M2 membrane camouflage magnetic nanoparticle composite hydrogel, preparation method thereof and application of M2 membrane camouflage magnetic nanoparticle composite hydrogel in tissue repair

Abstract

The invention discloses M2 membrane camouflage magnetic nanoparticle composite hydrogel as well as a preparation method and application thereof in tissue repair. The magnetic nanoparticle composite hydrogel comprises collagen modified by gallic acid and a double-network polymer matrix formed by oxidized methacrylic acid hyaluronic acid, M2 membrane camouflage magnetic nanoparticles are arranged in the double-network polymer matrix, and the magnetic nanoparticles are wrapped by M2 type macrophage membranes. The invention constructs a multifunctional integrated magnetic responsive composite hydrogel, the hydrogel comprises a double network polymer matrix formed by gallic acid modified collagen and oxidized methacrylic acid hyaluronic acid, magnetic nanoparticles are embedded in the matrix, and the matrix is coated with a membrane from M2 phenotype macrophages. Three core functions of antioxidation and removal of reactive oxygen species (ROS), active immune regulation and non-contact dynamic mechanical stimulation are integrated in a single system, and the problems that an existing dressing is single in function and insufficient in synergistic effect are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to an M2 membrane camouflaged magnetic nanoparticle composite hydrogel, its preparation method, and its application in tissue repair. Background Technology

[0002] Diabetic wound healing is a major challenge in clinical practice, with its core pathological mechanism lying in the persistent inflammation, impaired angiogenesis, and imbalance in extracellular matrix (ECM) remodeling caused by hyperglycemia. This pathological microenvironment is closely related to intracellular calcium (Ca²⁺) signaling disturbances—Ca²⁺ is a key second messenger regulating cell migration, proliferation, and angiogenesis, and its homeostasis severely affects the repair function of cells. Recent studies have further revealed that mechanosensitive channels (such as Piezo1 and TRPV4) and their downstream effector molecule Yes-associated protein (YAP) play a central role in sensing microenvironmental mechanical signals and transducing them into Ca²⁺ influx and regeneration gene expression. However, diabetic wounds lead to impaired Ca²⁺ homeostasis, which in turn disrupts mechanotransduction pathways, including RhoA / ROCK, reducing YAP activity and ultimately decreasing the efficiency of transduction of mechanical signals to regenerative signals, resulting in wound repair stagnation.

[0003] Currently used clinical treatment strategies have the following significant limitations:

[0004] 1. Limitations of Smart-Response Hydrogels: While thermosensitive, pH-responsive, or enzyme-triggered hydrogels can achieve controlled drug release or microenvironment regulation, their mechanisms of action are still primarily "passive responses," lacking the ability to actively and dynamically regulate the cellular mechanical microenvironment. These materials typically cannot mimic the dynamic mechanical stimuli experienced by cells under physiological conditions, making it difficult to activate key mechanosensitive signaling pathways.

[0005] 2. Limitations of cell therapy: Although cell therapy can promote reepithelialization and paracrine effects, transplanted cells have low survival rates in the harsh microenvironment of diabetic wounds, their function is easily affected, and there are problems such as immune rejection, ethical restrictions, and high costs.

[0006] 3. Limitations of negative pressure wound therapy (NPWT): Although devices such as NPWT can apply mechanical stimulation, their mechanism of action is unclear, and they are contact-based physical stimuli, which may cause discomfort to patients. They are also difficult to act on deep tissues, and more importantly, they cannot achieve precise mechanical control at the cellular level.

[0007] 4. Limitations of existing magnetically responsive materials: While the combination of magnetically responsive hydrogels and non-invasive magnetic fields has opened up new pathways for wireless mechanical regulation, current research mainly focuses on thermal effects or targeted drug delivery, with insufficient research on directly regulating cell behavior using non-thermodynamic signals. Most magnetically responsive materials serve only as drug carriers or rely on magnetocaloric effects, lacking the ability to specifically activate cellular mechanotransmission pathways.

[0008] 5. Lack of multidimensional regulatory capacity: Current treatment strategies often target only a single aspect of wound healing, lacking the comprehensive ability to simultaneously address multiple pathological factors such as oxidative stress, chronic inflammation, angiogenesis disorders, and biomechanical signal transduction disturbances. In particular, existing materials lack sufficient synergistic capabilities in antioxidation, immune regulation, and biomechanical signal transduction, making it difficult to effectively reverse the healing impairment of diabetic wounds.

[0009] 6. Insufficient utilization of mechanobiological mechanisms: Cellular behaviors crucial for wound healing, such as migration, proliferation, and matrix secretion, are themselves highly mechanosensitive processes. However, current treatments fail to effectively utilize mechanobiological principles and cannot activate endogenous repair programs through controlled mechanical stimulation.

[0010] Therefore, developing a smart material system that can simultaneously achieve multiple functions such as "antioxidation, immune regulation, and mechanical stimulation" and precisely regulate the microenvironment of diabetic wounds through non-contact methods has become a technical bottleneck that urgently needs to be overcome in the current field. Summary of the Invention

[0011] To overcome the problem of poor wound healing in diabetic patients, this invention proposes an M2 membrane-masked magnetic nanoparticle composite hydrogel, its preparation method, and its application in tissue repair. The innovative magnetically responsive composite hydrogel system proposed in this invention solves the comprehensive challenges of diabetic wound healing through a multi-dimensional synergistic strategy.

[0012] The first objective of this invention is to provide an M2 membrane-masked magnetic nanoparticle composite hydrogel, comprising a dual-network polymer matrix formed by gallic acid-modified collagen and oxymethacrylic acid hyaluronic acid, wherein M2 membrane-masked magnetic nanoparticles are disposed in the dual-network polymer matrix and the magnetic nanoparticles are encapsulated from the membrane of M2 macrophages.

[0013] The dual-network polymer matrix is ​​formed by the Schiff base reaction and Fe 3+ The hydrogel, composed of a first cross-linked network formed by coordination and a second cross-linked network formed by photopolymerization of methacrylate groups on OHAMA, exhibits reactive oxygen species (ROS) scavenging activity.

[0014] This invention proposes an ideal scaffold material with excellent biocompatibility, rapid injection molding capability, good hemostatic properties, and wound healing ability, providing long-term physical support and biochemical microenvironment for tissue regeneration.

[0015] This invention constructs a multifunctional, integrated, magnetically responsive composite hydrogel. The hydrogel comprises a double-network polymer matrix formed by gallic acid-modified collagen (CG) and oxymethacrylic acid hyaluronic acid (OHAMA). Magnetic nanoparticles are embedded within this matrix and encapsulated with membranes derived from M2 phenotype macrophages (Fe-M2NPs). This integrates three core functions—antioxidant scavenging of reactive oxygen species (ROS), active immune regulation, and non-contact dynamic mechanical stimulation—into a single system, addressing the limitations of existing dressings in terms of single-function application and insufficient synergistic effects.

[0016] Preferably, the magnetic nanoparticles include a superparamagnetic iron oxide core, and the M2 macrophage membrane is derived from interleukin-4 polarized macrophages.

[0017] Preferably, the concentration of M2 film-masked magnetic nanoparticles in the dual-network polymer matrix is ​​400-800 μg / mL.

[0018] A second objective of this invention is to provide a method for preparing the M2 film-masked magnetic nanoparticle composite hydrogel, comprising the following steps:

[0019] S1. Extract the membrane of M2 macrophages and prepare M2 membrane-masked magnetic nanoparticles (Fe-M2NPs) by extrusion with iron nanoparticles.

[0020] S2. Type I collagen extracted from tilapia skin is cross-linked with gallic acid via EDC / NHS to obtain gallic acid-modified collagen. Hyaluronic acid is oxidized with sodium periodate under light-protected conditions to obtain a mixture. The mixture is then dialyzed and freeze-dried sequentially to obtain oxidized hyaluronic acid solid. The oxidized hyaluronic acid solid obtained above is prepared into a solution, and methacrylic anhydride is added and reacted under ice-water bath conditions. The pH of the reaction system is adjusted to maintain at 8.0. After the reaction is completed, the mixed solution is dialyzed and freeze-dried sequentially to obtain oxidized methacrylamide hyaluronic acid solid. The gallic acid-modified collagen is dissolved in 0.01-0.03 M acetic acid to prepare a solution with a concentration of 8-12 mg / mL, and the pH is adjusted to neutral to obtain solution A. The oxidized methacrylamide hyaluronic acid solid is dissolved in water to prepare a solution B with a concentration of 140-160 mg / mL. The M2 type membrane-masked magnetic nanoparticles obtained in step S1 are dissolved in PBS to obtain a solution B with a concentration of 8-12 mg / mL. A solution of M2-type membrane-masked magnetic nanoparticles at a concentration of mg / mL was prepared. Solution A, solution B, the M2-type membrane-masked magnetic nanoparticle solution, and a 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (LAP photoinitiator) solution were mixed uniformly at 3℃-6℃ in the dark and allowed to stand at 37℃. The first network was formed through Schiff base reaction and Fe³⁺-induced CG self-assembly. Subsequently, the second network was photocrosslinked and strengthened in the presence of blue visible light and the photoinitiator LAP.

