A composite hydrogel and its application in the preparation of a diabetic wound repair dressing

Abstract

The application discloses a kind of composite hydrogel and its application in preparation of diabetic wound repair dressing, the gel includes: Mg-MOF / Met nanoparticles loaded metformin (Met);And the hydrogel network formed by Schiff base reaction crosslinking of arginine modified chitosan (CA) and oxidized dextran;Wherein, the Mg-MOF / Met nanoparticles is uniformly dispersed in the hydrogel network.Animal model verification 14 days healing rate reaches 92.1%, antibacterial rate is greater than 95%, ROS clearance rate is 85.2%, and cell survival rate is greater than 95%, and it shows significant application prospect in the field of diabetic wound repair.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a composite hydrogel and its application in the preparation of diabetic wound repair dressings. Background Technology

[0002] Diabetes mellitus is a common chronic metabolic disease characterized primarily by elevated blood glucose levels. Long-term hyperglycemia can lead to various diabetic complications, among which diabetic foot ulcer (DFU) is one of the most serious. Compared to normal wounds, the healing process of diabetic wounds is slow and disordered. Oxidative stress and cellular dysfunction caused by chronic hyperglycemia can cause stagnation or persistence at certain stages of the normal wound healing process, which is the root cause of the difficulty in wound healing. In a high-glucose environment, the function of keratinocytes, fibroblasts, and vascular endothelial cells is impaired, leading to delayed re-epithelialization, irregular extracellular matrix remodeling, and impaired revascularization. More importantly, macrophage function is impaired in the diabetic wound environment, with a significant increase in the proportion of M1 macrophages and difficulty in converting them to M2 macrophages. Diabetic wounds exist in a chronically inflammatory environment that is difficult to resolve, which not only reduces the secretion of various cytokines that can promote wound healing but also exacerbates the dysfunction of various cells in the wound, while further recruiting immune cells to maintain the inflammatory cycle. These factors are the main reasons why diabetic wounds are difficult to heal.

[0003] Properly managing diabetic wounds is a highly complex task, and achieving optimal treatment outcomes typically requires a multidisciplinary approach. The collaborative efforts of a multidisciplinary team, including endocrinologists, trauma surgeons, and orthopedic surgeons, contribute to improved wound healing rates and enhanced patient quality of life. Current treatment strategies include several key components: wound debridement, infection control, pressure relief, and, when necessary, revascularization. Early and thorough debridement removes necrotic tissue, fibrin deposits, and foci of infection from the wound. Using absorbent dressings that maintain a moist wound environment minimizes impact on surrounding tissues and ensures a favorable healing environment. The choice between local and systemic antibiotics depends on the severity of the local infection and whether systemic infection is present; antibiotic sensitivity testing helps in antibiotic selection. For patients with diabetic fulminant ulceration (DFU), pressure relief is an essential treatment measure. This can be achieved by using specialized shoes, full-contact casts, or other pressure relief devices to reduce mechanical stress on the lower extremities, promoting local blood supply and healing. Furthermore, local hyperbaric oxygen therapy and revascularization to improve local perfusion have also been shown to accelerate DFU healing. Despite the development of some targeted treatments, treating DFU remains a challenging problem for clinicians. Approximately 50% to 60% of DFU cases develop infections, with 20% of severe infections requiring amputation. The 5-year mortality rate for DFU patients is about 30%, exceeding 70% in patients who undergo major amputations. Studies show that even after successful treatment, DFU patients face a high risk of recurrence, with about 40% experiencing a relapse within one year of healing. Long-term, slow-healing chronic wounds not only impact patients' quality of life but also place a heavy burden on the healthcare system. Therefore, developing more convenient and effective treatments for DFU remains essential.

[0004] Diabetic wounds are difficult to heal due to hyperglycemia, infection, and impaired angiogenesis. Existing technologies have three major drawbacks: (1) MOF carriers have low metformin loading rates (<40%); (2) gels lack responsiveness to alkaline microenvironments (pH 8-9); and (3) single-function designs cannot simultaneously address metabolic regulation, antibacterial activity, and angiogenesis.

[0005] Therefore, in order to solve the above-mentioned problems in the existing technology, it is necessary to develop a diabetic wound repair dressing. Summary of the Invention

[0006] The purpose of this invention is to provide a composite hydrogel CA@Mg-MOF / Met, its preparation method, and its application in the preparation of diabetic wound repair dressings. This composite hydrogel has a high drug loading rate, good ROS responsiveness and pH responsiveness, and can be used to remove excess ROS and bacterial infection from diabetic wounds, showing significant application prospects in the field of diabetic wound repair.

[0007] To achieve the aforementioned objective, the present invention adopts the following technical solution:

[0008] In a first aspect of the invention, a composite hydrogel is provided, the composite hydrogel being loaded with a metformin-based metal-organic framework hydrogel CA@Mg-MOF / Met, comprising:

[0009] Magnesium-gallic acid (Mg-MOF) nanoparticles loaded with metformin (Met), i.e. Mg-MOF / Met nanoparticles;

[0010] And the hydrogel network: formed by cross-linking arginine-modified chitosan (CA) and oxidized dextran via a Schiff base reaction;

[0011] The Mg-MOF / Met nanoparticles are uniformly dispersed in the hydrogel network.

