Nb2C MXene-based composite functional film capable of promoting angiogenesis and preparation method of Nb2C MXene-based composite functional film

Abstract

The invention relates to the field of biomedical materials, in particular to an Nb2C MXene composite functional film capable of promoting angiogenesis and a preparation method of the Nb2C MXene composite functional film, the Nb2C MXene composite functional film is particularly suitable for diabetes wound healing, and the Nb2C MXene composite functional film comprises an Nb2C MXene functionalized collagen vitrified film layer, and a carboxymethyl chitosan vitrification film layer, wherein a nano interlocking interface is formed between the Nb2C MXene functionalized collagen vitrified membrane layer and the carboxymethyl chitosan vitrified membrane layer; the Nb2C MXene functionalized collagen vitrified membrane layer is hydrophobic, the carboxymethyl chitosan vitrified membrane layer is hydrophilic, through the double-layer asymmetric structural design, the inner layer is tightly attached to a wound, the outer layer prevents adhesion of bacteria and tissues, and the barrier function in the wound healing process is effectively guaranteed; the adopted Nb2CMXene has excellent ROS (reactive oxygen species) removal capacity, oxidative stress of wound parts can be effectively relieved, cell damage is avoided, and cell functions are recovered.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly to a composite functional membrane of Nb 2 C MXene capable of promoting angiogenesis and a preparation method thereof, which is particularly suitable for diabetic wound healing. Background Art

[0002] Diabetic wounds are characterized by a long healing period and a high recurrence rate. In severe cases, they may lead to non-traumatic amputation, posing a major threat to the health and quality of life of patients. One of the main inducements of diabetic complications is microvascular dysfunction caused by hyperglycemia, which can lead to ischemia and delayed wound healing. The cells involved in the wound healing process require nutrients and oxygen, which are provided by blood vessels. Therefore, enhancing blood vessel regeneration is crucial for accelerating diabetic wound healing. In addition, the loss of the inherent protective function of the skin increases the risk of bacterial infection in diabetic wounds. Since hyperglycemia can lead to excessive production and accumulation of reactive oxygen species (ROS) locally, causing cell and tissue damage and exacerbating tissue inflammation. These factors are closely related to the poor treatment effect of diabetic wounds.

[0003] Currently, commonly used wound dressings in clinics such as gauze, bandages, and medical cotton exhibit characteristics of insufficient tissue adhesion and limited drug loading efficiency. Although synthetic polymer hydrogels have appropriate strength and satisfactory adhesion properties, their potential toxicity, poor biodegradability, and excessive swelling behavior greatly limit their clinical applications. In contrast, the latest progress of natural polymers shows that they have better biocompatibility, degradability, viscoelasticity, and processability, and thus are more suitable for wound healing applications. However, problems such as insufficient mechanical strength and uncontrollable swelling of hydrogels still limit their in vivo applications.

[0004] MXenes are two-dimensional materials characterized by an ultrathin layer structure and have attracted increasing attention due to their potential biomedical applications. Two-dimensional niobium carbide (Nb 2 C) MXene nanosheets exhibit effective photothermal conversion, ideal photothermal stability, and good antibacterial properties. In addition, Nb 2 C MXene nanosheets also have significant antioxidant properties and intrinsic enzyme / H 2 O 2 responsive biodegradability. Niobium-containing biomaterials can significantly promote angiogenesis. It has been reported that niobium-containing bioactive glass can significantly increase the expression of vascular endothelial growth factor.

[0005] Therefore, there is an urgent need to develop an innovative wound dressing that can simultaneously solve key problems such as oxidative stress, bacterial infection, and angiogenesis disorders in diabetic wounds. Summary of the Invention

[0006] The object of the present invention is to provide Nb 2 C MXene-based composite functional membranes that can promote angiogenesis and a method for preparing the same. The composite functional membranes accelerate the healing of diabetic wounds through multiple action mechanisms of antioxidant, antibacterial, and angiogenesis promotion.

[0007] To achieve the above object, the present invention provides Nb 2 C MXene-based composite functional membranes that can promote angiogenesis. The composite functional membranes include: Nb 2 C MXene-functionalized collagen vitrified membrane layer; and carboxymethyl chitosan vitrified membrane layer; wherein, a nano-interlocking interface is formed between the Nb 2 C MXene-functionalized collagen vitrified membrane layer and the carboxymethyl chitosan vitrified membrane layer; the Nb 2 C MXene-functionalized collagen vitrified membrane layer is hydrophobic, and the carboxymethyl chitosan vitrified membrane layer is hydrophilic.