[0021] Preferably, step S1 specifically includes the following steps:

[0022] S11. RAW264.7 macrophages were stimulated with interleukin-4 to induce them to polarize into the M2 phenotype, which has anti-inflammatory and repair-promoting functions.

[0023] S12. Cells are broken up, and M2 cell membrane vesicles are separated and purified. The purified M2 cell membrane is mixed with iron nanoparticles to obtain a mixture. The mixture is extruded several times to form structurally stable magnetic nanoparticles Fe-M2NPs disguised as M2 macrophage membranes.

[0024] Further optimization involves stimulating RAW264.7 macrophages with interleukin-4 (IL-4, 40 ng / mL) for 24 h to induce them to polarize into the M2 phenotype, which has anti-inflammatory and repair-promoting functions (confirmed by high expression of the CD206 marker).

[0025] Further optimized, in step S12, cells are disrupted using a cell membrane protein extraction kit to separate and purify M2 cell membrane vesicles; the amount of iron nanoparticles added to the purified M2 membrane masquerading magnetic nanoparticles is 3-5 mg of iron nanoparticles per milligram of membrane protein, and the mixture is sequentially extruded several times through polycarbonate membranes with pore sizes of 400 nm and 200 nm. During this extrusion process, the M2 cell membrane spontaneously reorganizes under the action of fluid shear force, encapsulating the iron nanoparticles (Fe NPs) to form structurally stable Fe-M2NPs.

[0026] Preferably, step S2 specifically includes the following steps:

[0027] S21. First, lyophilized tilapia-derived type I collagen (Col) is dissolved in acetic acid solution to prepare a collagen solution with a concentration of 4-6 mg / mL. Gallic acid (GA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) are dissolved in deionized water in a molar ratio of 1.2:1:1, protected from light. The mass-to-volume ratio of gallic acid to deionized water is 1.25% (w / v, mg / mL). The mixture is slowly stirred at room temperature for 0.5-1.5 hours to obtain an activated GA / EDC / NHS mixed solution. This step aims to activate the carboxyl group of GA, making it easier for it to react with the amino group of collagen. The activated GA / EDC / NHS mixed solution is slowly added dropwise to the collagen solution, with a volume ratio of 1:3 between the mixed solution and the collagen solution. Subsequently, the pH of the mixture was adjusted to 5.0 with NaOH solution, and the reaction was carried out under a nitrogen atmosphere with stirring in the dark for 20-28 hours (this condition aims to promote the formation of stable amide bonds between GA and the amino groups of collagen side chains, achieving covalent grafting of GA). After the reaction, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 2-4 days to completely remove unreacted GA, EDC, NHS, and byproducts. Finally, the purified solution was freeze-dried to obtain a white, gallic acid-modified collagen (CG) sponge-like solid, which was then stored in a sealed container at -20°C.

[0028] S22. First, dissolve sodium hyaluronate (HA) in deionized water to obtain a sodium hyaluronate solution with a concentration of 0.005-0.015 g / mL. Then, dissolve sodium periodate in deionized water to obtain a sodium periodate solution with a concentration of 0.05-0.15 g / mL. Add the sodium periodate solution dropwise to the HA solution, with a mass ratio of sodium periodate to HA of 1:1.5-2.5. React at room temperature in the dark for 2.5-3.5 hours. Add ethylene glycol to terminate the reaction and continue stirring for 0.5-1.5 hours to consume the remaining sodium periodate. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed with deionized water for 2-4 days. After freeze-drying, oxidized hyaluronic acid (OHA) solid was obtained. The prepared OHA was dissolved in deionized water to prepare an 8-12 mg / mL OHA solution. Methacrylic anhydride was slowly added dropwise to the OHA solution at a volume ratio of 0.5-1.5:100 under an ice-water bath at 4°C. Subsequently, the pH of the reaction system was maintained at 8.0 with NaOH solution, and the reaction was continued for 10-14 hours. After the reaction was completed, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed with deionized water for 3-4 days. After freeze-drying, oxidized methacryloyl hyaluronic acid (OHAMA) white fibrous solid was obtained and stored at -20°C in the dark.

[0029] S23. The gallic acid-modified collagen was dissolved in 0.01-0.03 M acetic acid to prepare a solution with a concentration of 8-12 mg / mL, and the pH was adjusted to neutral with NaOH to obtain solution A; methacryloyl hyaluronic acid solid was dissolved in water to prepare solution B with a concentration of 140-150 mg / mL; the magnetic nanoparticles disguised as M2 macrophage membranes obtained in step S1 were dissolved in PBS to obtain a Fe-M2NPs solution with a concentration of 8-12 mg / mL; solutions A, B, Fe-M2NPs solution, and LAP photoinitiator solution were mixed evenly at 4°C in the dark, and allowed to stand at 37°C for 10 minutes to form the first network through Schiff base reaction; then, it was irradiated under 405 nm, 30 mW / cm² blue light for 90 seconds to form a double-network hydrogel (labeled as CGOHM).

[0030] Further optimized, in step S22, sodium hyaluronate (HA) is first dissolved in deionized water to obtain a sodium hyaluronate solution with a concentration of 0.01 g / mL, and sodium periodate is dissolved in deionized water to obtain a sodium periodate solution with a concentration of 0.1 g / mL. The sodium periodate solution is added dropwise to the HA solution, with a mass ratio of sodium periodate to HA of 1:2. The reaction is carried out at room temperature in the dark for 3 hours. Ethylene glycol is added to terminate the reaction, with a volume ratio of ethylene glycol to sodium periodate solution of 1:5. The mixture is stirred for another hour to consume the remaining sodium periodate. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed with deionized water for 3 days. After freeze-drying, oxidized hyaluronic acid (OHA) solid was obtained. The OHA obtained above was dissolved in deionized water to prepare a 10 mg / mL OHA solution. Methacrylic anhydride was slowly added dropwise to the OHA solution under ice-water bath conditions at 4°C. The volume ratio of methacrylic anhydride to OHA solution was 1:100. Subsequently, the pH of the reaction system was maintained at 8.0 with NaOH solution, and the reaction was continued for 12 hours.

[0031] Further preferred, in step S23, the concentration of the LAP photoinitiator solution is 15-25 mg / mL, and the mass ratio of CG, OHAMA, Fe-M2NPs, and LAP photoinitiator is (22.4-33.6):(43.68-46.8):(1.088-3.408):(2.25-3.75).

[0032] Further optimization involves the following: the concentration of the LAP photoinitiator solution is 20 mg / mL; gallic acid-modified collagen is dissolved in acetic acid to prepare a solution A with a concentration of 10 mg / mL; oxymethacrylamide hyaluronic acid solid is dissolved in water to prepare a solution B with a concentration of 144 mg / mL; the concentration of the Fe-M2NPs solution is 10 mg / mL; and the mass ratio of CG, OHAMA, Fe-M2NPs, and LAP photoinitiator is 28:45:(1.36-2.84):3.

[0033] A third objective of this invention is to provide the application of the aforementioned magnetic composite hydrogel in the preparation of pharmaceutical formulations for tissue repair.

[0034] Preferably, tissue repair is for the healing of diabetic wounds.

[0035] This invention constructs a multifunctional, integrated, magnetically responsive composite hydrogel. The hydrogel comprises a dual-network polymer matrix formed by gallic acid-modified collagen (CG) and oxymethyl methacrylate hyaluronic acid (OHAMA). Magnetic nanoparticles are embedded within this matrix and encapsulated with membranes from M2 phenotype macrophages (Fe-M2NPs). This integrates three core functions—antioxidant scavenging of reactive oxygen species (ROS), active immune regulation, and non-contact dynamic mechanical stimulation—into a single system, addressing the limitations of existing dressings in terms of single function and insufficient synergistic effects. It also provides a non-invasive, deep-tissue-accessible, and highly patient-compliant treatment solution. Controlled by an external magnetic field, it avoids direct contact and secondary damage to the wound and enables precise spatiotemporal therapeutic stimulation, making it suitable for long-term, safe treatment of patients with diabetes and other conditions accompanied by sensory impairment.

[0036] A fourth objective of this invention is to provide a pharmaceutical formulation for tissue repair, using the aforementioned magnetic composite hydrogel as the active ingredient.

[0037] Preferably, tissue repair specifically refers to the healing of diabetic wounds.

[0038] A fifth object of the present invention is to provide a method of using the aforementioned pharmaceutical preparation for tissue repair, wherein the pharmaceutical preparation for tissue repair is administered in conjunction with an external dynamic magnetic field.

[0039] Preferably, the operation steps are as follows: After applying the drug preparation, an external dynamic magnetic field (DMF) is applied to induce dynamic mechanical stimulation in the magnetic nanoparticles within the hydrogel. Specifically, after applying the drug preparation to the diabetic wound, an external dynamic magnetic field (DMF) is applied to induce dynamic mechanical stimulation in the magnetic nanoparticles within the hydrogel.