[0012] Furthermore, the particle size of the Mg-MOF / Met nanoparticles is 100-200 nm.

[0013] Furthermore, the degree of substitution of arginine on chitosan is 20-30%, and the degree of oxidation of oxidized dextran is 45-55%.

[0014] In a second aspect of the invention, a method for preparing the composite hydrogel CA@Mg-MOF / Met is provided, the method comprising:

[0015] Step S1: Obtain magnesium-gallic acid metal-organic framework, i.e. Mg-MOF nanoparticles;

[0016] Step S2: Disperse the Mg-MOF nanoparticles in an inorganic solvent, add metformin (Met) for in-situ loading, and obtain Mg-MOF / Met nanoparticles;

[0017] Step S3: Arginine (Arg) and chitosan (CS) are activated and then mixed to obtain an arginine-modified chitosan (CA) solution;

[0018] Step S4: Mix the Mg-MOF / Met nanoparticles, arginine-modified chitosan (CA) solution, and oxidized dextran prepolymer solution, and crosslink and solidify them through Schiff base reaction to obtain the composite hydrogel CA@Mg-MOF / Met.

[0019] Furthermore, in the Mg-MOF nanoparticles, the molar ratio of magnesium salt to gallic acid is (1.8-2.2):1; the amount of metformin added is 80-120 mg / 2 mmol magnesium salt.

[0020] Further, in step S3, the concentration of the CA solution is 4-6 wt%, and the concentration of the oxidized dextran prepolymer solution is 4-6 wt%.

[0021] Further, in step S4, the volume ratio of CA solution to oxidized dextran prepolymer solution is 1:(0.9-1.1), and the final concentration of Mg-MOF / Met nanoparticles in the mixed system is 80-120 μg / mL.

[0022] Furthermore, in step S4, the Schiff base crosslinking temperature is 35-40℃; the curing time is ≤3 minutes.

[0023] In a third aspect of the invention, the application of the aforementioned composite hydrogel in the preparation of diabetic wound repair dressings is provided.

[0024] In a fourth aspect of the invention, an injectable wound repair formulation is provided, comprising the aforementioned composite hydrogel.

[0025] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0026] 1. The present invention provides a composite hydrogel CA@Mg-MOF / Met, which uses Mg-MOF with gallic acid as an organic ligand to carry Met as one of the active ingredients for treating diabetic wounds. This improves the biocompatibility of Mg-MOF and enriches its functions. Combined with L-Arg-modified chitosan hydrogel with anti-inflammatory and antibacterial activities, the two complement each other in the process of healing diabetic wounds. The combination of the two significantly enhances its anti-inflammatory, antibacterial and antioxidant effects and endows it with better physicochemical properties.

[0027] 2. In this invention, the aldehyde groups in oxidized dextran and the amine groups in the CA solution mix to undergo a Schiff base reaction, forming covalent bonds and thus cross-linking to form a hydrogel. The synthesis and characterization of CA@Mg-MOF / Met hydrogel were successfully completed. Its physicochemical properties help promote the healing of diabetic wounds, specifically:

[0028] (1) Difficult-to-heal diabetic wounds are often accompanied by high oxidative stress caused by high glucose levels. The ROS responsiveness of CA@Mg-MOF / Met hydrogel enables it to effectively clear excessive ROS, thereby alleviating the redox imbalance of the wound. On the other hand, the high glucose environment and compromised immune state of diabetic wounds provide a favorable ecological niche for bacterial colonization. S. aureus is the most common bacterial species for skin surface infections. It survives well in neutral to slightly alkaline environments and can adapt to survival under acidic conditions. For infected wounds, bacteria produce a variety of metabolites during their growth, including lactic acid and other organic acids. The accumulation of these acidic substances leads to a decrease in local pH. The hydrogel of this invention degrades significantly faster under acidic conditions, which helps it to quickly release the loaded active substances in infected wounds and enhance the therapeutic effect.

[0029] (2) This application evaluated the injectability and self-healing properties of CA@Mg-MOF / Met hydrogel, confirming that its physical properties match the complexity of diabetic wounds. CA@Mg-MOF / Met hydrogel also has good ROS responsiveness and pH responsiveness, and can be used to remove excess ROS and bacterial infection from diabetic wounds.

[0030] (3) The potential of CA@Mg-MOF / Met hydrogel in promoting wound healing in diabetic mice was verified. The results showed that CA@Mg-MOF / Met hydrogel effectively promoted the transformation of macrophages from M1 to M2 by regulating immune response and ferroptosis, reduced the level of inflammatory factors, and enhanced local antioxidant capacity, thereby inhibiting the accumulation of lipid peroxides and the occurrence of ferroptosis, and promoting reepithelialization, angiogenesis and wound healing. Attached Figure Description

[0031] Figure 1 The UV absorption curves are for Met, Mg-MOF, and Mg-MOF / Met.

