, and 26 μl of polyethylene glycol diacrylate as an accelerator to the solution from Step 1. Stir at room temperature for 2 hours to obtain a prepolymer solution.

[0054] Step S3: Pour the prepolymer solution from step S2 into a mold, sonicate to remove all air bubbles, and react in a 60°C oven for 2 hours. Demold the polymerized gel and wash it three times with deionized water at 5°C to remove unreacted material. Hydrogel HP is obtained.

[0055] Comparative Example 3 Step S1: Add 0.1g of N-isopropylacrylamide, 20mg of HA, 10mg of PDA@Ppy, 40mg of glucose oxidase (Gox) and 1ml of AA to 4ml of deionized water, stir magnetically to dissolve, and form a transparent solution.

[0056] Step 2: Add 10 mg of N,N-methylenebisacrylamide, 10 mg of ammonium persulfate, and 13 μl of polyethylene glycol diacrylate to the solution in Step 1 and stir at room temperature for 2 hours to mix.

[0057] Step S3: Pour the prepolymer solution from step S2 into a mold, sonicate to remove all air bubbles, and react in a 60°C oven for 2 hours. Demold the polymerized gel and wash it three times with deionized water at 5°C to remove unreacted material. Hydrogel HPG is obtained.

[0058] Comparative Example 4 Step S1: Add 0.1g of N-isopropylacrylamide, 20mg of HA, 10mg of PDA@Ppy, 40mg of Gox, 40mg of cerium dioxide (CeO2) and 1ml of AA to 4ml of deionized water, stir magnetically to dissolve, and form a transparent solution.

[0059] Step 2: Add 10 mg of N,N-methylenebisacrylamide, 10 mg of ammonium persulfate, and 13 μl of polyethylene glycol diacrylate to the solution in Step 1 and stir at room temperature for 2 hours to mix.

[0060] Step S3: Pour the prepolymer solution from step S2 into a mold, sonicate to remove all air bubbles, and react in a 60°C oven for 2 hours. Demold the polymerized gel and wash it three times with 5°C deionized water to remove unreacted material. The resulting hydrogel is HPGC.

[0061] A series of performance tests were conducted on the hydrogel dressings prepared in the examples and comparative examples to evaluate their suitability as antibacterial dressings for wound treatment. The details are as follows: pH response performance test: The swelling behavior of the hydrogels obtained in Example 1 and Comparative Examples 1-4 was evaluated under different pH conditions. 2.5 g of each lyophilized and pre-weighed hydrogel sample was immersed in 10 mL of PBS buffer (pH=7.4) at 25°C. Samples were removed every 2 hours, and the surface moisture of the hydrogel was removed using filter paper. The actual weight of the hydrogel was measured according to the formula SR=(M S The swelling ratio (SR) was calculated as (-M0) / M0×100%, and the results are shown below. Figure 1 .

[0062] In the formula, M S M0 represents the mass of the hydrogel in its hydrated state, and M0 ... represent the mass of the hydrogel in its dry state.

[0063] Three pre-treated and pre-weighed hydrogel samples prepared in Example 1 were immersed in 10 mL of PBS buffer at pH=5, pH=7.4, and pH=10, respectively, at 25°C to determine the effect of different pH values ​​on the swelling efficiency of the hydrogel. The test results showed that the hydrogel exhibited the best water absorption and swelling performance simulating the human body environment when the ambient pH reached 10; its swelling performance was second best at the neutral pH of 7.4; and its swelling performance was relatively low in the acidic environment of pH 5.0. Figure 2Under alkaline conditions, the carboxyl groups in acrylic acid molecules reduce their negative charge density, which enhances the interaction forces between hydrogel networks, thereby promoting water absorption and gel expansion. Simultaneously, the hydroxyl groups in tannic acid undergo protonation in an alkaline environment, generating more free hydroxyl groups. These free hydroxyl groups can interact with water molecules through hydrogen bonds, further enhancing the hydrogel's water absorption capacity. This pH sensitivity makes this hydrogel valuable for applications in wound environment monitoring and biomedical sensing.