[0040] The drug formulation was applied to the wound site of the subject; then an external dynamic magnetic field (DMF) was applied to the wound site, thereby generating dynamic mechanical stimulation from magnetic nanoparticles within the hydrogel. This dynamic mechanical stimulation activated the Piezo1-YAP mechanotropic axis in cells at the wound site. Activation of the Piezo1-YAP mechanotropic axis induced therapeutic calcium deficiency. 2+ Inflow promotes fibroblast proliferation and migration, promotes angiogenesis and / or induces macrophage polarization toward the M2 phenotype.

[0041] This invention utilizes an external dynamic magnetic field (DMF) to precisely manipulate magnetic nanoparticles embedded in a hydrogel in a non-contact manner, converting the mechanical energy of the physical field into biological signals that act on cells. It specifically targets the mechanobiological pathway: aiming to specifically activate the mechanosensitive ion channel Piezo1 on the cell membrane through the aforementioned mechanical stimulation, thereby triggering downstream Ca²⁺ influx and nuclear translocation of the YAP transcriptional coactivator, thus restarting the regeneration process suppressed in diabetic wounds at the molecular level.

[0042] This invention achieves the following multiple biological effects through the synergy of the aforementioned materials and mechanisms: Reversing oxidative stress: Effectively clearing excess ROS at wound sites, protecting cells from oxidative damage, and restoring mitochondrial function. Reshaping the immune microenvironment: Actively guiding macrophages to polarize from the pro-inflammatory M1 phenotype to the anti-inflammatory, pro-repair M2 phenotype, terminating chronic inflammation. Promoting tissue regeneration: Directly enhancing the migration, proliferation, and collagen synthesis capabilities of fibroblasts, and promoting the formation of functional angiogenesis by endothelial cells, accelerating granulation tissue formation and re-epithelialization.

[0043] Further preferably, the magnetic flux density of the external dynamic magnetic field is 400-600 mt, and the oscillation frequency is 0.5-1.0 Hz.

[0044] In a further preferred embodiment, the magnetic flux density of the external dynamic magnetic field is 400 mt, and the oscillation frequency is 0.5-0.6 Hz.

[0045] Compared with the prior art, the beneficial effects of the present invention are:

[0046] 1. Multifunctional synergistic therapy, breaking through the limitations of single-mode action. Existing diabetic wound dressings have limited functions. This invention, through material innovation, integrates three major functions: anti-oxidation, immune regulation, and mechanical stimulation. Experiments have shown that when this hydrogel is combined with a dynamic magnetic field (DMF), the DPPH and ABTS free radical scavenging rates reach 53.33% and 72.99%, respectively; at the same time, it can effectively induce macrophages to polarize towards the reparative M2 phenotype (increasing the proportion of CD206+ cells to 16.3%), achieving synergistic regulation of chemical scavenging, immune remodeling, and physical stimulation.

[0047] 2. Non-contact deep biomechanical modulation with a clear mechanism. Unlike traditional contact-based physical stimulation, this invention uses an external magnetic field to drive Fe-M2NPs to generate micro-movements, achieving precise biomechanical stimulation of deep tissues. Studies have confirmed that this stimulation specifically activates the mechanosensitive channel Piezo1, triggering Ca²⁺ influx and YAP nuclear translocation, restarting the regeneration process at the molecular level. Inhibitor experiments show that inhibiting Piezo1 significantly weakens Ca²⁺ signaling, confirming the dominant role of this channel in signal transduction.

[0048] 3. Excellent healing effect, fully validated in vitro and in vivo. In a diabetic rat model of full-thickness skin defects, CGOHM... Fe-M2NPs The healing rate in the +DMF group reached 88.56% on day 7, significantly higher than that in the control group (65.22%), and the wound was basically closed by day 14. Histological examination showed that this treatment promoted re-epithelialization, increased collagen deposition (48.06% of the area stained by Masson on day 7), and facilitated the orderly progression of the wound through the inflammation, proliferation, and remodeling stages.

[0049] 4. Innovative biomimetic design enables active immune regulation. Employing an M2 macrophage membrane camouflage strategy, the material possesses active targeting and signal transduction capabilities. M2 membrane surface proteins can directly interact with M1 macrophages, transmitting anti-inflammatory signals and effectively reversing the chronic inflammatory state of diabetic wounds. This mechanism was verified using multiple methods, including flow cytometry and ELISA (increased IL-10 and TGF-β secretion).

[0050] 5. Excellent material properties, possessing both therapeutic and clinical application potential. The hydrogel proposed in this invention exhibits high biocompatibility (hemolysis rate <0.6%) and rapid hemostasis (in vivo hemostasis time 15 seconds). Its injectability and photocurable properties allow it to perfectly adhere to irregular wound surfaces, providing convenience for clinical use. Transcriptomic analysis further confirms at the systemic level that it promotes healing by regulating calcium signaling pathways, providing genomic evidence for its therapeutic efficacy.

[0051] In summary, this invention, through interdisciplinary innovation, is significantly superior to existing technologies in terms of functional integration, mechanism of action, therapeutic effect, and clinical applicability, providing a novel solution for the treatment of diabetic wounds. Attached Figure Description

[0052] Figure 1 The characterization results of Fe-M2NPs obtained in Example 3 are shown below; (A) Macrophage IL-4 stimulated polarization immunofluorescence, scale bar: 50 μm; (B) Magnetization process of Fe-M2NPS; (C) TEM images of Fe NPs, M2 macrophage membrane and Fe-M2NPS; (D) Particle size; (E) Zeta potential; (F) SDS-PAGE.

[0053] Figure 2 Characterization spectra of the composite hydrogel obtained in Example 3; (A) Infrared spectra of CG and OHAMA; (B) UV-Vis spectra of Col and CG; (C) Circular dichroism (CD) spectral analysis of Col and CG; (D) 1H NMR spectra of HA, OHA and OHAMA; (E) Different gel states of the hydrogel after UV irradiation and the microstructure of the hydrogel, scale bar: 500 μm.

[0054] Figure 3 The mechanical properties and in vitro free radical scavenging performance of the composite hydrogel obtained in Example 3 are as follows: (A) Real-time modulus curve during the gelation process under light irradiation; (B) Oscillatory strain scan of the hydrogel (before light irradiation); (C) Oscillatory strain scan of the hydrogel (after light irradiation); (DG) UV-Vis detection of DPPH and ABTS free radical scavenging capabilities after hydrogel scaffold treatment, solution color change and quantitative analysis.

[0055] Figure 4 The hemostatic properties and biocompatibility of the composite hydrogel obtained in Example 3 are as follows: (A) Blood compatibility; (B) Representative blood coagulation images; (C) Blood coagulation index; (D) In ​​vitro coagulation time of the hydrogel determined by the inverted method; (E) Representative SEM images of the interaction between whole blood and the hydrogel. Scale bar: 20 μm; (F) In vivo hemostasis; (G) Quantitative hemostasis time. (H) CCK-8 cell viability assay. (1) Representative live / dead staining images of L929 cells, scale bar: 200 μm.

[0056] Figure 5 The antioxidant properties of the composite hydrogel obtained in Example 3 are shown below: (A) Representative fluorescence images of ROS levels in L929 cells assessed by ROS-specific probe (DCFH-DA) and (B) Quantitative analysis by flow cytometry, scale bar: 200 μm; (C) Quantitative analysis of mitochondrial membrane potential of L929 cells by flow cytometry staining with tetramethylrhodamine methyl ester (TMRM) and (D) Representative fluorescence images, scale bar: 50 μm; (E) Apoptosis analysis of L929 cells by flow cytometry; (F) Representative fluorescence images of L929 cells stained with Edu and (G) Quantitative analysis, scale bar: 200 μm; (H) Representative images of L929 cell adhesion points and cytoskeleton immunofluorescence staining.

[0057] Figure 6 Example 3 illustrates in vitro immunomodulation of macrophages; (A) Schematic diagram of macrophage immunomodulation; (B) Representative immunofluorescence images of M2 (CD206, green) and M1 (CD86, red) markers. Scale bar: 50 μm; (C) Flow cytometry quantification of CD206 and CD86; (D) Representative immunofluorescence image of NF-κB p65 localization. Scale bar: 50 μm; (E) ELISA detection of IL-10 and (F) TGF-β secretion; (G) TEM image of mitochondria, scale bar: 2 μm and 200 nm.

[0058] Figure 7 Example 3 shows the effect of composite hydrogel on full-thickness skin wound healing in diabetic rats; (A) Schematic diagram of the full-thickness regeneration treatment process in diabetic rats; (B) Representative wound images on days 0, 3, 7 and 14, scale bar: 2 mm; (C) Quantitative analysis of wound healing rate and (D) Wound size thermogram; (E) Representative H&E staining images on days 3, 7 and 14 after treatment, left scale bar: 1 mm, right scale bar: 100 μm; (F) Representative Masson staining images on days 3, 7 and 14 after treatment, left scale bar: 1 mm, right scale bar: 100 μm; Quantitative analysis; (G) Wound edge; (H) Epidermal thickness and (I) Collagen deposition.