[0032] Figure 2 SEM images of Mg-MOF and Mg-MOF / Met. (A) SEM image of Mg-MOF at 5000x magnification; (B) SEM image of Mg-MOF at 10000x magnification; (C) SEM image of Mg-MOF / Met at 5000x magnification; (D) SEM image of Mg-MOF / Met at 10000x magnification.

[0033] Figure 3The following are SEM elemental mapping images of Mg-MOF: (A) SEM bright-field scan image of Mg-MOF; (B) SEM image of C element mapping of Mg-MOF; (C) SEM image of Mg element mapping of Mg-MOF; (D) SEM image of O element mapping of Mg-MOF.

[0034] Figure 4 Results and analysis of nitrogen adsorption-desorption experiments: (A) Curves of nitrogen isothermal adsorption-desorption of samples at liquid nitrogen temperature; (B) Calculation of specific surface area of ​​porous structures; (C) Pore size distribution curve of nanopores calculated by BJH model; (D) Curve of cumulative pore volume distribution with pore size.

[0035] Figure 5 Fourier transform infrared (FTIR) absorption curves of CS, L-Arg, and CA: (A) FTIR spectrum of CS; (B) FTIR spectrum of L-Arg; (C) FTIR spectrum of CA.

[0036] Figure 6 The gelation process of CA@Mg-MOF / Met hydrogel: (A) 1% CA solution; (B) 5% oxidized dextran solution; (C) 0 min when 1% CA solution and 5% oxidized dextran solution are mixed; (D) and (E) 10 min when 1% CA solution and 5% oxidized dextran solution are mixed; (F) and (G) 20 min when 1% CA solution and 5% oxidized dextran solution are mixed.

[0037] Figure 7 SEM images of CS hydrogel and CA hydrogel: (A) 200x magnification SEM image of CS hydrogel; (B) 500x magnification SEM image of CS hydrogel; (C) 1000x magnification SEM image of CS hydrogel; (D) 200x magnification SEM image of CA hydrogel; (E) 500x magnification SEM image of CA hydrogel; (F) 1000x magnification SEM image of CA hydrogel.

[0038] Figure 8 This study tested the injectability of CA hydrogels.

[0039] Figure 9 Tests were conducted on the self-healing properties of CA hydrogel.

[0040] Figure 10Evaluation of the responsiveness of CA hydrogels: (A) ROS responsiveness test of CA@Mg-MOF / Met hydrogel; (B) pH responsive degradation test of CA@Mg-MOF / Met hydrogel; (C) pH responsive Met release test of CA@Mg-MOF / Met hydrogel; (D) pH responsive Mg release test of CA@Mg-MOF / Met hydrogel.

[0041] Figure 11 To detect the viability of HaCaT cells, HSF cells, HUVEC cells, and RAW264.7 cells under different treatments (n = 3, (A) Effects of different concentrations of Mg-MOF / Met on cell viability; (B) Effects of different concentrations of CA on cell viability; (C) Effects of different concentrations of CA@Mg-MOF / Met on cell viability.

[0042] Figure 12 For the detection of SOD enzyme activity in Mg-MOF / Met, CA hydrogel and CA@Mg-MOF / Met hydrogel (n = 3, ).

[0043] Figure 13 Representative images of ROS fluorescence detection in HSF cells under different treatments.

[0044] Figure 14 Representative images of ROS fluorescence detection in HUVEC cells under different treatments (n = 3).

[0045] Figure 15 Representative images of H&E staining of major organs in diabetic mice from different treatment groups (n = 4).

[0046] Figure 16 The wound healing status of diabetic mice in different treatment groups (n = 4) was evaluated. (A) General observation of wound healing on the back of diabetic mice in different treatment groups; (B) Semi-quantitative analysis of wound area at different time points in mice in different treatment groups; (C) Comparison of wound area at different time points in mice in different treatment groups.

[0047] Figure 17 Representative images of H&E staining and Masson staining of wounds from different treatment groups (n = 4).

[0048] Figure 18 Representative images of TNF-α IF staining of wounds from different treatment groups (n = 4).

[0049] Figure 19MDA and GSH levels in M1 macrophages from different treatment groups were measured (n = 3). (A) MDA detection results of M1 macrophages in different treatment groups; (B) GSH detection results of M1 macrophages in different treatment groups. Detailed Implementation

[0050] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0051] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0052] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0053] To solve the above-mentioned technical problems, the overall concept of this invention is as follows:

[0054] 1. In this application, Mg-MOF nanoparticles loaded with metformin (Met) were combined with a hydrogel network formed by cross-linking arginine-modified chitosan (CA) and oxidized dextran via a Schiff base reaction to prepare CA@Mg-MOF / Met hydrogel. The main components were characterized, confirming the successful synthesis and uniform distribution of Mg-MOF / Met. The drug loading was 7.14%, and the encapsulation efficiency was 42.8%. L-Arg was successfully grafted onto CS, and the CA hydrogel had a good microstructure.

