Carbon oxide nanowhisker composite hydrogel, and preparation method and application thereof

Abstract

This invention relates to a carbon oxide nano-angle composite hydrogel, its preparation method, and its application. The carbon oxide nano-angle composite hydrogel comprises carbon oxide nano-angles, dopamine-grafted sodium alginate, and aminophenylboronic acid-grafted hyaluronic acid. The preparation method includes the steps of: preparing a carbon oxide nano-angle dispersion, preparing dopamine-grafted sodium alginate, preparing aminophenylboronic acid-grafted hyaluronic acid, and preparing the carbon oxide nano-angle composite hydrogel. This invention utilizes the synergistic effect of dynamic covalent and non-covalent bonds of phenylboronic esters to endow the hydrogel with dual responsive degradation behavior to high concentrations of glucose and excessive reactive oxygen species in the wound microenvironment. The carbon oxide nano-angles activate the AMPK signaling pathway, restore mitochondrial function of endothelial cells under high glucose conditions, and thus promote cell migration and tube formation, forming a cascade mechanism of "signaling pathway activation - mitochondrial repair - cell function recovery," which significantly accelerates the healing of diabetic wounds.

Description

Technical Field

[0001] This invention relates to the field of hydrogel technology, and in particular to a carbon oxide nano-angle composite hydrogel, its preparation method, and its application. Background Technology

[0002] Diabetic wounds pose a significant clinical challenge due to their complex pathological microenvironment. Wound healing depends on the precise coordination of cellular energy metabolism and functional regulation. In diabetes, mitochondrial energy metabolism is disordered, fundamentally weakening cell migration, proliferation, and angiogenesis. Defective angiogenesis further increases the risk of infection and inflammation, ultimately leading to prolonged wound healing. Therefore, the repair of diabetic wounds requires not only improving the external pathological microenvironment but also regulating cellular energy metabolism and angiogenesis levels, posing a severe challenge to the design of diabetic wound dressings.

[0003] In recent years, hydrogels have been widely used in the field of diabetic wound repair due to their unique advantages. Their three-dimensional porous network structure promotes gas exchange in the wound, while their high water content maintains an ideal moist environment. For the high-glucose microenvironment of diabetic wounds, hydrogel systems based on the dynamic covalent bonds of phenylboronic acid-cis-diol have attracted considerable attention. These hydrogels can improve the wound microenvironment by achieving local glucose consumption through glucose-responsive degradation. However, these hydrogels generally lack the ability to actively regulate wound cells, limiting their healing-promoting effects. Furthermore, traditional phenylboronic acid ester hydrogels have low intrinsic cohesion, are prone to over-swelling and exacerbating tissue damage, and are difficult to match the mechanical requirements of wound healing. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a carbon oxide nano-angle composite hydrogel. This hydrogel accelerates the healing of diabetic wounds through its nano-enhanced dynamic cross-linking network, excellent mechanical properties, and angiogenesis-promoting effects.

[0005] The technical solution of the present invention is as follows: A carbon oxide nano-angle composite hydrogel, characterized in that the carbon oxide nano-angle composite hydrogel comprises carbon oxide nano-angles, dopamine-grafted sodium alginate and aminophenylboronic acid-grafted hyaluronic acid. According to the concentration ratio, the carbon oxide nanoparticles: the dopamine-grafted sodium alginate: the aminophenylboronic acid-grafted hyaluronic acid is (0.006~0.025) wt% : (4~6) wt% : (2~3) wt%.

[0006] Single-walled carbon nanohorns are hollow, cone-shaped structures formed by rolling up a single layer of graphene. They can self-assemble into aggregates of 25–150 nm in size, forming a continuous three-dimensional network of channels. They possess excellent dispersibility and high specific surface area; their sidewalls and tips are rich in natural topological defects and active sites, and after oxidation, they readily graft functional groups such as hydroxyl and carboxyl groups, resulting in a functionalization efficiency far exceeding that of two-dimensional carbon materials. These structural characteristics allow oxidized single-walled carbon nanohorns (oxidized carbon nanohorns) to efficiently regulate the crosslinking density and cohesion of hydrogels through synergistic effects such as hydrogen bonding and π-π stacking. Simultaneously, their excellent bioactivity holds promise for precise regulation of cellular life activities. Based on this background, functionalizing phenylboronic acid-cis-diol dynamically covalently bonded hydrogels using oxidized carbon nanohorns is expected to improve the mechanical properties, responsiveness, and bioactivity of hydrogels, meeting the multi-dimensional needs of diabetic wound dressings. Based on single-walled carbon nano-angles, the present invention prepares carbon oxide nano-angle composite hydrogels that undergo responsive degradation in glucose and / or reactive oxygen environments. The adhesion strength to pigskin is 8-16 kPa, the tensile strength is 4-7 MPa, and the self-healing efficiency is greater than 90%.

[0007] In one embodiment, the concentration ratio of the carbon oxide nanoparticles: the dopamine-grafted sodium alginate: the aminophenylboronic acid-grafted hyaluronic acid is (0.006~0.025) wt%: 5 wt%: 2.5 wt%.

[0008] A second aspect of the present invention also provides a method for preparing the above-mentioned carbon oxide nano-angle composite hydrogel, comprising the following steps: Preparation of carbon oxide nano-angle dispersion: single-walled carbon nano-angles were added to concentrated nitric acid, refluxed, cooled, centrifuged to collect the precipitate, washed, and freeze-dried to obtain carbon oxide nano-angles; carbon oxide nano-angles were dispersed in phosphate buffer, sonicated in an ice bath, and homogenized under high pressure to obtain carbon oxide nano-angle dispersion. Preparation of dopamine-grafted sodium alginate: Sodium alginate was dissolved in buffer solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) and dopamine hydrochloride were added sequentially. The reaction was carried out under an inert gas atmosphere in the dark with stirring. The pH was adjusted during the reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain dopamine-grafted sodium alginate. Preparation of aminophenylboronic acid-grafted hyaluronic acid: Hyaluronic acid was dissolved in water, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM) was added and stirred to activate the reaction solution; 6-aminobenzo[c][1,2]oxaborphane-1(3H)-ol hydrochloride (ABO) was dissolved in dimethyl sulfoxide and added dropwise to the reaction solution. The pH was adjusted, the reaction was stirred, dialyzed, and freeze-dried to obtain aminophenylboronic acid-grafted hyaluronic acid. Preparation of carbon oxide nano-angle composite hydrogel: Dopamine-grafted sodium alginate was dissolved in phosphate buffer and mixed with carbon oxide nano-angle dispersion to obtain a first mixture; aminophenylboronic acid-grafted hyaluronic acid was dissolved in phosphate buffer to obtain a second mixture; the first mixture and the second mixture were stirred and mixed to obtain carbon oxide nano-angle composite hydrogel.

[0009] In one embodiment, in the preparation of the carbon oxide nanoparticle dispersion, the ratio of the single-walled carbon nanoparticle to the concentrated nitric acid is (1~5) mg: 1 mL. The reflux reaction is carried out at a temperature of 70-80°C for 1-3 hours.

[0010] In one embodiment, the concentration of concentrated nitric acid in the preparation of the carbon oxide nanoparticle dispersion is 68-70 wt%.

[0011] In one embodiment, the carbon oxide nanoparticles in the preparation of the carbon oxide nanoparticle dispersion have a particle size of 100 nm.

[0012] In one embodiment, in the preparation of dopamine-grafted sodium alginate, the mass ratio of sodium alginate: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide: N-hydroxysuccinimide: dopamine hydrochloride is 1:(0.95~1.05):(0.55~0.65):(0.95~1.05).

[0013] In one embodiment, the temperature of the light-protected stirring reaction in the preparation of dopamine-grafted sodium alginate is 22-25°C.