[0064] Scanning electron microscopy experiment: The microstructure of the hydrogels obtained in Example 1 and Comparative Examples 1-4 was observed using scanning electron microscopy. Before scanning electron microscopy observation, the hydrogel samples were frozen and broken in liquid nitrogen, and then dried in a freeze-drying oven for 48 hours to maintain their structural integrity.

[0065] Test results show that freeze-dried hydrogel samples, observed under scanning electron microscopy, exhibit a distinct porous structure. This three-dimensionally interconnected porous structure is beneficial to the mechanical and responsive properties of the hydrogel. Comparative Example 1 (H) has fewer and sparsely distributed pores; with compositional adjustments (such as the introduction of different modified components or composites), the number of pores significantly increases, the pore size distribution becomes more uniform, and a denser, more interconnected porous network structure is gradually formed (such as HP, HPG, and subsequent samples). Figure 3 The results show that the porous morphology of the hydrogel gradually improves with component optimization, and the abundance of pores and network integrity are continuously enhanced. This is mainly because the interaction between components and spatial effects synergistically regulate the network structure, continuously driving the evolution of the hydrogel network from "dense" to "porous interconnected".

[0066] Figure 4 The EDS elemental distribution confirms that C, N, O and Ce are highly uniformly dispersed in HPGCL without obvious agglomeration, indicating that Ce-based functional components have achieved nanoscale dispersion rather than simple physical mixing.

[0067] Figure 5 SEM and Figure 6 The results of DLS analysis showed that pure CeO2 was a monodisperse spherical nanoparticle with a main peak diameter of about 100 nm. A small number of shoulder peaks corresponded to slight agglomeration of the particles, which provided a basis for its uniform dispersion in the hydrogel network. Figure 7 and Figure 8The results show that PDA@Ppy exhibits porous, flower-like aggregates with a narrow DLS peak at approximately 100 nm, demonstrating good monodispersity and facilitating uniform mixing within the hydrogel precursor. In summary, HPGCL is a composite hydrogel composed of an organic hydrogel network, CeO2 nanoparticles, and the PDA@Ppy conductive polymer. The components are highly uniformly dispersed, and its three-dimensional porous structure, the catalytic properties of CeO2, and the photothermal properties of PDA@Ppy suggest promising applications in biomedical antibacterial and wound healing fields.

[0068] Mechanical performance testing: The mechanical properties of the hydrogels obtained in Example 1 and Comparative Examples 1-4 were evaluated by testing their toughness and elongation through tensile and compression tests.

[0069] Test results show that HPGCL hydrogel exhibits excellent deformability. When stretched in the horizontal direction, the hydrogel demonstrates superior ductility, thanks to its carefully designed network structure. Even after pre-tying and stretching, the hydrogel maintains excellent tensile properties, indicating its high flexibility and mechanical stability. Figure 9 Tensile testing was performed on a universal tensile testing machine. The hydrogel was cut into standard specimens, 20 mm in length and 4 mm in diameter, and stretched at a rate of 50 mm / min until fracture. The elongation at break of the HPGCL hydrogel reached 2700%. (See attached image.) Figure 10 The high tensile properties of hydrogels make them a promising material for everyday use and complex applications, especially in environments where materials need to withstand deformation or stress.

[0070] Furthermore, the significant reversibility of hydrogels during compression and relaxation processes, such as Figure 12 As shown, it can quickly recover its original shape after stress compression. Figure 11 The results indicate that the increase in material did not affect the compressive strength of the hydrogel, with the maximum pressure remaining around 350 kPa. This suggests that the construction of the multi-linked network significantly enhances its resistance to fracture. The tensile cyclic test (also known as the cyclic loading-unloading test, a classic method for evaluating the mechanical fatigue resistance of hydrogels, involves repeatedly subjecting the hydrogel sample to a "stretching → recovery → re-stretching" process to simulate the repeated stress scenarios in actual use and determine whether it can maintain stable performance and avoid damage after multiple deformations) curves were also analyzed. Figure 13 The results show that continuous cyclic tensile testing for five cycles at 400% tensile strain indicates good fatigue resistance. With the hydrogel strain value fixed at 60% of the original specimen length for five cycles, it can be observed that, except for the first cycle, subsequent cycles exhibit almost identical hysteresis curves, as shown in [Figure 1]. Figure 14The nearly identical hysteresis loops indicate that the prepared hydrogel exhibits significant self-healing ability during continuous stretching cycles. In conclusion, HPGCL hydrogel possesses excellent stretchability and extensibility, enabling it to adapt to the mechanical requirements of the physiological environment, effectively resist external bacteria, reduce the risk of infection, and demonstrates good feasibility for clinical application.