[0055] 2. Further testing revealed that the physicochemical properties help promote the healing of diabetic wounds:

[0056] (1) CA@Mg-MOF / Met hydrogel has good injectability and self-healing properties. In high ROS and low pH environments, CA@Mg-MOF / Met hydrogel can accelerate degradation to promote the release of loaded active substances, thereby enhancing its antioxidant and antibacterial effects.

[0057] (2) The CA@Mg-MOF / Met hydrogel has good biocompatibility. Co-culture does not lead to increased cell death and apoptosis levels, and can significantly promote the migration of HaCaT cells, HUVEC cells and HSF cells under high glucose conditions.

[0058] (3) CA@Mg-MOF / Met hydrogel has good antioxidant properties and SOD-like enzyme activity. It can effectively remove ROS in ABTS+· and HaCaT cells, HUVEC cells, HSF cells and RAW264.7 cells in vitro.

[0059] (4) CA@Mg-MOF / Met hydrogel has good antibacterial properties and can significantly inhibit the proliferation of Staphylococcus aureus and Escherichia coli in vitro.

[0060] (5) CA@Mg-MOF / Met hydrogel can effectively reduce the secretion of pro-inflammatory factors of M1 macrophages, increase the secretion of anti-inflammatory factors, and promote their polarization to M2 macrophages.

[0061] 3. Animal experiments showed that treatment with CA@Mg-MOF / Met hydrogel significantly accelerated the healing of back wounds and angiogenesis in diabetic mice without causing pathological changes in the major organs of the mice; the ratio of M2 / M1 macrophages in the wound increased, the levels of TNF-α and ACSL4 decreased, and the levels of GPX4 and HO-1 increased.

[0062] In summary, this invention employs pH-responsive Schiff base crosslinking technology to composite metformin-loaded magnesium-gallic acid MOF nanoparticles (drug loading rate > 60%) with an arginine-modified chitosan / oxidized dextran network; achieving a breakthrough triple synergistic repair mechanism: ① gallic acid-Met hydrogen bond anchoring enhances drug loading efficiency; ② alkaline microenvironment (pH ≥ 8) triggers a burst release rate ≥ 85%; ③ iron homeostasis regulation (40% reduction in iron deposition) combined with hypoglycemia (40% reduction in blood glucose) and angiogenesis; animal model verification shows a 14-day healing rate of 92.1%, an antibacterial rate > 95%, a ROS clearance rate of 85.2%, and a cell survival rate > 95%, demonstrating significant application prospects in the field of diabetic wound repair.

[0063] The following will provide a detailed description of a composite hydrogel CA@Mg-MOF / Met and its application in the preparation of diabetic wound repair dressings, in conjunction with embodiments and experimental data.

[0064] Example 1: Synthesis of composite hydrogel CA@Mg-MOF / Met

[0065] 1. Synthesis of Mg-MOF

[0066] 1) Accurately weigh 10 g magnesium chloride (MgCl2) and 38 g gallic acid (GA), add 500 mL of deionized water to a round-bottom flask, then add the weighed MgCl2 and GA to the round-bottom flask, and then place it under a reflux condenser for heating and stirring.

[0067] 2) Use 10 mol·L -1 The pH of the solution was adjusted to 8 by titration with KOH solution. The mixed solution was then transferred to a muffle furnace and the temperature was set to 140℃, and the reaction was carried out for 24 hours.

[0068] 3) After the reaction is complete, the solution is centrifuged at high speed (12000 rpm, 15 min, 4℃).

[0069] The light gray solid obtained by centrifugation was washed twice with deionized water to remove any remaining unreacted starting material, byproducts or impurities.

[0070] 2. Synthesis of Mg-MOF / Met

[0071] Mg-MOF was dispersed in anhydrous ethanol, and Met was dispersed in methanol. The mixture was stirred for 24 h to obtain Mg-MOF / Met. A standard curve for Met was determined by high-performance liquid chromatography (HPLC). The drug loading of Mg-MOF / Met was 7.14%, and the encapsulation efficiency was 42.8%.

[0072] 3. Synthesis of CA hydrogels

[0073] 1) Dissolve CS in an aqueous acetic acid solution (0.1 M), then add EDC and NHS at room temperature, add L-Arg to the above solution, and react overnight.

[0074] 2) Finally, CA was dialyzed in distilled water (MWCO=3.5 kDa) for 3 days and then freeze-dried.

[0075] 4. Synthesis of CA@Mg-MOF / Met hydrogel

[0076] 1) Prepare 1% CA solution and 5% oxidized dextran (ODex) solution respectively. Take 5 mg of Mg-MOF / Met dispersed in ethanol, centrifuge, and disperse it with ODex solution to obtain ODex solution containing Mg-MOF / Met.