[0014] In one embodiment, in the preparation of dopamine-grafted sodium alginate, the pH is adjusted to 5.3-5.7.

[0015] In one embodiment, in the preparation of dopamine-grafted sodium alginate, the dialysis is performed by first dialysis with deionized water containing NaCl, and then dialysis with deionized water.

[0016] In one embodiment, in the preparation of aminophenylboronic acid-grafted hyaluronic acid, the mass ratio of the hyaluronic acid to the 6-aminobenzo[c][1,2]oxaborane-1(3H)-ol hydrochloride (ABO) is 1:(0.23~0.27).

[0017] In one embodiment, in the preparation of hyaluronic acid grafted with aminophenylboronic acid, the stirring activation time is 20-40 min.

[0018] In one embodiment, in the preparation of aminophenylboronic acid-grafted hyaluronic acid, the mass ratio of the hyaluronic acid to the 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride is 1:(0.72~0.78).

[0019] In one embodiment, in the preparation of aminophenylboronic acid-grafted hyaluronic acid, the pH is adjusted to 6.3-6.7.

[0020] In one embodiment, in the preparation of the carbon oxide nano-angle composite hydrogel, the volume ratio of the first mixture to the second mixture is 1:1.

[0021] In one embodiment, the contents of carbon oxide nano-angle composite hydrogel, dopamine-grafted sodium alginate, and aminophenylboronic acid-grafted hyaluronic acid are (0.006~0.025) wt%, (4~6) wt%, and (2~3) wt%, respectively.

[0022] A third aspect of the present invention also provides the application of the above-described carbon oxide nano-angle composite hydrogel or the carbon oxide nano-angle composite hydrogel obtained by the above preparation method in the preparation of diabetic wound repair dressings.

[0023] In a fourth aspect, the present invention also provides a diabetic wound repair dressing, comprising the above-described carbon oxide nano-angle composite hydrogel or the carbon oxide nano-angle composite hydrogel prepared by the above-described preparation method.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention constructs a hydrogel system with a hierarchical energy dissipation mechanism through multiple synergistic interactions between carbon oxide nanoangles and a dynamic covalent cross-linked network. Specifically, the carbon oxide nanoangles act as multifunctional nano-crosslinking centers, forming multiple physical crosslinks with dopamine-grafted sodium alginate and aminophenylboronic acid-grafted hyaluronic acid, including hydrogen bonds, π-π stacking, and electrostatic interactions. Simultaneously, dynamic borate ester bonds endow the network with reversible covalent crosslinking properties. The synergistic effect of these multiple crosslinks significantly enhances the hydrogel's mechanical strength and toughness, tissue adhesion properties, and self-healing ability.

[0025] 2. This invention utilizes the synergistic effect of phenylboronic acid ester bonds and Schiff base bonds to endow the hydrogel with dual responsive degradation behavior to high concentrations of glucose and excessive reactive oxygen species in the wound microenvironment. Simultaneously, it can also reshape the local microenvironment of diabetic wounds by consuming glucose and reactive oxygen molecules in the microenvironment through dynamic reversible reactions.

[0026] 3. This invention reveals for the first time that carbon oxide nanoparticles restore mitochondrial function (membrane potential, ATP synthesis and oxidative phosphorylation) of endothelial cells under high glucose conditions by activating the AMPK signaling pathway, thereby promoting cell migration and tube formation, forming a cascade mechanism of "signaling pathway activation-mitochondrial repair-cell function recovery", which significantly accelerates the healing of diabetic wounds.

[0027] 4. The hydrogel of this invention exhibits excellent biocompatibility and in vivo safety. Live / dead cell staining results show that after co-culturing the hydrogel with HUVECs, Raw264.7, and L929 cells, the cell viability remained above 85%, and the hemolysis rate was <5%. No pathological abnormalities were observed in H&E staining of major organs in vivo, meeting the safety standards for biomedical materials.

[0028] 5. The preparation method of this invention is simple and mild (room temperature, near-neutral pH, aqueous phase reaction), requires no complicated equipment, and the hydrogel can be rapidly formed within 30 seconds to 5 minutes. It has good stability and repeatability and is suitable for large-scale production. Attached Figure Description

[0029] Figure 1 A schematic diagram illustrating the preparation process and mechanism of action of carbon oxide nano-angle composite hydrogel; Figure 2 Synthesis and characterization of carbon oxide nanoangles: A is a schematic diagram of the preparation process; B is a transmission electron microscope image; C is a particle size distribution map; D is a Zeta potential map; E is a high-angle annular dark-field STEM image and elemental distribution map; FH is an XPS spectrum; I is a Fourier transform infrared spectrum; J is an X-ray diffraction pattern; K is a Raman spectrum. Figure 3 Synthesis, characterization, and biocompatibility of carbon dioxide nanoparticle-based composite hydrogels: A shows a schematic diagram of the cross-linking strategy; B shows the sol-gel transition process; C shows the Fourier transform infrared spectrum; D shows the Zeta potential diagram; E shows the scanning electron microscope image; F shows the hydrogel pore size statistics; G shows the full X-ray photoelectron spectroscopy spectrum; H shows the elemental distribution diagram; IJ shows live / dead cell staining; KL shows the hemolysis experiment. Figure 4 The multifunctional properties of carbon dioxide nano-angle composite hydrogels are as follows: AC represents tensile properties; DF represents compressive properties; GH represents pigskin adhesion properties; I represents macroscopic visualization; J represents shape fidelity testing; KL represents rheological strain scanning and frequency scanning testing; M represents step strain testing; N represents the macroscopic self-healing process of cutting hydrogel blocks; and OQ represents degradation testing in different environments. Figure 5The in vivo wound healing performance of carbon dioxide nano-angle composite hydrogel in diabetic patients is shown in the following figures: A represents the in vivo experimental protocol; B represents a representative photograph of wound healing; C represents a simulated wound closure diagram; DE represents HE staining and Masson staining; F represents the wound healing rate; G represents the re-epithelialization rate; and H represents the collagen deposition rate. Figure 6 Bioinformatics analysis of wound tissue in the presence of carbon dioxide nanoparticle-coated composite hydrogel: A is Venn diagram; B is GSEA analysis; C is volcano diagram; D is GO enrichment analysis; E is KEGG enrichment analysis. Figure 7 Study on the mechanism of carbon dioxide nano-angles promoting angiogenesis (Part 1): AC represents immunofluorescence staining and quantitative analysis of CD31 (endothelial cells) and α-SMA (peripheral cells); DF represents tube formation assay; GJ represents migration assay; KM represents cytoskeleton fluorescence staining and quantitative analysis; N represents VEGF expression; O represents a schematic diagram of the mechanism (promoting endothelial migration). Figure 8 Study on the mechanism of carbon dioxide nanoangle promoting angiogenesis (Part 2): A is Venn diagram; B is GO enrichment analysis; C is a circular heatmap of mitochondrial DNA-encoded gene expression; D is a chord diagram; E is Mitotracker Green staining; F and H are JC-1 staining and quantitative analysis; G and I are MitoSOX staining and quantitative analysis; J is ATP level; K is NAD+ / NADH ratio; L is mitochondrial OXPHOS subunit expression; M is a schematic diagram of the mechanism (restoration of energy metabolism). Figure 9 Study on the mechanism of carbon dioxide nanoparticles promoting angiogenesis (Part 3): A shows KEGG pathway analysis; B shows AMPK, AKT, LATS1 / 2 phosphorylation and total protein levels; C shows AMPK phosphorylation and mitochondrial OXPHOS subunit expression; D and F show JC-1 staining; E and G show MitoSOX staining; H shows Mitotracker staining; I shows cytoskeleton fluorescence staining; J shows VEGF expression; and KM shows tube formation assay and quantitative analysis. Figure 10 The graph shows the cell proliferation activity assay of carbon oxide nanoparticles: A represents cell activity after treatment with carbon oxide nanoparticles under normal culture conditions; B represents cell activity after treatment with carbon oxide nanoparticles under high glucose culture conditions. Figure 11 The staining images of live / dead cells in the carbon oxide nano-angle composite hydrogel are shown, with a scale bar of 100 μm. Figure 12 The results of H&E staining of major organs are shown, with a safety assessment diagram and a scale bar of 200 μm. Figure 13 This is a diagram of the wet adhesion test of biological tissues; Figure 14Rheological test diagrams of carbon oxide nano-angle composite hydrogels with different contents: where A is strain scan; B is frequency scan; C is step strain test; Figure 15 The graph shows the ROS scavenging ability of composite hydrogels containing (or without) carbon oxide nanoangles: A represents fluorescence staining with a scale bar of 100 μm; B represents quantitative analysis of fluorescence intensity. Figure 16 The in vivo wound healing evaluation diagrams for nano-angle composite hydrogels with different carbon oxide contents are shown below: A is a representative photograph of wound healing; B is the wound healing rate. Detailed Implementation