[0008] Preferably, the content of Nb 2 C MXene in the Nb 2 C MXene-functionalized collagen vitrified membrane layer is 0.125 wt% to 1.0 wt%.

[0009] Preferably, the water contact angle of the Nb 2 C MXene-functionalized collagen vitrified membrane layer is 80° to 90°, and the water contact angle of the carboxymethyl chitosan vitrified membrane layer is 20° to 30°.

[0010] Preferably, the thickness of the composite functional membrane is 400 um to 500 um, wherein the thickness of the Nb 2 C MXene-functionalized collagen vitrified membrane layer is 250 μm to 300 μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer is 150 μm to 200 μm.

[0011] The present invention also provides a method for preparing the above-mentioned Nb 2 C MXene-based composite functional membranes that can promote angiogenesis. The method includes the following steps:

[0012] (1) Prepare Nb 2 C MXene nanosheets;

[0013] (2) Mix the Nb 2 C MXene nanosheets with a collagen solution, adjust the pH value to 6.8 ± 0.1, and enable the collagen to self-assemble to form a hydrogel;

[0014] (3) Vitrify the hydrogel at 10 ± 0.5 °C for 48 hours to form Nb2 Nb C MXene-functionalized collagen vitrified membrane;

[0015] (4) Uniformly coat the carboxymethyl chitosan solution on the Nb 2 C MXene-functionalized collagen vitrified membrane;

[0016] (5) Vitrify the coated structure for 48 hours at 10 ± 0.5 °C to form the composite functional membrane of Nb 2 C MXene that can promote angiogenesis.

[0017] Preferably, the method for preparing the Nb 2 C MXene nanosheets in step (1) includes:

[0018] (a) Add Nb 2 AlC MAX phase powder into a 50% concentration hydrofluoric acid solution, and stir and react at 0 - 4 °C for 24 hours;

[0019] (b) Centrifuge and wash the obtained product until the pH value of the supernatant reaches about 6;

[0020] (c) Disperse the product obtained in step (b) in a 25% concentration tetrapropylammonium hydroxide solution, and stir at 37 °C for 3 days;

[0021] (d) Centrifuge and wash the obtained product until the pH value of the supernatant is close to neutral;

[0022] (e) Disperse the product obtained in step (d) in deionized water, and perform ultrasonic treatment to obtain Nb 2 C MXene nanosheets with an average lateral size of 150 - 200 nm and a thickness of 0.4 - 0.6 nm.

[0023] Preferably, the collagen solution in step (2) is a type I collagen solution prepared by the following method:

[0024] (a) Immerse the rat tail tendon in a 0.5 M glacial acetic acid solution, and stir and extract at 4 °C for 48 hours;

[0025] (b) Filter and centrifuge the extract, and add a 5 M sodium chloride solution to the supernatant to a final concentration of 0.9 M;

[0026] (c) Let it stand overnight at 4 °C, centrifuge to collect the precipitate and redissolve it in 0.5 M glacial acetic acid;

[0027] (d) Dialyze and freeze-dry the solution to obtain purified type I collagen;

[0028] (e) Dissolve the purified type I collagen in 0.01 M glacial acetic acid (pH 3.0) at a concentration of 5 mg / mL.

[0029] Preferably, the final concentration of collagen in the mixed solution in step (2) is 2 mg / mL, and the mixture is evenly distributed into the mold at a volume density of 0.22 mL / cm 2 and incubated at 37 °C for 24 hours to allow the collagen to fully self-assemble.

[0030] Preferably, the concentration of the carboxymethyl chitosan solution in step (4) is 2 wt%, and the dosage is 0.15 mL / cm 2 , and an oil-free air stream with a pressure of 0.5 MPa is used to assist its penetration into the gaps between collagen fibers, and it is left standing at room temperature for 1 hour.

[0031] Preferably, the vitrification processes in steps (3) and (5) are carried out under the condition of a relative humidity of 30 - 40%, where it is pre-dried at room temperature for 2 hours before the first vitrification, and after the second vitrification is completed, the Nb 2 C MXene-promoted angiogenesis composite functional membrane is sterilized by ultraviolet light for 20 minutes.

[0032] The beneficial effects of the present invention are as follows:

[0033] 1. Through the double-layer asymmetric structure design of the composite functional membrane of the present invention, the inner layer adheres tightly to the wound, and the outer layer prevents bacteria and tissue adhesion, effectively ensuring the barrier function during the wound healing process.

[0034] 2. The Nb 2 C MXene adopted in the present invention has excellent ROS scavenging ability, can effectively reduce the oxidative stress at the wound site, avoid cell damage, and restore cell function.