[0071] Adhesion performance test: The adhesion of the hydrogel to different substrates (such as glass, plastic, metal, and wood) was evaluated. The hydrogel (15mm × 10mm × 4mm) was sandwiched between the overlapping areas of two glass slides (4.5mm × 1.5mm × 3mm), and a 500g weight was applied for 30 minutes to cure. Then, a single-lap tensile shear test was performed on a universal testing machine at a rate of 50mm / min. The maximum force was recorded and divided by the overlap area (150mm²) to obtain the shear strength.

[0072] Test results show that HPGCL hydrogels achieve substrate adhesion through multi-mode interactions, including dipole-dipole, hydrogen bonding, hydrophobicity, and electrostatic interactions, provided by multiple components such as PDA@PPy, GOx, CeO2, and L-arg, which include catechol and carboxyl groups. It exhibits the best adhesion performance to substrates such as glass and plastics (shear strength reaching 11–13 kPa). Figure 17 It has relatively weak adhesion to substrates such as wood and skin. Figure 18 Simultaneously, this hydrogel possesses excellent self-healing and stretchability, and can stably adhere to various biological tissues such as the heart and kidneys. Its multimodal adhesion and synergistic self-healing effects demonstrate its promising application potential in wound healing scenarios requiring strong adhesion and rapid repair. (See...) Figure 15 This multimodal interaction mechanism endows hydrogels with excellent self-adhesive properties, making them potentially useful in wound healing, especially in applications requiring strong adhesion and rapid repair.

[0073] Self-healing performance test: To test the self-healing ability of hydrogels, the hydrogel was cut in half, and the broken surfaces were brought into contact. The results were then observed to see if the hydrogel could heal itself and restore its structure and function without external intervention.

[0074] Test results show that this self-healing property is mainly due to the fact that HPGCL hydrogel, through the synergistic interaction of dynamic hydrogen bonds, electrostatic interactions, and π-π stacking, utilizes the catechol in PDA@PPy, the protein functional groups in GOx, the surface hydroxyl groups in CeO2, and the multifunctional groups in L-arg to re-establish intermolecular interactions and drive network structure reorganization after damage, thereby achieving self-healing. This provides structural and functional durability support for biomedical applications such as wound healing. This process not only strengthens the internal connections of the hydrogel network but also successfully achieves structural reconstruction across the cutting interface, endowing the hydrogel with excellent self-healing capabilities. Figure 19 Thanks to this special self-healing mechanism, hydrogels can quickly regain their original function and integrity after being damaged, greatly enhancing their durability and reliability in practical use.

[0075] Photothermal and nitric oxide release tests: The photothermal properties of the hydrogel were evaluated by irradiating it with near-infrared light at a wavelength of 808 nm and determining the photothermal properties by the heating rate. The amount of nitric oxide released from the hydrogel was detected using a nitric oxide kit to assess its nitric oxide release capacity.