[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0033] Example I. Experimental Methods In the in vitro cell culture and treatment method of this invention, umbilical vein endothelial cells (HUVECs) are cultured in an endothelial cell culture system using a medium containing 5% fetal bovine serum (FBS) at 37°C and 5% CO2. To establish a high glucose injury model, cell samples are pretreated with high glucose medium (35 mM) for 48 hours before further incubation. Cells in the normal group (untreated group) are cultured in normal glucose medium (5.5 mM). After initial incubation, the medium is changed: the high glucose group receives fresh high glucose medium, while the treatment group cells are cultured for 48 hours with 2 µg / mL carbon dioxide nanoparticles added to the high glucose medium. For AMPK inhibition experiments, cells are pretreated with a 2 µM AMPK inhibitor (dosomofen) for 30 minutes. Subsequently, cells are cultured under conditions containing a 1 µM AMPK inhibitor.

[0034] II. This invention provides a method for preparing carbon oxide nano-angle composite hydrogel, comprising the following steps: 1. Preparation of carbon oxide nano-angle dispersion (1) The original single-walled carbon nanoparticle powder was added to 68-70wt% concentrated nitric acid at a mass-volume concentration of 1-5 mg / mL and refluxed at 70-80℃ for 1-3 hours to obtain the oxidation product; (2) Centrifuge at high speed, discard the supernatant waste acid, and collect the bottom precipitate; (3) Wash the precipitate with deionized water until the supernatant is neutral; (4) Collect and dry the precipitate to obtain purified carbon oxide nanoparticles; (5) Disperse the purified carbon oxide nanoparticles at a concentration of 1 mg / mL in phosphate buffer; (6) The carbon oxide nano-angle dispersion is subjected to ultrasonic treatment; (7) The dispersion is subjected to high-pressure homogenization.

[0035] 2. Synthetic dopamine-grafted sodium alginate (1) Sodium alginate was dissolved in MES buffer to prepare a 1 wt% solution; (2) Add EDC, NHS and dopamine hydrochloride in sequence. The mass ratio of sodium alginate, EDC, NHS and dopamine hydrochloride is 1:(0.95~1.05):(0.55~0.65):(0.95~1.05). Stir to dissolve. (3) Under an inert atmosphere, stir the reaction at room temperature in the dark for 24 hours, and maintain the pH at 5.3~5.7 with NaOH or HCl; (4) After the reaction is complete, the reaction solution is transferred to a dialysis bag (molecular weight cutoff 3.5kDa), first dialyzed with deionized water containing NaCl, and then dialyzed with deionized water. (5) Freeze-dry to obtain dopamine-grafted sodium alginate.

[0036] 3. Synthesis of hyaluronic acid grafted with aminophenylboronic acid (1) Hyaluronic acid (molecular weight 1.5~2.5 MDa) was dissolved in deionized water to prepare a 1 wt% solution; (2) Add DMTMM and stir to activate for 20-40 minutes. The mass ratio of hyaluronic acid to DMTMM is 1:(0.72-0.78). (3) Add a dimethyl sulfoxide solution containing ABO, with the mass ratio of hyaluronic acid to ABO being 1:(0.23~0.27). (4) Adjust the pH to 6.3-6.7 with NaOH or HCl, and stir the reaction at room temperature for 24 hours; (5) Transfer the reaction solution to a dialysis bag (molecular weight cutoff 3.5kDa) and dialyze with deionized water; (6) Freeze-dry to obtain hyaluronic acid grafted with aminophenylboronic acid.

[0037] 4. Mix the carbon dioxide nanoparticle dispersion, dopamine-grafted sodium alginate, and aminophenylboronic acid-grafted hyaluronic acid to form a hydrogel. (1) The dopamine-grafted sodium alginate was dissolved in phosphate buffer and mixed with the carbon dioxide nanoparticle dispersion. The concentration of the dopamine-grafted sodium alginate was 5 wt%. (2) Dissolve the aminophenylboronic acid-grafted hyaluronic acid in phosphate buffer to prepare a 2.5 wt% solution; (3) Then the two solutions are mixed at a volume ratio of 1:1 at room temperature to form a hydrogel; the concentration of the carbon oxide nano-angle in the hydrogel is 0.006~0.025wt%.

[0038] from Figure 1 As can be seen, the hydrogel of this invention is formed by a covalent-non-covalent dual dynamic network structure, combining carbon dioxide nano-angles with dopamine-grafted sodium alginate and aminophenylboronic acid-grafted hyaluronic acid polymers. This design gives the hydrogel the following properties: (1) It possesses excellent mechanical strength, superior tissue adhesion, self-healing ability, and a dual-response degradation system: it can simultaneously degrade reactive oxygen species and glucose. It is suitable for regulating the microenvironment of hyperglycemic wounds.

[0039] (2) Inside the cell, carbon nanoparticles activate the AMPK signaling pathway, thereby restoring function.

[0040] (3) Carbon oxidized nanoangles can regulate mitochondrial oxidative phosphorylation. This metabolic reprogramming can promote cytoskeleton remodeling, enhance endothelial cell migration and angiogenesis, and ultimately accelerate the healing of diabetic wounds.

[0041] Figure 2 The physicochemical characterization of carbon oxide nanoangles is presented.

[0042] Figure 2 B represents the morphological evolution of carbon nanohorns under transmission electron microscopy (TEM). The original carbon nanohorns in the left image appear as dense, large-scale aggregates. In the right image, after oxidation, the structure on the outer periphery of the polymer undergoes partial defolding, while the inner core retains its original conformation. This morphological evolution stems from the synergistic effect of selective oxidation etching. Figure 2Dynamic light scattering (DLS) analysis results shown in Figure C indicate that oxidation significantly reduces the aggregate size of carbon nanohorns: the average hydrodynamic diameter decreases significantly from approximately 170 nm (carbon nanohorn) to approximately 100 nm (oxidized carbon nanohorn). This size reduction may be attributed to the selective etching removal of carbon atoms by high-concentration nitric acid, and the electrostatic repulsion and enhanced hydrophilicity generated upon the introduction of anionic groups, thereby reducing the aggregation degree of oxidized carbon nanohorns. Figure 2 The Zeta potential measurement results shown in Figure D confirm this mechanism. The value changed significantly from -17mV (carbon nanoangle) to -35mV (carbon oxide nanoangle), indicating a significant increase in the surface negative charge density, which is beneficial to improving dispersion stability.