[0035] 3. The composite functional membrane of the present invention also has good antibacterial properties, especially under near-infrared light irradiation, the antibacterial effect is further enhanced, and it can effectively prevent wound infection.

[0036] 4. The present invention first reveals the mechanism by which Nb 2 C MXene promotes angiogenesis by activating the SDF-1α / CXCR4 signaling axis and its downstream MAPK and PI3K / AKT pathways, effectively promoting wound vascularization and tissue regeneration.

[0037] 5. The dual-vitrification process adopted in the present invention is simple and safe, does not require the use of toxic cross-linking agents, and at the same time endows the composite functional membrane with excellent mechanical properties and appropriate degradation behavior. Description of the Drawings

[0038] Figure 1Nb of the present invention 2 Schematic diagram of the preparation process flow and mechanism of action of the composite functional membrane of Nb

[0039] Figure 2 Nb 2 Synthesis and characterization of Nb 2 C MXene nanosheets: A. Schematic diagram of the preparation process of Nb 2 C MXene; B. Transmission electron microscope (TEM) image showing the flaky structure of Nb

[0040] Figure 3 C MXene; C. Elemental mapping analysis; D. Atomic force microscope (AFM) image and thickness analysis; E - G. X - ray diffraction (XRD) and X - ray photoelectron spectroscopy (XPS) analysis; H - J. High - resolution XPS analysis showing the valence states and chemical environments of Nb, C, and O elements.

[0041] Figure 4 Preparation and property analysis of bilayer asymmetric membranes: A. Schematic diagram of the preparation process of bilayer membranes; B. Scanning electron microscope (SEM) image showing the CMC layer, collagen (COL) layer, and cross - sectional structure; C. Energy - dispersive X - ray spectroscopy (EDS) analysis showing the distribution of niobium elements in the collagen layer; D - E. XPS and Fourier transform infrared spectroscopy (FTIR) analysis; F - K. Test results of mechanical properties, swelling properties, and degradation characteristics. 2 O 2 Test results of ROS scavenging ability: A. Schematic diagram of the ROS scavenging mechanism; B - D. Test of the scavenging ability against H

[0042] Figure 5 O 2 C MXene content samples; D - J. Antibacterial performance tests, including plate culture, live / dead staining, and quantitative analysis; K - L. Bacterial adhesion experiment and schematic diagram of the antibacterial mechanism.

[0043] Figure 6 In vitro angiogenesis ability test: A - C. Results and quantitative analysis of scratch experiments; D - G. Transwell migration experiment and quantitative analysis; E - G. Tube formation experiment and quantitative analysis; H - J. Immunofluorescence staining analysis of F - actin and CD31 expression; K. qRT - PCR analysis of angiogenesis - related gene expression.

[0044] Figure 7In vivo wound healing experiment: A. Schematic diagram of animal experiment design; B. Digital photos of wounds at different time points; C. Quantitative analysis of wound size; D. Results of H&E staining and Masson staining; E - G. Quantitative analysis of wound healing rate, granulation tissue thickness, and collagen deposition; H - L. Immunofluorescence staining analysis showing CD31 expression (neovascularization) and DHE staining (ROS level).

[0045] Figure 8 Nb 2 Study on the mechanism of Nb

[0046] Figure 9 Nb 2 C MXene in promoting angiogenesis (Part I): A - D. Results of RNA sequencing analysis, including volcano plot of differentially expressed genes, KEGG pathway enrichment analysis, gene expression heat map, etc.; E - F. Results of qRT - PCR and Western blot of SDF - 1α and CXCR4 expression. 2 C MXene in promoting angiogenesis (Part II): A - D. Effects of CXCR4 inhibitor (AMD 3100) on the migration of HUVEC; E - I. Involvement of ERK and AKT signaling pathways in the mechanism of Nb 2 C MXene - promoted angiogenesis mechanism verification; J. Schematic diagram of pro - angiogenesis signaling pathway.

[0047] Figure 10 SEM images of CMC hydrogel.

[0048] Figure 11 Digital photo of Nb@CCJM lifting a 500 - gram weight, demonstrating its mechanical strength.

[0049] Figure 12 Stress - displacement curve of lap - shear test for measuring tissue adhesion strength.

[0050] Figure 13 Cumulative curve of niobium element release from Nb@CCJM.

[0051] Figure 14 Nb 2 C MXene in scavenging H 2 O 2 Performance test.

[0052] Figure 15 Performance test of Nb 2 C MXene in scavenging hydroxyl radicals, corresponding to the description of ROS scavenging ability in the specification.