[0076] Test results show that under 808 nm near-infrared light irradiation, the HPGCL hydrogel (containing PDA@PPy) exhibits a significant photothermal heating effect, while comparative examples 1 and 2 only show weak temperature changes. Figure 20 , Figure 22 This stems from the synergistic photothermal mechanism of PDA@PPy: the rigid conjugated backbone of PPy efficiently absorbs near-infrared light energy and converts it into heat energy through π-π* transitions and charge transfer; PDA, through near-infrared absorption of catechol / quinone groups and its π-π stacked heterojunction structure with PPy, further enhances light capture and energy conversion efficiency, enabling the hydrogel to achieve a significant temperature rise within 5 minutes and exhibiting good photothermal cycling stability. (See...) Figure 21 Simultaneously, the CeO2 nanozyme mimics nitric oxide synthase activity, catalyzing the oxidation of L-arginine guanidinyl groups to NO, see [link to relevant documentation]. Figure 24 The HPGCL hydrogel released approximately 85 μM of NO within 60 min, significantly higher than that of Comparative Example 1. Figure 23 Its release behavior is regulated by CeO2 catalytic kinetics and also depends on the synergistic effect of swelling and diffusion of the hydrogel three-dimensional network and wound microenvironment (such as H2O2 and pH), which realizes the time-controlled photothermal response and NO delivery, providing support for the synergistic treatment of antibacterial, anti-inflammatory and angiogenesis.

[0077] Antibacterial performance test: The photothermal properties of the hydrogel were evaluated, and its direct antibacterial effect against Gram-negative *Escherichia coli* and Gram-positive *Staphylococcus aureus* was quantitatively assessed using a plate count method. 100 mg of sterile hydrogel sample was added to 500 μL of bacterial suspension (10⁵ CFU / mL). To investigate the effect of photothermal properties on the antibacterial effect of the hydrogel, the co-incubated bacterial suspension was irradiated with an 808 nm laser for 5 min. Negative and positive controls were set up, one with near-infrared (NIR) illumination (+) and the other without NIR illumination (-). The mixture was co-cultured at 37°C for 12 h. Subsequently, the co-cultured bacterial suspension was evenly spread onto agar plates. The agar plates were incubated at 37°C for 14 h. The basal control group was treated the same as the sample group, but without the addition of hydrogel. After 14 hours, observe and photograph the number of colonies on the plate, record the number of colonies on the solid culture medium, and calculate the antibacterial rate using the formula Antibacterial rate (%) = (CFU0 - CFU) / CFU0 × 100%, where CFU0 is the number of viable colonies in the control sample and CFU is the number of viable colonies in the antibacterial sample. Repeat the test 3 times.

[0078] Test results show that the antibacterial effect of H hydrogels, whether exposed to light or not, against Staphylococcus aureus and Escherichia coli is negligible. This is because H hydrogels do not contain photothermal components and have poor inherent antibacterial properties. With the addition of photothermal materials, HP and HPG hydrogels, after light exposure, exhibited certain antibacterial effects against Staphylococcus aureus and Escherichia coli, with inhibition rates reaching approximately 50%. However, the antibacterial effect of unexposed HP and HPG hydrogels against Staphylococcus aureus and Escherichia coli was almost negligible. The addition of cerium dioxide nanoparticles resulted in a certain antibacterial effect in unexposed HPGC hydrogels, while the antibacterial effect of exposed HPGC hydrogels was significantly enhanced. This is because the newly added nano-sized CeO2 contains interconvertible Ce particles on its surface. 3+ and Ce 4+ Its valence state allows it to mimic the action of catalase, catalyzing the decomposition of H₂O₂. This catalytic activity originates from the redox reaction on its surface—Ce. 4+ Acceptable electrons are reduced to Ce 3+ Simultaneously, H2O2 is decomposed into •OH (hydroxyl radical) and H2O; subsequently, Ce 3+ It can be oxidized back to Ce. 4+ Continuously participating in the catalytic cycle, ·OH has a rapid bactericidal effect. The photo-exposed HPGCL hydrogel exhibited excellent antibacterial effects, which is attributed to the photothermal effect of PDA@Ppy, the antibacterial effect of arginine, and the synergistic effect of cerium dioxide catalyzing the release of NO from arginine. The inhibitory effect against Staphylococcus aureus and Escherichia coli both reached over 90%. Figure 25-27 .

[0079] Animal experimental testing: Ideal wound dressings should have the ability to prevent infection and promote wound healing. Based on the comprehensive properties of HPGCL hydrogel, including its antibacterial and antioxidant activities, an in vivo wound healing model was established in a full-thickness skin defect infection model in diabetic mice.