[0043] The surface chemical properties of carbon oxide nanoangles were further characterized. Figure 2 The STEM-EDS mapping analysis shown in Figure E indicates that oxygen molecules are uniformly distributed within the carbon oxide nano-angle aggregates. Figure 2 As can be seen from F, XPS analysis shows a significant enhancement of the Ols signal and an increased oxygen / carbon atom ratio. From Figure 2 The G signal indicates that the Cl signal shows four distinct components, corresponding to C=C / CC (284.8 eV), CO (286.2 ± 231 eV), C=O (287.8 eV), and carboxyl (289.0 eV). Figure 2 As can be seen from H, the corresponding Ol signal spectrum also shows the same characteristics, with characteristic peaks attributable to C=O (531.2 eV), CO (532.5 eV), and carboxyl (533.8 eV). From Figure 2 As can be seen from I, the FTIR spectrum at 1705 cm⁻¹ -1 A new absorption band appears at 228 cm⁻¹ (C=O stretching vibration peak). -1 (CO stretching vibration peak), and at 1300~1400 cm⁻¹ -1 Enhanced regional absorption confirms the successful introduction of oxygen-containing functional groups that adhere to the carbon oxide nanocorner surface. Figure 2 JK further evaluated the effect of oxidation on the carbon framework structure using XRD. Raman spectroscopy analysis showed that both carbon nanoangle and carbon oxide nanoangle exhibited two broad diffraction peaks at 26° and 43°, corresponding to the (002) and (l00) crystal planes of graphitic carbon, respectively. After oxidation treatment, the intensity of the peaks decreased and the peak shape broadened, indicating a decrease in the order of graphite. Raman spectroscopy showed an increase in the ID / IG ratio, from 1.8 (carbon oxide nanoangle) to 2.0 (carbon oxide nanoangle), confirming the formation of the structure.

[0044] This invention further conducted cell proliferation experiments to verify and screen the biosafe concentration range of carbon dioxide nanoparticles and their regulatory effect on the activity of normal and high-glucose-damaged cells. The corresponding detection results are as follows: Figure 10 As shown in the figure, A represents cell proliferation activity data after intervention with carbon oxide nano-angles in a conventional culture system, used to clarify the cell compatibility of carbon oxide nano-angles in the physiological microenvironment; B represents cell proliferation activity results after treatment with carbon oxide nano-angles in a high-glucose culture system simulating the pathological state of diabetes, directly reflecting the ability of carbon oxide nano-angles to improve high-glucose-induced cell proliferation inhibition and maintain normal cell survival and proliferation, thus providing biological evidence at the cellular level for the biological advantages of the carbon oxide nano-angle composite hydrogel of this invention for the repair of diabetic wounds.

[0045] Example 1 A carbon oxide nano-angle composite hydrogel comprises the following components in mass fractions: 0.025 wt% carbon oxide nano-angles, 5 wt% dopamine-grafted sodium alginate, and 2.5 wt% aminophenylboronic acid-grafted hyaluronic acid; wherein, the dopamine-grafted sodium alginate and the aminophenylboronic acid-grafted hyaluronic acid form a dynamic covalent cross-linked network through Schiff base bonds and borate ester bonds, and the carbon oxide nano-angles form a nano-locked physical cross-link with the dynamic covalent cross-linked network through topological entanglement, hydrogen bonding, electrostatic interaction, and π-π stacking.

[0046] The preparation method includes the following specific steps: (1) Preparation of carbon dioxide nano-angle dispersion 100 mg of raw single-walled carbon nanoparticle powder was added to 50 mL of 70 wt% concentrated nitric acid and stirred under reflux at 80 °C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature. The reaction solution was centrifuged at 35000 × g for 20 minutes using an ultracentrifuge. The supernatant waste acid was discarded, and the bottom precipitate was collected. The precipitate was washed 5 times with deionized water by centrifugation until the pH of the supernatant was close to neutral. Finally, it was washed with anhydrous ethanol. The precipitate was collected and freeze-dried for 24 hours to obtain purified carbon oxide nanoparticles.

[0047] The purified carbon oxide nanoparticles were dispersed in phosphate buffer (pH=7.4) at a concentration of 1 mg / mL to obtain a preliminary suspension. A JY88-IIN probe-type ultrasonic pulverizer was used with 80% amplitude and intermittent ultrasonication (3 seconds on, 1 second off) under ice bath conditions for 10 minutes. After ultrasonication, the nanoparticles were homogenized at 1000 bar for 15 minutes using a high-pressure homogenizer, and the process was repeated 3 times to obtain a uniformly dispersed carbon oxide nanoparticle dispersion. The obtained carbon oxide nanoparticles had a particle size of approximately 100 nm.

[0048] (2) Synthesis of dopamine-grafted sodium alginate Dissolve 1.00 g of sodium alginate in 100 mL of MES buffer (pH ~6.0, 99%). 0.98 g EDC (98.5%), 0.59 g NHS (98%), and 0.98 g dopamine hydrochloride (98%) were added sequentially and stirred until dissolved. Under argon protection, the reaction was carried out at room temperature (approximately 22-25°C) in the dark with magnetic stirring for 24 hours. During the reaction, the pH of the reaction solution was monitored, and the pH was slowly adjusted with 1 M NaOH or HCl at a rate of approximately 0.05 pH units / minute to maintain the pH at 5.4-5.5. After the reaction was completed, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 3.5 kDa). The solution was first dialyzed with deionized water containing 10 mM NaCl for 72 hours, with the dialysate changed every 6 hours. Then, the solution was dialyzed with deionized water for 12 hours, with the dialysate changed every 6 hours. The solution was freeze-dried for 48 hours to obtain dopamine-grafted sodium alginate, which was a light gray, fluffy, flocculent solid.

[0049] (3) Synthesis of hyaluronic acid grafted with aminophenylboronic acid Dissolve 1.00 g of hyaluronic acid (molecular weight 1.5~2.5 MDa) in 95 mL of deionized water; add 0.75 g of DMTMM (97%) and stir vigorously for 30 minutes; dissolve 0.25 g of ABO (97%, molecular weight 185.42) in 5 mL of dimethyl sulfoxide and add it dropwise to the reaction solution; slowly adjust the pH to 6.5 with 1 M NaOH or HCl at a rate of about 0.05 pH units / min, and stir magnetically at room temperature for 24 hours; transfer the reaction solution to a dialysis bag (molecular weight cutoff 3.5 kDa), dialyze with deionized water for 72 hours, changing the dialysate every 6 hours; freeze-dry for 48 hours to obtain aminophenylboronic acid-grafted hyaluronic acid, which is a light yellow blocky solid.

[0050] (4) Preparation of carbon oxide nano-angle composite hydrogel The dopamine-grafted sodium alginate synthesized above was dissolved in phosphate buffer to prepare a concentrated solution; the carbon oxide nanoparticle dispersion (1 mg / mL) prepared above was mixed with the dopamine-grafted sodium alginate concentrated solution so that the mass fraction of dopamine-grafted sodium alginate in the mixed solution was 5 wt% and the final concentration of carbon oxide nanoparticles was 0.25 mg / mL (corresponding to 0.025 wt%), thus obtaining the first mixture.

[0051] The synthesized aminophenylboronic acid-grafted hyaluronic acid was dissolved in phosphate buffer to prepare a 2.5 wt% solution, thus obtaining the second mixture.

[0052] The first mixture and the second mixture were stirred together at a volume ratio of 1:1 at room temperature. Figure 3 As can be seen from B, after thorough mixing, a hydrogel is formed through sol-gel conversion; Figure 3The FTIR spectrum of C confirmed this result, from Figure 3 As can be seen from E, the hydrogel has a three-dimensional porous network structure. Figure 3 As can be seen from F, the average pore size is 89.14 μm (SEM cross-sectional observation). Figure 3 As can be seen from D, the average Zeta potential is 23.78 mV. Figure 3 The XPS spectrum of G further confirmed the successful integration of nitrogen-boron functional groups, which showed obvious C 1s, N 1s, O 1s and B 1s signals, respectively. Figure 3 The elemental distribution diagram of H shows that nitrogen (N), carbon (C), oxygen (O) and other elements are uniformly distributed, and the particles and B particles are uniformly distributed throughout the hydrogel matrix, confirming that it has uniform dispersion characteristics.

