Application of a Schiff base-metal ion synergistically cross-linked sodium alginate cryogel in preparation of an infectious diabetic wound repair material

Abstract

The application of a Schiff base-metal ion synergistically cross-linked sodium alginate cryogel in the preparation of an infectious diabetic wound repair material solves the defects of traditional ion cross-linked alginate active components burst release and insufficient long-acting activity. In vitro tests show that the cryogel has excellent mechanical elasticity and controllable slow-release properties, and can release L-arginine, nitric oxide and zinc ions in a long-acting and stable manner. The cryogel has multiple biological activities, such as strong and broad-spectrum antibacterial activity, multi-channel antioxidant activity, regulation of macrophage polarization from pro-inflammatory M1 to repair M2, promotion of fibroblast migration and endothelial angiogenesis. In vivo experiments on a Staphylococcus aureus infected diabetic rat wound model show that the cryogel can remove pathogenic bacteria from the wound, inhibit excessive inflammation, accelerate blood vessel neogenesis and orderly collagen deposition, significantly improve the wound healing rate, and the new tissue has an intact epidermis, mature collagen, no inflammation residue and scar hyperplasia. The cryogel has good clinical conversion prospects in the field of chronic and difficult-to-heal skin damage such as infectious diabetic foot.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering technology, specifically to the application of a Schiff base-metal ion synergistic crosslinked sodium alginate cryogel in the preparation of wound repair materials for infectious diabetic diseases. Background Technology

[0002] Diabetic wounds are one of the most serious complications of diabetes, with about a quarter of diabetic patients developing chronic, non-healing wounds during their disease course. Unlike ordinary wounds, infectious diabetic wounds have an extremely complex pathological microenvironment, mainly manifested as: (1) persistent bacterial infection and biofilm formation, making it difficult for antibiotics to penetrate; (2) high levels of oxidative stress, with excessive reactive oxygen species (ROS) damaging the extracellular matrix and hindering cell migration; (3) chronic inflammatory response, with macrophages remaining stationary in a pro-inflammatory phenotype (M1 type) for a long time and unable to transform into a pro-repair phenotype (M2 type); and (4) impaired angiogenesis, leading to tissue hypoxia and insufficient nutrient supply. Traditional medical dressings (such as gauze, cotton pads, etc.) can only provide passive physical barriers and moisture absorption functions, lacking the biological activity to actively regulate the wound microenvironment and promote tissue regeneration, making it difficult to meet the treatment needs of such complex wounds.

[0003] Cryogels are a class of macroporous hydrogels prepared below their freezing point via freeze polymerization. Because ice crystals act as pore-forming templates during freezing and form interconnected macroporous structures after thawing, cryogels possess high porosity, excellent elastic recovery, and good mass transfer properties, making them ideal wound dressing matrices. Sodium alginate, a natural anionic polysaccharide, is widely used in biomedical materials due to its good biocompatibility, abundant carboxyl sites, and mild gelation conditions.

[0004] L-arginine is a precursor in the biosynthesis of nitric oxide (NO). NO, as an important gaseous signaling molecule, plays multiple roles in wound healing, including broad-spectrum antibacterial activity, promoting vasodilation and angiogenesis, regulating inflammatory responses, and stimulating collagen deposition. Zinc ions (Zn²⁺) + It is an essential trace element for the human body, with significant broad-spectrum antibacterial activity, and can promote the proliferation and migration of epithelial cells, accelerating the re-epithelialization of wounds.

[0005] However, existing alginate-based dressings face significant technical bottlenecks when simultaneously loading arginine and zinc ions: in systems lacking dynamic covalent bond stability, zinc ions bind only to the carboxyl groups on the alginate chain via ionic bonds, resulting in weak binding and susceptibility to competitive displacement by other cations in the physiological environment. This leads to rapid diffusion and ion exchange between zinc ions and arginine, causing a large loss of active components within a short period. This not only fails to maintain a long-lasting therapeutic concentration but may even cause cytotoxicity due to a sudden increase in local concentration.

[0006] The construction of a dual crosslinked cryogel system that can both stabilize and anchor arginine and zinc ions through Schiff base bonds to achieve long-term sustained release and enhance network toughness and structural stability by utilizing the coordination effect of zinc ions and carboxyl groups has not yet been reported.

[0007] Therefore, developing a cryogel dressing that can synergistically deliver NO donors and zinc ions, possesses antioxidant, antibacterial, immunomodulatory, and angiogenesis-promoting functions, and has controllable release behavior is of significant clinical importance and application value for the repair of infectious diabetic wounds. Summary of the Invention

[0008] To address the aforementioned technical deficiencies in existing technologies, this invention provides an application of Schiff base-metal ion synergistic crosslinked sodium alginate cryogel in the preparation of infectious diabetic wound repair materials. This addresses the technical problems of limited binding sites, excessively rapid release of active components, and lack of multiple synergistic repair functions in existing zinc ion crosslinked alginate hydrogels.

[0009] The technical solution adopted in this invention is: the application of a Schiff base-metal ion synergistic crosslinked sodium alginate cryogel in the preparation of infectious diabetic wound repair materials. The synergistic crosslinked sodium alginate cryogel contains a Schiff base / metal coordination double crosslinked network structure. The Schiff base / metal coordination double crosslinked network structure includes a Schiff base bond formed by the covalent bonding of the amino group of L-arginine and the aldehyde group of oxidized sodium alginate, and a coordination bond formed by the coordination of zinc ions with the carboxyl group on the alginate chain and the imine nitrogen atom in the Schiff base structure.

