Near-infrared responsive injectable MXene / fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nano composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses a near-infrared responsive injectable MXene / fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nano composite hydrogel SCO (at) M, and relates to the technical field of biomedical materials. The method disclosed by the invention comprises the following steps: 1) preparing an MXene single-layer nanosheet by adopting LiF-HCl in-situ HF etching; (2) modifying the fibroin through glycidyl methacrylate to obtain SF-MA; 3) oxidizing hyaluronic acid with sodium periodate to obtain oxidized hyaluronic acid OHA; (4) uniformly dispersing MXene in the SF-MA solution, and sequentially adding carboxymethyl chitosan CMCS and OHA, so as to prepare the injectable nano composite soft hydrogel with the shear thinning characteristic; and 5) through ultraviolet irradiation crosslinking, rapid in-situ curing of the hydrogel is realized, and the mechanical strength of the hydrogel is improved. The hydrogel provided by the invention has excellent injectability and mechanical property, has good photo-thermal antibacterial property, oxidation resistance and cell compatibility, can be used as a carrier of cells, medicines and bioactive substances in the field of biological medicines, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a photocrosslinked injectable hydrogel material, a preparation method and applications thereof; more specifically, the present invention relates to a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel, a preparation method and applications thereof. Background Art

[0002] With the continued increase in the aging and obese population, chronic diabetic wounds are becoming a serious public health threat. The pathological environment of diabetic wounds is more complex than that of acute wounds, with the following key issues: ① Continuously elevated oxidative stress levels lead to excessive inflammatory responses; impaired vascular regeneration leads to tissue hypoxia; ② More than 90% of diabetic wounds are bacterially infected, resulting in slow or even incomplete wound healing; and ③ Uncontrolled inflammation and abnormal macrophage phenotypes in diabetic wounds are important factors hindering wound healing.

[0003] Injectable hydrogels have good fluidity, can wrap the wound area well and isolate it from the external environment without affecting the permeability of the wound. They are an ideal basic material for diabetic wound repair. Injectable hydrogels are usually prepared using reversible physical cross-linking or dynamic chemical cross-linking strategies to maintain good fluidity. However, these relatively weak cross-linking reactions result in insufficient mechanical properties of the hydrogel, limiting its ability to provide continuous support and protection for the wound. The inherent contradiction between the fluidity and mechanical strength of injectable hydrogels is a major challenge.

[0004] In addition, to accelerate diabetic wound healing, injectable hydrogels should also possess antioxidant and antibacterial properties, and be able to regulate the immune microenvironment, promoting macrophage polarization toward the reparative M2 phenotype, thereby balancing the inflammatory response. Common antioxidant strategies include the introduction of natural polyphenolic antioxidants, antioxidant enzymes or enzyme-mimicking materials, and reducing drugs; while approaches to impart antibacterial properties involve antibiotics, antimicrobial peptides, metal / metal oxide nanoparticles, cationic polymers, and photothermal materials. However, natural polyphenols and small molecule drugs have poor stability and are easily inactivated, enzyme-mimicking materials have dose-related toxicity, and antibiotics and antimicrobial peptides face challenges in drug resistance and dosage control.

[0005] Based on the above reasons, this application is hereby filed. Summary of the Invention

[0006] The applicant's research suggests that photothermal materials not only possess dual antioxidant and antibacterial properties but can also be stably bound to the hydrogel network through covalent or physical means, enhancing their overall performance. Therefore, using photothermal materials to construct multifunctional hydrogels based on photothermal responses is a viable strategy for balancing safety and functional integration.

[0007] Therefore, based on the above reasons and in view of the problems or defects in the prior art, the object of the present invention is to provide a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel and its preparation method and application, so as to solve or at least partially solve the above technical defects in the prior art:

[0008] The present invention constructs a double network structure through in situ photocrosslinking, which can significantly improve the mechanical strength of the prepared injectable hydrogel; at the same time, the introduction of MXene nanosheets with photothermal effect, antibacterial and antioxidant properties achieves the synergistic optimization of structural enhancement and functional integration, and constructs a new injectable hydrogel with clinical transformation potential.

[0009] In order to achieve the above-mentioned first object of the present invention, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M) comprises the following steps:

[0011] 1) Preparation of MXene monolayer nanosheets by in situ HF etching of LiF-HCl;

[0012] 2) Modifying silk fibroin with glycidyl methacrylate (GMA) to obtain freeze-dried vinyl-modified silk fibroin (SF-MA);

[0013] 3) oxidizing hyaluronic acid with sodium periodate to obtain oxidized hyaluronic acid (OHA);

[0014] 4) dispersing the MXene monolayer nanosheets described in step 1) in deionized water to obtain a MXene dispersion; dissolving the SF-MA described in step 2) in deionized water to obtain a SF-MA solution; dissolving the OHA described in step 3) in deionized water to obtain an OHA solution; dissolving carboxymethyl chitosan (CMCS) in deionized water to obtain a CMCS solution; and sequentially adding the MXene dispersion, CMCS solution, photoinitiator, and OHA solution to the SF-MA solution according to a ratio, and mixing thoroughly to obtain a Schiff base cross-linked soft hydrogel;

[0015] 5) The Schiff base cross-linked soft hydrogel in step 4) is irradiated under ultraviolet light for 10-60 seconds to obtain a double cross-linked hydrogel, namely the SCO@M.

[0016] Furthermore, in the above technical solution, the preparation method of the MXene single-layer nanosheet in step 1) is as follows:

[0017] Lithium fluoride (LiF) and concentrated hydrochloric acid are mixed according to a ratio, stirred and reacted at 35-45°C for 10-20 minutes to obtain a mixed etching solution; then, aluminum titanium carbide (Ti3AlC2) is added to the mixed etching solution, stirred and etched at 40-45°C for 12-48 hours. After the reaction is completed, the mixture is washed and centrifuged to obtain MXene multilayer nanosheets; the MXene multilayer nanosheets are then subjected to low-temperature ultrasound and low-speed centrifugation in sequence, the supernatant is collected, and freeze-dried to obtain the MXene single-layer nanosheets.

[0018] Furthermore, in the above technical solution, the molar ratio of the lithium fluoride to HCl in concentrated hydrochloric acid is 1:3.

[0019] Furthermore, in the above technical solution, the stirring speed is 3000-5000r / min.

[0020] Furthermore, in a preferred embodiment of the present invention, the rotation speed of the low-speed centrifugation is 1000-2000 r / min, and the time of the low-speed centrifugation is 20-40 min.

