A near-infrared light-mediated hybrid hydrogel multi-bioactivity synergistic tissue repair material and a preparation method and application thereof

Abstract

The application provides a near-infrared light-mediated hybrid hydrogel multi-biological activity synergistic tissue repair material and a preparation method and application thereof, and relates to the technical field of biological materials. The method comprises the following steps: 1) dispersing calcium peroxide in a solvent, adding 3-hydroxy tyramine hydrochloride solution for reaction to obtain CaO2@PDA nanoparticles; reacting the CaO2@PDA nanoparticles with catalase solution to obtain ORN oxygen-releasing microparticles; 2) mixing the surface thiol-rich ORN oxygen-releasing microparticles and the surface selenol-rich Se-NPs subjected to reduction treatment under the protection of an inert atmosphere to obtain ORN@Se composite microparticles; dissolving hyaluronic acid methacrylate in deionized water, uniformly mixing the ORN@Se composite microparticles, adding a photoinitiator, and performing ultraviolet light curing to obtain an ORN@Se-HAMA hydrogel, which can realize local response oxygen supply for an anoxic wound and has antibacterial and antioxidant properties.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more specifically, to a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material, its preparation method, and its application. Background Technology

[0002] Diabetes is a chronic metabolic disorder affecting millions of people worldwide. One of the common complications of diabetes, diabetic foot ulcer (DFU), can cause prolonged pain, decreased mobility, and in severe cases, amputation. Chronic diabetes wound healing faces significant challenges due to rampant bacterial infection, persistent inflammation, interrupted oxygen delivery due to impaired angiogenesis, and excessive reactive oxygen species (ROS).

[0003] Diabetic wound healing is a complex process with three overlapping and influential phases: the inflammatory phase, the proliferative phase, and the remodeling phase. During the inflammatory phase, open wounds are highly susceptible to bacterial attack, and the hyperglycemic environment further promotes bacterial proliferation. Following induced injury, during the proliferative and remodeling phases, the damaged vascular system immediately hinders oxygen delivery to the wound, further exacerbating the prolonged hypoxic environment in surrounding tissues and leading to increased recruitment of oxygen-consuming inflammatory cells. Oxygen is essential for crucial physiological processes such as angiogenesis, granulation tissue formation, epithelial regeneration, and extracellular matrix synthesis, which typically require two weeks or longer. However, microcirculatory disturbances and chronic inflammation caused by diabetes often result in insufficient local oxygen supply, significantly delaying these repair processes.

[0004] In recent years, oxygen therapy, especially hyperbaric oxygen therapy (HBOT), has been frequently used in the clinical treatment of diabetic wounds to promote healing. However, as a systemic oxygen supply strategy, HBOT may pose risks of tissue hyperoxia, such as oxygen toxicity-induced seizures. To avoid the risks and side effects of systemic oxygen supply, it is essential to improve the local tissue hypoxic microenvironment. Currently, hydrogel-based oxygenation is a cutting-edge approach to continuously supply oxygen to improve cell survival under local ischemia. Various oxygen release systems have been developed, based on substances such as hydrogen peroxide (H2O2) and calcium peroxide (CaO2), or by converting excess ROS from the wound into oxygen to achieve local oxygen supply. However, during tissue regeneration, excessive oxygen may induce the formation of reactive oxygen species, leading to cell apoptosis; as the number of blood vessels gradually increases, the tissue oxygen content gradually rises, and regenerating cells require less exogenous oxygen. Existing systems not only cannot dynamically regulate oxygen release according to the healing process, but also struggle to address the bacterial infection problems that occur during wound repair, resulting in persistent inflammatory responses that severely hinder tissue regeneration. Therefore, it is crucial to develop hybrid hydrogels that can provide local oxygen supply to hypoxic wounds and also have antibacterial properties. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material, which can prepare a hybrid hydrogel that can provide local oxygen supply to hypoxic wounds and has antibacterial and antioxidant properties.

[0006] Another objective of this invention is to provide a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material that can control oxygen release and has dual antioxidant and antibacterial effects.

[0007] Another objective of this invention is to provide an application of near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material in the preparation of diabetic wound repair materials. By utilizing the interaction between photothermal effect and nanocomposite particles, it can both regulate the oxygen release rate through NIR-mediated regulation to dynamically adapt to changes in wound oxygen demand, and use the high temperature generated by photothermal to destroy the bacterial biofilm structure, thereby enhancing the antibacterial effect, thus achieving the dual therapeutic goals of locally responsive photothermal accelerated oxygen supply and bacterial growth inhibition.

[0008] This invention is achieved through the following technical solution:

[0009] A method for preparing a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material includes the following steps:

[0010] 1) Calcium peroxide was dispersed in a mixed solution of deionized water and ethanol at a volume ratio of 1-1.5:1, and the pH was adjusted to about 10 with ammonia. 3-hydroxytyramine hydrochloride solution was added to react with the solution. The mass ratio of calcium peroxide to 3-hydroxytyramine hydrochloride was 3-5:1 to obtain CaO2@PDA nanoparticles. CaO2@PDA nanoparticles were then reacted with catalase solution at a ratio of 6-8:1 mg / mL to obtain ORN oxygen-releasing microparticles.

[0011] 2) ORN oxygen-releasing microparticles with thiol-rich surfaces are mixed with Se-NPs with selenol-rich surfaces after reduction treatment under an inert atmosphere to obtain ORN@Se composite microparticles; hyaluronic acid methacrylate is dissolved in deionized water, and ORN@Se composite microparticles are added and mixed evenly. The mass ratio of hyaluronic acid methacrylate to ORN@Se composite microparticles is 1.5-3.5:1. A photoinitiator is added, and the mixture is photographed at 365nm and 10W / cm. 2 The ORN@Se-HAMA hydrogel was obtained by UV curing for 50-70 seconds.

[0012] The preparation of calcium peroxide involved dissolving calcium nitrate (Ca(NO3)2) and polyvinylpyrrolidone (PVP) in a mixed solution containing 40 mL of deionized water, 7.5 mL of ammonia (NH3·H2O), and 20 mL of ethanol. Then, under vigorous ultrasonic stirring at 0°C, a mixed solution of 7.5 mL of hydrogen peroxide (H2O2) and 15 mL of deionized water was slowly added dropwise. After continuous stirring for 3 hours, a milky white calcium peroxide (CaO2) precipitate was obtained by centrifugation and washing.

[0013] Preparation of selenium nanoparticles (Se-NPs): PVP, sodium selenite (Na2SeO3) and 50 mL of deionized water were mixed and stirred for 1 h. Then, 20 mL of 0.11 g / mL ascorbic acid solution was slowly added dropwise to the system. After reacting for 18 h, a dark red solution was obtained. Finally, the solution was washed three times by centrifugation with deionized water and filtered through a 0.22 μm filter membrane to obtain selenium nanoparticles (Se-NPs).