[0010] The synergistically crosslinked sodium alginate cryogel is prepared by the following steps: S1. Precursor synthesis: Sodium alginate is reacted with methacrylic anhydride to prepare alginate methacrylate; another portion of sodium alginate is oxidized with an oxidizing agent and then reacted with methacrylic anhydride to prepare oxidized alginate methacrylate. S2. Preparation of precursor solution: Dissolve the alginate methacrylate, oxidized alginate methacrylate and L-arginine in physiological saline to form a Schiff base; adjust the pH to 7.0, add a zinc ion source, then add an initiator and a catalyst, and mix well to obtain the precursor solution; S3. Cryogelation: The precursor solution is injected into a mold and frozen to obtain the Schiff base-metal ion synergistic crosslinked sodium alginate cryogel.

[0011] Furthermore, the reaction conditions for forming Schiff base in step S1 are a reaction at 37°C for 6 hours.

[0012] Furthermore, in step S1, when preparing alginate methacrylate, the molar ratio of methacrylic anhydride to sodium alginate is 1:4; the reaction is carried out at room temperature, and the pH is maintained at 8-9 by controlling the alkaline solution, and the reaction time is 6 hours; after the reaction is completed, the product is subjected to dialysis and lyophilization.

[0013] Furthermore, in step S1, when preparing the precursor of oxidized alginate methacrylate, sodium alginate, the molar ratio of oxidant to sodium alginate in the oxidation reaction is 1:2, and the reaction time is 24 hours; excess ethylene glycol is used to quench the oxidation reaction.

[0014] Furthermore, in step S1, the molar ratio of oxidized sodium alginate to methacrylic anhydride is 1:2, the reaction pH is 8-9, the reaction time is 6 hours, and the reaction is carried out at room temperature; after the reaction is completed, the mixture is subjected to dialysis and lyophilization.

[0015] Furthermore, in step S1, the degree of substitution of methacrylate in alginate methacrylate is 10%-15%, the degree of substitution of methacrylate in oxidized alginate methacrylate is 20-25%, and the degree of oxidation of oxidized alginate methacrylate is 35-40%.

[0016] Furthermore, in step S2, the initiator is ammonium persulfate and the catalyst is tetramethylethylenediamine.

[0017] Furthermore, the concentrations of each component in the precursor solution in step S2 are as follows: alginate methacrylate 2.5% w / v, oxidized alginate methacrylate 2.5% w / v, L-arginine 1% w / v, zinc ion source 0.015% w / v, ammonium persulfate 0.5% w / v, and tetramethylethylenediamine 0.1% v / v.

[0018] Furthermore, in step S3, the freezing temperature is -20°C and the freezing time is 24 hours.

[0019] The beneficial effects of this invention are as follows: This invention provides an application of Schiff base-metal ion synergistic crosslinked sodium alginate cryogel in the preparation of infectious diabetic wound repair materials, which has the following beneficial effects: (1) Structural stability and sustained release performance: By constructing a Schiff base / metal coordination double cross-linking network, the continuous and controllable release of arginine and zinc ions was achieved, which effectively overcame the burst release effect caused by ion exchange in traditional zinc ion cross-linked hydrogels and avoided the risk of cytotoxicity caused by a sudden increase in local concentration.

[0020] (2) Excellent physical properties: The prepared cryogel has a highly interconnected porous structure, good mechanical properties, high water absorption and suitable moisture permeability, which can provide an ideal moist healing microenvironment for the wound.

[0021] (3) Synergistic effects of multiple bioactivities: This cryogel integrates multiple functions such as antibacterial, antioxidant, immunomodulatory, angiogenesis and tissue regeneration. It can induce macrophages to polarize from a pro-inflammatory phenotype (M1 type) to a repair phenotype (M2 type), reshaping the immune microenvironment of the wound; at the same time, it promotes the migration and proliferation of fibroblasts and accelerates the formation of new blood vessels.

[0022] (4) Significant in vivo repair effect: In a full-thickness skin defect model of diabetic rats infected with Staphylococcus aureus, the cryogel can effectively reduce the bacterial load on the wound and alleviate the inflammatory response, significantly accelerate wound closure, promote re-epithelialization and dense collagen deposition, and provide a novel treatment strategy with clinical translational potential for the repair of infectious diabetic wounds. Attached Figure Description

[0023] Figure 1 Here is a chemical structure characterization diagram of the key material of this invention; wherein Figure 1 (A) in the figure is the Fourier transform infrared (FTIR) spectrum of alginate methacrylate (AlgMA) and oxidized alginate methacrylate (OAlgMA); Figure 1 (B) in the image shows the ¹H NMR spectra of AlgMA and OAlgMA. Figure 1 (C) in the figure is the X-ray photoelectron spectroscopy (XPS) spectrum of the ArgZn@AO cryogel. Figure 2 The microstructure and compressibility of the ArgZn@AO cryogel prepared in Example 1 of this invention are shown in the diagram. Figure 2 Image (A) in the image is a scanning electron microscope (SEM) image; Figure 2 Image (B) in the image is a laser confocal microscope (CLSM) image; Figure 2 (C) in the figure is a statistical diagram of the pore size distribution of ArgZn@AO cryogel in dry and wet states; Figure 2 The stress-strain curve of 10 cycles of compression of (D)ArgZn@AO cryogel.