[0059] Figure 8 To obtain the signaling pathways related to the promotion of diabetic wound healing by the composite hydrogel in Example 3, the following were observed: (A) Principal component analysis (PCA) of transcriptomics data; (B) Volcano plot of differentially expressed genes; (C) Gene ontology (GO) enrichment analysis of biological processes (BP), cellular components (CC), and molecular functions (MF) of differentially expressed genes in the CGOHMFe-M2NPs group; (D) KEGG enrichment analysis of differentially expressed genes in the CGOHMFe-M2NPs group; (E) Calcium-mediated signaling gene set enrichment analysis (GSEA); (F) Schematic diagram of the pathway of calcium ion entry into cells; (G) Representative images of calcium ion imaging in fibroblasts treated with Hv1a and (H) GsMTX4, scale bar: 50 μm; (I) Statistical analysis of calcium ion correlation in fibroblasts treated with Hv1a and (J) GsMTX4; (K) Immunofluorescence image of YAP in fibroblasts treated with GsMTX4, scale bar: 25 μm; (L) Relative expression of YAP and (M) Piezo1 mRNA.

[0060] Figure 9 The biocompatibility of composite hydrogels with different Fe-M2 NPs concentrations was obtained for Examples 1-3. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to the embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are considered to be commercially available materials and reagents that can be obtained through conventional markets and other commercial channels.

[0062] Comparative Example 1

[0063] A method for preparing an gallic acid-modified collagen / oxidized methacrylamide hyaluronic acid (CG / OHAMA) dual-network hydrogel (CGOHM hydrogel for short) includes the following steps:

[0064] S1, CG: This preparation, containing type I collagen extracted from tilapia skin and gallic acid (GA) cross-linked with EDC / NHS, possesses antioxidant activity. The specific steps are as follows: First, freeze-dried tilapia-derived type I collagen is dissolved in 0.02 M acetic acid solution to prepare a collagen solution with a concentration of 5 mg / mL. GA, EDC, and NHS are dissolved in 50 mL of deionized water at a molar ratio of 1.2:1:1, protected from light, and activated by slow stirring at room temperature for 1 hour. This step aims to activate the carboxyl groups of GA, making it easier for them to react with the amino groups of collagen. The activated GA / EDC / NHS mixed solution is then slowly added dropwise to the collagen solution, with a volume ratio of the mixed solution to the collagen solution of 1:3. Subsequently, the pH of the mixture is adjusted to 5.0 with NaOH solution. The reaction is carried out under a nitrogen atmosphere with light-protected stirring for 24 hours. This condition aims to promote the formation of stable amide bonds between GA and the amino groups of the collagen side chains, achieving covalent grafting of GA. After the reaction was complete, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3 days to completely remove unreacted GA, EDC, NHS, and byproducts. Finally, the purified solution was freeze-dried to obtain a white, sponge-like solid CG, which was then sealed and stored at -20°C.

[0065] S2. For OHAMA, first dissolve 0.5 g of sodium hyaluronate in 50 mL of deionized water to obtain an HA solution. Dissolve 0.5 g of sodium periodate in 5 mL of deionized water, then add it dropwise to the HA solution. React at room temperature in the dark for 3 hours. Add 1 mL of ethylene glycol to terminate the reaction, and continue stirring for 1 hour to consume the remaining sodium periodate. Transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 14000 Da, dialyze against deionized water for 3 days, and freeze-dry to obtain oxidized hyaluronic acid (OHA) solid. Dissolve the OHA obtained above in deionized water to prepare a 10 mg / mL OHA solution. Slowly add 1 mL of methacrylic anhydride to 100 mL of OHA solution in an ice-water bath at 4°C. Then, maintain the pH of the reaction system at 8.0 with NaOH solution and continue the reaction for 12 hours. After the reaction is complete, transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyze against deionized water for 3-4 days. After freeze-drying, OHAMA white fibrous solid is obtained and stored at -20°C in the dark.

[0066] S3, Hydrogel: Take 2.8 mL of neutral CG solution (10 mg / mL), 0.312 mL of OHAMA solution (144 mg / mL), and 0.15 mL of LAP photoinitiator solution (20 mg / mL), mix them thoroughly at 4℃ in the dark, and let them stand at 37℃ for 10 minutes to form the first network through Schiff base reaction. Then, irradiate them under 405 nm, 30 mW / cm² blue light for 90 seconds to form a double network hydrogel (labeled as CGOHM).

[0067] Example 1 (Low concentration Fe-M2NPs)

[0068] A method for preparing a magnetic composite hydrogel includes the following steps:

[0069] S1 and M2 membrane-masked magnetic nanoparticles (Fe-M2NPs): These were prepared by extruding M2 macrophage membranes with iron nanoparticles (Fe NPs). First, RAW264.7 macrophages were stimulated with interleukin-4 (IL-4, 40 ng / mL) for 24 h, inducing polarization to the M2 phenotype, which possesses anti-inflammatory and pro-repair functions (confirmed by high expression of the CD206 marker). Subsequently, cells were disrupted using a cell membrane protein extraction kit, and M2 cell membrane vesicles were isolated and purified. The purified M2 cell membranes were mixed with iron nanoparticles (Fe NPs) at a specific protein ratio (0.1 mg membrane protein: 0.4 mg Fe NPs). Using a liposome extruder, the mixture was extruded 20 times sequentially through polycarbonate membranes with 400 nm and 200 nm pore sizes. During this process, the M2 cell membrane spontaneously reorganized under fluid shear force, encapsulating the Fe NPs to form structurally stable Fe-M2NPs.

[0070] S2, Gallic acid-modified collagen / oxymethylacrylamide hyaluronic acid (CG / OHAMA) dual-network hydrogel (CGOHM hydrogel):

[0071] S21, CG: This preparation, containing type I collagen extracted from tilapia skin and gallic acid (GA) cross-linked with EDC / NHS, possesses antioxidant activity. The specific steps are as follows: First, freeze-dried tilapia-derived type I collagen is dissolved in 0.02 M acetic acid solution to prepare a collagen solution with a concentration of 5 mg / mL. GA, EDC, and NHS are dissolved in 50 mL of deionized water at a molar ratio of 1.2:1:1, protected from light, and activated by slow stirring at room temperature for 1 hour. This step aims to activate the carboxyl groups of GA, making it easier for them to react with the amino groups of collagen. The activated GA / EDC / NHS mixed solution is then slowly added dropwise to the collagen solution, with a volume ratio of the mixed solution to the collagen solution of 1:3. Subsequently, the pH of the mixture is adjusted to 5.0 with NaOH solution. The reaction is carried out under N2 atmosphere with light-protected stirring for 24 hours. This condition aims to promote the formation of stable amide bonds between GA and the amino groups of the collagen side chains, achieving covalent grafting of GA. After the reaction was complete, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3 days to completely remove unreacted GA, EDC, NHS, and byproducts. Finally, the purified solution was freeze-dried to obtain a white, sponge-like solid CG, which was then sealed and stored at -20°C.

[0072] S22. For OHAMA, first dissolve 0.5 g of sodium hyaluronate in 50 mL of deionized water to obtain an HA solution. Dissolve 0.5 g of sodium periodate in 5 mL of deionized water, then add it dropwise to the HA solution and react at room temperature in the dark for 3 hours. Add 1 mL of ethylene glycol to terminate the reaction, and continue stirring for 1 hour to consume the remaining sodium periodate. Transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 14000 Da, dialyze against deionized water for 3 days, and freeze-dry to obtain oxidized hyaluronic acid (OHA) solid. Dissolve the OHA obtained above in deionized water to prepare a 10 mg / mL OHA solution. Slowly add 1 mL of methacrylic anhydride to 100 mL of OHA solution under an ice-water bath at 4°C. Subsequently, maintain the pH of the reaction system at 8.0 with NaOH solution and continue the reaction for 12 hours. After the reaction is complete, transfer the mixed solution to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyze against deionized water for 3-4 days. After freeze-drying, OHAMA white fibrous solid is obtained and stored at -20°C in the dark.

[0073] S23. Hydrogel: Gallic acid-modified collagen (CG) was dissolved in 0.02 M acetic acid to prepare a solution with a concentration of 10 mg / mL, and the pH was adjusted to neutral with NaOH to obtain solution A (neutral CG solution); methacryloyl hyaluronic acid solid was dissolved in water to prepare solution B (OHAMA solution) with a concentration of 144 mg / mL; the M2 membrane masquerading as magnetic nanoparticles obtained in step S1 was dissolved in PBS to obtain a Fe-M2NPs solution with a concentration of 10 mg / mL. LAP photoinitiator was dissolved in water to prepare a LAP photoinitiator solution with a concentration of 200 mg / mL.

[0074] Take 2.8 mL of neutral CG solution (10 mg / mL), 0.312 mL of OHAMA solution (144 mg / mL), 0.136 mL of Fe-M2NPs solution (10 mg / mL), and 0.15 mL of LAP photoinitiator solution (20 mg / mL) and mix them thoroughly at 4℃ in the dark to make the final concentration of Fe-M2NPs 400 μg / mL. Let it stand at 37℃ for 10 minutes, and then irradiate it under 405 nm, 30 mW / cm² blue light for 90 seconds to make the final concentration of Fe-M2NPs 400 μg / mL, thus obtaining a magnetic composite hydrogel.