[0014] Preparation of hyaluronic acid methacrylate (HAMA): 7 mL of methacrylic anhydride was added to 100 mL of a 0.02 g / mL hyaluronic acid solution. The pH was adjusted to above 8 with sodium hydroxide aqueous solution, and the mixture was stirred continuously at 0 °C for 24 h. The resulting white, opaque reaction solution was dialyzed in deionized water for 5 days, and then freeze-dried for 48 h to obtain a white, spongy, dried HAMA.

[0015] Furthermore, the specific steps for preparing ORN@Se composite microparticles are as follows:

[0016] 2.1) Disperse the ORN oxygen-releasing microparticles in PBS buffer at pH 7.4 to prepare a dispersion with a concentration of 1 mg / mL. Then, dilute the dispersion to a concentration of 0.5 mg / mL with PBS buffer at pH 8.0. Add Traut's reagent dropwise with stirring. The mass ratio of Traut's reagent to ORN oxygen-releasing microparticles is 10-20:1. React at room temperature in the dark for 1-2 hours to introduce additional -SH groups. Under N2 protection at 4°C, centrifuge and wash at least 5 times with pre-cooled degassed PBS buffer (pH 7.4) to completely remove small molecule impurities and obtain thiolized ORN. Resuspend the thiolized ORN in pre-cooled degassed PBS buffer (pH 7.4) and sonicate to obtain a thiolized ORN dispersion.

[0017] 2.2) Se-NPs were dispersed in ultrapure water to prepare a dispersion with a concentration of 0.5 mg / mL. Then, a 0.01 mol / L NaBH4 solution was slowly added dropwise under ice-water bath and nitrogen protection, with n(NaBH4):n(Se) = 2-5:1. The reaction was carried out in the dark for 30-60 min. Under N2 protection at 4℃, the mixture was washed three times with degassed ultrapure water to completely remove the remaining NaBH4, borate and byproducts, to obtain Se-NPs with a surface rich in selenool. The Se-NPs with a surface rich in selenool were resuspended in ice-pre-cooled degassed ultrapure water and ultrasonically dispersed to obtain a Se-NPs dispersion with a surface rich in selenool.

[0018] 2.3) Under continuous N2 protection, the Se-NPs dispersion rich in selenool on the surface was added to the thiolized ORN dispersion and mixed. The ORN oxygen-releasing particles rich in thiol on the surface and the Se-NPs rich in selenool after reduction treatment were mixed at a molar ratio of n(SH):n(SeH)=2:1. The reaction was carried out at 4℃, in the dark, under N2 protection with shaking for 12-24h. Low temperature helps to inhibit the oxidative decomposition of -SeH and maintain biological activity. After the reaction was completed, the precipitate was centrifuged at 6000-8000r / min for 15-20min and carefully washed 3 times with pre-cooled, degassed PBS buffer (pH 7.4) to remove unreacted Se-NPs and byproducts, and ORN@Se composite particles were obtained.

[0019] Hydrogels deliver oxygen directly to the wound microenvironment via ORN oxygen-releasing microparticles, significantly improving endothelial cell survival rates in vitro and promoting tubular structure formation. Selenium exerts its antioxidant effect through the synthesis of selenoproteins; this enzyme catalyzes the decomposition of hydrogen peroxide and organic peroxides into harmless substances, effectively scavenging free radicals and protecting cells from oxidative damage. As a key supplement for tissue repair and regeneration, selenium can significantly improve wound healing efficacy by enhancing the in vitro expansion capacity of mesenchymal stem cells and maintaining their stem cell characteristics. Hydrogels can utilize the synergistic effect of selenium and oxygen to enhance cellular antioxidant capacity and promote the dynamic balance of redox reactions. This synergistic effect not only improves cellular energy metabolism and enhances immune function but also significantly improves the migration and angiogenesis of HUVECs by reducing oxidative stress damage, thus promoting wound repair.

[0020] In this invention, Se nanoparticles (Se-NPs) and catalase (CAT) on the surface of ORN oxygen-releasing microparticles form a composite system through disulfide bonds (-Se-S-). This connection method can exert its effects in the following four dimensions:

[0021] 1. Enhance the stability of the composite system

[0022] Free catalase (CAT), as a protein-based biocatalyst, is easily deactivated by environmental factors (such as temperature, pH, and protease degradation) and has a short half-life; while Se-NPs, although having good biocompatibility and antioxidant properties, may lose their function when alone due to aggregation or excessively rapid metabolism.

[0023] After bridging by disulfide bonds: (1) Enzyme structure protection: Disulfide bonds can anchor CAT to the surface of Se-NPs, reducing disordered folding or conformational damage of enzyme molecules, maintaining the integrity of their active sites (such as iron porphyrin structures), thereby improving the stability of CAT in complex biological environments (such as prolonged half-life at physiological pH or body temperature). (2) Improved nanoparticle dispersibility: Grafting of CAT molecules can inhibit the aggregation of Se-NPs through steric hindrance, maintain their nanoscale dispersibility, and ensure the exposure of their surface active sites.

[0024] 2. Achieve functional synergy and efficiency enhancement

[0025] Both Se-NPs and CAT have antioxidant activity, but their mechanisms are different: Se-NPs scavenge reactive oxygen species (ROS) through the redox properties of selenium (such as its participation in the synthesis of glutathione peroxidase); while CAT specifically catalyzes the decomposition of hydrogen peroxide (H2O2) into H2O and O2, directly eliminating one of the most cytotoxic ROS.

[0026] Disulfide bond bridging enables the two to form a synergistic effect of "1+1>2": (1) Efficient ROS removal: The composite system can simultaneously deal with multiple ROS (such as H2O2 and superoxide anion), especially in oxidative stress environments (such as inflammation and cell damage sites), the antioxidant capacity of Se-NPs and the catalytic activity of CAT are superimposed, significantly improving antioxidant efficiency. (2) Optimization of catalytic efficiency: The nanoscale effect of Se-NPs (large specific surface area and surface charge) can enhance the adsorption capacity of substrate (H2O2), providing a local high-concentration substrate environment for CAT, indirectly improving its catalytic rate (reducing the Michaelis constant Km).

[0027] 3. Impart responsive release functionality to the system.

[0028] Disulfide bonds are common dynamic chemical bonds in living organisms and can be specifically broken in reducing environments (such as high concentrations of glutathione GSH). This characteristic makes the composite system responsive to pathological microenvironments: (1) Targeted release: In normal tissues, disulfide bonds are stable, the composite system remains intact, and the functional waste caused by premature release of CAT is avoided; while in pathological sites (such as inflamed tissues, where the GSH concentration is significantly higher than in normal tissues), disulfide bonds break, CAT is released and plays a catalytic role locally, improving bioavailability. (2) Controllable degradation: After the disulfide bonds break, Se-NPs can be gradually degraded into low-toxicity selenium ions (participating in human metabolism), avoiding long-term accumulation of nanoparticles in the body and reducing potential toxicity.