[0021] Furthermore, in the above technical solution, the specific preparation method of SF-MA in step 2) is as follows:

[0022] The raw silk fiber is degummed and dried to obtain degummed silk fibroin fiber; the degummed silk fibroin fiber is then added to a lithium bromide (LiBr) solution and heated to dissolve; glycidyl methacrylate (GMA) is immediately added dropwise to the dissolved degummed silk fibroin fiber according to a ratio, and after the dropwise addition is completed, the obtained reaction system A is stirred and reacted at 55-65° C. for 1-5 hours; after the reaction is completed, the obtained product is dialyzed and centrifuged in sequence, and the obtained supernatant is frozen and then dried to obtain the said SF-MA.

[0023] Furthermore, in the above technical solution, the concentration of the lithium bromide solution is 5 to 15 mol / L.

[0024] Furthermore, in the above technical solution, the heating temperature is 40-80° C., and the heating time is 0.5-1.5 h.

[0025] Furthermore, in the above technical solution, the ratio of the degummed silk fibroin fiber to the lithium bromide solution is (10-30) g:100 mL. In a preferred embodiment of the present invention, the ratio of the degummed silk fibroin fiber to the lithium bromide solution is 20 g:100 mL.

[0026] Furthermore, in the above technical solution, the usage ratio of the degummed silk fibroin fiber to GMA is 10 parts by mass: (1 to 5) parts by volume; wherein: the mass parts and volume parts are based on g:mL.

[0027] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the stirring reaction temperature is 60° C., and the stirring reaction time is 3 hours.

[0028] Furthermore, in the above technical solution, the stirring speed is 100-500r / min.

[0029] Furthermore, in the above technical solution, the temperature of the frozen supernatant is -100 to -50°C, and the time for freezing the supernatant is 10 to 15 hours.

[0030] Furthermore, in the above technical solution, the preparation method of OHA in step 3) is as follows:

[0031] Hyaluronic acid (HA) is dissolved in deionized water to obtain an HA solution; sodium periodate is then added to the HA solution according to a ratio, and the resulting reaction system B is stirred and reacted in the dark at room temperature for 20-30 hours; after the reaction, the resulting product is dialyzed and freeze-dried to obtain the OHA.

[0032] Furthermore, in the above technical solution, the mass ratio of HA to sodium periodate is 2:1.

[0033] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the stirring reaction time is 24 hours.

[0034] Furthermore, in the above technical solution, the concentration of the MXene dispersion in step 4) is 1-10 mg / mL. In a preferred embodiment of the present invention, the concentration of the MXene dispersion is 5 mg / mL.

[0035] Furthermore, in the above technical solution, the concentration of the SF-MA solution in step 4) is 10-20% (w / v).

[0036] Furthermore, in the above technical solution, the concentration of the OHA solution in step 4) is 1-5% (w / v).

[0037] Furthermore, in the above technical solution, the concentration of the CMCS solution in step 4) is 1-5% (w / v).

[0038] Furthermore, in the above technical solution, in step 4), the volume ratio of the MXene dispersion to the SF-MA solution is (10-30) μL:1 mL, preferably 20 μL:1 mL.

[0039] Furthermore, in the above technical solution, in step 4), the volume ratio of the CMCS solution to the SF-MA solution is (0.1-1) mL:1 mL, preferably 0.5 mL:1 mL.

[0040] Furthermore, in the above technical solution, in step 4), the volume ratio of the OHA solution to the SF-MA solution is (0.1-1) mL:1 mL, preferably 0.5 mL:1 mL.

[0041] Furthermore, in the above technical solution, in step 4), the ratio of the photoinitiator to the SF-MA solution is (1-10) parts by mass: 1 part by volume; preferably 5 parts by mass: 1 part by volume; wherein: the mass parts and volume parts are based on mg:mL.

[0042] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the photoinitiator in step 4) is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, referred to as LAP.

[0043] The second object of the present invention is to provide a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M) prepared by the above method.

[0044] The third object of the present invention is to provide the use of the near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M) prepared by the above-mentioned method in the preparation of diabetic wound repair materials.

[0045] Furthermore, in the above technical solution, the wound surface can be a complex wound surface, such as a joint wound or a chronic wound.

[0046] Furthermore, in the above technical solution, the diabetic wound repair material is in the form of an injectable preparation.

[0047] Furthermore, in the above technical solution, the diabetic wound repair material is used in combination with near-infrared laser irradiation.

[0048] The fourth object of the present invention is to provide a diabetic wound repair material, comprising the near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M) prepared by the above method.

[0049] The main raw materials used in the present invention play the following roles in the present invention:

[0050] The silk fibroin (SF) used in the present invention has excellent biocompatibility, degradability, and certain cell adhesion and proliferation activities. It has a good support effect on the wound healing environment, does not trigger adverse immune reactions, and helps the regeneration and repair of skin tissue. SF contains amino groups that can react with the aldehyde groups on OHA to form a Schiff base to form a dynamic reversible covalent network, constituting the first network (physical / dynamic chemical cross-linking network). SF-MA forms a main chemical cross-linking skeleton network through a photocross-linking reaction, constituting the second network, which significantly enhances the mechanical properties and structural stability of the hydrogel, especially in a moist and dynamic wound environment, helping to improve its durability and closure.

[0051] The CMCS employed in this invention possesses natural broad-spectrum antimicrobial properties, moderate hemostatic ability, good biocompatibility, moisturizing properties, and viscosity-adjusting properties, helping to regulate the rheological behavior of the system. Furthermore, CMCS is rich in primary amine groups, which react with the aldehyde groups on OHA via a Schiff base reaction to form dynamic covalent bonds, thereby forming a primary network with SF-MA.

[0052] The HA used in this invention is one of the main components of the extracellular matrix (ECM). It has good biocompatibility and degradability, and can promote cell adhesion, migration, and proliferation, which is beneficial for epithelial regeneration and tissue repair. OHA also retains these properties and can provide aldehyde groups to react with the amino groups (-NH2) in CMCS and SF-MA to undergo a Schiff base reaction, quickly forming an injectable soft gel and constructing the first network of the hydrogel (dynamic reversible cross-linking network), achieving good injectability and adaptability to irregular wound surfaces.

[0053] The MXene nanosheets used in the present invention have excellent near-infrared (NIR) photothermal conversion capabilities, which can achieve drug-free photothermal antibacterial effects; they have good free radical scavenging capabilities and can effectively remove excess reactive oxygen species (ROS); they have the function of regulating macrophage polarization, exerting an immunomodulatory effect, and helping to alleviate chronic inflammation; they can form physical fillings or weak interactions (such as hydrogen bonds) in the network, which can enhance the structural integrity, toughness and stability of the hydrogel to a certain extent, and synergistically improve the material performance.