[0075] Example 2 (Medium concentration Fe-M2NPs)

[0076] Same as in Example 1, except that in step S23, 0.208 mL of Fe-M2NPs dispersion (10 mg / mL) was added to make the final concentration of Fe-M2NPs 600 μg / mL, thus obtaining the composite hydrogel.

[0077] Example 3 (High concentration Fe-M2NPs)

[0078] Same as in Example 1, except that in step S23, 0.284 mL of Fe-M2NPs dispersion (10 mg / mL) was added to make the final concentration of Fe-M2NPs 800 μg / mL, thus obtaining the composite hydrogel.

[0079] Test case

[0080] The composite hydrogels obtained in Examples 1-3 and Comparative Example 1 were characterized and tested as follows:

[0081] 1. Characterization of Fe-M2 NPs

[0082] Macrophage characterization was confirmed by iNOS (M1 phenotype) and CD206 (M2 phenotype) immunofluorescence. Dynamic light scattering (Malver, Zetasizer Pro, UK) facilitated the measurement of the size and zeta potential of the acquired nanoparticles. Morphological images were captured using transmission electron microscopy (TEM, Hitachi, HT7800, Japan). Nanoparticle membrane protein analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0083] 2. Structural characterization of CG and OHMA

[0084] The prepared materials were characterized by Fourier transform infrared spectroscopy (FITR, Nicolet iS20, ThermoFisher, USA), spectrophotometry (PerkinElmer, Lambda 1050+, UK), circular dichroism (CD, JASCO, J-1500, Japan) and nuclear magnetic resonance spectroscopy (1H NMR, AVANCE III HD 600, Bruker, Germany).

[0085] 3. Scanning electron microscope (SEM)

[0086] The morphology of the hydrogel cross sections was characterized using emission scanning electron microscopy (FE-SEM; Zeiss, Sigma, Germany).

[0087] 4. Rheological properties

[0088] Rheological tests were performed using a rotational rheometer (MCR302e, Antor Paar, Austria). Cylindrical hydrogels with a diameter of 25 mm were placed on the rheometer plate at 37°C. Oscillatory strain amplitude scans were performed at a frequency of 1 rad / s, with strain amplitudes ranging from 0.1% to 150%. For time-of-flight rheological scans, a custom-designed photopolymerization device was mounted on the base of the rheometer. CGOHM Fe-M2NPs Preserved for 60 s under wireless irradiation, and then irradiated with blue light (405 nm) for 120 s under a photocuring device (strain: 1%, angular frequency: 10 rad s⁻¹).

[0089] 5. In vitro reactive oxygen species scavenging performance

[0090] The antioxidant efficiency of the hydrogel was evaluated using DPPH and ABTS radical scavenging activity methods. For DPPH radical scavenging, 1 mL of the hydrogel scaffold and 2 mL of DPPH solution were incubated together in the dark at room temperature for 30 minutes, and the absorbance of the mixture was recorded at 517 nm using a spectrophotometer (PerkinElmer, Lambda 1050+, UK). For DPPH radical scavenging, 1 mL of the hydrogel scaffold and 1 mL of ABTS solution were incubated together in the dark at room temperature for 30 minutes, and the absorbance of the mixture was recorded at 734 nm using a spectrophotometer. The DPPH and ABTS scavenging activities were calculated as follows: DPPH / ABTS scavenging ability (%) = (A0-A1) / A0. A0 and A1 are the absorbances of the control (no sample) and the sample, respectively.

[0091] 6. DMF stimulation

[0092] In order to CGOOHM Fe-M2NPs Dynamic magnetomechanical stimulation was performed using hydrogels, with a 400 mT neodymium permanent magnet (length: 100 mm, width: 50 mm, height: 20 mm) mounted on a reciprocating actuator. The hydrogel construct was attached to the bottom of well plates or culture dishes, and the cyclic magnetic force was converted into mechanical perturbation of the loaded cells, stimulating them at a frequency of 0.5 Hz. All cell experiments were stimulated for 30 min / day, three times daily.

[0093] 7. Cell compatibility

[0094] Cell compatibility of L929 cells was determined using the Cell Counting Kit-8 (CCK-8) assay. L929 cells were counted at 3 × 10⁶ cells per well. 3 L929 cells were seeded at a density of 1 × 10⁶ cells per well on the surface of a hydrogel scaffold and co-cultured for 1, 3, and 5 days. After each co-culture, 10 μL of CCK-8 solution was added, followed by incubation for 1 hour. The absorbance at 450 nm was measured in triplicate. In the live / dead staining assay, L929 cells were seeded at a density of 1 × 10⁶ cells per sample. 4 Cells were seeded at a density of [number] cells onto different hydrogel scaffold surfaces in 24-well plates and cultured for 1, 3, and 5 days. After culturing in predetermined liquids for 1, 3, and 5 days, live and dead cells were stained green and red, respectively, with Calcein AM / PI. The samples were then observed using a high-content imaging system (Operetta CLS, PerkinElmer, UK).

[0095] 8. In vitro antioxidant capacity

[0096] First, an H2O2-induced oxidative stress model was established, and the H2O2 concentration was determined to be 400 μM. The extracellular ROS scavenging capacity of the hydrogel was detected using a DCFH-DA probe (S0033S, Beyotime, China). The DCFH-DA probe was then added, and the cells were incubated for 30 min. Fluorescence intensity was observed using a high-content imaging system, and the stained cells were analyzed by flow cytometry (BD FACS, Celesta, USA) according to the kit instructions. Furthermore, the membrane potential of mitochondria in L929 cells was examined by TMRM staining (C2001S, Beyotime, China). The stained cells were analyzed by flow cytometry according to the kit instructions. The effect of the hydrogel on L929 cell proliferation under oxidative stress was detected using an Edu kit. In addition, L929 cells were subjected to YAP (bs-3605R, Bioss) and Vinculin (bs-23650R, Bioss) immunofluorescence staining to detect the effect of magnetic response-induced mechanical stimulation on cell adhesion properties.

[0097] 9. Immune regulation

[0098] To simulate oxidative stress and macrophage activation in the diabetic microenvironment, RAW264.7 cells were first treated with LPS (100 ng / mL) and IFN-γ (20 ng / mL) for 1 day and co-cultured in a hydrogel for 2 days. For immunofluorescence staining, cells were stained with CD86 (AB119857, abcam) and CD206 (AB300621, abcam) to investigate macrophage polarization. Simultaneously, P65 staining was performed to investigate the immunomodulatory mechanisms. Furthermore, macrophages were treated with C86 antibody (561962, BDPharmingen) and CD206 antibody (568273BD, Pharmingen), and the stained cells were analyzed by flow cytometry according to the manufacturer's protocol. Similarly, the membrane potential of RAW cell mitochondria was examined by TMRM staining. The stained cells were analyzed by flow cytometry according to the kit method, and macrophage mitochondria were observed by TEM. To investigate the concentrations of IL-4 and TGF-β secreted by cells in the collected supernatant, an enzyme-linked immunosorbent assay (ELISA) kit (Fintest, Shanghai, China) was used.

[0099] 10. Establishment of animal models

[0100] All animal experiments were approved by the Animal Experiment Center of Hunan University and the Hunan University User Committee (No.: HNU-IACUC-2024-114) and conducted in accordance with the guidelines of the National Health and Medical Research Commission of the People's Republic of China. Six- to eight-week-old male SD rats were used, and STZ (65 mg / kg) was administered. −1 Intraperitoneal injection was administered. Rats were observed daily. After 3 days, blood was collected from the tail vein to measure random blood glucose levels. Random blood glucose levels >16.7 mmol / L were recorded. -1 The rats were considered diabetic rats. For a diabetic full-thickness dorsal skin wound model, diabetic rats were randomly divided into four groups: control group (no hydrogel scaffold treatment), CGOHM group (comparative example 1), CGOHM... Fe-M2NPs Group (Example 1) and CGOHM Fe-M2NPs (Example 1) +DMF (magnetic flux density of 400 mt, oscillation frequency of 0.5 Hz) group. A full-thickness wound was made on the skin of the back using a 10 mm diameter perforated biopsy device. The wound healing process was photographed with a digital camera, and the wound size was quantified using ImageJ software.