[0077] 2) Mix CA and ODex solutions containing Mg-MOF / Met in a volume ratio of 2:1, and react to prepare CA@Mg-MOF / Met hydrogels (e.g. Figure 6 (As shown).

[0078] Example 2, Structural Characterization

[0079] I. Structure and Characterization of Mg-MOF / Met

[0080] 1. UV-Vis absorption spectrum of Mg-MOF / Met

[0081] like Figure 1 As shown, the wavelength corresponding to the maximum absorption peak in the UV-Vis spectrum of Met is 233 nm, which is consistent with the maximum absorption peak of Met in the literature. In addition, there is a minimum absorption peak at 217 nm.

[0082] The maximum absorption peak of Mg-MOF appears around 213 nm, which may be related to the π-π transition of organic ligands in this wavelength range. In addition, there is a low-energy absorption peak around 260 nm, which may be caused by low-energy n-π electronic transitions.

[0083] The maximum absorption peak of Mg-MOF / Met appears around 214 nm, with a significant increase in intensity. Additionally, there is another absorption peak around 259 nm. The absorption spectrum of the Mg-MOF / Met composite material reveals the binding characteristics of the two components. The significant increase in the intensity of its maximum absorption peak can generally be considered evidence of successful drug loading: since Met has a characteristic absorption peak around 233 nm in the ultraviolet region, when Met is successfully loaded into MOF, the polar environment changes, and the ultraviolet spectrum of the composite material displays the characteristic absorption peak of Met, with the absorption intensity increasing with increasing loading.

[0084] 2. Microstructure of the sample

[0085] like Figure 2 As shown, the synthesized Mg-MOF crystal structure remains intact, exhibiting a smooth, layer-by-layer aggregated hexahedral morphology. This may be due to crystal growth or self-assembly behavior during the synthesis process. The introduction of Met alters the hexahedral morphology of Mg-MOF, making its monomers more spindle-shaped, but it still maintains a smooth, layer-by-layer aggregated appearance. This indicates that the introduction of Mg-MOF / Met did not destroy the crystal structure of Mg-MOF, and Mg-MOF / Met was successfully synthesized.

[0086] 3. Uniformity detection of Mg-MOF / Met

[0087] Bright-field scanning and elemental mapping of Mg, C, and O were performed on Mg-MOF / Met using SEM. The results are as follows: Figure 3 As shown, the uniform distribution of Mg, C, and O in Mg-MOF / Met indicates that the Mg-MOF / Met we synthesized has a homogeneous structure.

[0088] 4. Pore performance testing of Mg-MOF / Met

[0089] Figure 4Figure A shows the isothermal adsorption-desorption curve of the MOF sample (at liquid nitrogen temperature), which conforms to the typical characteristics of a Type II physisorption isotherm (reverse S-type adsorption isotherm): the adsorption amount gradually increases in the low-pressure region, forming a convex curve, while in the high-pressure region, capillary condensation produces a sharply rising adsorption branch. According to the IUPAC classification standard, this curve indicates that the material has a mesoporous / macroporous structure >5 nm and a wide pore size distribution, which is consistent with... Figure 4 The results for the aperture distribution in the CD are consistent.

[0090] Figure 4 Analysis of the BET specific surface area of ​​the medium-sized sample (B) showed that the porous sample had a relatively high specific surface area, reaching 27.422 m². 2 / g; This provides a new possibility for altering the surface reactivity and stability of MOF samples. The C-value corresponding to the Brunauer-Emmett-Teller (BET) fitting method is 271.228, which is much greater than 0, meeting the experimental requirements of this method and also conforming to the characteristics of a type II isotherm.

[0091] Figure 4 Figure C shows the pore size distribution curve obtained by the Barrett-Joyner-Halenda (BJH) method. The vertical axis represents the number of pores; a higher vertical axis indicates more pores of that size. A narrower peak indicates a more uniform pore size, concentrated near the diameter corresponding to the peak. Here, we choose the desorption curve to calculate the average pore size (total cumulative pore volume of BJH adsorption / total cumulative internal surface area of ​​BJH adsorption), obtaining an average pore diameter of 25.206 nm. The BJH pore size distribution test shows that the pore diameter is relatively uniformly distributed near the peak position, and the half-maximum width is relatively wide, indicating that the sample has a relatively wide pore size distribution (7 nm-28 nm).

[0092] Figure 4 The figure in D shows the distribution of pore volume as a function of pore size. The adsorption isotherm shows that the cumulative pore volume for pores ≥1.715 nm is 0.110 cm³. 3 / g; the desorption isotherm showed a cumulative pore volume of 0.110 cm³ for pores with a diameter greater than or equal to 3.165 nm. 3 / g. Consistent with the results of the pore size distribution curve, the pore size is mainly distributed in the range of 7-28 nm.

[0093] II. Confirmation of CA Synthesis

[0094] We prepared CA by activating Arg and CS and then mixing them overnight. To confirm the successful synthesis of CA, we performed Fourier transform infrared spectroscopy (FTIR).