[0029] 4. Optimize biocompatibility and delivery efficiency

[0030] Low biotoxicity: Se-NPs themselves are low in toxicity and biodegradable. Disulfide bonds are naturally occurring chemical bonds in living organisms, and this linkage method avoids the use of chemical cross-linking agents (such as glutaraldehyde), reducing the immunogenicity and cytotoxicity of the complex system. Enhanced targeted delivery: Se-NPs can achieve targeted recognition of specific cells (such as oxidatively damaged cells) through surface modification (such as targeting peptides and antibodies), and the disulfide-linked CAT can be endocytosed along with Se-NPs, improving the enzyme's enrichment efficiency at the target site.

[0031] Further, in 3), the photoinitiator includes a solution of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone with a mass concentration of 0.1 g / mL.

[0032] A near-infrared light-mediated hybrid hydrogel material with multiple bioactive synergistic effects on tissue repair.

[0033] Application of a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material in the preparation of diabetic wound repair materials.

[0034] The technical solution of the present invention has at least the following advantages and beneficial effects:

[0035] This invention utilizes near-infrared light to trigger hydrogels to exert synergistic therapeutic effects through multiple bioactivities. By leveraging the interaction between photothermal effects and nanocomposite particles, it can both regulate the oxygen release rate through NIR-mediated regulation to dynamically adapt to changes in the oxygen demand of the wound, and use the high temperature generated by photothermal light to destroy the bacterial biofilm structure, thereby enhancing the antibacterial effect. This achieves the dual therapeutic goals of locally responsive photothermal accelerated oxygen supply and bacterial growth inhibition.

[0036] This invention provides a smart oxygen-releasing microparticle (ORN) with a three-layer core-shell structure (CaO2@PDA@CAT) exhibiting photothermal effects. ORN achieves multifunctional integration by depositing a layer of polydopamine (PDA) on the surface of calcium peroxide nanoparticles via a self-polymerization reaction and coating the outermost layer with catalase. The innermost layer of the ORN microparticle consists of PVP and CaO2, with CaO2 providing the oxygen source. The photothermal effect of the middle PDA layer generates a mild thermal stimulus to achieve a smart response that accelerates oxygen release and promotes cell proliferation. The catalase in the outermost layer enables the conversion of calcium peroxide into oxygen within the microparticle, thereby avoiding chemical damage to tissues caused by the reaction byproduct hydrogen peroxide.

[0037] In this invention, ORN oxygen-releasing microparticles and Se-NPs are bridged by disulfide bonds to form a composite system. This composite system can release CAT in response to GSH at the site of inflammation or injury, decompose H2O2 produced by inflammation, and accelerate tissue repair. ORN@Se composite microparticles, using dynamic chemical bonds as a "bridge," integrate the advantages of nanomaterials and biological enzymes, achieving synergistic optimization of stability, functionality, and biocompatibility, providing an efficient tool for precise catalysis and targeted therapy. Furthermore, the photothermal effect of Se-NPs combined with ORN oxygen-releasing microparticles can effectively achieve dual antibacterial effects of biological inhibition and photothermal sterilization at a temperature that does not affect surrounding healthy tissue, ultimately providing a therapeutic pathway that links oxygen supply, fatty acid metabolism, and energy metabolism to jointly promote wound repair. Attached Figure Description

[0038] Figure 1 A schematic diagram illustrating the preparation and application of the ORN@Se-HAMA hydrogel provided by this invention;

[0039] Figure 2 Analysis diagrams of CaO2 and CaO2@PDA nanoparticles provided for this invention.

[0040] Figure 3 An analytical diagram of the Se-NPs nanoparticles provided by this invention;

[0041] Figure 4 Macroscopic and microscopic analysis diagrams of different groups of hydrogels provided for this invention;

[0042] Figure 5 A schematic diagram illustrating the photothermal conversion capability of the ORN@Se-HAMA hydrogel provided by this invention;

[0043] Figure 6 A schematic diagram illustrating the oxygen-responsive release performance of the ORN@Se-HAMA hydrogel provided by this invention;

[0044] Figure 7A schematic diagram illustrating the free radical scavenging performance of the ORN@Se-HAMA hydrogel provided by this invention;

[0045] Figure 8 A schematic diagram illustrating the in vitro cell proliferation capacity of the ORN@Se-HAMA hydrogel provided by this invention;

[0046] Figure 9 A schematic diagram illustrating the toxicity detection of the ORN@Se-HAMA hydrogel provided by this invention;

[0047] Figure 10 A schematic diagram illustrating the in vitro antibacterial properties of the ORN@Se-HAMA hydrogel provided by this invention;

[0048] Figure 11 A schematic diagram illustrating the in vitro cell migration ability of the ORN@Se-HAMA hydrogel provided by this invention;

[0049] Figure 12 A schematic diagram illustrating the angiogenesis capability of the ORN@Se-HAMA hydrogel provided by this invention;

[0050] Figure 13 This is a schematic diagram illustrating the in vivo wound healing effect of the ORN@Se-HAMA hydrogel provided by this invention.

[0051] Figure 14 This is a schematic diagram illustrating the in vivo wound healing process of the ORN@Se-HAMA hydrogel provided by this invention. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise stated, the raw materials and reagents used in the embodiments of the present invention are conventionally purchased raw materials and reagents.

[0053] Example 1

[0054] Preparation of calcium peroxide: 3.19 g of calcium nitrate (Ca(NO3)2) and 3 g of polyvinylpyrrolidone (PVP) were dissolved in a mixed solution containing 40 mL of deionized water, 7.5 mL of ammonia (NH3·H2O), and 20 mL of ethanol. Then, under vigorous ultrasonic stirring at 0 °C, a mixed solution of 7.5 mL of hydrogen peroxide (H2O2) and 15 mL of deionized water was slowly added dropwise. After continuous stirring for 3 h, a milky white calcium peroxide (CaO2) precipitate was obtained by centrifugation and washing.

[0055] Preparation of selenium nanoparticles (Se-NPs): 0.5 g PVP, 1.1 g sodium selenite (Na2SeO3), and 50 mL deionized water were mixed and stirred for 1 h. Then, 20 mL of 0.11 g / mL ascorbic acid solution was slowly added dropwise to the system. After reacting for 18 h, a dark red solution was obtained. Finally, the solution was washed three times with deionized water by centrifugation and filtered through a 0.22 μm filter membrane to obtain selenium nanoparticles (SeNPs).

[0056] Preparation of hyaluronic acid methacrylate (HAMA): 7 mL of methacrylic anhydride was added to 100 mL of a 0.02 g / mL hyaluronic acid solution. The pH was adjusted to above 8 with sodium hydroxide aqueous solution, and the mixture was stirred continuously at 0 °C for 24 h. The resulting white, opaque reaction solution was dialyzed in deionized water for 5 days, and then freeze-dried for 48 h to obtain a white, spongy, dried HAMA.