[0054] In summary, the SF, OHA, CMCS, and MXene nanosheets used in the present invention each play a unique role in the injectable hydrogel and achieve a high degree of synergy at both the structural and biological functional levels. The synergistic effect is mainly reflected in the following aspects:

[0055] (1) The synergistic effect of each component at the structural level constructs a dual-network hydrogel with both injectability and mechanical strength, which is suitable for complex wounds (such as joints or chronic wounds). OHA and SF-MA / CMCS form a dynamic reversible cross-linked network through Schiff base reaction, achieving good injectability and conformability; SF-MA provides double bond groups to form a covalent cross-linked network through ultraviolet irradiation, improving mechanical strength and stability; MXene nanosheets form physical filling and interfacial interactions in the network to improve the toughness of the network.

[0056] (2) The synergistic effect of each component at the biological function level integrates the multifunctionality of photothermal antibacterial, antioxidant, immunomodulatory, and cell adhesion and migration. Among them, the OHA, SF-MA, and CMCS composite components simulate ECM, enhancing cell adhesion and migration and tissue repair capabilities; MXene nanosheets provide photothermal antibacterial, antioxidant, and immunomodulatory functions, enhancing anti-infection ability, effectively alleviating the oxidative stress microenvironment, and promoting the reconstruction of the immune microenvironment. The efficient integration and synergistic enhancement of natural polymers and functional nanomaterials significantly enhances the comprehensive therapeutic potential of hydrogels in treating difficult-to-heal diabetic wounds.

[0057] The near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel SCO@M prepared by the present invention has photothermal antibacterial and antioxidant functions, and exhibits good cell compatibility and blood compatibility. It can be used for the repair of diabetic wounds and as a carrier of drugs and bioactive substances, and has broad application prospects.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) The present invention uses SF, HA, CMCS and MXene nanosheets as raw materials to construct an injectable hydrogel with excellent photothermal antibacterial properties, antioxidant properties, degradability and excellent biocompatibility.

[0060] (2) The implementation strategy of the injectable hydrogel provided by the present invention overcomes the inherent contradiction between its fluidity and mechanical strength. In the first step, the first dynamic cross-linking network is constructed through the Schiff base reaction between OHA and SF-MA / CMCS, and the hydrogen bonding between MXene nanosheets and the above macromolecules, forming a soft hydrogel with shear thinning ability and conformability, ensuring the good fluidity of the hydrogel while avoiding excessive penetration and diffusion due to excessive fluidity, effectively covering the wound area. In the second step, the soft hydrogel is irradiated with short-term ultraviolet light (no more than 60s) to construct a second stable covalent cross-linking network, strengthen the network structure of the hydrogel, effectively improve the mechanical properties, and enhance the sealing of the wound. Moreover, the hydrogel integrates multiple functions that are beneficial to wound repair, including photothermal antibacterial, antioxidant, and immunomodulatory functions, which can effectively improve the efficiency of wound repair.

[0061] (3) The preparation process of the injectable hydrogel provided by the present invention is simple and easy, the conditions are easy to control, the raw materials are widely available, and the cost is low. It has important application value, especially for chronic diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0063] Figure 1 Schematic diagram of the preparation method of the hydrogel SCO@M according to Example 1 of the present invention.

[0064] Figure 2 In the figure: a is a flow chart of the preparation process of the MXene single-layer nanosheet prepared in Example 1 of the present invention; b is a Tyndall effect diagram of the MXene single-layer nanosheet prepared in Example 1 of the present invention; c is a diagram of the SCO@M hydrogel prepared in Example 1 of the present invention, the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, and the soft hydrogel SCO prepared in Comparative Example 3 none-UV d is the SCO@M hydrogel prepared in Example 1 of the present invention, the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, and the soft hydrogel SCO prepared in Comparative Example 3 none-UV a is the SEM image; e is the DLS particle size distribution diagram of the MXene single-layer nanosheets prepared in Example 1 of the present invention.

[0065] Figure 3 Middle: a is the soft hydrogel SCO prepared in Comparative Example 3 none-UVA picture of letters written with a syringe; b is the SF-MA solution prepared in Example 1 and the soft hydrogel SCO prepared in Comparative Example 3 none-UV Comparison of viscosity change curves with increasing shear force; c is the soft hydrogel SCO prepared in comparative example 3 none-UV d is a comparison diagram of the rheological properties of the hydrogel SCO prepared in comparative example 2 before and after ultraviolet irradiation.

[0066] Figure 4 a is a comparison diagram of the representative tensile stress-strain curves of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively; Figure 4 b is a comparison chart of the tensile elastic modulus of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively; Figure 4 c is a comparison diagram of the tensile fracture strain of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively; Figure 4 d is a comparison diagram of the tensile breaking stress of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively.

[0067] Figure 5 a is a comparison diagram of the representative compressive stress-strain curves of the hydrogels prepared in Example 1 and Comparative Examples 1-2; Figure 5 b is a comparison chart of the compressive elastic modulus of the hydrogels prepared in Example 1 and Comparative Examples 1-2.

[0068] Figure 6 The DPPH free radical scavenging rate comparison chart of the hydrogels prepared in Example 1 and Comparative Examples 1-2, wherein SCO@M+L in the figure represents the SCO@M synergistic near-infrared light irradiation group.

[0069] Figure 7 a is the MXene single-layer nanosheet dispersion prepared in Example 1, the SCO hydrogel prepared in Comparative Example 2, and the SCO@M hydrogel prepared in Example 1, respectively, under near-infrared light (808 nm, 1 W / cm 2 ) after 5 minutes of irradiation. Figure 7 b shows the MXene single-layer nanosheet dispersion prepared in Example 1, the SCO hydrogel prepared in Comparative Example 2, the SCO@M hydrogel prepared in Example 1, and deionized water, respectively, under near-infrared light (808 nm, 1 W / cm 2 ) temperature change curve comparison after 5 minutes of irradiation under the conditions. Figure 7 c is the temperature change curve of SCO@M prepared in Example 1 under cyclic light irradiation.