[0024] Figure 3The graph shows the rheological performance test results of Example 1 (ArgZn@AO) and Comparative Examples 1 (AO), 2 (Arg@AO), and 3 (ArgZn@A) of the present invention; wherein Figure 3 (A) in the figure represents the frequency scanning curve; Figure 3 (B) in the figure represents the strain scanning curve.

[0025] Figure 4 The in vitro cumulative release curves of the active components of Example 1 (ArgZn@AO) and Comparative Example 3 (ArgZn@A) are shown; wherein Figure 4 (A) in the figure represents the L-arginine release curve; Figure 4 (B) in the figure represents the hydrogen peroxide-induced nitric oxide release curve; Figure 4 (C) in the text represents Zn² + Release curve.

[0026] Figure 5 The antioxidant performance test results of Example 1 (ArgZn@AO) and Comparative Examples 1 (AO), 2 (Arg@AO), and 3 (ArgZn@A) are presented; wherein Figure 5 (A) in the figure is a bar chart of ABTS free radical scavenging rate; Figure 5 (A) and (B) in the figure are bar charts of DPPH free radical scavenging rate; Figure 5 (A) and (C) in the figure are bar charts of superoxide anion scavenging rate.

[0027] Figure 6 The results show the effects of different groups of materials on the migration-promoting effect and cell compatibility evaluation of L929 fibroblasts; among them Figure 6 (A) in the figure shows the results of the cell scratch migration experiment; Figure 6 (B) in the figure is a quantitative bar chart of scratch migration rate; Figure 6 (C) in the figure represents the bar chart of relative fibroblast survival rate on day 3.

[0028] Figure 7 The results show the effects of different groups of materials on the tube-forming ability of human umbilical vein endothelial cells (HUVECs); among them Figure 7 (A) in the figure shows the original and analytical images of the tube formation experiment. Figure 7 (B), (C), and (D) in the figure are quantitative statistical charts of total capillary length, vascular branch points, and vascular mesh number, respectively.

[0029] Figure 8 The results of the evaluation of the antibacterial properties of different groups of materials against Escherichia coli and Staphylococcus aureus are shown; among them Figure 8 (A) in the image is a SEM image of Escherichia coli morphology; Figure 8 (B) in the figure is a bar chart showing the survival rate of E. coli; Figure 8(C) in the image is a SEM image of Staphylococcus aureus bacterial morphology. Figure 8 (D) in the figure represents the bar chart of Staphylococcus aureus survival rate.

[0030] Figure 9 The results show the effects of different groups of materials on the polarization phenotype of RAW264.7 macrophages; among them Figure 9 (A) in the figure is a scatter plot of CD86 / CD206 dual-color flow cytometry; Figure 9 (B) in the text refers to type M1 (CD86). + (Bar chart showing the percentage of macrophages) Figure 9 (C) in the text refers to type M2 (CD206). + (Bar chart showing the percentage of macrophages)

[0031] Figure 10 The wound healing status of each group of infected diabetic rats at different time points after treatment of the wounds was observed; among them Figure 10 (A) in the image represents representative wound photographs taken on days 0, 3, 7, and 14. Figure 10 (B) in the figure is a statistical chart of the relative area of ​​the wound on day 14.

[0032] Figure 11 Histological analysis results of the wounds of infectious diabetic rats treated with materials in each group on days 3 and 7; including Giemsa stained sections (day 3), TNF-α inflammatory factor immunofluorescence stained sections (day 3), CD68 / CD86 immunofluorescence stained sections (day 3), CD206 / CD86 immunofluorescence stained sections (day 3), CD31 immunofluorescence stained sections (day 7), and VEGF immunohistochemical stained sections (day 7).

[0033] Figure 12 Based on Figure 11 Quantitative analysis results of histological sections; among which Figure 12 (A) represents the relative number of bacterial colonies; Figure 12 (B) represents the TNF-α expression level; Figure 12 (C) represents the fluorescence intensity of M1 macrophages; Figure 12 (D) represents the fluorescence intensity of M2 macrophages; Figure 12 (E) represents the CD31 expression level; Figure 12 (F) in the figure represents the VEGF expression level.

[0034] Figure 13 Histological analysis results of the wounds of infectious diabetic rats treated with materials from each group on day 14; including H&E stained sections, Masson trichrome collagen stained sections, Sirius red (PRS) polarized light stained sections and α-SMA immunohistochemical stained sections.

[0035] Figure 14 Based on Figure 13 Quantitative analysis results of histological sections; among which Figure 14 In this context, (A) represents the thickness of the newborn epidermis; Figure 14 In the figure, (B) represents the relative area of ​​collagen deposition; Figure 14 (C) represents the ratio of type I to type III collagen; Figure 14 (D) in the figure represents the expression level of α-SMA. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Preparation of ArgZn@AO cryogel 1. Synthesis of precursor materials 1.1 Synthesis of Alginate Methacrylate (AlgMA) Sodium alginate was completely dispersed in deionized water and stirred continuously at room temperature until the system became clear, transparent, and completely dissolved. Methacrylic anhydride was slowly added dropwise to the sodium alginate solution, while the pH was simultaneously adjusted in real time using 5 M sodium hydroxide solution to maintain a stable pH of approximately 8.0. The reaction was carried out under constant temperature and light conditions for 6 hours. After the reaction, the resulting mixture was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed against deionized water for 3 days, changing the deionized water three times daily to thoroughly remove unreacted monomers, small molecule impurities, and byproducts. After dialysis, the purified solution was freeze-dried to obtain a white, spongy solid AlgMA product, which was then stored in a sealed, light-protected environment at -20 °C for later use.