[0053] The composite hydrogels of this invention exhibit excellent biocompatibility and in vivo safety. From... Figure 3 I, Figure 11 and Figure 3 As can be seen from the KL results, compared with the untreated group, the live / dead cell staining results showed that after co-culturing the hydrogel with HUVECs, Raw264.7, and L929 cells, the cell viability was maintained above 85%, and the hemolysis rate was <5%. Figure 3 J showed that cells maintained a survival rate of >90% after 24 hours of exposure to this condition. Figure 12 It can be seen that no pathological abnormalities were found in the H&E staining of the major organs in the body, which meets the safety standards for biomedical materials.

[0054] from Figure 4 As can be seen from the AC, the tensile strength of the composite hydrogel in this embodiment is approximately 6.22 MPa, and the tensile modulus is approximately 15.95 MPa; both compressive strength and modulus are improved. Figure 4 The DF (dielectric strength) indicates a compressive strength of approximately 1.38 MPa, meaning the material is not prone to breakage or excessive deformation in dynamic environments. It can withstand a certain load without collapsing, yet it is not so hard as to cause mechanical damage to fragile granulation tissue. Figure 4 The GH results show that the pigskin overlap shear test indicates an adhesion strength of approximately 15.08 kPa, higher than that of the hydrogel without carbon dioxide nanoparticles, indicating its effective fixation on moist diabetic wounds. Furthermore, Figure 13 The image visually demonstrates the adhesion ability of hydrogels to different fresh, moist organ tissues.

[0055] The composite hydrogel in this embodiment exhibits excellent microstructural stability. For example... Figure 4 As shown in Figure I, the composite hydrogel did not fracture during the tensile test and could adapt to curved surfaces (such as finger joints). When bent and twisted, it maintained its structural integrity and no visible cracks appeared. Figure 4 The results showed that the composite hydrogel could be directly extruded through a needleless syringe, forming a stable “SMU” structural pattern on the substrate without obvious collapse or diffusion.

[0056] from Figure 4 As can be seen from K, strain scanning experiments show that the critical strain (gel-sol transition point) of the composite hydrogel is 949%, exhibiting excellent resistance to deformation; from Figure 4 As can be seen from L, the frequency scanning results show that the composite hydrogel maintains a high storage modulus throughout the frequency range of 1~100 rad / s and exhibits excellent stability in the high-frequency region; from Figure 4 As can be seen from M, the step strain test shows that the composite hydrogel has rapid self-healing properties and a modulus recovery rate of over 90%.

[0057] like Figure 4 As shown in N, the two separate halves of the composite hydrogel fused seamlessly and remained stable upon contact, and no fracture occurred during the tensile test.

[0058] from Figure 4 The OQ results show that the composite hydrogel of this invention exhibits dual glucose and reactive oxygen species (ROS) responsive degradation behavior: it remains stable in phosphate buffered saline (PBS) but shows significant degradation in both glucose and ROS conditions, with the fastest degradation observed under combined glucose and ROS treatment. In the high-glucose, high-ROS diabetic microenvironment, the borate ester bonds in the hydrogel undergo reversible breakage, gradually loosening the structure and exposing more active sites, thereby further enhancing its ability to capture and scavenge excess ROS. DCFH assay results show that the intracellular ROS fluorescence intensity is 58.47% under high-glucose, high-ROS culture conditions; from Figure 15 As can be seen, the reactive oxygen species (ROS) level was significantly reduced to 9.32% after treatment with the composite hydrogel of this invention, compared to 2.23% under normal culture conditions. This indicates that the composite hydrogel of this invention can reduce the ROS level in a high-sugar, high-ROS environment by more than 84% and bring it close to normal physiological levels, thereby effectively alleviating oxidative stress damage at the wound site in diabetic patients.

[0059] To further evaluate the in vivo efficacy of Example 1 in treating diabetic wounds, according to Figure 5 Animal experiments were conducted according to protocol A shown in Figure A. Control group 2 consisted of healthy controls. Figure 5 BC analysis showed that, compared with the diabetic control group (control group 1), the group without composite hydrogel carrier (comparative example 1), and the commercial hydrogel group (comparative example 2), the composite hydrogel (Example 1) significantly accelerated wound closure. Histological analysis was performed on days 7 and 14 using hematoxylin-eosin (H&E) staining and Masson's staining methods, respectively. Figure 5DE studies showed that on day 7, composite hydrogel treatment reduced the acute inflammatory cellular response and increased the density of newly formed microvessels in granulation tissue. From Figure 5 The effect of the composite hydrogel of this invention on significantly promoting wound healing in diabetic mice was quantified by the wound healing rate. The wound healing rate reached 77.31% on day 7 and 99.54% on day 14. Furthermore, Figure 5 G and H were used to quantify wound healing quality from epithelialization rate and relative collagen deposition rate, respectively. On day 14, the epithelialization rate of wound tissue in the Example 1 group reached 99.79%, and the relative collagen deposition rate reached 79.09%, which was significantly better than other groups except the healthy control group (control group 2).

[0060] To further investigate the therapeutic advantages of introducing carbon oxide nanoparticles as a key component of the composite hydrogel in this embodiment, differentially expressed genes (DEGs) were screened for subsequent analysis. Figure 6 The Venn diagram shown in Figure A identified 1952 overlapping differentially expressed genes. The upregulation / downregulation trends of these genes in Example 1 and Comparative Example 1 were determined by... Figure 6 The C-volcano map shows... Figure 6 The GSEA analysis shown in Figure B indicates that angiogenesis is a key mechanism behind the therapeutic advantages of composite hydrogels. Figure 6 The GO enrichment analysis shown by D indicates that DEGs are enriched in vascular-related biological processes. Figure 6 KEGG pathway enrichment analysis of E also showed that DEGs were enriched in multiple signaling pathways closely related to angiogenesis and cell migration, including the VEGF signaling pathway, PI3K-Akt signaling pathway, HIF-1 signaling pathway, regulation of the actin cytoskeleton, and calcium signaling pathway. These results provide transcriptomic evidence that oxidized carbon nanohorns can promote angiogenesis in diabetic wounds.

[0061] Figure 7 The AC further provided tissue-level evidence for the above bioinformatics analysis results. On day 7 post-treatment, immunofluorescence staining was performed on wound tissue sections from different groups using CD31 (an endothelial cell marker) and α-SMA (a perivascular cell marker). Compared with the healthy control group (control group 2), the untreated diabetic wound (control group 1) showed severe vascular structure destruction, with almost complete lumen loss and only a very small number of intact lumens remaining. No significant improvement was observed in the commercial dressing group (comparative example 2) and the hydrogel carrier group alone (comparative example 1). In contrast, the carbon oxide nanoparticle-composite hydrogel group (Example 1) demonstrated a significant vascular regeneration and maturation process.

[0062] To elucidate the cellular basis of this effect, human umbilical vein endothelial cells (HUVECs) were cultured under high glucose conditions. Figure 7The tube formation experiments of DF showed that carbon oxide nanocorner components can directly reverse angiogenesis disorders caused by high glucose treatment. Figure 7 Scratch assays with GI confirmed that high sugar treatment significantly inhibited HUVEC migration; however, carbon oxide nanocorner treatment significantly accelerated HUVEC migration. Figure 7 Transwell migration experiments with H and J further confirmed the migration-promoting effect of carbon oxide nanocorner treatment. Figure 7 K showed that high sugar treatment conditions led to the depolymerization and structural disorder of F-actin, while carbon oxide nanohorns rescued this effect. Figure 7 Quantitative analysis of L showed that carbon oxide nanocorner treatment could enhance the formation level of F-actin. Figure 7 The Western blot results shown in M ​​further confirmed the increased expression of VEGF (a key regulator of tubular structure formation) (the protein loading amount was calibrated using the internal reference gene β-actin). Figure 7 The N in the diagram represents a schematic of this mechanism, demonstrating that carbon oxide nanohorns can promote F-actin polymerization and stress fiber formation, and reverse the high glucose-induced impairment of endothelial cell migration and tubular structure formation, thereby endowing the hydrogel with a strong pro-angiogenic ability.