[0070] Figure 8a is the colony images of the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, the SCO@M hydrogel prepared in Example 1, SCO@M+L, and the blank control group after co-culture with Escherichia coli and Staphylococcus aureus, respectively; Figure 8 b is a comparison of the antibacterial rates of SF-MA hydrogel prepared in Comparative Example 1, SCO hydrogel prepared in Comparative Example 2, SCO@M hydrogel prepared in Example 1, SCO@M+L, and the blank control group against Escherichia coli; Figure 8 c is the SEM images of the colonies of the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, the SCO@M hydrogel prepared in Example 1, SCO@M+L, and the blank control group after co-culture with Escherichia coli and Staphylococcus aureus, respectively; Figure 8 d is a comparison chart of the antibacterial rates of SF-MA hydrogel prepared in comparative example 1, SCO hydrogel prepared in comparative example 2, SCO@M hydrogel prepared in example 1, SCO@M+L, and the blank control group against Staphylococcus aureus.

[0071] Figure 9 1 is a comparison chart of the cell proliferation activity of NIH3T3 cells after culturing for 24 hours and 48 hours using the SF-MA hydrogel extract prepared in Comparative Example 1, the SCO hydrogel extract prepared in Comparative Example 2, the SCO@M hydrogel extract prepared in Example 1, and the blank control group.

[0072] Figure 10 The in vivo evaluation results of the hydrogel of the specific embodiment of the present invention on the repair of diabetic wounds in SD rats are as follows: Figure 10 a is a schematic diagram of the establishment of diabetic rat model and treatment process, Figure 10 b is a representative photo of the wound during the wound recovery process. Figure 10 c is a schematic diagram of the wound area. Figure 10 d is the wound healing rate of the SCO hydrogel prepared in comparative example 2, the SCO@M hydrogel prepared in example 1, and the SCO@M+L group during the healing process; Tegaterm TM The treated wounds served as the control group. Figure 10 e is a H&E staining picture (red arrows: blood vessels, yellow arrows: hair follicles, green arrows: epithelialized areas, blue dotted line: unclosed epidermis). DETAILED DESCRIPTION

[0073] The present invention is further described in detail below through an implementation case. This implementation case is implemented based on the technology of the present invention. Detailed implementation methods and specific operating procedures are now given to illustrate the creativity of the present invention, but the protection scope of the present invention is not limited to the following implementation case.

[0074] Based on the information contained in this application, it will be readily apparent to those skilled in the art that various changes can be made to the precise description of the present invention. It should be understood that the scope of the present invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are provided only to illustrate specific aspects of the present invention.

[0075] In order to better understand the present invention and not to limit the scope of the present invention, all numbers used in this application to express amounts, percentages, and other numerical values ​​should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0076] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following examples are all conventional methods in the art unless otherwise specified.

[0077] The raw silk fibers used in the following examples of the present invention were purchased from Zhejiang Shengzhou Xiehe Silk Co., Ltd., and were 4A grade.

[0078] The photoinitiator LAP used in the following examples of the present invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and its CAS number is 85073-19-4.

[0079] Glycidyl methacrylate (GMA) used in the following examples of the present invention is a colorless transparent liquid with a purity of 99% and a CAS number of 106-91-2.

[0080] The Escherichia coli (E. coli) used in the antibacterial performance test of the present invention was purchased from BNCC (Beina Biological) with a serial number of BNCC185253; the Staphylococcus aureus (S. aureus) used in the antibacterial performance test of the present invention was purchased from BNCC (Beina Biological) with a serial number of BNCC381462.

[0081] The SCO@M+L involved in the present invention represents SCO@M hydrogel synergistically irradiated with near-infrared light.

[0082] Example 1

[0083] The present embodiment provides a method for preparing a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M), the method specifically comprising the following steps:

[0084] (1) Preparation of MXene monolayer nanosheets: A polytetrafluoroethylene beaker was placed in a fume hood, and 3.2 g of LiF and 40 mL of HCl solution (9 mol / L) were added and reacted at 40 °C for 15 min. Subsequently, 2 g of Ti3AlC2 was gradually added and reacted at 40-45 °C for 48 h. After the reaction, the mixture was repeatedly washed with deionized water and centrifuged (at a speed of 4000 rpm) until the pH was ≈ 6, and then vacuum filtered. The precipitate was further washed and centrifuged until a viscous residue was obtained. The residue was added to 100 mL of deionized water, purged with nitrogen, and sonicated in an ice water bath for 1 h. The unpeeled large particles were then removed using low-speed centrifugation (1500 rpm, 30 min), and the supernatant was collected to obtain a dark green MXene monolayer nanosheet dispersion with a concentration of 200 μg / mL.

[0085] (2) Preparation of SF-MA: The raw silk fibers were degummed three times in a 0.205 wt% Na2CO3 solution at 98 ± 2°C for 30 min each time to remove the sericin. After air drying, 20 g of the degummed silk fibers were dissolved in 100 mL of lithium bromide (LiBr) solution (9.3 mol / L) at 60°C for 1 h. After the silk fibers were completely dissolved, 6 mL of glycidyl methacrylate (GMA) was immediately added dropwise, and the mixture was stirred at 60°C for 3 h for grafting reaction at a speed of 300 rpm. After the reaction was completed, the resulting solution was dialyzed with deionized water for 4 days. Finally, the reaction product was centrifuged to remove the precipitate, and the supernatant was frozen at -80°C for 12 h and then freeze-dried for 48 h to obtain the SF-MA, which was stored at -20°C for future use.

[0086] (3) Preparation of OHA: Dissolve 2 g of hyaluronic acid (HA) in 100 mL of deionized water and heat at 50°C with stirring to completely dissolve the HA. After the solution cools to room temperature, add 1 g of sodium periodate to the HA solution and continue stirring. The reaction is allowed to proceed at room temperature in the dark for 24 h. After the reaction is complete, the reaction mixture is dialyzed against deionized water for 3 days and then freeze-dried to obtain freeze-dried OHA, which is stored at -20°C until use.

[0087] (4) Preparation of SCO@M: The MXene monolayer nanosheet dispersion prepared in step (1) was diluted to obtain a MXene monolayer nanosheet dispersion with a concentration of 5 mg / mL; the SF-MA freeze-dried in step (2) was dissolved in deionized water to obtain a 20% (w / v) SF-MA solution; the OHA freeze-dried in step (3) was dissolved in deionized water to obtain a 3% (w / v) OHA solution; carboxymethyl chitosan (CMCS) was dissolved in deionized water to obtain a 2% (w / v) CMCS aqueous solution; 20 μL of the 5 mg / mL MXene monolayer nanosheet dispersion was added to 1 mL of the 20 wt% SF-MA solution, and after shaking evenly, 0.5 mL of the 2% CMCS solution was added to the above solution, and then 5 mg of the photoinitiator LAP was added, and complete dissolution was ensured by stirring and homogenization. Subsequently, 0.5 mL of the 3% OHA solution prepared in step (3) was added and mixed thoroughly to obtain a Schiff base cross-linked soft hydrogel. The composite hydrogel was then exposed to ultraviolet light for 30 seconds to generate a double-crosslinked hydrogel (SCO@M). In these experiments, the concentration of the photoinitiator LAP in the hydrogel prepolymer was 0.25% (w / v).