[0038] 1.2 Synthesis of Oxyalgin Methacrylate (OAlgMA) Sodium alginate was dispersed in deionized water and stirred at room temperature until completely dissolved. Sodium periodate was separately dissolved in an appropriate amount of deionized water. The sodium periodate solution was then slowly added dropwise to the sodium alginate solution under light-protected conditions (sodium periodate:sodium alginate = 1:2). The mixture was stirred continuously at room temperature under light-protected conditions for 24 hours to ensure complete oxidation of the sodium alginate. After the oxidation reaction was complete, ethylene glycol of an equimolar amount to sodium periodate was added to the reaction system, and the mixture was stirred thoroughly for 30 minutes to completely quench any remaining sodium periodate and terminate the oxidation reaction. The reaction mixture was then dialyzed against deionized water for 2 days to remove oxidation byproducts and residual ethylene glycol.

[0039] The purified sodium alginate solution was placed in a dark environment again, and a measured amount of methacrylic anhydride was added. The pH of the system was adjusted to 8.0 using 5 M sodium hydroxide solution, and the reaction was carried out at a constant temperature and in the dark for 6 hours to complete the acylation modification. After the reaction, dialyzing was continued for 3 days, with the water changed 3 times a day. After purification, the solid OAlgMA product was obtained by freeze drying and stored in a sealed container at -20 °C in the dark for later use.

[0040] 2. Preparation of cryogels The AlgMA and OAlgMA prepared above were mixed at a mass ratio of 1:1 and uniformly dispersed in sterile physiological saline. The mixture was stirred until completely dissolved to prepare a homogeneous solution with a total polymer concentration of 5% w / v. L-arginine was added to this polymer solution to achieve a final concentration of 1% w / v. After mixing, the solution was placed in a constant temperature environment of 37 °C for 6 h to ensure that L-arginine and the aldehyde groups on the OAlgMA molecular chain fully undergo a Schiff base reaction to generate a stable dynamic covalent intermediate.

[0041] After the reaction was complete, the pH of the system was slowly adjusted to 7.0 (physiological neutral environment) using 1 M hydrochloric acid solution. Then, zinc chloride was added to control the final concentration of the system to 0.015% w / v. The mixture was stirred continuously at room temperature for 30 min to allow zinc ions to fully coordinate with the gel precursor. Finally, ammonium persulfate (APS, final concentration 0.5% w / v) and tetramethylethylenediamine (TEMED, final concentration 0.1% v / v) were added sequentially, and the mixture was quickly vortexed to mix thoroughly. The homogenized precursor solution was then immediately injected into a pre-cooled cylindrical silicone mold.

[0042] The filled mold was placed in a -20 ℃ low temperature freezer for 24 h to fully solidify and form the gel by relying on the pore-forming effect of ice crystals and the double cross-linking system. After demolding, the Schiff base-arginine-zinc ion synergistic cross-linked sodium alginate cryogel was obtained and named ArgZn@AO.

[0043] Comparative Example 1: Preparation of zinc ion-free and arginine-free sodium alginate cryogel (AO) 1. Precursor material synthesis Following the same process parameters and operating steps as in Example 1, AlgMA and OAlgMA precursor materials were synthesized and purified respectively.

[0044] 2. Cryogel preparation AlgMA and OAlgMA were dissolved in sterile physiological saline at a 1:1 mass ratio to prepare a homogeneous solution with a total polymer concentration of 5% w / v. APS with a final concentration of 0.5% w / v and TEMED with a final concentration of 0.1% v / v were added directly to the solution, and the mixture was quickly stirred and mixed. The mixture was then poured into a silicone mold and frozen in a -20 ℃ environment for 24 h. After demolding, a blank sodium alginate cryogel with a single cross-linked network was obtained, denoted as AO.

[0045] Comparative Example 2: Preparation of Zinc Ion-Free Arginine-Modified Sodium Alginate Cryogel (Arg@AO) 1. Precursor material synthesis Following the same process parameters and operating steps as in Example 1, AlgMA and OAlgMA precursor materials were synthesized and purified respectively.

[0046] 2. Cryogel preparation AlgMA and OAlgMA were dissolved in sterile physiological saline at a 1:1 mass ratio to prepare a homogeneous solution with a total polymer concentration of 5% w / v. L-arginine was added and the final concentration was controlled at 1% w / v. The mixture was reacted at 37 °C for 6 h to allow the Schiff base reaction to occur fully. After the reaction was completed, APS with a final concentration of 0.5% w / v and TEMED with a final concentration of 0.1% v / v were added directly to the solution. The mixture was quickly stirred and mixed, then poured into a silicone mold and frozen at -20 °C for 24 h. After demolding, a modified cryogel containing only arginine Schiff base crosslinking and without zinc ion coordination was obtained, denoted as Arg@AO.

[0047] Comparative Example 3: Preparation of Schiff base-free zinc ion arginine-modified sodium alginate cryogel (ArgZn@A) 1. Precursor material synthesis AlgMA precursor materials were synthesized and purified according to the process in Example 1 only.

[0048] 2. Cryogel preparation AlgMA was dissolved in sterile physiological saline to prepare a homogeneous solution with a concentration of 5% w / v. L-arginine was added to a final concentration of 1% w / v, and the mixture was stirred and shaken at 37 °C for 6 h. The pH of the system was adjusted to 7.0 using 1 M hydrochloric acid, and zinc chloride was added to a final concentration of 0.015% w / v. The mixture was stirred for 30 min to ensure complete coordination. APS (0.5% w / v) and TEMED (0.1% v / v) were added directly to the solution, and the mixture was rapidly stirred until homogeneous. The mixture was then poured into a silicone mold and frozen at -20 °C for 24 h. Demolding yielded a freeze gel containing arginine that was dynamically cross-linked without Schiff bases and only modified by zinc ion coordination, denoted as ArgZn@A.