[0057] A method for preparing a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material includes the following steps:

[0058] 1) Disperse 3g of calcium peroxide in a mixed solution of 27mL of deionized water and 20mL of ethanol, and adjust the pH to about 10 with ammonia. Dissolve 0.7g of 3-hydroxytyramine hydrochloride in 3mL of deionized water and add it to the above system for reaction. Stir continuously for 12h, and finally wash with ethanol by centrifugation more than 3 times. Collect the yellow-brown precipitate and dry it at 60℃ for 24h to obtain CaO2@PDA nanoparticles. Mix 40mg of CaO2@PDA nanoparticles with 6mL of catalase solution, stir at 4℃ for 4h, and centrifuge to remove unreacted enzyme to obtain ORN oxygen-releasing microparticles.

[0059] 2) Preparation of ORN@Se composite microparticles:

[0060] 2-1) Disperse ORN oxygen-releasing microparticles in PBS buffer at pH 7.4 to prepare a dispersion with a concentration of 1 mg / mL. Then, dilute the dispersion to a concentration of 0.5 mg / mL with PBS buffer at pH 8.0. Add Traut's reagent dropwise with stirring. The mass ratio of Traut's reagent to ORN oxygen-releasing microparticles is 10:1. Incubate at room temperature in the dark for 1 h to introduce additional -SH groups. Under N2 protection at 4 °C, centrifuge and wash at least 5 times with pre-cooled degassed PBS buffer (pH 7.4) to completely remove small molecule impurities and obtain thiolized ORN. Resuspend the thiolized ORN in pre-cooled degassed PBS buffer (pH 7.4) and sonicate to obtain a thiolized ORN dispersion.

[0061] 2-2) Se-NPs were dispersed in ultrapure water to prepare a dispersion with a concentration of 0.5 mg / mL. Then, a 0.01 mol / L NaBH4 solution was slowly added dropwise under ice-water bath and nitrogen protection, with n(NaBH4):n(Se) = 3:1. The reaction was carried out in the dark for 50 min. Under N2 protection at 4 °C, the mixture was washed three times with degassed ultrapure water to completely remove the remaining NaBH4, borate and byproducts, to obtain Se-NPs with a surface rich in selenool. The Se-NPs with a surface rich in selenool were resuspended in ice-pre-cooled degassed ultrapure water and ultrasonically dispersed to obtain a Se-NPs dispersion with a surface rich in selenool.

[0062] 2-3) Under continuous N2 protection, the Se-NPs dispersion rich in selenool on the surface was added to the thiolized ORN dispersion and mixed. The ORN oxygen-releasing particles rich in thiol on the surface and the Se-NPs rich in selenool after reduction treatment were mixed at a molar ratio of n(SH):n(SeH)=2:1. The reaction was carried out at 4℃, in the dark, under N2 protection and shaking for 20h. Low temperature helps to inhibit the oxidative decomposition of -SeH and maintain biological activity. After the reaction was completed, the precipitate was centrifuged at 7000r / min for 15min and carefully washed 3 times with pre-cooled, degassed PBS buffer (pH7.4) to remove unreacted Se-NPs and byproducts, and ORN@Se composite particles were obtained.

[0063] 2-4) Dissolve 0.12g of hyaluronic acid methacrylate in 6mL of deionized water, add 36mg of ORN@Se composite microparticles and mix well, then add 300μL of photoinitiator (a 0.1g / mL solution of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone). Use a wavelength of 365nm and a wavelength of 10W / cm². 2 After UV curing for 60 s, ORN@Se-HAMA hydrogel was obtained, as per reference. Figure 1 .

[0064] The names of each group of hydrogels and the corresponding component ratios are shown in Table 1.

[0065] Table 1

[0066]

[0067] Example of effect

[0068] 1. Physicochemical properties of ORN oxygen-releasing particles and Se-NPs

[0069] The morphology and chemical structure of the three nanoparticles in Example 1—CaO2, CaO2@PDA nanoparticles, and Se-NPs—were analyzed. The surface morphology and elemental distribution of the samples were observed using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscopy. Elemental distribution curves of the CaO2@PDA particles were obtained using line scan analysis to verify the particle structure. The particle size distribution was determined using dynamic light scattering (DLS).

[0070] Figure 2 In the figure, a represents transmission electron microscopy and scanning electron microscopy images of CaO2 and CaO2@PDA, b represents the particle size distribution of CaO2 and CaO2@PDA determined by dynamic light scattering, c represents the elemental surface scan analysis of CaO2@PDA, and d represents the dark field transmission electron microscopy image and line scan analysis of CaO2@PDA.

[0071] Observations can be made using SEM and TEM, as shown in the reference. Figure 2 a. Both CaO2 and CaO2@PDA nanoparticles exhibit a regular spherical structure, with the CaO2@PDA surface uniformly coated with a translucent PDA layer.

[0072] DLS analysis shows that, referring to Figure 2 b. CaO2 particles have a narrow particle size distribution, good uniformity and excellent dispersibility, while CaO2@PDA nanoparticles have a wider particle size distribution. This may be due to the strong adsorption of the PDA layer, which leads to the aggregation of microparticles. However, the particle size of both is mainly concentrated around 1 μm.

[0073] To further confirm that the PDA layer is uniformly coated on the CaO2 surface, TEM elemental surface scanning analysis was performed on the CaO2@PDA nanoparticles. Figure 2 c), confirming the presence of nitrogen in the PDA composition. A line scan of one particle was then performed to verify the elemental distribution, revealing a characteristic peak for nitrogen in the outer PDA layer. Figure 2 d), confirming that the PDA layer has been uniformly coated on the surface of the CaO2 particles.

[0074] Figure 3 In the image, a is the TEM image of Se-NPs, b is the dark-field TEM image and elemental distribution map, c is the size distribution of Se-NPs in DLS, and d is the ultraviolet-near-infrared absorption spectrum of Se-NPs.

[0075] Selenium nanoparticles (Se-NPs) were prepared using a one-step redox strategy with ascorbic acid as the reducing agent. Transmission electron microscopy (TEM) images clearly show that the Se-NPs exhibit a regular spherical structure, good dispersion, and an average particle size stable at approximately 100 nanometers. Figure 3 a). Elemental analysis via TEM ( Figure 3(b) It can be seen that selenium is uniformly distributed on the particle surface, comprehensively verifying the successful preparation of selenium nanoparticles. For example... Figure 3 DLS particle size analysis of c showed that the size distribution of Se-NPs was mainly concentrated in the range of 68-105 nm.

[0076] To avoid the influence of Se-NPs on the photothermal effect of ORN, their spectral absorption characteristics in the wavelength range of 200-1400 nm were characterized. The results showed that Se-NPs had a significant absorption peak in the ultraviolet (UV) region at 290 nm, but no absorption peak in the near-infrared (NIR) region. This indicates that selenium nanoparticles do not produce a photothermal effect under near-infrared light irradiation. This demonstrates that the presence or absence of Se-NPs does not have any other impact when investigating the photothermal properties of the subsequent hydrogel.