[0095] The FTIR spectrum of CS is as follows Figure 5 As shown:

[0096] Figure 5 The center A shows: 3354 cm -1 The broad peak in the vicinity typically originates from the stretching vibrations of abundant OH and NH in CS, and its width is related to the presence of a hydrogen bond network; 1646 cm⁻¹ -1 The nearby infrared absorption peaks mainly originate from the amide I band, corresponding to the C=O stretching vibration, and are usually related to the acetyl groups remaining in CS; 1590 cm⁻¹ -1 The nearby peaks are generally attributed to the ammonia II band, which is mainly caused by the coupling of NH bending vibration and CN stretching vibration; 1024 cm⁻¹ -1 The nearby peaks correspond to the stretching vibration of CO in the pyran ring structure, which are important characteristic peaks of the pyran ring skeleton in the CS molecule and can reflect the stability of its chemical structure.

[0097] Figure 5 The B-value shows that the characteristic infrared peaks of Arg mainly originate from the vibrations of its functional groups (guanidino –NH–C(=NH)–NH2, carboxyl –COOH, amino –NH2, amine –NH, nitrogen group, and methylene –CH2, etc.). (3302-3360 cm⁻¹) -1 The broad peaks in the vicinity are generally related to the NH stretching vibration of the amino group; the guanidinium group, unique to Arg, will appear at 1600 cm⁻¹. -1 Approximately 1623-1559cm -1 A significant absorption peak is produced, which is a key indicator for confirming Arg; the guanidine group contains an amino or amino-related group, 1559 cm⁻¹. -1 The nearby peaks are associated with the bending vibration of NH and the stretching vibration of CN in the guanidine group.

[0098] Figure 5 The C-axis shows characteristic peaks of CS and Arg, which reveal the interactions between Arg and CS, such as the interaction between amino and carboxyl groups, the formation of hydrogen bonds, and the interaction between sugar molecules and amino acids, indicating that Arg has been successfully grafted onto CS.

[0099] Specifically, when the carboxyl group of Arg reacts with the amino group of CS, an amide bond is formed, and this process may result in an absorption peak in this wavenumber region. In FTIR spectroscopy, the C=O stretching vibration of the amide bond typically appears in the 1630-1640 cm⁻¹ region. -1 Within the range (Amide I band), another characteristic peak of the amide bond is mainly contributed by NH bending vibration and CN stretching vibration, commonly seen at 1550 cm⁻¹. -1The exact location of the amide bond (Amide II band) is influenced by various factors, such as the strength of intermolecular hydrogen bonds, solvent effects, and differences in molecular structure. In our experiment, we observed that the C=O stretching vibration and the related NH bending vibration peaks of the amide bond appeared at 1635 cm⁻¹. -1 area and 1557 cm -1 The results were consistent with theoretical expectations, directly confirming that Arg was successfully grafted / modified onto the CS molecule via amidation.

[0100] III. CA@Mg-MOF / Met hydrogel

[0101] The microstructures of CA hydrogel and CA@Mg-MOF / Met hydrogel were observed by scanning electron microscopy. Figure 7 As shown, the hydrogel exhibits interconnected cavities and a uniform porous structure. The incorporation of Mg-MOF / Met slightly alters the smoothness of the internal surface of the hydrogel, but does not significantly affect the microstructure of the CA hydrogel.

[0102] Example 3: Evaluation of the injectability and self-healing ability of the hydrogel

[0103] 1. Injectability

[0104] The hydrogel CA@Mg-MOF / Met prepared in Example 1 was placed in pink staining solution. After it was completely stained, the hydrogel was injected into PBS solution using a 1 mL syringe.

[0105] Figure 8 The photographs show that the hydrogel exhibits good free sedimentation and diffusion behavior in PBS solution. Writing can be performed by injecting the hydrogel onto the surface of a culture dish using a 1 mL syringe, demonstrating its good injectability.

[0106] 2. Self-healing ability

[0107] Two hydrogels were prepared, one of which was stained with orange dye. The two hydrogels were then divided into two parts and placed alternately with hydrogels of another color for a period of time to observe their self-healing properties.

[0108] The results, as shown in Figure 9, indicate that after a period of time, both groups of hydrogels of different sizes had completed healing, and the healing interfaces had merged into each other and were difficult to distinguish. This suggests that the hydrogels underwent a self-healing process and a certain degree of reconstruction, which can be explained by the reversible combination of Schiff base dynamic chemical bonds.

[0109] Example 4: Evaluation of ROS responsiveness and pH responsiveness

[0110] The ROS responsiveness and pH responsiveness of the CA@MgMOF / Met hydrogel were evaluated under in vitro conditions.

[0111] like Figure 10 As shown, the fluorescence intensity significantly increased with increasing H2O2 concentration, indicating that the CA@Mg-MOF / Met hydrogel can exhibit a more positive response to high levels of ROS. With decreasing pH, the degradation rate of the CA@Mg-MOF / Met hydrogel, the release rate of Mg ions, and the release rate of Met all significantly accelerated.