[0049] Table 1

[0050] Application examples and performance characterization experiments To fully verify the chemical structure, microstructure, mechanical properties, sustained-release properties, antioxidant activity, biocompatibility, pro-repair activity, antibacterial ability, and in vivo wound repair effect of the precursor materials and target cryogels prepared in this invention, systematic physicochemical characterization, in vitro functional evaluation, and in vivo animal experiments were carried out on the AlgMA and OAlgMA precursor materials and the ArgZn@AO cryogel prepared in Example 1. The specific test methods and experimental procedures are as follows.

[0051] 1. Chemical structure characterization of precursor materials (FTIR testing) Chemical functional groups and structures of dried and purified AlgMA and OAlgMA powder samples were characterized using a Bruker Tensor II Fourier transform infrared spectrometer with an attenuated total reflectance (ATR) accessory. All samples were thoroughly dried before testing to completely eliminate interference from infrared absorption peaks caused by adsorbed moisture. The spectral scanning range was set to 4000–400 cm⁻¹. - ¹, spectral data were acquired using transmission mode.

[0052] 2. Quantitative structural characterization of precursor materials (¹H NMR testing) The molecular structure, degree of substitution, and oxidative modification effect of AlgMA and OAlgMA were quantitatively characterized using a Weinheim Bruker Avance-I 400 MHz quantitative nuclear magnetic resonance (HMR) spectrometer. The sample concentration was 10 mg / mL, with heavy water (D₂O) as the deuterated solvent and TSP as the internal standard (δ = 0 ppm). The test temperature was kept constant at 25 ℃, the spectral width was set to 12 ppm, 16 scans were performed, and the relaxation delay time (D₁) was set to 10 s to ensure a stable spectral baseline and accurate integration quantification.

[0053] 3. Elemental and chemical characterization of the cryogel surface (XPS testing) The surface elemental composition, elemental content, and chemical bonding state of freeze-dried ArgZn@AO cryogel samples were analyzed using a Thermo Fisher Scientific K-Alpha X-ray photoelectron spectrometer (TPS). Al Kα rays were used as the excitation source, and a high-resolution narrow scan mode was employed with a pass energy of 50 eV and a step size of 0.1 eV.

[0054] 4. Microstructure characterization of cryogels (SEM testing) ArgZn@AO cryogel samples were pre-frozen to -80 °C and then rapidly fractured along the central longitudinal axis in a liquid nitrogen environment to obtain a smooth gel fracture cross-section. A platinum (Pt) conductive thin film was sputtered onto the fracture cross-section using a Leica EM ACE600 high-vacuum ion sputtering system (Germany) to eliminate the sample's charge effect. Finally, the interior of the gel was observed using a Hitachi SU8010 field emission scanning electron microscope (Japan) at an accelerating voltage of 5 kV.

[0055] 5. Three-dimensional structural imaging characterization of cryogels (CLSM test) The cryogel samples were immersed in sterile physiological saline and allowed to swell to equilibrium. The gels were then thoroughly stained with 0.1% rhodamine solution, and after adsorption saturation in the dark, they were rinsed to remove residual dye. Tomographic scanning and three-dimensional imaging of the stained gels were performed using a Nikon A1 laser confocal scanning microscope (Japan).

[0056] 6. Cryogel Compression Mechanical Properties Testing The cryogel samples were immersed in physiological saline until they reached complete swelling equilibrium. Room temperature compression performance tests were conducted using a Massachusetts Instron 5944 universal testing machine. Before testing, free moisture was gently absorbed from the sample surface. The sample was placed centered between parallel compression plates, and a preload of 0.02 N was applied to ensure complete contact between the plates and the sample. The initial contact height was taken as the original height of the sample. Displacement control mode was used, with the maximum compressive strain set to 80%, and both loading and unloading rates at 10 mm / min. Ten consecutive compression cycles were performed.

[0057] 7. Cryogel Rheological Properties Testing Oscillatory rheological tests were performed on fully swollen, equilibrium-bound cryogel samples using a TA Instruments DHR-2 rotational rheometer to evaluate the gel's viscoelasticity and structural stability. The tests were conducted entirely at room temperature. First, a fixed oscillation frequency of 1 Hz was used to perform strain amplitude scanning within the strain range of 0.1%–10%, defining the gel's linear viscoelastic range. Within this linear range, a fixed strain γ = 0.2% was selected for frequency scanning tests, with a scanning frequency of 0.1–10 Hz.

[0058] 8. Evaluation of the sustained-release performance of the active components in cryogels To verify the long-lasting sustained-release properties of the gel of this invention, L-arginine, nitric oxide (NO), and zinc ions (Zn²⁺) were respectively tested. + The in vitro release behavior of the substance was quantitatively detected.

[0059] L-arginine release assay: Immerse the cryogel sample in 1 mL of sterile physiological saline and incubate at 37 ℃ in a constant temperature shaker; collect the supernatant at preset time points, and use the Beijing Boxbio arginine content detection kit to quantitatively detect the L-arginine concentration in the supernatant according to the standard instructions, and plot the cumulative release curve.