[0077] 2. Physicochemical properties of ORN@Se-HAMA hydrogel

[0078] Pure HAMA was used as the control group, and different concentrations of ORN were used as experimental groups (corresponding to ORN / L-HAMA, ORN / M-HAMA, and ORN / H-HAMA). SEM was used to observe the macroscopic morphology and microporous structure of the hydrogels in different groups.

[0079] Reference Figure 4 Macroscopic images of the hydrogel show that its color gradually deepens to brownish-red with increasing ORN particle concentration. SEM images reveal that the hydrogel exhibits a network-like porous structure at the microscale, with smooth pore walls, mimicking the extracellular matrix and providing a suitable three-dimensional environment for cell proliferation. Upon magnification, it is clearly observed that ORN particles are uniformly adsorbed on the surface of the hydrogel matrix, and their distribution becomes more dense with increasing concentration.

[0080] 3. Photothermal conversion capability of ORN@Se-HAMA hydrogel

[0081] Se-NPs show no significant absorption in the near-infrared (NIR) region, indicating that they have almost no effect on the final photothermal effect of the hydrogel. Therefore, evaluating the photothermal performance of the ORN-loaded hydrogel can represent the final performance of the ORN@Se-HAMA hydrogel. Infrared images were recorded and captured using a thermal imaging camera to analyze the differences in photothermal conversion performance of the hydrogel after loading with different concentrations of ORN oxygen-releasing particles. Figure 5 .

[0082] Figure 5 In the figure, a is the real-time infrared thermal image of different hydrogels, b is the photothermal temperature rise curve of the hydrogel, c is the photothermal temperature rise curve of ORN / H-HAMA hydrogel under different laser power densities, and d is the photothermal effect of ORN / H-HAMA hydrogel in four laser switching cycles.

[0083] Hydrogels of identical size were irradiated under 808nm near-infrared light, and their temperature changes were recorded in real time using an infrared thermal imager to plot time-temperature curves. Figure 5 a, 5b). The results showed that at 1.5 W / cm 2 Under high-power NIR irradiation, the hydrogel temperature rose rapidly within 10 seconds, and all groups reached a stable temperature at approximately 20 seconds. With increasing ORN concentration, the final temperature of the hydrogel increased significantly (ORN / H-HAMA reached 52.5℃, ORN / M-HAMA reached 42.7℃, and ORN / L-HAMA reached 37.2℃), while the temperature of the control group HAMA showed no significant change.

[0084] Further research revealed that, as shown in Figure 5c, under NIR irradiation with different powers, the temperature of ORN / H-HAMA gradually increased with increasing power, reaching 52.5℃ (1.5 W / cm²) at 63 s. 2 ), 45.8℃ (1.2W / cm) 2 ), 43.9℃ (1.0W / cm) 2 ), 39.7℃ (0.8W / cm) 2 ) and 31.9℃ (0.5W / cm) 2 Furthermore, the photothermal stability of ORN / H-HAMA was verified through four "on / off" laser cycle experiments, revealing that the temperature could reach above 50℃ in each cycle, indicating its excellent photothermal stability. Figure 5 d). Among them, 1.2W / cm 2 At the specified power, ORN / H-HAMA can be stabilized at approximately 45°C, a temperature that allows for photothermal therapy without causing significant damage to surrounding tissues.

[0085] 4. Oxygen response and release performance

[0086] ORN’s three-layered core-shell structure endows it with a variety of properties: the calcium peroxide (CaO2) in the core and the catalase (CAT) in the outer layer together form an ideal oxygen release system, as shown in the formula; while the polydopamine (PDA) in the middle layer serves as a carrier for photothermal effect and reactive oxygen species (ROS) scavenging function.

[0087]

[0088]

[0089] 2 mg of CaO2 or ORN microparticles were added to 20 mL of PBS buffer and incubated in a 37°C constant temperature shaking incubator. The oxygen concentration in the solution was measured at different time points using a dissolved oxygen meter to indirectly verify the oxygen release from the particles. For near-infrared light-based intelligent response oxygen release, under NIR illumination, the local temperature of the particles in the solution was observed using an infrared thermal imager until it reached approximately 45°C. This temperature was maintained for 3 minutes, and the oxygen concentration was then re-measured and compared with a control group. Finally, a dissolved oxygen concentration-time curve was plotted. Figure 6 .

[0090] Figure 6 In the diagram, a is a schematic diagram of the mechanism of near-infrared (NIR) controlled release of oxygen and removal of reactive oxygen species (ROS), b is the determination of oxygen concentration in PBS buffer, and c is the determination of oxygen concentration in PBS buffer under near-infrared light irradiation.

[0091] A schematic diagram illustrating the dual conversion function of ORN microparticles is shown below. Figure 6 As shown in a. In the microenvironment of diabetic wounds, bacterial growth can disrupt local blood circulation, leading to tissue hypoxia. Therefore, continuous local oxygen supply is crucial for wound healing. CaO2 and ORN microparticles were added to PBS buffer, and the dissolved oxygen content in the water was monitored periodically to plot oxygen release curves (6b).

[0092] The results showed a significant early oxygen burst in CaO2, with the oxygen content consistently lower than that of ORN in the later stages. This indicates that the three-layer structure of ORN effectively prevents premature and excessive oxygen release, ensuring a stable and continuous oxygen supply. To verify that ORN microparticles can achieve controllable enhanced oxygen release characteristics through NIR intelligent response, ORN was subjected to periodic NIR irradiation for a short period (<50 min), and the changes in oxygen content before and after each irradiation were measured. Figure 6 As shown in Figure c, the oxygen content significantly increased after NIR irradiation. The oxygen content of the ORN+NIR group was consistently significantly higher than that of the unirradiated ORN group. This is because the solution temperature increased and cooled slowly after NIR irradiation, resulting in a consistently higher oxygen release rate compared to the ORN group alone. Based on the above experimental measurements, it can be verified that ORN can achieve NIR-responsive accelerated oxygen release, laying a theoretical foundation for subsequent animal experiments.

[0093] 5. Free radical scavenging performance

[0094] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazole-6-sulfonic acid) (ABTS) were used to evaluate the in vitro free radical scavenging ability of hydrogels. Hydrogels from different groups were prepared into cylindrical shapes of equal size (h=0.5cm, d=1cm) and reacted with DPPH and ABTS solutions, respectively.

[0095] Reference Figure 7 , a is a schematic diagram of the DPPH removal mechanism, d is a schematic diagram of the ABTS removal mechanism, bc is the DPPH absorbance curve and removal rate of the hydrogel, and ef is the ABTS absorbance curve and removal rate of the hydrogel.

[0096] Inflammation and bacterial infection in diabetic wounds can lead to excessive free radical production, triggering an overactive inflammatory response that hinders tissue repair. For example... Figure 7 As shown in a and 7d, the free radical scavenging performance of the hydrogel was verified using two widely used models for evaluating the ability of nanoparticles to scavenge reactive nitrogen (RNS): 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and 2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). DPPH appears deep purple in ethanol solution and has a distinct characteristic absorption peak at 517 nm. After hydrogels loaded with different concentrations of ORN reacted with DPPH, it was clearly observed that the intensity of the characteristic peak decreased with increasing ORN content, indicating enhanced scavenging ability. Figure 7 b).