[0080] The test results showed that the wounds in both the control and experimental groups decreased in size over time. However, the control group exhibited significant wound suppuration in the early stages, while the experimental group only showed mild infection. Figures 28-30 This indicates that the HPGCL group maintained a high wound contraction rate throughout the treatment period. The wound closure rate on day 7 in the HPGCL group was approximately 79.19%, significantly higher than that in the control group (47.93%) and other experimental groups. By day 14, the wounds treated in the HPGCL group had essentially healed completely, with a wound closure rate of 99.36%.

[0081] These results indicate that HPGCL hydrogel effectively promotes the healing of infectious diabetic wounds. Figures 28-30 .

[0082] The hydrogel obtained in this invention, with its synergistic photothermal response and nitric oxide release, offers multiple advantages for diabetic wound healing. Its PDA@PPy generates heat under near-infrared light, effectively killing pathogenic bacteria (including drug-resistant bacteria) and improving wound microcirculation. L-arginine slowly releases NO via CeO2 catalysis, promoting angiogenesis, reducing chronic inflammation, and synergistically enhancing antibacterial activity with photothermal stimulation. Simultaneously, it possesses strong adhesion and self-healing properties, adhering tightly to the wound and maintaining its effectiveness even after damage. Furthermore, its functional strength can be adjusted to adapt to the entire healing cycle, providing highly efficient support for diabetic wound healing.

[0083] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A photothermal response and nitric oxide release synergistic hydrogel and a preparation method thereof, characterized in that, Includes the following: Step 1: Add N-isopropylacrylamide, hyaluronic acid, photothermal nanoparticles, nitric oxide donor, nanozyme, acrylic acid and glucose oxidase to deionized water, and dissolve by magnetic stirring to form a mixed solution. Step 2: Add crosslinking agent, catalyst and accelerator to the mixed solution, stir and mix well to obtain prepolymer solution; Step 3: Pour the prepolymer solution into the mold, defoam it by ultrasonication, and then carry out the polymerization reaction under constant temperature conditions. Demold and wash the polymerized gel to obtain the hydrogel.

2. The production method according to claim 1, characterized by, The nanozyme is cerium dioxide, and the nitric oxide donor is L-arginine.

3. The production method according to claim 1, characterized by, The photothermal nanoparticles are polypyrrole coated with polydopamine.

4. The method of claim 1, wherein, The amounts of each component used in step 1 are as follows: N-isopropylacrylamide 0.1g, acrylic acid 1mL, hyaluronic acid 0.02g, photothermal nanoparticles 0.01g, glucose oxidase 0.04g, nanozyme 0.04g, and nitric oxide donor 0.02g.

5. The preparation method according to claim 1, characterized in that, In step 2, the crosslinking agent is 10 mg of N,N-methylenebisacrylamide, the catalyst is 10 mg of ammonium persulfate, and the accelerator is 13 μl of polyethylene glycol diacrylate.

6. The photothermally responsive and nitric oxide releasing hydrogel prepared by any of the methods of claims 1-5, wherein, include: The hydrogel framework is formed by copolymerization of N-isopropylacrylamide and acrylic acid in the presence of a crosslinking agent; Photothermal nanoparticles, dispersed in a hydrogel matrix, are used to generate a photothermal effect under near-infrared light irradiation; Nitric oxide donors are dispersed in a hydrogel matrix; And nanozymes, used to catalyze the production of nitric oxide from nitric oxide donors.

7. The hydrogel according to claim 6, characterized in that, The hydrogel exhibits an inhibition rate of over 90% against both Staphylococcus aureus and Escherichia coli.

8. The hydrogel according to claim 6, characterized in that, The hydrogel has a maximum tensile strength at break of 2700%, and it maintains structural integrity after being cyclically stretched 5 times at a tensile strength of 400%.

9. The application of a photothermal responsive and nitric oxide release synergistic hydrogel in the preparation of a medicament or dressing for treating chronic diabetic wounds.

Images