[0088] Example 2

[0089] The present embodiment provides a method for preparing an injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M). The method is substantially the same as that of Example 1, except that the concentration of the SF-MA solution used in step (4) of the present embodiment is 10% (w / v).

[0090] Example 3

[0091] The present embodiment provides a method for preparing an injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel (SCO@M). The method is substantially the same as that of Example 1, except that the concentration of the SF-MA solution used in step (4) of the present embodiment is 15% (w / v).

[0092] Comparative Example 1

[0093] The preparation method of a photocrosslinked hydrogel SF-MA in this comparative example is basically the same as that in Example 1, except that this comparative example only contains steps (2) and (4), and the raw materials MXene monolayer nanosheets, CMCS and OHA are not used in step (4).

[0094] Comparative Example 2

[0095] The preparation method of a silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid photocrosslinked hydrogel (SCO) in this comparative example is basically the same as that in Example 1, except that this comparative example does not contain step (1), and the raw material MXene monolayer nanosheet is not used in step (4).

[0096] Comparative Example 3

[0097] A soft hydrogel SCO in this comparative example that has not been photocrosslinked none-UV The preparation method is basically the same as that of Comparative Example 2, except that the photocrosslinking agent LAP is not used in step (4) of this comparative example.

[0098] The structures of the hydrogels prepared in Example 1 and Comparative Examples 1-3 of the present invention were tested. Figure 2 shown.

[0099] Figure 2 In the figure: a is a flow chart of the preparation process of MXene single-layer nanosheets prepared in Example 1 of the present invention; b is a 200μg / mL MXene single-layer nanosheet solution prepared in Example 1 of the present invention and its Tyndall effect diagram. The nanosheet solution appears dark green. When illuminated from one side with a laser pen, a clear red light channel can be seen, showing the Tyndall effect, confirming that the MXene single-layer nanosheets are well dispersed in the aqueous solution. c is the SCO@M hydrogel prepared in Example 1 of the present invention, the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, and the soft hydrogel SCO prepared in Comparative Example 3. none-UV The actual picture shows that the hydrogel without UV irradiation is very soft, so that it cannot maintain a cylindrical shape after demolding. The strength of the hydrogel after UV irradiation curing increases and has good plasticity. Among them, the hydrogel in Example 1 is dark gray, which is due to the retention of the unique color of the MXene single-layer nanosheet. d is the SCO@M hydrogel prepared in Example 1 of the present invention, the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, and the soft hydrogel SCO prepared in Comparative Example 3. none-UVSEM image. As can be seen from Figure d, all samples show a uniform and interconnected porous structure. The pore size of the hydrogel in Comparative Example 3 is larger, with an average pore size of 105.65±22.60μm. After 30s of ultraviolet irradiation, the pore size of the hydrogel in Comparative Example 2 is significantly reduced to 71.76±10.18μm. The SF-MA hydrogel prepared with the same mass concentration as SCO shows a similar pore structure with a pore size of 77.31±11.28μm. The reason for the reduction in SCO pore size after ultraviolet cross-linking may be that the SF-MA photocross-linking reaction enhances the density of the hydrogel network, thereby reducing the pore size. Moreover, the addition of MXene monolayer nanosheets does not affect the formation of the hydrogel. The average pore size of the SCO@M hydrogel prepared in Example 1 is 52.03±7.33μm. e is a DLS particle size distribution diagram of the MXene monolayer nanosheets prepared in Example 1 of the present invention. It can be seen that the average particle size of the MXene monolayer nanosheets is 890nm.

[0100] The properties of the hydrogels prepared in Example 1 and Comparative Examples 1-3 of the present invention were tested.

[0101] (1) Injectability test

[0102] The injectability of the hydrogel was evaluated macroscopically. The Rhodamine B-stained hydrogel was placed in a syringe for injection testing. The hydrogel was squeezed out and the word TZU was written on a whiteboard. The shear thinning rheological behavior of the hydrogel was measured using a dynamic rheometer (Anton Paar DynamicRheometer). A 24 mm parallel sample stage was used, the temperature was 25°C, and the gap distance between the two plates was fixed at 1 mm. The angular frequency was fixed at 6.28 rad / s, the sample was placed between the parallel sample stages, and a dynamic amplitude strain scan from 1% to 1000% was used to measure the viscosity change of the sample in this range. In order to evaluate the rheological behavior during the gelation process, the strain was fixed at 1% and the frequency was 1 Hz. The sample was scanned for about 600 seconds using a rheometer, and the curves of G' and G" changing with time were recorded. For experimental results, see Figure 3 .

[0103] Figure 3 a is the prepared SCO none-UV Image of letters written using a syringe. SCO using rhodamine B none-UV The surface was dyed and the word "TZU" was written with a syringe, indicating that the hydrogel had good fluidity and that the soft gel could still maintain a certain shape after extrusion to prevent flowing and spreading. Figure 3 b is SF-MA solution and SCO none-UV Viscosity changes with increasing shear force. When the shear rate increases gradually, SCO none-UVThe viscosity of the SF-MA solution decreased significantly, from 473 Pa·s to 0.168 Pa·s, demonstrating its excellent shear-thinning ability, enabling it to pass smoothly through fine needles during injection without clogging. The SF-MA solution, on the other hand, only decreased from 0.0398 to 0.00797 Pa·s, indicating that the SF-MA solution has good fluidity, similar to that of water, but is prone to diffusion when injected directly into a wound. Figure 3 c is SCO none-UV Rheological properties of the formation process, Figure 3 d is the rheological properties before and after SCO irradiation. During the gelation process, between 60 and 100 s, SCO none-UV The G' of SCO increases rapidly from 1Pa to 85Pa, reaching a plateau at 100Pa. The rapid increase in G' is much greater than G", indicating that it has transformed from solution to hydrogel, but the relatively low G' indicates that the hydrogel is very soft. In contrast, the G' of SCO increases sharply from 100Pa to 1570Pa after 30s of UV irradiation, indicating that the strength of the hydrogel is significantly improved. This rapid solidification of SCO can be attributed to the SF-MA component in the hydrogel, which induces rapid free radical polymerization under UV light and enhances the stability of the SF-MA hydrophobic domain.