[0060] NO release test: Immerse the cryogel sample in 1 mM hydrogen peroxide (H2O2) solution and incubate at 37 ℃ in a shaker; collect the supernatant periodically, and use the Shanghai Beyotime nitric oxide detection kit (Cat. No. S0021M) to strictly follow the instructions to detect the NO release concentration in the medium.

[0061] Zn² + Release test: The zinc-loaded cryogel sample was immersed in 1 mL of deionized water and incubated at 37 ℃. The supernatant was collected at preset time points, filtered through a 0.22 μm aqueous microporous membrane, and appropriately diluted according to the concentration requirements. The zinc ion concentration in the supernatant was determined using an Agilent 7850 inductively coupled plasma mass spectrometer (ICP-MS) to accurately characterize the zinc ion release kinetics.

[0062] 9. Evaluation of the in vitro antioxidant activity of cryogels The antioxidant capacity of the cryogel was comprehensively evaluated using ABTS radical scavenging assay, DPPH radical scavenging assay, and superoxide anion radical scavenging assay. The radical scavenging rate was calculated using the following formula: Clearance rate (%) = (Absorbance of control group) (Absorbance of sample group) / Absorbance of control group × 100%.

[0063] ABTS free radical scavenging experiment: An equal volume of 7 mmol / L ABTS stock solution was mixed with 2.45 mmol / L potassium persulfate solution and allowed to stand in the dark for 12–16 h to prepare ABTS• + Free radical stock solution; the stock solution was diluted with PBS buffer to obtain an absorbance of 0.70±0.02 at 734 nm, thus obtaining the ABTS working solution. The cryogel was immersed in the working solution and incubated at room temperature in the dark for 5 min. The absorbance of the supernatant at 734 nm was measured, and the free radical scavenging rate was calculated.

[0064] DPPH radical scavenging experiment: A 0.1 mmol / L DPPH working solution was prepared using anhydrous ethanol and kept in the dark. The absorbance at 517 nm was measured to ensure system stability. The cryogel was placed in the DPPH working solution and incubated at room temperature in the dark for 30 min. The absorbance of the supernatant at 517 nm was measured to calculate the DPPH radical scavenging capacity.

[0065] Superoxide anion radical scavenging assay: A superoxide anion generation system was constructed using an NBT-NADH-PMS reaction system. The working solution was freshly prepared in pH 7.4 PBS buffer, containing 336 μmol / L NBT, 936 μmol / L NADH, and 120 μmol / L PMS. After adding the cryogel sample, the mixture was incubated at 37 ℃ in the dark for 5 min. The absorbance of the supernatant at 560 nm was measured, and the superoxide anion radical scavenging rate was calculated.

[0066] 10. Evaluation of cell compatibility in cryogels The cytotoxicity and biocompatibility of cryogels were evaluated using an L929 fibroblast indirect co-culture system with Transwell. L929 fibroblasts were cultured at 5 × 10⁶ cells / mL. 4 Cells were seeded at a density of [number] cells / well in 24-well plates. After routine culture until the cells were stable and adhered, Transwell chambers loaded with sterile cryogel samples were placed in the corresponding wells of the plates. Complete culture medium was added to establish a co-culture system, and the plates were incubated at 37 ℃ in a 5% CO2 incubator. Cell viability was assessed on day 3: old culture medium was aspirated, cells were gently washed with PBS, and DMEM medium containing 10% (v / v) CCK-8 reagent was added to each well. Cells were incubated at 37 ℃ in the dark for 2 h. Absorbance at 450 nm was measured using a BioTek EP0CH2 microplate reader. A blank group (containing only 10% CCK-8 medium, no cells) and a control group (normally cultured cells, no gel) were set up. The relative cell viability was calculated using the following formula: Relative cell viability (%) = (absorbance of sample group – absorbance of blank group) / (absorbance of control group – absorbance of blank group) × 100%.

[0067] 11. Evaluation of the cell migration-promoting ability of cryogels The cell scratch assay was used to evaluate the migration-promoting effect of cryogels on L929 fibroblasts. L929 cells were sputtered at 1×10⁶ cells / cells. 5 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured until a dense, uniform monolayer was formed. A sterile 200 μL pipette tip was used to vertically and uniformly scratch the cells. Exfoliated cells were removed by washing with PBS, and the medium was replaced with serum-free medium. Sterile cryogels were placed in Transwell chambers and co-cultured with the scratched cells for 48 h. Images of the scratched areas were acquired using a BDS400 inverted microscope from Chongqing Optoelectronics, China.

[0068] 12. Evaluation of the angiogenesis-promoting capacity of cryogels The angiogenesis-promoting activity of the materials was evaluated using a human umbilical vein endothelial cell (HUVECs) tube-forming assay. 24-well plates were pre-coated with matrix gel (Beyotime, C0372), and HUVECs were cultured at 1.5 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of cells / well in well plates; an indirect co-culture system of gel samples and endothelial cells was established using Transwell chambers and incubated at 37 ℃ and 5% CO2 for 8 h. After incubation, images of cell tube formation in each group were acquired under a microscope, and the total tube length, number of branch points, and number of grids were quantitatively analyzed using the ImageJ angiogenesis analysis plugin.