[0101] 11. Immunohistochemistry and immunofluorescence

[0102] Samples collected on days 3, 7, and 14 were fixed in 4% paraformaldehyde for 24 h, then embedded in paraffin, cut into 40 μm thick sections, and stained for analysis. On days 3, 7, and 14, the wound closure area was determined using hematoxylin and eosin (H&E) staining, and collagen deposition was quantified using Masson's trichrome staining. Immunohistochemistry using CD86 (561962, BD Pharmingen) and CD206 (568273BD, Pharmingen) immunofluorescence staining for IL-6 (DF6087, Affinity), IL-10 (DF6894, Affinity), TNF-α (AF7014, Affinity), and Arg-1 (93668S, Cell Signaling) was used to observe macrophage infiltration and differentiation in the wound. For the proliferation and remodeling stages, immunohistochemistry and immunofluorescence staining were performed using CD31 (AF6191, Affinity), VEGF (AF5131, Affinity), α-SMA (AF1032, Affinity), Ki67 (GB111499, Servicebio), COLⅠ (AF9001, Affinity), and COLⅢ (22734-1, Proteintech), and quantitative analysis was performed using Imagej.

[0103] 12. Transcriptomic analysis of regenerated wound tissue

[0104] Transcriptomic analysis was performed on diabetic wound tissues from the control group and the CGOOHMFe-M2NPs+DMF group by Sewell Biotech (Hangzhou). In short, total RNA was extracted from the diabetic wound sites of rats on day 14 using TRIzol reagent. The size and integrity of the extracted products were confirmed by detection using an Agilent 4150 TapeStation. Differential gene expression, gene ontology terminology, and signaling pathway terminology were analyzed.

[0105] The test results are as follows:

[0106] 1. Characterization of Fe-M2 NPs:

[0107] The M2 polarization status of macrophages was assessed by detecting the expression of iNOS (M1 marker) and CD206 (M2 marker). The results showed that IL-4 treatment increased CD206 expression, indicating that cells successfully polarized to the M2 type (iNOS, M1 marker, and CD206, M2 marker). Figure 1 A). Subsequently, the extracted M2 cell membrane and Fe NPS were extruded through a continuous porous membrane to obtain functionalized Fe-M2NPS. These nanoparticles were black and attracted to a magnet, indicating good paramagnetism. Figure 1 B). TEM images further revealed that Fe-M2NPS has a typical core-shell structure ( Figure 1 C), with Fe as the core and an outer layer of a cell membrane double shell, has an average particle size of approximately 200 nm, consistent with the DLS results. Figure 1 D). Zeta potential analysis showed that the potential of Fe NPs was -10.6 mV, while after successful coating of the M2 cell membrane, the potential of Fe-M2NPS became -15.1 mV. Figure 1 E). Furthermore, as described by polyacrylamide gel electrophoresis (SDS-PAGE), Fe-M2 NPS and M2 macrophage membranes exhibited similar protein bands (E). Figure 1 F) indicates that the membrane surface protein has been successfully transferred to the nanoparticle surface.

[0108] 2. Synthesis and structural characterization of hydrogels:

[0109] First, CG and OHAMA were synthesized, and the structure of CG was characterized by FTIR, with the spectrum at 3325 cm⁻¹. -1 The peak at 1640 cm⁻¹ represents the stretching vibration of the phenolic hydroxyl group (–OH), while the amide I band extends from 1640 cm⁻¹. -1 Displaced to 1632 cm -1 This indicates that GA has been successfully grafted onto collagen. Figure 2 A). The UV-Vis spectrum further reveals the characteristic absorption peak of the conjugated structure of GA at 265 nm. Figure 2B). Circular dichroism (CD) spectroscopy analysis confirmed that the modified collagen still maintained its complete triple helix structure. Figure 2 C). For OHAMA, at 1730 cm -1 An enhanced C=O extension is observed at 1614 cm⁻¹, and the characteristic C=C absorption peak of methacrylate appears at 1614 cm⁻¹. 1 1H NMR spectroscopy further confirmed the successful methacrylation modification, with new peaks appearing at 5.7 and 6.2 ppm, corresponding to vinyl protons ( Figure 2 D). To construct the CGOHMFe-M2NPs hydrogel scaffold, the three main components, CG, OHAMA, and Fe-M2NPs, were rapidly mixed. First, a first-layer network was formed through a Schiff base reaction and Fe³⁺-induced CG self-assembly. Subsequently, a second-layer network was photocrosslinked and enhanced in the presence of blue visible light and the photoinitiator LAP. The microstructure was observed using SEM (Self-Eye Microscopy). Figure 2 The CGOHM framework (E) is a porous structure that provides ample space for the loading of Fe-M2NPs. Interestingly, the addition of Fe-M2NPs can further bind with gallic acid, enhancing the cross-linking network.

[0110] 3. Mechanical properties and in vitro free radical scavenging properties of the hydrogel:

[0111] The gelation process of CGOHMFe-M2NPs was monitored in real time using an optical rheometer. Figure 3 A). Before irradiation (0–60 s), the system was in a pre-gel state with low storage modulus (G′) and loss modulus (G″). After irradiation, G′ and G″ increased rapidly, entering a rheologically stable phase after 100 s. At 0 s and 100 s of irradiation, G′ was 9.6 Pa and 467.2 Pa, respectively, an increase of 48.6 times, indicating that the system had completed photocrosslinking and formed a stable gel.

[0112] Further verification of the dual-network structure was achieved through amplitude scanning. Figure 3 B, C). Under light-protected conditions at 37℃, G′ was already higher than G″, indicating the formation of the first network through collagen self-assembly and Schiff base reaction. After light exposure, G′ increased further, reflecting photocrosslinking of the methacrylamide groups in OHAMA, constructing the second network. After introducing Fe-M2NPs, the linear viscoelastic region G′ increased from 106.6 Pa to 322.8 Pa, indicating that it significantly enhances the stiffness and structural stability of the hydrogel through coordination with the tea polyphenol groups in gallic acid, which is beneficial for providing long-term mechanical support. Its antioxidant properties were evaluated by DPPH and ABTS free radical scavenging experiments. Figure 3(D–G). CGOHM already possesses certain antioxidant capabilities due to the presence of gallic acid phenolic hydroxyl groups, while CGOHM Fe-M2NPs further enhance the free radical scavenging ability. This is attributed to the Fe-M2NPs conferring peroxidase / catalase-like activity to the system, synergistically working with gallic acid. When combined with DMF treatment, the antioxidant efficacy was highest, with DPPH and ABTS scavenging rates reaching 53.33% and 72.99%, respectively. The solution color also significantly lightened, suggesting that DMF may synergistically enhance antioxidant performance by promoting electron transfer and increasing the exposure of active sites.

[0113] 4. Hemostasis and biocompatibility of hydrogels

[0114] In clinical wound management, rapid hemostasis is particularly crucial for patients with coagulation disorders such as diabetes. Ideal hemostatic materials must possess both high coagulation efficiency and good biocompatibility. This study systematically evaluated the hemostatic properties of CGOHM and CGOHMFe-M2NPs hydrogels. Both exhibited good blood compatibility, with a hemolysis rate of <0.6%. Figure 4 A). CGOHMFe-M2NPs showed superior hemostatic ability, with a coagulation index (BCI) of 28.2%, lower than CGOHM (31.24%) and commercial reagents (38.65%). Figure 4 B, C). In vitro coagulation time assays showed that blood in the PBS group did not clot after 120 seconds, while both hydrogels promoted blood coagulation within 11 seconds, faster than commercial reagents (B, C). Figure 4 D). Scanning electron microscopy images show that CGOHMFe-M2NPs adsorb erythrocytes and form dense aggregates on their surface, indicating that they have a stronger ability to induce erythrocyte aggregation. Figure 4 E. Verification of in vivo hemostasis using a rodent tail hemorrhage model ( Figure 4 F,G), CGOHMFe-M2NPs hydrogel shortens bleeding time to 15 s. Its rapid hemostasis mechanism includes: 1) photocuring forms a physical barrier, concentrating coagulation components; 2) iron ions in Fe-M2NPs and the GA catechol structure synergistically mimic natural cofactors, enhancing erythrocyte and platelet aggregation. Furthermore, the hydrogel exhibits good biocompatibility; CCK-8 and live / dead cell staining both support high cell viability (…). Figure 4 H,I), and the composite hydrogels with Fe-M2 NPs concentrations of 400-800 μg / mL all exhibited good biocompatibility (H,I), Figure 9 ).

[0115] 5. Antioxidant properties of hydrogels

[0116] To address the issue of oxidative stress delaying wound healing in diabetic wounds, this study systematically evaluated the antioxidant and repair-promoting functions of hydrogels under dynamic magnetic field (DMF) intervention. In a 400 μM H2O2-induced oxidative stress model, detection using the DCFH-DA fluorescent probe revealed (… Figure 5 A) Intracellular ROS levels significantly increased after H2O2 stimulation, while hydrogel treatment effectively removed ROS. Flow cytometry quantitative analysis further demonstrated that ( Figure 5 B), CHOMH Fe-M2NPs Combined use with DMF restored ROS levels to near the normal control group.