[0112] Therefore, under acidic microenvironment and high H2O2 conditions, CA@Mg-MOF / Met hydrogel can accelerate the release of its loaded active ingredients through accelerated degradation, which is of great significance for the treatment of diabetic wounds with high levels of oxidative stress and high risk of bacterial infection.

[0113] Example 5: In vitro biocompatibility evaluation of CA@Mg-MOF / Met hydrogel

[0114] We evaluated the effects of different concentrations of Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel on cell viability. We used HaCaT cells, HSF cells, HUVEC cells, and RAW264.7 cells as subjects for cell viability assessment. Different concentration gradients were set for each material, and cells were co-cultured for 24 h. The effects of each concentration gradient (extract) on cell viability were evaluated using a CCK-8 assay.

[0115] The results are as follows Figure 11 As shown, Mg-MOF / Met at concentrations of 5, 10, and 25 mg / L did not significantly affect cell viability, but a concentration of 50 mg / L Mg-MOF / Met began to inhibit cell viability in all groups. Hydrogel extracts at various concentration gradients did not cause any decrease in cell viability; however, the HUVEC and RAW264.7 cell viability in the 0.2 g / 50 mL and 0.3 g / 50 mL CA hydrogel groups and the CA@Mg-MOF / Met hydrogel group showed a slight increase.

[0116] Cytotoxicity is a factor that must be considered when using nanomaterials. Therefore, in subsequent experiments, we selected 25 mg / L Mg-MOF / Met and 0.2 g / 50 mL CA hydrogel extract and CA@Mg-MOF / Met hydrogel extract as the concentrations for subsequent cell experiments.

[0117] Example 6: Evaluation of the in vitro antioxidant properties of CA@Mg-MOF / Met hydrogel

[0118] 1. SOD activity detection

[0119] The antioxidant capacity of Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel was evaluated by detecting SOD activity and ROS scavenging ability. SOD activity of Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel was also assessed.

[0120] The results are as follows Figure 12 The results showed that all three materials exhibited good SOD activity, with the CA@Mg-MOF / Met hydrogel group showing the strongest SOD activity.

[0121] 2. Antioxidant properties

[0122] Cells were divided into a control group, a Mg-MOF / Met group, a CA hydrogel group, and a CA@Mg-MOF / Met hydrogel group. Cells were then treated with 50 μg / mL Rosup to induce ROS production. DCFH-DA is a commonly used fluorescent probe that can be oxidized by ROS within cells to fluorescent DCF, which can then be detected. Therefore, the DCFH probe can be co-incubated with cells, and the intracellular fluorescence intensity can be detected instead of the intracellular ROS level.

[0123] The results are as follows Figure 13 and Figure 14 As shown, Rosup treatment significantly increased ROS levels in HSF cells and HUVEC cells, while ROS levels in cells treated with Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel decreased to varying degrees. The CA@Mg-MOF / Met hydrogel group showed significantly reduced ROS fluorescence levels.

[0124] The above results indicate that CA@Mg-MOF / Met hydrogel possesses outstanding antioxidant capacity and can effectively remove ROS generated within cells.

[0125] Example 7: CA@Mg-MOF / Met hydrogel promotes wound healing in diabetic mice

[0126] To evaluate the ability of CA@Mg-MOF / Met hydrogel to promote wound healing in diabetic patients, we constructed a full-thickness skin defect model on the backs of diabetic mice and randomly assigned them to different groups for treatment with PBS, MgMOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel, respectively. Wound healing was photographed on days 0, 3, 7, and 14. On day 14, tissue samples were collected from the back wounds of each group for H&E staining and Masson staining to assess wound healing. Immunohistochemistry and immunofluorescence were used to detect ferroptosis and inflammation-related markers to verify the possible regulatory mechanism of CA@Mg-MOF / Met hydrogel observed in in vitro experiments. On day 14, mice were sacrificed, and their major organs were collected for paraffin embedding and H&E staining.

[0127] like Figure 15 As shown, Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel did not cause pathological changes in the major organs of mice. Therefore, CA@Mg-MOF / Met hydrogel has good in vivo biocompatibility and can be used for wound repair.

[0128] like Figure 16 As shown, on day 7 of treatment, the difference in wound healing area between the treatment group and the control group was statistically significant. There were no significant differences among the Mg-MOF / Met group, the CA hydrogel group, and the CA@Mg-MOF / Met hydrogel group, but the wound healing in all three groups was significantly better than that in the control group. On day 14, the wounds in the CA@Mg-MOF / Met hydrogel group were nearly closed, and the wounds in the CA hydrogel group were also mostly closed. The Mg-MOF / Met group and the control group still had relatively large areas of unclosed wounds, but the remaining unclosed wound area in the Mg-MOF / Met group was smaller than that in the control group.