[0097] After quantitative calculations, ORN / H-HAMA showed the highest DPPH scavenging rate, increasing it fourfold, exhibiting a significant concentration-dependent effect. Figure 7 c). Furthermore, ABTS exhibits a blue-green color and a distinct characteristic absorption peak at 734 nm. Figure 7 e) The characteristic absorption peak also gradually decreased with increasing ORN concentration, with ORN / H-HAMA showing a scavenging efficiency of up to 89.5% for RNS.

[0098] 6. In vitro cell proliferation capacity

[0099] In the treatment of diabetic wounds, oxygen, as a key factor for cell proliferation, can alleviate the hypoxic state of the microenvironment and is of great significance for tissue regeneration.

[0100] To evaluate whether the oxygen released by the hydrogel could improve cell survival under hypoxic conditions, human umbilical vein endothelial cells (HUVECs) were co-cultured with ORN in a 1% oxygen environment, and the effect was verified by measuring double-stranded DNA (dsDNA) content. Figure 8 a.

[0101] Reference Figure 8 a is a schematic diagram of cell hypoxia culture; b is the proliferation capacity of HUVECs co-cultured with ORN-HAMA and irradiated with near-infrared light on days 1, 3, and 5 as shown by CCK8 assay; c is the determination of double-stranded DNA (dsDNA) content of HUVECs cultured under hypoxia.

[0102] like Figure 8 As shown in b, the dsDNA content in the ORN group was significantly higher than that in the control group. This indicates that the released oxygen can effectively improve cell survival. The three-layered nucleoshell structure of ORN continuously supplies oxygen through layer-by-layer reactions, mimicking the effect of hyperbaric oxygen therapy while avoiding its burst release risk, and laying the foundation for subsequent cell proliferation. Simultaneously, gentle thermal stimulation (approximately 45°C) can promote cell transformation and proliferation; therefore, 808nm near-infrared light (with a power of 1.2W / cm² selected based on photothermal conversion capacity experiments) was used. 2 The effect of irradiating ORN-containing hydrogels on the proliferation of HUVECs was investigated. Figure 8 c).

[0103] CCK8 assays showed a positive correlation between ORN concentration and cell proliferation capacity, with the ORN / H-HAMA group exhibiting significantly better proliferation performance than other groups. Based on its effect on cell proliferation capacity, we selected ORN / H-HAMA and renamed it ORN-HAMA for subsequent loading of selenium nanoparticles (Se-NPs) to explore its antibacterial properties. The addition of Se-NPs complements the 45°C temperature window (which does not cause thermal damage to surrounding tissues) required for antibacterial performance (>55°C) in the treatment of diabetic wounds.

[0104] 7. In vitro antibacterial properties

[0105] Different concentrations (1 μg / mL, 2 μg / mL, 4 μg / mL) of Se-NPs were co-loaded with ORN into HAMA hydrogels, corresponding to ORN / H-Se / L-HAMA, ORN / H-Se / M-HAMA, and ORN / H-Se / H-HAMA. After co-culturing the hydrogels with HUVECs for 2 days, live / dead cell viability staining was performed (red fluorescent markers for dead cells, green markers for live cells).

[0106] Reference Figure 9 a is the live / dead staining image of S. aureus treated with different concentrations of Se-NPs, b is the porosity of the hydrogel, c is the swelling ratio of the hydrogel in PBS solution, d is the DPPH absorbance curve, e is the DPPH free radical scavenging rate, f is the ABTS absorbance curve, and g is the ABTS free radical scavenging rate.

[0107] The results showed that the ORN / H-Se / H-HAMA group (4 μg / mL) had a significantly increased number of red fluorescent cells, indicating that high concentrations of Se were more toxic; while the ORN / H-Se / M-HAMA group (2 μg / mL) had fewer red fluorescent cells and a higher cell survival rate. Figure 9a). Therefore, we ultimately chose 2 μg / mL as the effective working concentration for cell biocompatibility, which retains the antibacterial effect and allows for the sustained release of Se-NPs through the porous structure of the HAMA hydrogel, reducing local toxicity. The final selected composite hydrogel ORN / H-Se / M-HAMA was named ORN@Se-HAMA.

[0108] The physicochemical properties of the hydrogels loaded with Se-NPs were verified, and the influence of Se-NPs on other properties was further analyzed. After loading Se-NPs, the porosity of each group of hydrogels stabilized at approximately 70%. Figure 9 b) indicates that the introduction of Se-NPs did not significantly alter the porous structure of the material. For wound dressing hydrogels, a swelling ratio greater than 3000% after freeze-drying is essential for rapid absorption of exudate and maintenance of a moist environment. Measurements showed no significant difference in swelling ratio among the groups, all reaching approximately 30 g / g (3000%) after 48 hours. Figure 9 c) This indicates that the loading of Se-NPs did not affect the water absorption and swelling properties of the hydrogel. Furthermore, the antioxidant properties were investigated again, such as... Figure 9 As shown in d-9g, free radical scavenging experiments using DPPH and ABTS revealed that the addition of Se-NPs had no significant effect on the free radical scavenging ability of the hydrogel.

[0109] To evaluate the in vitro antibacterial properties of ORN@Se-HAMA, Gram-positive Staphylococcus aureus (S. aureus) was selected as a model, and experiments were conducted under different treatment groups (near-infrared laser irradiation / irradiation for 5 min, power 1.2 W / cm2). The colony plating results showed ( Figure 10 a) Regardless of whether NIR was applied, the number of bacterial colonies in the blank group and the ORN-HAMA group was significantly higher than that in the ORN@Se-HAMA group. Among them, ORN@Se-HAMA under near-infrared light synergy showed the strongest antibacterial activity and the lowest bacterial survival rate. SEM observation of bacterial morphology revealed ( Figure 10 b) Bacteria treated with HAMA and ORN-HAMA maintained their intact spherical shape and smooth cell walls; while bacteria in the ORN@Se-HAMA group showed obvious shrinkage on the surface, and bacteria in the ORN@Se-HAMA group treated with near-infrared light showed lysis and leakage of intracellular substances, further verifying the photothermal synergistic antibacterial effect.

[0110] In addition, optical density measurement and live / dead staining experiments were also conducted. Figure 10 c) Verification revealed that ORN@Se-HAMA significantly inhibited bacterial proliferation under near-infrared light irradiation, resulting in a marked decrease in the proportion of viable bacteria. This synergistic effect stemmed from the combined action of membrane structure disruption caused by photothermal effects and the chemical antibacterial mechanism of Se-NPs. The optical density (OD450) values ​​of the bacterial suspension were measured after co-culturing for 0, 24, and 48 hours, respectively. Figure 10 d). The results showed that the ORN@Se-HAMA group effectively inhibited the growth and proliferation of high concentrations of *S. aureus* at both 24h and 48h, while the bacterial proliferation rate of the ORN@Se-HAMA group irradiated with NIR was significantly lower. Live / dead staining experiments further demonstrated ( Figure 10 e) Among all groups, ORN@Se-HAMA and ORN@Se-HAMA-NIR groups had the highest bactericidal rate against S. aureus, and the antibacterial effect of ORN-HAMA and ORN@Se-HAMA was significantly enhanced under the synergistic effect of near-infrared light.