[0104] (2) Mechanical properties testing

[0105] Long strip hydrogel samples (20×40×1.5mm) were prepared and the tensile mechanical properties of the hydrogels were evaluated by tensile tests. The tensile test was performed using the tensile mode, with the fixture controlled at 30mm from the initial distance and stretched at a rate of 2mm / min until the material broke. The stress-strain curve was plotted and the fracture strength and elongation were calculated. The slope of the initial linear region of the stress-strain curve was calculated to determine the tensile modulus (E). Each group of samples was repeated more than 3 times and the average value was taken. The experimental results can be found in Figure 4 .

[0106] Figure 4 a is a comparison diagram of the representative tensile stress-strain curves of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively; Figure 4 b is a comparison chart of the tensile elastic modulus of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively; Figure 4 c is a comparison diagram of the tensile fracture strain of the hydrogels prepared in Example 1 and Comparative Examples 1-2, respectively; Figure 4 d is a comparison of the tensile stress at break of the hydrogels prepared in Example 1 and Comparative Examples 1-2. The tensile stress-strain results show that SCO@M exhibits the highest tensile stress at break, strain, and tensile elastic modulus, reaching 10.84 kPa, 76%, and 19.02 kPa, respectively. These results demonstrate that the dual-crosslinked hydrogel effectively enhances its mechanical properties, while the addition of MXene monolayer nanosheets improves the hydrogel's ductility.

[0107] Cylindrical samples (approximately 8 mm in diameter and 10 mm in height) were prepared and the compressive mechanical properties of the hydrogels were evaluated by compression testing. The compression rate was set at 5 mm / min. Based on the initial dimensions of the sample, the load and displacement data were converted to stress-strain values. The compression modulus (E) was determined by calculating the slope of the initial linear region of the stress-strain curve. Each group of samples was repeated at least three times and the average value was taken. The experimental results are shown in [ 1 ]. Figure 5 .

[0108] Figure 5 a is a comparison diagram of the representative compressive stress-strain curves of the hydrogels prepared in Example 1 and Comparative Examples 1-2; Figure 5 b is a comparison chart of the compressive elastic modulus of the hydrogels prepared in Example 1 and Comparative Examples 1-2 under different strain conditions. The compressive stress-strain curves show that all hydrogels can withstand at least 80% strain, showing good compressibility. Among them, SCO hydrogel and SCO@M hydrogel both exhibit better compression resistance. In contrast, SF-MA hydrogel is more easily deformed under the same stress, indicating that its mechanical stability is poor. In addition, the compressive elastic modulus increases with strain. When the compressive strain is 60%, the compressive elastic modulus corresponding to SCO hydrogel and SCO@M hydrogel is 186kPa and 204kPa, which is significantly higher than SF-MA (19kPa), indicating that the double cross-linking strategy effectively improves the compressive strength of the hydrogel.

[0109] (3) Antioxidant performance test

[0110] The antioxidant capacity of the hydrogel was tested using DPPH. 1 mL of hydrogel was gently crushed with a tissue grinder to form a gel dispersion. 0.3 mL of DPPH solution (1 mM) and the above dispersion were then dispersed in 3.0 mL of ethanol. For the near-infrared irradiation group, near-infrared light (808 nm, 1 W / cm 2 ) irradiate the hydrogel for 1 minute. The mixture was incubated in the dark for 30 minutes, and the absorbance of the supernatant was read at 517 nm using a microplate reader. The DPPH radical scavenging efficiency was calculated as follows:

[0111]

[0112] Where A b , A h The absorbance of blank group and sample group at 517nm respectively. Figure 6 .

[0113] Figure 6Comparison of the DPPH radical scavenging rates of the hydrogels prepared in Example 1 and Comparative Examples 1-2. Compared to hydrogels without MXene nanosheets (SF-MA and SCO), SCO@M exhibits strong antioxidant activity, both with and without near-infrared irradiation. At a concentration of 125 μg / mL of a MXene monolayer nanosheet solution, the DPPH radical scavenging rate exceeds 60%, and near-infrared irradiation does not affect the hydrogel's antioxidant capacity.

[0114] (IV) Photothermal conversion performance measurement

[0115] In order to investigate the photothermal conversion performance of the material, near-infrared light (808 nm, 1 W / cm2) was emitted by an MDL-III-808-2W laser (Changchun New Industry Optoelectronics Technology Co., Ltd.). 2 ) irradiate the hydrogel for 5 minutes, and record the temperature change within 5 minutes with an infrared imager. In addition, a photothermal cycle test was performed to evaluate the photothermal stability of the hydrogel under cyclic illumination. Take 1 mL of the hydrogel precursor solution and place it in a 10 mL transparent glass bottle. After gelation, irradiate the hydrogel with near-infrared light (808 nm, 1 W / cm 2 ) After irradiation for 3 minutes, it was naturally cooled to room temperature. The heating-natural cooling cycle was repeated 5 times, and the temperature change was recorded with an infrared imager. Figure 7 .

[0116] Figure 7 a is the MXene single-layer nanosheet dispersion prepared in Example 1, the SCO hydrogel prepared in Comparative Example 2, and the SCO@M hydrogel prepared in Example 1, respectively, under near-infrared light (808 nm, 1 W / cm 2 ) after 5 minutes of irradiation. Figure 7 b shows the MXene single-layer nanosheet dispersion prepared in Example 1, the SCO hydrogel prepared in Comparative Example 2, the SCO@M hydrogel prepared in Example 1, and deionized water, respectively, under near-infrared light (808 nm, 1 W / cm 2 ) temperature change curve comparison after 5 minutes of irradiation under the conditions. Figure 7 c is the temperature change curve of SCO@M prepared in Example 1 under cyclic light irradiation.

[0117] Under the same near-infrared light exposure time, Figure 7a Infrared thermal imaging results show that the temperature of the MXene monolayer nanosheet dispersion and SCO@M hydrogel increased significantly. With the extension of irradiation time, the materials containing MXene monolayer nanosheets all showed a clear temperature increase trend. After 5 minutes of near-infrared light irradiation, the temperatures of deionized water, SCO, SCO@M, and MXene monolayer nanosheet dispersion with equivalent concentration increased by 1.4°C, 3.65°C, 21.1°C, and 22.3°C, respectively, indicating that SCO@M has excellent photothermal conversion properties. In addition, the number of cyclic illumination cycles had no effect on the temperature increase of SCO@M, indicating that the photothermal effect of SCO@M is stable under cyclic illumination.