[0117] Mitochondrial membrane potential detection showed ( Figure 5 C, D), H2O2 significantly reduced membrane potential, while the magnetically responsive hydrogel effectively restored the membrane potential upon DMF stimulation. This functional restoration not only ensured ATP synthesis but also significantly reduced cell death by inhibiting mitochondrial apoptosis. Annexin V / PI flow cytometry confirmed ( Figure 5 E), CHOMH Fe-M2NPs +DMF reduced the H2O2-induced total apoptosis rate from 27.4% to 2.8%. Cell proliferation capacity was assessed using an EdU assay. Figure 5 The proportion of EdU-positive cells in the F,G)CGOHMFe-M2NPs+DMF group reached 52.65%, which was 2.03 times higher than that in the H2O2 group, indicating that it significantly promoted cell proliferation. Cytoskeleton fluorescence staining showed ( Figure 5 H), H2O2 stimulation caused cell contraction and blurred actin structure, while cells on hydrogel scaffolds exhibited better spreading morphology. In CGOHM Fe-M2NPs In the +DMF group, cells further exhibited significant aggregation of actin stress fibers and nysin at focal adhesion sites. These results indicate that CGOHM Fe-M2NPs When used in combination with DMF, it significantly enhances the protective effect against oxidative stress damage and promotes repair by effectively clearing ROS, restoring mitochondrial function, inhibiting apoptosis, and promoting cell proliferation and spread.

[0118] 6. In vitro immunomodulatory capacity of hydrogels

[0119] To evaluate the immunomodulatory function of CGOHMFe-M2NPs hydrogel, this study established an M1 polarization model by inducing RAW264.7 macrophages with LPS / IFN-γ, and then performed interventions on different hydrogel samples. Figure 6 A).

[0120] Immunofluorescence and flow cytometry analysis showed that ( Figure 6B, C), CGOMHFe-M2NPs combined with DMF intervention effectively reversed M1 polarization, significantly reduced the expression of the M1 marker CD86 (from 20.4% to 5.77%), and increased the proportion of M2 marker CD206-positive cells to 16.3%, indicating that it successfully promoted the conversion of macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. At the mechanistic level, immunofluorescence analysis showed ( Figure 6 (D) This combined treatment significantly inhibited LPS / IFN-γ-induced p65 nuclear translocation, indicating that NF-κB pathway activation was effectively suppressed. ELISA assays further confirmed this. Figure 6 Combined treatment with E and F significantly increased the secretion levels of the anti-inflammatory factor IL-10 and the repair-related factor TGF-β, functionally validating the establishment of the M2 phenotype. To explore the metabolic basis of phenotypic transformation, this study assessed mitochondrial function and ultrastructure. The results showed that ( Figure 6 G), CGOHMFe-M2NPs+DMF treatment can effectively restore the decrease in mitochondrial membrane potential induced by LPS / IFN-γ. TEM observation results ( Figure 6 H) Further, this treatment significantly improved mitochondrial structural integrity and promoted cristae structure recovery, confirming its metabolic regulatory role at the ultrastructural level. These results collectively indicate that CGOHMFe-M2NPs combined with DMF can effectively regulate the M1-M2 phenotypic transition of macrophages by inhibiting the NF-κB pathway and improving mitochondrial function, thereby exerting immunomodulatory and repair-promoting effects.

[0121] 7. In vivo evaluation of diabetic regeneration

[0122] To evaluate the comprehensive therapeutic effect of this hydrogel system under complex pathological conditions, we constructed an STZ-induced full-thickness skin defect model in diabetic rats, and established a control group, a CGOHM group, and a CGOHM group. Fe-M2NPs Group and CGOHM Fe-M2NPs +DMF joint group intervention ( Figure 7 A). Macroscopic healing observation showed that on day 14, CGOHM Fe-M2NPs The wound in the +DMF combined group was almost completely closed, while unhealed areas remained in the other groups. Figure 7 B). Quantitative analysis showed that by day 7, the healing rate of the combined group had reached 88.56%, significantly higher than that of other groups (65.22%-75.82%). By day 14, only the combined group achieved complete healing. Figure 7 C, D). Histological analysis showed that the combined group had the lowest degree of inflammatory cell infiltration on day 3, indicating that it effectively alleviated early excessive inflammation. Figure 7 E). On day 7, the wound edge distance in the combined group (3.22 mm) was significantly smaller than that in the control group (5.47 mm). Figure 7G). By day 14, the combined group had achieved complete reepithelialization, and the degree of epithelialization was the best. Figure 7 H). Masson staining results showed that on day 7, the combined group had a collagen deposition area of ​​48.06%, significantly higher than other groups (26.28%-40.21%), and the collagen fibers were arranged in a mature network. Figure 7 F,I). On day 14, the combined group still maintained the highest collagen deposition and maturity. These results indicate that CGOHM Fe-M2NPs When used in combination with DMF, it significantly accelerates the healing process of diabetic wounds by effectively reducing inflammation, promoting epithelialization, and guiding orderly ECM remodeling.

[0123] 8. Enrichment of signaling pathways related to hydrogel-induced diabetic wound healing

[0124] To elucidate the molecular mechanism by which CGOHMFe-M2NPs combined with dynamic magnetic field (DMF) promotes the repair of diabetic wounds, we performed RNA sequencing analysis on rat wound tissue. Principal component analysis showed that the intervention induced significant transcriptome remodeling. Figure 8 A), the combined treatment group identified a total of 363 upregulated and 51 downregulated genes (A). Figure 8 B). Consistent GO and KEGG enrichment analyses showed that the calcium signaling pathway was deeply activated ( Figure 8 C,D), including key components such as calcium ion transport, voltage-gated calcium channel complexes, and calmodulin binding. GSEA further confirmed a significant upregulation of calcium-mediated signaling (C,D). Figure 8 E). Based on the mechanosensing function of Piezo1, we propose that CGOHMFe-M2NPs+DMF activates Piezo1 channels to induce initial Ca²⁺ influx, which in turn activates voltage-gated calcium channels (VGCCs) through membrane potential depolarization, forming a signal amplification loop. This was validated using the Piezo1 inhibitor GsMTX4 and the VGCC inhibitor ω-hexatoxin-Hv1a. Figure 8 F), immunofluorescence and quantitative results showed ( Figure 8 GJ), inhibiting Piezo1 significantly attenuates the Ca²⁺ signal, while inhibiting VGCCs only causes partial attenuation, confirming that Piezo1 plays a dominant role in signal initiation. Further research revealed that this mechanosensitive Ca²⁺ signal can drive YAP / TAZ nuclear translocation ( Figure 8 K), and the mRNA expression of YAP and Piezo1 was synchronously upregulated ( Figure 8 L,M), thus constructing a complete signaling axis from mechanosensory to gene regulation: Piezo1 / VGCCs / Ca²⁺ / YAP.

[0125] The above examples demonstrate that the addition of Fe-M2NPs is the key to achieving the magnetic response function and enhancing the antioxidant performance of this invention. Compared with pure Fe NPs, Fe-M2NPs have better biocompatibility and are still non-biotoxic to cells at 800 μg / mL.

[0126] Example 4

[0127] Same as Example 2, except that:

[0128] S1. The amount of iron nanoparticles added is 3 mg of iron nanoparticles per milligram of membrane protein.

[0129] S2, Gallic acid-modified collagen / oxymethylacrylamide hyaluronic acid (CG / OHAMA) dual-network hydrogel (CGOHM hydrogel):

[0130] S21. First, the freeze-dried tilapia-derived type I collagen (Col) was dissolved in acetic acid solution to prepare a collagen solution with a concentration of 4 mg / mL. Gallic acid (GA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were dissolved in deionized water in a molar ratio of 1.2:1:1, protected from light. The mass-to-volume ratio of gallic acid to deionized water was 1.25% (w / v). The solution was slowly stirred at room temperature for 0.5 hours to obtain an activated GA / EDC / NHS mixed solution. The activated GA / EDC / NHS mixed solution was then slowly added dropwise to the collagen solution, with a volume ratio of 1:3 between the mixed solution and the collagen solution. Subsequently, the pH of the mixture was adjusted to 5.0 with NaOH solution. The mixture was then stirred and reacted for 20 hours under a nitrogen atmosphere in the dark. After the reaction, the solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 2 days to completely remove unreacted GA, EDC, NHS, and byproducts. Finally, the purified solution was freeze-dried to obtain a white, gallic acid-modified collagen (CG) sponge-like solid, which was then stored in a sealed container at -20°C.

[0131] S22. First, dissolve sodium hyaluronate (HA) in deionized water to obtain a sodium hyaluronate solution with a concentration of 0.005 g / mL. Then, dissolve sodium periodate in deionized water to obtain a sodium periodate solution with a concentration of 0.05 g / mL. Add the sodium periodate solution dropwise to the HA solution, with a mass ratio of sodium periodate to HA of 1:1.5. React at room temperature in the dark for 2.5 hours. Add ethylene glycol to terminate the reaction and continue stirring for 0.5 hours to consume the remaining sodium periodate. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed against deionized water for 2 days. After freeze-drying, oxidized hyaluronic acid (OHA) solid was obtained. The prepared OHA was dissolved in deionized water to prepare an 8 mg / mL OHA solution. Methacrylic anhydride was slowly added dropwise to the OHA solution at a volume ratio of 0.5:100 under an ice-water bath at 4°C. Subsequently, the pH of the reaction system was maintained at 8.0 with NaOH solution, and the reaction was continued for 10 hours. After the reaction was completed, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed against deionized water for 3 days. After freeze-drying, oxidized methacryloyl hyaluronic acid (OHAMA) white fibrous solid was obtained and stored at -20°C in the dark.