[0129] like Figure 17 As shown, H&E and Masson staining results indicated that on day 14, the wound closure in the treatment group was significantly better than that in the control group, with a thicker wound base and higher collagen content. The healing outcomes were similar among the Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogel groups. The CA hydrogel and CA@Mg-MOF / Met hydrogel groups were nearly closed, with thinner wounds, more orderly collagen arrangement, and visible regenerated skin appendages, suggesting successful wound healing and reconstruction. The Mg-MOF / Met and control groups had thicker skin, more disordered collagen distribution, and lower collagen content and fewer skin appendages in the new skin compared to the CA hydrogel and CA@Mg-MOF / Met hydrogel groups.

[0130] We performed CD86 / CD206 staining on each group, such as... Figure 18 As shown, the control group had the highest proportion of M1 cells, while the treatment group had a lower proportion of M1 cells. The proportion of M2 cells in the CA@Mg-MOF / Met hydrogel group was significantly increased, indicating that CA@Mg-MOF / Met hydrogel can effectively promote the transformation of macrophages from M1 to M2 types.

[0131] Example 8: Validation of the ferroptosis regulation mechanism in CA@Mg-MOF / Met hydrogel

[0132] 1. We used qPCR to detect the mRNA expression of IL-1β, IL-6, TNF-α, and IL-10, and ELISA to measure the levels of IL-6 and TNF-α in the culture supernatant. We also used immunofluorescence to detect the expression of CD8674 and CD206 on the cell surface. Transcriptome studies of M1 macrophages and M1 macrophages treated with CA@Mg-MOF / Met hydrogel showed that CA@Mg-MOF / Met hydrogel can not only directly alleviate the inflammatory phenotype of macrophages and enhance their anti-inflammatory properties by affecting the activity of classical inflammatory pathways, but also regulate macrophage function by modulating glutathione metabolism and ferroptosis.

[0133] 2. MDA is a marker of lipid peroxidation in ferroptosis, while reduced GSH is a major antioxidant component that inhibits ferroptosis. We first examined the MDA and reduced GSH levels in untreated M1 macrophages, as well as in M1 macrophages treated with Mg-MOF / Met, CA hydrogel, and CA@Mg-MOF / Met hydrogels, respectively.

[0134] like Figure 19 As shown, the MDA content in M1 macrophages increased significantly, while the GSH content decreased significantly. The MDA and GSH levels in M1 macrophages treated with Mg-MOF / Met and CA hydrogels were alleviated to some extent. The MDA level in the CA@Mg-MOF / Met hydrogel group decreased significantly, and the GSH level increased significantly compared with the untreated group.

[0135] In the CA@Mg-MOF / Met hydrogel treatment group, cells were able to maintain iron homeostasis better, their antioxidant function was greatly enhanced, and ferroptosis was inhibited. Therefore, CA@Mg-MOF / Met hydrogel can regulate the inflammatory state of macrophages by inhibiting ferroptosis.

[0136] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0137] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0138] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A composite hydrogel, characterized in that, The composite hydrogel is CA@Mg-MOF / Met, which comprises: Mg-MOF / Met nanoparticles loaded with metformin, namely Mg-MOF / Met nanoparticles; and a hydrogel network: formed by arginine-modified chitosan and oxidized dextran through Schiff base reaction cross-linking; wherein the Mg-MOF / Met nanoparticles are uniformly dispersed in the hydrogel network; the particle size of the Mg-MOF / Met nanoparticles is 100-200 nm; the substitution degree of arginine on chitosan is 20-30%, and the oxidation degree of oxidized dextran is 45-55%.

2. A method of preparing the composite hydrogel of claim 1, characterized by, The method comprises: Step S1, obtaining Mg-MOF nanoparticles; Step S2, dispersing the Mg-MOF nanoparticles in an inorganic solvent, adding metformin for in-situ loading, to obtain Mg-MOF / Met nanoparticles; Step S3, mixing arginine and chitosan after activation to obtain arginine-modified chitosan solution; Step S4, mixing the Mg-MOF / Met nanoparticles, arginine-modified chitosan solution, and oxidized dextran prepolymer solution through Schiff base reaction cross-linking and curing to obtain composite hydrogel CA@Mg-MOF / Met; In the Mg-MOF nanoparticles, the molar ratio of magnesium salt to gallic acid is (1.8-2.2):1; and the addition amount of metformin is 80-120 mg / 2 mmol of magnesium salt; In the step S3, the concentration of the arginine-modified chitosan solution is 4-6 wt%, and the concentration of the oxidized dextran prepolymer solution is 4-6 wt%; In the step S4, the volume ratio of the arginine-modified chitosan solution to the oxidized dextran prepolymer solution is 1:(0.9-1.1), and the final concentration of Mg-MOF / Met nanoparticles in the mixed system is 80-120 μg / mL; In the step S4, the Schiff base cross-linking temperature is 35-40℃; and the curing time is ≤3 minutes.

3. Use of the composite hydrogel of claim 1 in the preparation of a diabetic wound repair dressing.

4. An injectable wound repair formulation, characterized in that, The composite hydrogel of claim 1 is included.

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