[0111] The results show that NIR significantly enhances the antibacterial properties of ORN@Se-HAMA. This is likely due to the following mechanism: under mild thermal conditions of 45°C, the photothermal antibacterial properties of ORN are activated, producing a synergistic effect with the chemical antibacterial activity of Se-NPs. In summary, ORN@Se-HAMA-NIR possesses excellent antibacterial properties, and its mechanism mainly relies on the synergistic effect between the chemical bactericidal activity of Se-NPs and the photothermal properties of ORN.

[0112] 8. Evaluation of in vitro cell migration and angiogenesis capabilities

[0113] Diabetic wounds are chronically exposed to a microenvironment of hyperglycemia, hypoxia, and high oxidative stress, severely hindering skin cell migration and survival. Angiogenesis, a crucial step in dermal regeneration, is difficult to sustain in chronic hypoxia, although acute hypoxia can mediate pro-angiogenic signals through hypoxia-inducible factor-1α (HIF-1α). Numerous studies have confirmed that angiogenesis is essential for wound healing and reconstruction, promoting new skin formation by accelerating the transport of nutrients and signaling molecules. Therefore, biomaterials must possess excellent pro-angiogenic activity to meet the needs of wound repair. The potential of ORN@Se-HAMA hydrogel to induce HUVEC migration under NIR treatment was evaluated using a Transwell migration assay, referring to... Figure 11 .

[0114] HUVEC migration was assessed using Transwell assays. 1×10⁻⁶ 4 Cells were seeded in the upper compartment of a Transwell plate, and different groups of hydrogels were directly immobilized in the lower compartment. The control group used α-MEM medium without material. Cells were incubated at 37°C for 24 hours, during which time they were irradiated with NIR. The upper compartment was then removed, and the lower compartment was slowly washed with PBS buffer. Cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and finally stained with 0.5% crystal violet solution for 10 minutes. Cells were observed and photographed using an inverted microscope, and cell counts were performed in five fields of view (center, top, bottom, left, and right) for each filter membrane. The average value was used to determine the number of migrating cells.

[0115] HUVECs were seeded in 24-well plates and incubated until 90% confluence. The culture medium was then changed, and a horizontal line was drawn by scraping the cells with a 200 μL pipette tip. The cells were then co-cultured with different groups of hydrogels or PBS buffer for 24 h, during which time NIR irradiation was performed. Subsequently, the cells were washed three times with PBS buffer, fixed with 4% paraformaldehyde for 30 min, and stained with 0.05% crystal violet for 20 min. Scratch images were taken under a microscope, and the cell migration ability was evaluated by the degree of confluence of the scratch marks.

[0116] HUVECs were 5 × 10 4 HUVECs were seeded into 6-well plates and pretreated with conditioned medium and α-MEM at 37°C for 2 days. The conditioned medium consisted of HUVECs co-cultured with ORN@Se-HAMA hydrogel for 48 hours, followed by supernatant collection. 96-well plates were pre-cooled to 4°C, and 50 μL Matrigel was added to each well, followed by incubation at 37°C for 30 minutes to form a homogeneous gel layer. Then, 10⁴ cells per well were seeded into Matrigel-coated 96-well plates and cultured at 37°C with 5% CO₂ for 6 hours. The formation of luminal structures was observed using an inverted microscope. The number of nodes and the length of tubular structures in each region were determined using ImageProPlus software.

[0117] Figure 11 In the figure, a is a crystal violet staining image of the Transwell migration assay, b is the quantitative analysis result of the Transwell migration assay, c is a schematic diagram of the mechanism of the effect of ORN@Se-HAMA hydrogel on wound healing and angiogenesis of HUVECs under NIR irradiation, d is a representative image of cell migration of HUVECs from 0 to 24 h, and e is the quantitative statistics of scratch closure rate of HUVECs at different time points.

[0118] like Figure 11 As shown in Figure a, after co-culturing different groups of hydrogels for 24 h, the ORN@Se-HAMA group significantly promoted the migration of HUVECs from the upper to the lower layer of the Transwell chamber. Quantitative analysis further indicated that this group had a superior chemotactic recruitment effect on HUVECs compared to other groups. Figure 11 b). Furthermore, the cell migration activity was verified using a wound scratch assay. Figure 11 c) In the ORN@Se-HAMA group, the scratch area was almost completely closed at 24 hours, significantly better than the ORN-HAMA group (11 days). Quantitative analysis confirmed ( Figure 11e) The scratch closure rate of the ORN@Se-HAMA group was higher than that of ORN-HAMA, HAMA and control group at 24h. This is mainly attributed to the synergistic sustained-release effect of oxygen and Se triggered by near-infrared light periodic irradiation. The two promote endothelial cell migration and angiogenesis by activating the HIF-1α / VEGF signaling pathway.

[0119] To further evaluate the angiogenesis-promoting ability of the prepared hydrogel, an in vitro angiogenesis experiment was conducted using Matrigel matrix gel, referring to... Figure 12 .

[0120] Figure 12 In the figure, a represents in vitro angiogenesis experimental images of HUVECs treated with different ORN@Se-HAMA groups, and b represents a quantitative analysis of the number of grids, nodes, connection points, and total length of blood vessels formed in the vascular network structure.

[0121] like Figure 12 As shown in Figure a, only a small number of tubular structures were observed to form in the control group, while both the ORN-HAMA and ORN@Se-HAMA groups induced HUVECs to construct a tubular network framework. The ORN@Se-HAMA group showed more mature and complete vascular structures with a significantly increased cell connection density. Quantitative analysis indicated that ( Figure 12 b) The number of grids, nodes, connection points, and total vessel length in the ORN@Se-HAMA group were significantly higher than those in the ORN-HAMA group and the HAMA group.

[0122] In summary, the ORN@Se-HAMA group demonstrated a stronger ability to promote endothelial cell migration and angiogenesis compared to other groups.

[0123] 9. Assessment of internal wound healing

[0124] Based on the excellent properties of ORN@Se-HAMA-NIR in antibacterial, antioxidant, oxygen release, HUVEC growth promotion, and biocompatibility, its in vivo therapeutic effects in anti-inflammation and wound healing were further evaluated, referring to... Figures 13-14 .

[0125] in, Figure 13 a is a schematic diagram of the construction of a full-thickness skin defect model for diabetes. Figure 13 b represents the experimental protocol for treatment with ORN@Se-HAMA hydrogel NIR irradiation. Figure 13 c represents SD rats implanted with hydrogel after 10 seconds of near-infrared light irradiation (1.2 W / cm²). 2 Real-time infrared thermal image at 808nm. Figure 14 Image d shows representative images of the wound healing process in SD rats from different treatment groups on days 0, 3, 6, 9, 12, and 15. The colored areas indicate the wound area at different time points. Figure 14 e represents the quantitative analysis of wound area at each time point and the relative percentage statistics of wound closure rate on day 15 (n=15).