[0069] 13. Evaluation of the immunomodulatory properties of cryogel macrophages The inflammatory and immunomodulatory capabilities of the gel were evaluated using a RAW264.7 macrophage inflammatory polarization model. RAW264.7 macrophages were sputtered at a concentration of 1.5 × 10⁻⁶ cells. 5 Macrophages were seeded in 24-well plates and cultured overnight. They were stimulated for 24 h with 100 ng / mL lipopolysaccharide (LPS) and 20 ng / mL interferon-γ (IFN-γ) to induce macrophage polarization toward the pro-inflammatory M1 phenotype. The control group did not receive inflammatory stimulation.

[0070] Sterile cryogel samples were placed in Transwell chambers and indirectly co-cultured with polarized model macrophages for 24 h. The proportion of positive cells for M1 marker CD86 and M2 marker CD206 was detected using a Beckman Coulter CytoFLEX flow cytometer.

[0071] 14. Evaluation of the in vitro antibacterial properties of cryogels Staphylococcus aureus and Escherichia coli were used as model strains, and the antibacterial properties of the gel were evaluated by colony counting and bacterial morphology observation, respectively.

[0072] Colony quantification experiment: Take 100 μL of a concentration of 1×10⁻⁶ 6 CFU / mL bacterial suspension was evenly added to the surface of the gel sample and incubated at 37 ℃ for 8 h. The residual bacterial suspension was collected, serially diluted 10³ times with sterile PBS, and 100 μL of the diluted bacterial suspension was spread on an agar plate and incubated upside down at 37 ℃ for 12 h. The number of colonies in each group was counted and the antibacterial efficiency was quantitatively analyzed.

[0073] Bacterial morphology characterization experiment: After co-incubation, residual bacteria on the gel surface were collected, and the bacterial pellet was collected by centrifugation at 8000 rpm for 3 min. The pellet was fixed with 2.5% glutaraldehyde solution at 4 ℃ for 12 h, washed 3 times with PBS, dehydrated stepwise with 30% to 100% graded ethanol, replaced with tert-butanol, and air-dried. The integrity of bacterial morphology was observed by scanning electron microscopy.

[0074] 15. Animal Model Construction and Intervention Experiment of Diabetic Infected Wounds All animal experiments in this study were approved by the Laboratory Animal Ethics Committee of the Wenzhou Institute of the University of Chinese Academy of Sciences (Approval No.: WIUCAS25101603). All experimental procedures strictly followed the "Regulations on the Feeding and Use of Laboratory Animals" and the institution's ethical requirements to minimize animal suffering and experimental dosage.

[0075] Eight-week-old male SD rats weighing 220–280 g were used in the experiment. After 7 days of acclimatization, the rats were used to establish the type 1 diabetes model. After fasting for 12 hours, streptozotocin (STZ) solution, freshly prepared in 0.1 mol / L, pH 4.5 citrate-sodium citrate buffer, was injected intraperitoneally at a dose of 60 mg / kg body weight. Fasting blood glucose was monitored daily after injection. A type 1 diabetes model was considered successfully established when fasting blood glucose remained stable above 16.7 mmol / L for three consecutive days and was accompanied by typical diabetic symptoms of polyuria, polydipsia, polyphagia, and weight loss.

[0076] The rats were randomly divided into 6 groups, with 3 rats in each group: Control group, CMP group (commercial dressing, Urgo 552342), AO group, Arg@AO group, ArgZn@AO group, and ArgZn@A group.

[0077] Rats were anesthetized by isoflurane inhalation. After the back was prepared and disinfected, symmetrical full-thickness skin defects with a diameter of 6 mm were constructed on the lateral dorsal side. 20 μL of 1×10⁻⁶ solution was instilled into each wound. 9 A stable infectious diabetic wound model was constructed by incubating a CFU / mL Staphylococcus aureus suspension with a sterile transparent dressing for 24 h.

[0078] According to the group, the corresponding sterile cryogel or commercial dressing was applied. The blank group was not treated. All wounds were uniformly covered with sterile Tegaderm™ dressing and fixed and sealed. The dressing application time was recorded as day 0 of the experiment.

[0079] 16. Wound healing evaluation and histological analysis Wounds in each group were photographed and recorded under anesthesia on days 0, 3, 7, and 14 of the experiment. The wound area was measured using ImageJ software, and the relative wound area was calculated using the following formula: Relative wound area (%) = Remaining wound area at the corresponding time point / Initial wound area × 100%.

[0080] Rats were sacrificed on postoperative days 3, 7, and 14, and wound tissues were collected. After fixation with 4% paraformaldehyde, the tissues were dehydrated in a gradient manner, cleared with xylene, and embedded in paraffin to prepare 4 μm continuous tissue sections. The sections were then stained with H&E, Masson's trichrome, Giemsa bacterial staining, Sirius red (PSR) staining, and immunofluorescence staining.

[0081] The system performance of the gels from Example 1 and each comparative example was characterized and their in vitro and in vivo functional verification was performed. The results are as follows: 1. Structural and morphological characterization: such as Figure 1 As shown, the ArgZn@AO cryogel of this invention exhibits a uniform and interconnected three-dimensional macroporous structure with a uniform pore size distribution and a dense and stable double cross-linked network structure. XPS, FTIR, and ¹H NMR characterization results confirm that Schiff base bonds and zinc ion coordination bonds were successfully constructed, and each active component was effectively combined in the gel system.

[0082] 2. Mechanical and rheological properties: such as Figure 2 and 3 As shown, the rheological test and cyclic compression test results indicate that the ArgZn@AO gel has excellent storage modulus, structural stability and elastic recovery performance. The double cross-linked network can effectively improve the gel toughness and deformation resistance, and can withstand repeated deformation of the wound surface without easily breaking down.