[0132] S23. Gallic acid-modified collagen was dissolved in acetic acid to prepare a solution A with a concentration of 8 mg / mL. Oxymethylacrylamide hyaluronic acid solid was dissolved in water to prepare a solution B with a concentration of 140 mg / mL. M2 membrane-disguised magnetic nanoparticles were dissolved in water to obtain a Fe-M2NPs solution with a concentration of 8 mg / mL. The concentration of LAP photoinitiator solution was 15 mg / mL. The mass ratio of CG, OHAMA, Fe-M2NPs, and LAP photoinitiator was 22.4:43.68:0.448:2.25. Solutions A, B, Fe-M2NPs solution, and LAP photoinitiator solution were mixed evenly at 4℃ in the dark, and then allowed to stand at 37℃ for 10 minutes. Subsequently, the mixture was irradiated under 405 nm, 30 mW / cm² blue light for 90 seconds to form a magnetic composite hydrogel.

[0133] Example 5

[0134] Same as Example 2, except that:

[0135] S1. The amount of iron nanoparticles added is 5 mg of iron nanoparticles per milligram of membrane protein.

[0136] S2, Gallic acid-modified collagen / oxymethylacrylamide hyaluronic acid (CG / OHAMA) dual-network hydrogel (CGOHM hydrogel):

[0137] S21. First, the freeze-dried tilapia-derived type I collagen (Col) was dissolved in acetic acid solution to prepare a collagen solution with a concentration of 6 mg / mL. Gallic acid (GA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were dissolved in deionized water in a molar ratio of 1.2:1:1, protected from light. The mass-volume ratio of gallic acid to deionized water was 1.25% (w / v). The solution was slowly stirred at room temperature for 1.5 hours to obtain an activated GA / EDC / NHS mixed solution. The activated GA / EDC / NHS mixed solution was then slowly added dropwise to the collagen solution, with a volume ratio of 1:3 between the mixed solution and the collagen solution. Subsequently, the pH of the mixture was adjusted to 5.0 with NaOH solution. The mixture was then stirred and reacted for 28 hours under a nitrogen atmosphere in the dark. After the reaction, the solution was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 4 days to completely remove unreacted GA, EDC, NHS, and byproducts. Finally, the purified solution was freeze-dried to obtain a white, gallic acid-modified collagen (CG) sponge-like solid, which was then stored in a sealed container at -20°C.

[0138] S22. First, dissolve sodium hyaluronate (HA) in deionized water to obtain a sodium hyaluronate solution with a concentration of 0.015 g / mL. Dissolve sodium periodate in deionized water to obtain a sodium periodate solution with a concentration of 0.15 g / mL. Add the sodium periodate solution dropwise to the HA solution. The mass ratio of sodium periodate to HA is 1:2.5. React at room temperature in the dark for 3.5 hours. Add ethylene glycol to terminate the reaction. Continue stirring for 1.5 hours to consume the remaining sodium periodate. The mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed against deionized water for 2-4 days. After freeze-drying, oxidized hyaluronic acid (OHA) solid was obtained. The prepared OHA was dissolved in deionized water to prepare a 12 mg / mL OHA solution. Methacrylic anhydride was slowly added dropwise to the OHA solution at a volume ratio of 1.5:100 under an ice-water bath at 4°C. Subsequently, the pH of the reaction system was maintained at 8.0 with NaOH solution, and the reaction was continued for 14 hours. After the reaction was completed, the mixed solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyzed against deionized water for 4 days. After freeze-drying, oxidized methacryloyl hyaluronic acid (OHAMA) white fibrous solid was obtained and stored at -20°C in the dark.

[0139] S23. Gallic acid-modified collagen was dissolved in acetic acid to prepare a solution A with a concentration of 12 mg / mL. Oxymethylacrylamide hyaluronic acid solid was dissolved in water to prepare a solution B with a concentration of 150 mg / mL. The M2 film disguised as magnetic nanoparticles obtained in step S1 was dissolved in water to obtain a Fe-M2NPs solution with a concentration of 12 mg / mL. The concentration of the LAP photoinitiator solution was 25 mg / mL. The mass ratio of CG, OHAMA, Fe-M2NPs, and LAP photoinitiator was 33.6: 46.8: 1.344: 3.75. Solutions A, B, Fe-M2NPs solution, and LAP photoinitiator solution were mixed evenly at 4°C in the dark. The mixture was then allowed to stand at 37°C for 10 minutes and then irradiated under 405 nm, 30 mW / cm² blue light for 90 seconds to form a magnetic composite hydrogel.

[0140] The above description of the embodiments is only for the purpose of helping to understand the technical solution and core idea of ​​the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A composite hydrogel with M2 film disguised as magnetic nanoparticles, characterized in that, The invention comprises a dual-network polymer matrix formed by gallic acid-modified collagen and oxymethacrylic acid hyaluronic acid, wherein M2 membrane-masked magnetic nanoparticles are disposed in the dual-network polymer matrix and the magnetic nanoparticles are encapsulated from the membrane of M2 macrophages.

2. The M2 film-masked magnetic nanoparticle composite hydrogel according to claim 1, characterized in that, The magnetic nanoparticles include a superparamagnetic iron oxide core and an M2-type macrophage membrane derived from interleukin-4 polarized macrophages.

3. The M2 film-masked magnetic nanoparticle composite hydrogel according to claim 1 or 2, characterized in that, The concentration of M2 film-masked magnetic nanoparticles in the dual-network polymer matrix is ​​400-800 μg / mL.

4. The method for preparing the M2 film camouflaged magnetic nanoparticle composite hydrogel according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Extract the membrane of M2 macrophage cells and prepare M2 membrane-masked magnetic nanoparticles by extrusion with iron nanoparticles. S2. Type I collagen extracted from tilapia skin is cross-linked with gallic acid via EDC / NHS to obtain gallic acid-modified collagen. Hyaluronic acid is oxidized with sodium periodate under light-protected conditions to obtain a mixture. The mixture is then dialyzed and freeze-dried sequentially to obtain oxidized hyaluronic acid solid. The obtained oxidized hyaluronic acid solid is prepared into a solution, and methacrylic anhydride is added and reacted under ice-water bath conditions. The pH of the reaction system is adjusted to maintain 8.

0. After the reaction, the mixed solution is dialyzed and freeze-dried sequentially to obtain oxidized methacrylamide hyaluronic acid solid. The gallic acid-modified collagen is dissolved in 0.01-0.03 M acetic acid to prepare a solution with a concentration of 8-12 mg / mL, and the pH is adjusted to neutral to obtain solution A. The oxidized methacrylamide hyaluronic acid solid is dissolved in water to prepare a solution B with a concentration of 140-150 mg / mL. The M2 membrane-masked magnetic nanoparticles obtained in step S1 are dissolved in PBS to obtain a solution with a concentration of 8-12 mg / mL. A solution of M2 membrane camouflaged magnetic nanoparticles was prepared by mixing solution A, solution B, M2 membrane camouflaged magnetic nanoparticle solution, and 2,4,6-trimethylbenzoyl-diphenylphosphine oxide solution at 3℃-6℃ in the dark, allowing the mixture to stand at 37℃, and then irradiating it under blue light to form the magnetic composite hydrogel.

5. The preparation method according to claim 4, characterized in that, Step S1 specifically includes the following steps: S11. RAW264.7 macrophages were stimulated with interleukin-4 to induce them to polarize into the M2 phenotype, which has anti-inflammatory and repair-promoting functions. S12. The cells were broken up, and the M2 cell membrane vesicles were separated and purified. The purified M2 cell membrane was mixed with iron nanoparticles to obtain a mixture. The mixture was extruded several times to form structurally stable M2 membrane disguised magnetic nanoparticles Fe-M2NPs.

6. The preparation method according to claim 5, characterized in that, In step S12, the amount of iron nanoparticles added to the M2 membrane camouflaged magnetic nanoparticles is 3-5 mg of iron nanoparticles per milligram of membrane protein, and the mixture is extruded several times through polycarbonate membranes with pore sizes of 400 nm and 200 nm.

7. The application of the M2 film camouflaged magnetic nanoparticle composite hydrogel according to any one of claims 1-3 or the preparation method according to any one of claims 4-6 in the preparation of pharmaceutical formulations for tissue repair.

8. A pharmaceutical preparation for tissue repair, characterized in that, The active ingredient is the M2 film camouflage magnetic nanoparticle composite hydrogel obtained by any one of claims 1-3 or by any one of claims 4-6.

9. The method of using the pharmaceutical preparation for tissue repair according to claim 8, characterized in that, The drug formulation for tissue repair is administered in conjunction with an external dynamic magnetic field.

10. The method of use according to claim 9, characterized in that, The magnetic flux density of the external dynamic magnetic field is 400-600 mt, and the oscillation frequency is 0.5-1.0 Hz.

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