[0126] Animal experiments were conducted using 4-week-old male Sprague-Dawley (SD) rats. The effect of ORN@Se-HAMA hydrogel on wound healing was determined using an in vivo diabetic wound model.

[0127] First, a type 1 diabetic mouse model was established: The model was induced using chemical drugs. Streptozotocin solution (STZ) was prepared using cold citrate-sodium citrate buffer and administered to rats via intraperitoneal injection for 3 days. After one week of observation, blood glucose was monitored by collecting blood samples from the rat tails. Rats with stable blood glucose (>16.7 mmol / L) were selected as the type 1 diabetic model. Next, a diabetic wound model was established: Rats were anesthetized via intraperitoneal injection of sodium pentobarbital (3wt%) (40 mg / kg). After complete anesthesia, the back area was disinfected with 75% ethanol, and the rat hair was removed with an electric shaver to expose the skin. A pair of bilaterally symmetrical circular (d=1 cm) full-thickness skin defects were created using a skin biopsy sampler. Equal volumes of different groups of hydrogels were injected into the wounds and cured with UV light, followed by treatment with 3M... TM Tegaderm TM A transparent wound dressing was used to cover and fix the wound, and the gel dressing was changed every other day for the rats. Figure 13 a-13b.

[0128] Mice were randomly divided into 5 groups: 1) Control group; 2) HAMA group; 3) ORN-HAMA group; 4) ORN@Se-HAMA group; 5) ORN@Se-HAMA-NIR group (808nm, 1.2W / cm²). 2 To investigate the tissue healing ability of PTT treatment, the hydrogel treatment area of ​​rats in the fifth group was subjected to NIR irradiation (808nm) for 5 minutes, and images were captured using an infrared thermal imaging camera. Wound photographs were taken on days 0, 3, 6, 9, 12, and 15 to observe the healing effect.

[0129] Thermal imaging images ( Figure 13 c) shows that after 10 seconds of NIR irradiation, the local wound temperature in the ORN@Se-HAMA group and the ORN-HAMA group rapidly increased to 46.8℃ and 45.3℃, respectively, with consistent in vitro photothermal conversion capacity; while the local temperatures in the control group and the HAMA group were 35.4℃ and 35.8℃, respectively.

[0130] Macroscopic images of the wound, wound closure trajectory, and quantitative data on wound area at different time points, such as... Figure 14 As shown in d and 14e. On day 3, some rats in the control and HAMA groups showed significant wound infection, while no significant infection was observed in the ORN@Se-HAMA and ORN@Se-HAMA-NIR groups. The ORN@Se-HAMA-NIR group showed a better healing trend than the other groups. On day 6, the wound area in the ORN@Se-HAMA-NIR group was significantly reduced, while the wound size in the control, HAMA, ORN-HAMA, and ORN@Se-HAMA groups remained as high as 81%, 55%, 45%, and 40%, respectively. On day 9, the wound in the ORN@Se-HAMA-NIR group was close to healing (closure rate 90%), and by day 15 it was almost completely healed (closure rate 99.3%). In contrast, the control and HAMA groups still had large wound areas, while the ORN@Se-HAMA and ORN-HAMA groups had better healing rates than the former two. The results indicate that the NIR-treated ORN@Se-HAMA hydrogel can effectively promote the healing of infectious diabetic wounds.

[0131] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material, characterized in that, Includes the following steps: 1) Calcium peroxide was dispersed in a solvent and reacted with 3-hydroxytyramine hydrochloride solution to obtain CaO2@PDA nanoparticles; CaO2@PDA nanoparticles were reacted with catalase solution to obtain ORN oxygen-releasing microparticles. 2) ORN oxygen-releasing microparticles with thiol-rich surfaces are mixed with Se-NPs with selenool-rich surfaces after reduction treatment under an inert atmosphere to obtain ORN@Se composite microparticles; Hyaluronic acid methacrylate was dissolved in deionized water, ORN@Se composite microparticles were added and mixed evenly, a photoinitiator was added, and the mixture was cured under ultraviolet light to obtain ORN@Se-HAMA hydrogel.

2. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, In 1), the solvent is a mixed solution of deionized water and ethanol with a volume ratio of 1-1.5:1, the mass ratio of calcium peroxide to 3-hydroxytyramine hydrochloride is 3-5:1, and the ratio of CaO2@PDA nanoparticles to catalase solution is 6-8:1 mg / mL.

3. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, The specific steps for preparing Se-NPs are as follows: polyvinylpyrrolidone and sodium selenite are added to deionized water, and ascorbic acid solution is slowly added dropwise to the system after stirring. After the reaction, a dark red solution is obtained. After centrifugation, washing and filtration with deionized water, Se-NPs are obtained.

4. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, The specific steps for preparing ORN@Se composite microparticles are as follows: 2.1) Disperse ORN oxygen-releasing microparticles in PBS buffer, add Traut's reagent dropwise while stirring, react at room temperature in the dark for 1-2 h, centrifuge and wash to obtain thiolized ORN; 2.2) Disperse Se-NPs in ultrapure water, then add NaBH4 solution dropwise, react in the dark for 30-60 min, centrifuge and wash to obtain Se-NPs with selenool-rich surface; 2.3) The Se-NPs dispersion rich in selenol on the surface was added to the mercaptoized ORN dispersion and mixed. The mixture was shaken and reacted for 12-24 hours. After centrifugation and washing, ORN@Se composite microparticles were obtained.

5. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, The specific steps for preparing hyaluronic acid methacrylate are as follows: add methacrylic anhydride to the hyaluronic acid solution, adjust the pH value to above 8 with sodium hydroxide aqueous solution, and stir continuously at 0℃ for 24 hours. The resulting white opaque reaction solution is dialyzed in deionized water for 5 days and then freeze-dried to obtain white sponge-like dried HAMA.

6. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, In 2), the molar ratio of ORN oxygen-releasing microparticles rich in thiol groups on the surface to Se-NPs rich in selenols after reduction treatment is n(SH):n(SeH)=2:1, and the mass ratio of hyaluronic acid methacrylate to ORN@Se composite microparticles is 1.5-3.5:

1.

7. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, In 2), the photoinitiator includes a solution of 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone with a mass concentration of 0.1 g / mL.

8. The method for preparing the near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material according to claim 1, characterized in that, In section 2), 365nm and 10W / cm were used. 2 UV curing for 50-70 seconds.

9. A near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material prepared by the preparation method according to any one of claims 1-8.

10. The application of near-infrared light-mediated hybrid hydrogel multi-bioactive synergistic tissue repair material prepared by the preparation method according to any one of claims 1-8 in the preparation of diabetic wound repair materials.

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