[0118] (5) Antibacterial performance testing

[0119] Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used as model bacteria to evaluate the antibacterial properties of the hydrogel. A single colony was picked and activated in a shaker at 37°C at 200 rpm for 12 hours. The bacterial solution was diluted with saline to 4×10 8 CFU / mL (OD 600 The hydrogel was made into a 1.5 mm diameter disc, placed at the bottom of a 48-well plate, and sterilized. 10 μL of bacterial dilution was added to the surface of the hydrogel, and the SCO@M synergistic illumination group (SCO@M+L) was illuminated under near-infrared light (808 nm, 1 W / cm 2 ) conditions for 1 minute. All samples were placed in a 37°C constant temperature incubator for incubation for 4 hours. Then, 1 mL of sterile PBS was used to resuspend the bacterial solution. The blank control group was 1 mL of sterile PBS with 10 μL of bacterial dilution added. 50 μL of the above bacterial solution was smeared on LB solid culture medium and cultured at 37°C for 12 hours. Photos were taken and recorded. At the same time, another 100 μL of the above bacterial solution was cultured with LB liquid culture medium at 37°C for 12 hours. The OD value was measured at 600 nm using a microplate reader and the inhibition rate was calculated. The inhibition rate calculation formula is as follows:

[0120]

[0121] Where OD positivecontrol is the absorbance of the blank control group without gel at 600 nm, OD samples is the absorbance of the hydrogel sample group at 600 nm, OD samples The absorbance of the untreated culture medium and the LB liquid culture medium at 600 nm. Figure 8 .

[0122] Figure 8a is the colony images of the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, the SCO@M hydrogel prepared in Example 1, SCO@M+L, and the blank control group after co-culture with Escherichia coli and Staphylococcus aureus, respectively; Figure 8 b is a comparison of the antibacterial rates of SF-MA hydrogel prepared in Comparative Example 1, SCO hydrogel prepared in Comparative Example 2, SCO@M hydrogel prepared in Example 1, SCO@M+L, and the blank control group against Escherichia coli; Figure 8 c is the SEM images of the colonies of the SF-MA hydrogel prepared in Comparative Example 1, the SCO hydrogel prepared in Comparative Example 2, the SCO@M hydrogel prepared in Example 1, SCO@M+L, and the blank control group after co-culture with Escherichia coli and Staphylococcus aureus, respectively; Figure 8 d is a comparison chart of the antibacterial rates of SF-MA hydrogel prepared in comparative example 1, SCO hydrogel prepared in comparative example 2, SCO@M hydrogel prepared in example 1, SCO@M+L, and the blank control group against Staphylococcus aureus.

[0123] Figure 8 The test results showed that colony imaging confirmed the antibacterial activity of the hydrogel. After co-incubation with SCO@M, the number of colonies was significantly reduced, and no colonies were observed on the agar plate after synergistic near-infrared illumination. Compared with E. coli, the number of S. aureus colonies was lower after co-incubation with the same hydrogel. In addition, SEM images showed that S. aureus and E. coli co-incubated with SCO and SCO@M lost membrane integrity, with wrinkles and ruptures visible on the cell surface. Shrinkage and damage to the bacterial cell membrane were more pronounced in the co-treated group. The SCO@M hydrogel exhibited excellent broad-spectrum antibacterial efficacy, with an inhibition rate of 92.67±0.49% against S. aureus and 78.36±5.80% against E. coli. The antibacterial activity of SCO@M hydrogel against S. aureus was slightly higher than that against E. coli, which may be attributed to the unique outer membrane (OM) structure of Gram-negative bacteria such as E. coli, which forms an additional barrier to enhance resistance to antimicrobial factors. Near-infrared irradiation increased the inhibition rate of SCO@M against Escherichia coli to 93.67±1.23%, which is the result of photothermal antibacterial effect. Comprehensive experimental results show that SCO@M hydrogel exhibits excellent broad-spectrum antibacterial properties in conjunction with near-infrared irradiation, mainly due to the antibacterial properties of MXene and CMCS components, as well as the synergistic antibacterial effect caused by the photothermal effect.

[0124] (IX) Cytotoxicity assay

[0125] The hydrogel was sterilized with 75% alcohol and repeatedly washed with PBS to remove the ethanol. Complete culture medium was added at 60 mg / mL and incubated in a shaker at 37°C at 100 r / min for 24 h. The supernatant was sterilized using a sterile needle filter (0.22 μm, PES material). The filtered liquid was the hydrogel extract.

[0126] Mouse embryonic fibroblast cell line (NIH3T3) was cultured in 96-well plates at 37°C and 5% CO2, with 2×10 cells seeded per well. 3 After culturing for 24 hours, different hydrogel extracts were added to each well. Four replicates were set up for each group. After continuing to culture for 24 and 48 hours, working solution (culture medium: CCK-8 working solution = 10:1, volume ratio) was added to each well and incubated for 3 hours. The absorbance at a wavelength of 450 nm was measured using a microplate reader and the cell proliferation activity was calculated. The calculation formula is as follows:

[0127]

[0128] Where OD sample is the absorbance of the experimental group at 450 nm, OD control is the absorbance of the complete culture medium group at 450nm. For experimental results, see Figure 9 .

[0129] Figure 9 1 is a comparison chart of the cell proliferation activity of NIH3T3 cells after culturing for 24 hours and 48 hours using the SF-MA hydrogel extract prepared in Comparative Example 1, the SCO hydrogel extract prepared in Comparative Example 2, the SCO@M hydrogel extract prepared in Example 1, and the blank control group. Figure 9 The CCK-8 test results showed that the cell survival rate of NIH3T3 after treatment with different hydrogel extracts for 24 hours and 48 hours was above 90%, indicating that the hydrogel has good cell compatibility and no obvious cytotoxicity, which meets the national standard (GB / T16886.5-2017: a relative survival rate ≥70% indicates that the material has no obvious cytotoxicity).