[0063] To further elucidate the biomolecular regulatory targets of carbon oxide nanoparticles as a key component, we identified genes that were significantly suppressed under high glucose conditions (compared to the untreated group), as well as the groups treated with carbon oxide nanoparticles (comparison between carbon oxide nanoparticles + high glucose treatment and high glucose treatment), and performed an intersection analysis of the two sets of data. Figure 8 Venn diagram analysis of A showed that 579 of them were overlapping differentially expressed targets. Figure 8 GO enrichment analysis of B clearly points to mitochondrial biological mechanisms: "oxidative phosphorylation" and "mitochondria" showed significant enrichment at both the biological process and cellular levels. Figure 8 The C-values ​​showed that oxidized carbon nanohorns restored the expression of 12 mitochondrial oxidative phosphorylation (OXPHOS) subunit genes. Notably, these subunits are encoded by mitochondrial DNA rather than nuclear DNA, suggesting that oxidized carbon nanohorns have a crucial impact on mitochondrial homeostasis. Figure 8 KEGG pathway analysis of oxidized carbon nanohorns revealed significant enrichment of OXPHOS, AMPK, FoxO, PI3K-Akt, and cell cycle pathways, all of which are closely related to energy metabolism. These findings suggest that the tube-promoting function of oxidized carbon nanohorns may stem from their ameliorative effect on mitochondrial energy metabolism.

[0064] Further experimental evidence supports the above inferences. Figure 8The results showed that high sugar levels can induce mitochondrial fragmentation, while carbon dioxide nanohorn treatment can restore the mitochondrial network structure. Figure 8 JC-1 staining of F and H showed that high glucose can lead to loss of mitochondrial membrane potential (ΔΨm), and this index was significantly reversed after treatment with carbon oxidized nanohorns (3.97±1.19). Figure 8 G and I data showed that carbon dioxide nano-angle treatment can reduce mitochondrial oxidative stress levels. Figure 8 J and K showed that carbon oxide nanoangles rescued the reduced intracellular ATP levels and NAD+ / NADH ratio under high glucose conditions. Figure 8 Western blot analysis of L confirmed the corresponding increase in the protein level of the OXPHOS subunit, consistent with the transcriptomic results. Figure 8 The M-axis visualized this "mitochondrial function-energy supply" axis, through which carbon dioxide nanoangles provide bioenergy support for endothelial cells to perform their physiological functions.

[0065] To identify the key upstream signaling pathways mediating pro-angiogenic effects, we first performed KEGG pathway enrichment analysis on the samples rescued by carbon oxide nanoparticles in the examples. Figure 9 A represents a gene pathway whose expression is downregulated under high-glucose culture conditions and upregulated after treatment with carbon dioxide nanoparticles. Figure 9 B was analyzed using Western blotting to determine which pathway was dominant. The results showed that the protective effect of carbon oxide nanohorns was dominated by the p-AMPK / AMPK (AMPK pathway), while the p-AKT / AKT (PI3K-AKT pathway) and p-LATS1 / 2 / LATS1 / 2 (Hippo pathway) showed no significant changes. Figure 9 The C values ​​showed that, after AMPK inhibition, the expression of OXPHOS-related subunits induced by carbon oxide nanohorns in the examples was downregulated. Figure 9 The DH study demonstrated that carbon dioxide nanoparticles rescue mitochondrial function and network morphology by upregulating the AMPK pathway. Figure 9 The IM further indicates that inactivation of the AMPK pathway will inhibit the regulatory effect of carbon oxidized nanohorns on endothelial cell cytoskeleton rearrangement and angiogenesis.

[0066] In summary, the carbon oxide nanoparticle-based hydrogel of this invention possesses excellent mechanical properties, self-healing properties, tissue adhesion, and glucose / ROS dual-response degradation performance, while meeting biosafety standards. It can remove excess reactive oxygen species from wounds, alleviate oxidative stress in diabetic wounds, efficiently accelerate wound healing, and promote collagen deposition and epithelial regeneration. Mechanistically, the carbon oxide nanoparticles activate the AMPK pathway to repair mitochondria damaged by high glucose, remodel the endothelial cell cytoskeleton, upregulate angiogenesis-related genes, and significantly induce microvascular angiogenesis, achieving multi-dimensional synergistic repair of chronic diabetic wounds.

[0067] Example 2 The carbon oxide nano-angle composite hydrogel in this embodiment has the same composition and preparation method as in Example 1, except that the content of carbon oxide nano-angles in the carbon oxide nano-angle composite hydrogel in this embodiment is 0.0125wt%.

[0068] In preparing the carbon oxide nano-angle composite hydrogel, the carbon oxide nano-angle dispersion was mixed with a dopamine-grafted sodium alginate concentrate solution to achieve a final concentration of carbon oxide nano-angles of 0.125 mg / mL (corresponding to 0.0125 wt%) in the mixed solution. The remaining steps were the same as in Example 1. Figure 3 As can be seen from F, the average pore size of the prepared composite hydrogel is 120.84 μm. Figure 3 As can be seen from D, the average value of the Zeta potential is 26.15 mV.

[0069] from Figure 14 As can be seen from A, the critical strain of the composite hydrogel of this invention is 705%, exhibiting strong resistance to deformation; from Figure 14 As can be seen from B, frequency scanning shows a slight fluctuation in storage modulus, initially increasing and then decreasing with increasing frequency, but the complete gel network structure is still maintained; from Figure 14 As can be seen from C, the step strain test shows that it can still maintain a high modulus recovery rate after undergoing multiple failure-recovery cycles; from Figure 4 As can be seen from B, in terms of mechanical properties, the average tensile strength of the composite hydrogel is 4.60 MPa. Figure 4 As can be seen from C, the average tensile modulus is 11.43 MPa; from Figure 4 As can be seen from E, the average compressive strength is 1.10 MPa; from Figure 4 As can be seen from H, the average adhesion strength is 12.65 kPa, exhibiting balanced reinforcement characteristics and load-bearing capacity.

[0070] from Figure 16 In vivo wound healing experiments showed that the composite hydrogel promoted wound repair, but the effect was weaker than that in Example 1.

[0071] Example 3 The carbon oxide nano-angle composite hydrogel in this embodiment has the same composition and preparation method as in Example 1, except that the content of carbon oxide nano-angles in the carbon oxide nano-angle composite hydrogel in this embodiment is 0.00625wt%.

[0072] In preparing the carbon oxide nano-angle composite hydrogel, the carbon oxide nano-angle dispersion was mixed with a dopamine-grafted sodium alginate concentrate solution to achieve a final concentration of carbon oxide nano-angles of 0.0625 mg / mL (corresponding to 0.00625 wt%) in the mixed solution. The remaining steps were the same as in Example 1. Figure 3 As can be seen from F, the average pore size of the prepared composite hydrogel is 155.32 μm. Figure 3 As can be seen from D, the average value of the Zeta potential is 30.70 mV.