[0083] 3. Release performance of active components: such as Figure 4 As shown, in vitro release experiments confirmed that ArgZn@AO gel can achieve stable and long-term release of zinc ions, L-arginine and downstream signaling molecule NO, without obvious burst release phenomenon, and can continuously provide active repair factors for wounds.

[0084] 4. Antioxidant properties: such as Figure 5 As shown, ArgZn@AO gel has excellent scavenging ability against ABTS, DPPH and superoxide anion free radicals, can effectively remove excess ROS from wounds, alleviate oxidative stress damage, and its antioxidant effect is significantly better than that of a single cross-linked gel system.

[0085] 5. Cell compatibility and repair-promoting activity: such as Figure 6 and 7 As shown in the cell experiments, the gel of the present invention is non-cytotoxic and has excellent biocompatibility; it can significantly promote the migration and proliferation of fibroblasts and induce endothelial cell tube formation, effectively assisting in wound tissue repair and angiogenesis.

[0086] 6. Antibacterial and immunomodulatory properties: such as Figure 8 and 9As shown, antibacterial experiments confirmed that ArgZn@AO gel has excellent broad-spectrum antibacterial effects against Escherichia coli and Staphylococcus aureus, and can effectively inhibit the proliferation of pathogenic bacteria in wounds; flow cytometry analysis showed that the gel can significantly promote the polarization of macrophages from the pro-inflammatory M1 phenotype to the repair M2 phenotype, effectively balancing the inflammatory immune microenvironment of wounds.

[0087] 7. In vivo infection-related diabetic wound repair effect: such as... Figure 10 and 11 As shown, an in vivo pharmacodynamic experiment was conducted based on a streptozotocin-induced Staphylococcus aureus-infected full-thickness skin wound model in diabetic rats. The results showed that, compared with all control dressing materials, ArgZn@AO gel significantly reduced the bacterial load of the wound, inhibited the expression of inflammatory factors, promoted angiogenesis and collagen deposition, accelerated wound re-epithelialization, significantly improved the wound healing rate 14 days after surgery, and demonstrated excellent wound repair quality with no scar hyperplasia or residual inflammation.

[0088] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0089] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. The application of a Schiff base-metal ion synergistic crosslinked sodium alginate cryogel in the preparation of infectious diabetic wound repair materials, characterized in that, The synergistic crosslinked sodium alginate cryogel comprises a Schiff base / metal coordination dual crosslinked network structure, which includes a Schiff base bond formed by the covalent bonding of the amino group of L-arginine and the aldehyde group of oxidized sodium alginate, and a coordination bond formed by the coordination of zinc ions with the carboxyl group on the alginate chain and the imine nitrogen atom in the Schiff base structure.

2. The application according to claim 1, characterized in that, The synergistically crosslinked sodium alginate cryogel is prepared by the following steps: S1. Precursor synthesis: Sodium alginate is reacted with methacrylic anhydride to prepare alginate methacrylate; another portion of sodium alginate is oxidized with an oxidizing agent and then reacted with methacrylic anhydride to prepare oxidized alginate methacrylate. S2. Preparation of precursor solution: Dissolve the alginate methacrylate, oxidized alginate methacrylate and L-arginine in physiological saline to form a Schiff base; adjust the pH to 7.0, add a zinc ion source, then add an initiator and a catalyst, and mix well to obtain the precursor solution; S3. Cryogelation: The precursor solution is injected into a mold and frozen to obtain the Schiff base-metal ion synergistic crosslinked sodium alginate cryogel.

3. The application according to claim 2, characterized in that, The reaction conditions for forming Schiff bases in step S1 are: reaction at 37°C for 6 hours.

4. The application according to claim 2, characterized in that, In step S1, when preparing alginate methacrylate, the molar ratio of methacrylic anhydride to sodium alginate is 1:4; the reaction is carried out at room temperature, and the pH is maintained at 8-9 by controlling the alkaline solution, and the reaction time is 6 hours; after the reaction is completed, the product is subjected to dialysis and lyophilization.

5. The application according to claim 2, characterized in that, In step S1, when preparing the precursor of oxidized alginate methacrylate, sodium alginate, the molar ratio of oxidant to sodium alginate in the oxidation reaction is 1:2, and the reaction time is 24 hours; excess ethylene glycol is used to quench the oxidation reaction.

6. The application according to claim 2, characterized in that, In step S1, the molar ratio of oxidized sodium alginate to methacrylic anhydride is 1:2, the reaction pH is 8-9, the reaction time is 6 hours, and the reaction is carried out at room temperature; after the reaction is completed, the mixture is subjected to dialysis and lyophilization.

7. The application according to claim 2, characterized in that, In step S1, the degree of substitution of methacrylate in alginate methacrylate is 10%-15%, the degree of substitution of methacrylate in oxidized alginate methacrylate is 20-25%, and the degree of oxidation of oxidized alginate methacrylate is 35-40%.

8. The application according to claim 2, characterized in that, In step S2, the initiator is ammonium persulfate and the catalyst is tetramethylethylenediamine.

9. The application according to claim 8, characterized in that, The concentrations of each component in the precursor solution in step S2 are as follows: alginate methacrylate 2.5% w / v, oxidized alginate methacrylate 2.5% w / v, L-arginine 1% w / v, zinc ion source 0.015% w / v, ammonium persulfate 0.5% w / v, and tetramethylethylenediamine 0.1% v / v.

10. The application according to claim 2, characterized in that, In step S3, the freezing temperature is -20℃ and the freezing time is 24 hours.

Images