[0130] (10) In vivo evaluation of hydrogel repair of diabetic wounds

[0131] In vivo experiments were conducted using SD rats (male, 8 weeks old, 180-200g). In order to establish a diabetic wound model, streptozotocin (60mg / kg) solution was injected into rats via the peritoneal cavity until the blood glucose level was ≥16.7mmol / L, and the rats were randomly divided into four groups. Then, the rats were anesthetized with isoflurane gas, and two full-thickness wounds were caused on both sides of the rat's back with a needle biopsy device (10mm in diameter) under anesthesia. The wounds were randomly treated with 3M Tegaderm film, SCO hydrogel prepared in Comparative Example 2, SCO@M hydrogel prepared in Example 1, and SCO@M hydrogel in combination with near-infrared light irradiation (SCO@M+L). The experimental group used sterilized hydrogel, and the SCO@M+L group continuously received near-infrared light (808nm, 1W / cm 2 The wounds were irradiated for 1 minute each day for three consecutive days. The dressing was changed every two days. The control group was 3M Tegaderm film. The wounds were photographed on days 4, 6, 8, 12, and 14 to record the wound recovery process. The wound photos were processed using ImageJ software to calculate the area, and the wound contraction rate was calculated using the following formula (11):

[0132]

[0133] Area0: initial wound area; Area n : The area corresponding to different time points.

[0134] Wound tissue was collected on days 6 and 12 and evaluated histologically by H&E.

[0135] Figure 10 a is a schematic diagram of the establishment of diabetic rat model and treatment process, Figure 10 b is a representative photo of the wound during the wound recovery process. Figure 10 c is a schematic diagram of the wound area. Figure 10 d is the wound healing rate of SCO hydrogel prepared in comparative example 2, SCO@M hydrogel prepared in example 1, and SCO@M+L group during the healing process, Tegaterm TM The treated wounds served as the control group. Figure 10e is an H&E-stained image (red arrows: blood vessels, yellow arrows: hair follicles, green arrows: epithelialized areas, blue dashed line: unclosed epidermis). Representative wound images preliminarily demonstrate that wounds treated with SCO, SCO@M, and SCO@M+L healed significantly faster than those in the control group. Among them, diabetic wounds treated with SCO@M+L recovered the fastest. Wound closure rates showed that on day 6, the SCO@M+L group had a 57.54% wound closure rate, higher than the SCO@M group (46.80%) and the SCO group (46.42%). In contrast, the Tegaderm group had a wound closure rate of only 27.78%. After 14 days of treatment, the wounds in the SCO@M+L group were largely healed and scar-free, while the other groups still had minor scars, indicating that the wounds were not yet fully repaired. Six days after treatment, H&E-stained images revealed that the regenerated skin in the SCO@M+L group had a higher degree of epithelialization, abundant neovascularization (red arrows), denser granulation tissue, and more hair follicles (blue arrows). After 12 days of treatment, the wound surfaces of all groups were completely re-epithelialized. Among them, the SCO@M+L group showed a significant decrease in granulation tissue, skin regeneration entered the remodeling stage, hair follicles increased, and collagen deposition under the epidermis was dense and orderly, very similar to the structure and morphology of the normal skin layer. These results highlight the effectiveness of SCO@M synergistic near-infrared therapy in diabetic wound repair.

[0136] Although the embodiments of the present invention have been described in detail above, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and purpose of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel SCO@M, characterized by: The method specifically comprises the following steps: 1) Preparation of MXene monolayer nanosheets by in situ HF etching of LiF-HCl; 2) Modifying silk fibroin with glycidyl methacrylate (GMA) to obtain freeze-dried vinyl-modified silk fibroin SF-MA; 3) oxidizing hyaluronic acid with sodium periodate to obtain oxidized hyaluronic acid (OHA); 4) dispersing the MXene monolayer nanosheets described in step 1) in deionized water to obtain a MXene dispersion; dissolving the SF-MA described in step 2) in deionized water to obtain a SF-MA solution; dissolving the OHA described in step 3) in deionized water to obtain an OHA solution; dissolving carboxymethyl chitosan (CMCS) in deionized water to obtain a CMCS solution; and sequentially adding the MXene dispersion, CMCS solution, photoinitiator, and OHA solution to the SF-MA solution according to a ratio, and mixing thoroughly to obtain a Schiff base cross-linked soft hydrogel; 5) The Schiff base cross-linked soft hydrogel in step 4) is irradiated under ultraviolet light for 10-60 seconds to obtain a double cross-linked hydrogel, namely the SCO@M.

2. The preparation method according to claim 1, wherein: The preparation method of the MXene single-layer nanosheet in step 1) is as follows: Lithium fluoride (LiF) and concentrated hydrochloric acid are mixed according to a ratio, stirred and reacted at 35-45°C for 10-20 minutes to obtain a mixed etching solution; then, aluminum titanium carbide Ti3AlC2 is added to the mixed etching solution, stirred and etched at 40-45°C for 12-48 hours. After the reaction is completed, the solution is washed and centrifuged to obtain MXene multilayer nanosheets; the MXene multilayer nanosheets are then subjected to low-temperature ultrasound and low-speed centrifugation in sequence, the supernatant is collected, and freeze-dried to obtain the MXene single-layer nanosheets.

3. The preparation method according to claim 1, wherein: The specific preparation method of the SF-MA in step 2) is as follows: The raw silk fiber is degummed and dried to obtain degummed silk fibroin fiber; the degummed silk fibroin fiber is then added to a lithium bromide (LiBr) solution and heated to dissolve; glycidyl methacrylate (GMA) is immediately added dropwise to the dissolved degummed silk fibroin fiber according to a ratio, and after the addition is completed, the obtained reaction system A is stirred and reacted at 55-65° C. for 1-5 hours; after the reaction is completed, the obtained product is dialyzed and centrifuged in sequence, and the obtained supernatant is frozen and then dried to obtain the described SF-MA.

4. The preparation method according to claim 1, wherein: The preparation method of OHA in step 3) is as follows: Hyaluronic acid HA is dissolved in deionized water to obtain an HA solution; sodium periodate is then added to the HA solution according to a ratio, and the resulting reaction system B is stirred and reacted in the dark at room temperature for 20-30 hours; after the reaction, the resulting product is dialyzed and freeze-dried to obtain the OHA.

5. The preparation method according to claim 4, characterized in that: The mass ratio of HA to sodium periodate is 2:

1.

6. The near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel SCO@M prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel SCO@M prepared by the preparation method according to any one of claims 1 to 5 in the preparation of diabetic wound repair materials.

8. The use according to claim 7, characterized in that: The diabetic wound repair material is in the form of an injection preparation.

9. The use according to claim 7, characterized in that: The diabetic wound repair material is used in combination with near-infrared laser irradiation.

10. A diabetic wound repair material, characterized by: The invention relates to a near-infrared responsive injectable MXene / silk fibroin / carboxymethyl chitosan / oxidized hyaluronic acid nanocomposite hydrogel SCO@M prepared by the preparation method according to any one of claims 1 to 5.

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