[0073] The composite hydrogel of this invention exhibits a stable network structure. Figure 14 As can be seen from A, rheological tests show that its critical strain is 519%; from Figure 14 As can be seen from B, frequency scanning indicates that its energy storage modulus is always greater than its loss modulus; from Figure 14 As can be seen from C, the step strain test shows that it has a certain self-healing ability; from Figure 4 As can be seen from B, in terms of mechanical properties, its average tensile strength is 3.72 MPa. Figure 4 As can be seen from C, the average tensile modulus is 8.89 MPa; from Figure 4 As can be seen from E, the average compressive strength is 0.69 MPa; from Figure 4 As can be seen from H, the average adhesion strength is 8.65 kPa.

[0074] from Figure 16 As can be seen from the in vivo wound healing experiment, the composite hydrogel of the present invention can promote wound healing, but the stability of the effect is worse than that of Examples 1 and 2.

[0075] As can be seen from the above embodiments, the carbon oxide nano-angle composite hydrogel of the present invention, through a dual structural design of dynamic covalent cross-linked network and nano-locking physical cross-linking, achieves synergistic effects of mechanical enhancement, glucose / ROS dual-responsive degradation, anti-oxidation, and angiogenesis promotion. The results of the embodiments show that the composite hydrogel network structure of the present invention is stable and reliable, possessing excellent mechanical properties and a significant healing-promoting effect. This invention overcomes the limitations of traditional dressings in terms of single function and insufficient mechanical properties, providing an intelligent and precise treatment strategy for the efficient repair of difficult-to-heal diabetic wounds.

[0076] To further verify the superior performance of the carbon oxide nano-angle composite hydrogel of the present invention, the following comparative examples were designed and systematic comparative tests were conducted.

[0077] Comparative Example 1 (AH) A hydrogel without carbon oxide nano-angles is prepared in the same way as in Example 1, but carbon oxide nano-angles are not added in step (4), and the remaining steps remain unchanged.

[0078] Comparative Example 2 3M Tegaderm is commonly used in business TM Hydrocolloid Thin Dressing, 10cm x 10cm, use as per instructions.

[0079] Implementation effect evaluation 1. Mechanical performance testing Tensile tests were conducted on a uniaxial testing machine (Dongguan Kejian) using dumbbell-shaped specimens (gauge length 35 mm, width 6 mm, thickness 2 mm) at a tensile rate of 10 mm / min until fracture. Compression tests were conducted on a universal testing machine (Shenzhen Sansi) using cylindrical specimens (diameter 10 mm, height 6 mm) at a compression rate of 5 mm / min until 80% strain was achieved. Tensile / compression moduli were calculated based on the initial linear region (0–10% strain).

[0080] 2. Adhesion test The adhesion between the hydrogel and tissue was evaluated using a fresh pigskin strip (30mm × 10mm × 0.3mm) with an overlap shear test. The adhesion area was 16mm². 2 (100 μL hydrogel). Tests were conducted on the Shenzhen Sansi universal testing system at a rate of 5 mm / min. The adhesive shear strength (τ) was calculated using the formula: τ = Fmax / A, where Fmax is the maximum load (N) and A is the overlap area (m²). 2 ), τ is in Pa.

[0081] 3. Rheological testing Rheological measurements were performed using a rotational rheometer (Thermo Fisher Scientific, Germany) with a 20 mm parallel plate. Hydrogel discs (20 mm in diameter, 2 mm thick) were tested at 25°C. Frequency scans (1–100 rad / s) were performed at 1% strain. Strain scans (1–1000%) were performed at 10 rad / s. Self-healing capability was assessed using alternating step strain tests (1% and 800% strain, 1 Hz, 3 cycles), monitoring the recovery of G' and G''.

[0082] 4. Glucose and ROS-responsive degradation experiment The degradation behavior of the hydrogel was evaluated in four different media: PBS (pH=7.4), glucose (50 mM), H2O2 (1 mM), and mixtures thereof. Pre-weighed hydrogel samples (W0) were immersed in 2 mL of each medium at 37°C. At predetermined time intervals (0–48 h), the supernatant was collected and replaced with fresh medium. The concentration of the released components was quantified by measuring the absorbance at 320 nm using a microplate reader (BioTek, USA). The cumulative degradation rate was calculated using the formula: W... released / W0×100%, where W released From a pre-established calibration curve.

[0083] 5. ROS removal capability test Endothelial cells were pretreated with high glucose and incubated with 100 μM H2O2 at 37°C in the dark for 1.5 h. Then, DCFH-DA (Beyotime, China) and Hoechst33342 (Solepro, China) were added and incubated for 30 min. Fluorescence images were acquired using an inverted fluorescence microscope (LeicaSTELLARIS5, Germany) (DCF: Ex / Em = 488 / 525 nm; Hoechst33342: Ex / Em = 350 / 461 nm).

[0084] 6. Internal wound healing effect test Animal Model: A diabetic model was induced in male C57BL / 6 mice (6-8 weeks old, weighing 20-25g) using streptozotocin (50mg / kg, dissolved in citrate buffer, administered intraperitoneally every other day on an empty stomach, for a total of 5 times). Successful establishment of the diabetic model was confirmed by a fasting blood glucose level ≥16.8mmol / L for three consecutive days. Mice were anesthetized, their backs were shaved and disinfected, and full-thickness wounds were created using an 8mm diameter skin punch. Mice were randomly divided into 5 groups (n=5 / group): diabetic control group (control group 1), healthy control group (control group 2), comparative example group 1, comparative example group 2, and example 1 group. Dressings were changed every 3 days until day 14.

[0085] Wound healing rate determination: Wound photographs were taken on days 0, 3, 7, 10, and 14, and the wound area was measured using ImageJ software. Healing rate calculation formula: Wound healing rate (%) = (A0 - A...) t ) / A0×100%. Where A0 is the initial wound area, A t The wound area at a specified time point.

[0086] On days 7 and 14 post-modeling, a random number of mice were euthanized, and wound tissue was harvested. The tissue was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and cut into 5 μm thick sections. The sections were stained with hematoxylin and eosin (H&E) to observe general morphology, and Masson's trichrome staining to detect collagen deposition. Reepithelialization rate was measured using ImageJ software based on H&E-stained sections. Based on Masson's trichrome-stained sections, the proportion of blue-stained area to the total wound area was used to quantify the collagen deposition area.

[0087] Table 1 shows a comparison of the mechanical properties, microenvironment response, and regulation performance between Example 1 and Comparative Example 1.

[0088] Table 1 Comparison of mechanical properties, microenvironment response, and regulation performance between Example 1 and Comparative Example 1

[0089] The comparison results of in vivo wound healing effects between the examples and the comparative examples are shown in Table 2.

[0090] Table 2 Comparison of in vivo wound healing effects among different groups of samples

[0091] Based on the above comparative test results, the following analytical conclusions can be drawn: 1. Mechanical Properties, Self-Healing, and Structural Stability: The carbon oxide nano-angle composite hydrogel of the present invention (Example 1) significantly outperforms Comparative Example 1 (without carbon oxide nano-angles) in terms of mechanical strength, deformation resistance, and self-healing efficiency. This is mainly attributed to the fact that the carbon oxide nano-angles, as multifunctional nanofillers, form stable interfacial coupling with the polymer matrix through multiple non-covalent interactions such as topological entanglement, hydrogen bonding, and π-π stacking. This multi-scale network can preferentially dissipate energy through the breaking of non-covalent bonds under stress, avoiding sudden destruction of the covalent skeleton, and rapidly rebuild the interactions after stress relief, thereby achieving efficient self-healing and significantly improved critical strain. In contrast, Comparative Example 1, lacking such nano-reinforcement and energy dissipation mechanisms, has insufficient mechanical properties and self-repair capabilities.

[0092] 2. Stimulus-Responsive Degradation Behavior: Example 1 exhibits ideal responsive degradation characteristics in a diabetic microenvironment. Under normal physiological conditions, the non-specific degradation rate of Example 1 is significantly lower than that of Comparative Example 1 due to the increased diffusion tortuosity caused by the topological entanglement and interfacial interactions introduced by the carbon oxide nanocorners, demonstrating better structural stability. However, in a simulated diabetic microenvironment characterized by high glucose, oxidative stress, or both, the degradation rate of Example 1 is significantly accelerated, with both response sensitivity and degradation extent far exceeding those of Comparative Example 1. This behavior stems from the reversible breaking mechanism of borate ester bonds in the hydrogel matrix: competitive binding by high glucose leads to a decrease in crosslinking density, and reactive oxygen species attack further blocks bond reformation. The presence of carbon oxide nanocorners does not weaken this responsiveness; instead, it accelerates degradation on demand by regulating the network topology, facilitating the safe removal of the hydrogel from the body after wound healing.

[0093] 3. Extracellular microenvironment improvement and metabolic regulation: Example 1 outperforms Comparative Example 1 in its ability to scavenge intracellular reactive oxygen species (ROS), exhibiting synergistically enhanced antioxidant activity. Example 1 acts as a "smart scavenger," effectively reducing ROS levels in diabetic wounds by consuming excess glucose and ROS locally through borate ester bonds; and restoring intracellular redox homeostasis by releasing carbon dioxide nanoparticles to regulate the intracellular "AMPK-mitochondrial OXPHOS" axis. This dual approach is one of the key features distinguishing Example 1 from ordinary hydrogels, laying a favorable microecological foundation for its promotion of diabetic wound healing.

[0094] 4. In vivo wound healing effect in diabetic mice: In a full-thickness skin defect model in diabetic mice, the wound healing speed, reepithelialization degree, collagen deposition, and neovascularization density of Example 1 were significantly better than those of Comparative Example 1 (AH), Comparative Example 2 (3M commercial dressing), and the diabetic control group (Control Group 1), even reaching levels comparable to the healthy control group. Histological analysis further showed that Example 1 could effectively reduce early inflammatory response, promote granulation tissue maturation, and reconstruct the epidermis. This excellent in vivo efficacy is the result of the synergistic effects of multiple functions, including mechanical adaptation, microenvironment regulation, on-demand degradation, and angiogenesis promotion. In contrast, Comparative Example 1, lacking the enhancement and functionalization of nanofillers, and Comparative Example 2, as a traditional commercial dressing, lacking the ability to actively regulate the pathological microenvironment, both significantly lagged behind Example 1 in overall healing quality.

[0095] In summary, through multi-dimensional comparative testing, the carbon oxide nano-angle composite hydrogel of this invention (Example 1) exhibits significant advantages in mechanical adaptability, microenvironment-responsive degradation, oxidative stress regulation, angiogenesis promotion, and in vivo wound healing. This advantage primarily stems from the synergistic coupling between the carbon oxide nano-angle nanofiller and the AH dynamic covalent network: the carbon oxide nano-angle not only structurally enhances and stabilizes the hydrogel network but also participates in energy dissipation and network reconstruction through multiple intermolecular interactions; while the dynamic covalent chemistry provided by the AH matrix provides the molecular basis for microenvironment-responsive degradation and ROS consumption. Together, they construct an intelligent hydrogel system capable of actively adapting to and regulating the pathological microenvironment of diabetes, fully demonstrating the innovation and practical value of this invention in the field of chronic wound repair in diabetes.

[0096] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0097] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A carbon oxide nano-angle composite hydrogel, characterized in that, The carbon oxide nano-angle composite hydrogel includes carbon oxide nano-angles, dopamine-grafted sodium alginate, and aminophenylboronic acid-grafted hyaluronic acid. According to the concentration ratio, the carbon oxide nanoparticles: the dopamine-grafted sodium alginate: the aminophenylboronic acid-grafted hyaluronic acid is (0.006~0.025) wt% : (4~6) wt% : (2~3) wt%.

2. The carbon oxide nano-angle composite hydrogel according to claim 1, characterized in that, According to the concentration ratio, the carbon oxide nanoparticles: the dopamine-grafted sodium alginate: the aminophenylboronic acid-grafted hyaluronic acid is (0.006~0.025) wt%: 5 wt%: 2.5 wt%.

3. The method for preparing the carbon oxide nano-angle composite hydrogel according to any one of claims 1-2, characterized in that, Includes the following steps: Preparation of carbon oxide nano-angle dispersion: single-walled carbon nano-angles were added to concentrated nitric acid, refluxed, cooled, centrifuged to collect the precipitate, washed, and freeze-dried to obtain carbon oxide nano-angles; carbon oxide nano-angles were dispersed in phosphate buffer, sonicated in an ice bath, and homogenized under high pressure to obtain carbon oxide nano-angle dispersion. Preparation of dopamine-grafted sodium alginate: Sodium alginate was dissolved in buffer solution, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and dopamine hydrochloride were added sequentially. The reaction was carried out under an inert gas environment with stirring in the dark. The pH was adjusted during the reaction. After the reaction was completed, the solution was dialyzed and freeze-dried to obtain dopamine-grafted sodium alginate. Preparation of aminophenylboronic acid-grafted hyaluronic acid: Hyaluronic acid was dissolved in water, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added and stirred to activate the reaction solution; 6-aminobenzo[c][1,2]oxaborphane-1(3H)-ol hydrochloride was dissolved in dimethyl sulfoxide and added dropwise to the reaction solution. The pH was adjusted, the reaction was stirred, dialyzed, and freeze-dried to obtain aminophenylboronic acid-grafted hyaluronic acid; Preparation of carbon oxide nano-angle composite hydrogel: Dopamine-grafted sodium alginate was dissolved in phosphate buffer and mixed with carbon oxide nano-angle dispersion to obtain a first mixture; aminophenylboronic acid-grafted hyaluronic acid was dissolved in phosphate buffer to obtain a second mixture; the first mixture and the second mixture were stirred and mixed to obtain carbon oxide nano-angle composite hydrogel.

4. The preparation method according to claim 3, characterized in that, In the preparation of carbon dioxide nanoparticle dispersion, the ratio of single-walled carbon nanoparticles to concentrated nitric acid is (1~5) mg: 1 mL. The reflux reaction is carried out at a temperature of 70-80°C for 1-3 hours.

5. The preparation method according to claim 3, characterized in that, In the preparation of dopamine-grafted sodium alginate, the mass ratio of sodium alginate: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide: N-hydroxysuccinimide: dopamine hydrochloride is 1:(0.95~1.05):(0.55~0.65):(0.95~1.05).

6. The preparation method according to claim 3, characterized in that, In the preparation of hyaluronic acid grafted with aminophenylboronic acid, the mass ratio of the hyaluronic acid to the 6-aminobenzo[c][1,2]oxaborane-1(3H)-ol hydrochloride is 1:(0.23~0.27).

7. The preparation method according to claim 3, characterized in that, In the preparation of carbon oxide nano-angle composite hydrogels, the volume ratio of the first mixture to the second mixture is 1:

1.

8. The preparation method according to claim 3, characterized in that, In the carbon oxide nano-angle composite hydrogel, the contents of carbon oxide nano-angle, dopamine-grafted sodium alginate, and aminophenylboronic acid-grafted hyaluronic acid are (0.006~0.025) wt%, (4~6) wt%, and (2~3) wt%, respectively.

9. The application of the carbon oxide nano-angle composite hydrogel according to any one of claims 1-2 or the carbon oxide nano-angle composite hydrogel obtained by the preparation method according to any one of claims 3-8 in the preparation of diabetic wound repair dressings.

10. A diabetic wound repair dressing, characterized in that, The carbon oxide nano-angle composite hydrogel includes the carbon oxide nano-angle composite hydrogel according to any one of claims 1-2 or the carbon oxide nano-angle composite hydrogel obtained by the preparation method according to any one of claims 3-8.

Images