[0053] Figure 16 Nb 2 Performance test of Nb

[0054] Figure 17 The hemolysis experiment tests blood compatibility.

[0055] Figure 18 Nb with different concentrations 2 Thermal imaging diagrams of the composite functional membranes of Nb

[0056] Figure 19 H&E staining results of major organs showing safety assessment. Specific implementation manners

[0057] The present invention will be further described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0058] Please refer to Figure 1 - 19 , the present invention will be further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0059] Example 1

[0060] Nb 2 The composite functional membrane of Nb 2 C MXene-functionalized collagen vitrification membrane layer and carboxymethyl chitosan vitrification membrane layer, wherein the Nb 2 C MXene-functionalized collagen vitrification membrane layer and the carboxymethyl chitosan vitrification membrane layer form a nano-interlocking interface; the Nb 2 C MXene-functionalized collagen vitrification membrane layer is hydrophobic with a water contact angle of 85.4°, and the carboxymethyl chitosan vitrification membrane layer is hydrophilic with a water contact angle of 26.9°. The Nb 2 C MXene in the Nb 2 C MXene-functionalized collagen vitrification membrane layer has a content of 0.5 wt%, and the total thickness of the composite functional membrane is 400 μm, wherein the Nb 2 C MXene-functionalized collagen vitrification membrane layer has a thickness of 250 μm, and the carboxymethyl chitosan vitrification membrane layer has a thickness of 150 μm.

[0061] The preparation method of the composite functional membrane includes the following steps:

[0062] (1) Prepare Nb 2 C MXene nanosheets:

[0063] Weigh 1 g of Nb 2The AlC MAX phase powder was slowly added to 20 mL of a 50% hydrofluoric acid solution, and magnetically stirred at a speed of 600 rpm for 24 hours at 2 °C while introducing a trace amount of argon gas to prevent oxidation. After etching, the excess HF was removed by centrifugal washing (3500 rpm, 10 minutes each time), washed 5 times with deionized water until the pH value of the supernatant reached 6, and finally washed 2 times with absolute ethanol. The above-mentioned etched product was dispersed in 20 mL of a 25% tetrapropylammonium hydroxide solution, and stirred at a speed of 300 rpm in a constant temperature water bath at 37 °C under an inert atmosphere for 3 days. After intercalation, the product was collected by centrifugation (8000 rpm, 15 minutes each time), washed at least 5 times with deionized water until the pH value of the supernatant was close to neutral, and finally washed 2 times with ethanol. The product was vacuum dried at 60 °C for 12 hours. The dried intercalated material was dispersed in deionized water at a concentration of 1 mg / mL, and ultrasonic treatment was carried out at a power of 300 W and a frequency of 40 kHz. First, ultrasonic treatment was carried out for 1 hour, and then intermittent ultrasonic treatment (working for 2 minutes and pausing for 1 minute) was carried out under ice bath conditions for another 5 hours. After ultrasonic treatment, the supernatant was collected by centrifugation at 12000 rpm for 15 minutes and filtered through a 0.22 μm filter membrane to obtain Nb 2 C MXene nanosheets with an average lateral size of 175 nm and a thickness of 0.5 nm.

[0064] (2) Preparation of type I collagen solution:

[0065] Fresh SPF-grade SD rat tails were taken, surface disinfected with 70% ethanol, and the tail tendons were stripped and cut into small pieces about 1 cm long under sterile conditions. The tail tendon fragments were washed 3 times with PBS solution to remove blood and other tissue residues. The tail tendon fragments were soaked in 50 times the volume of 0.5 M glacial acetic acid solution and stirred at a speed of 100 rpm at 4 °C for 48 hours for extraction. The extract was filtered through a 120-mesh nylon mesh to remove undissolved tissue fragments, and then centrifuged at 25000 g at 4 °C for 1 hour. The supernatant was collected, and pre-cooled 5 M NaCl solution was added to a final concentration of 0.9 M, and left standing overnight at 4 °C to precipitate the collagen. The precipitate was collected by centrifugation at 10000 g for 30 minutes and redissolved in 0.5 M glacial acetic acid. Using a dialysis bag with a molecular weight cut-off value of 14 kDa, dialysis was carried out against 0.1 M glacial acetic acid for 24 hours (changing the dialysis solution 3 times), and then against 0.01 M glacial acetic acid for 24 hours (changing the dialysis solution 3 times). The dialyzed collagen solution was first pre-frozen at -80 °C for 4 hours and then freeze-dried for 48 hours to obtain purified type I collagen. The purified type I collagen was dissolved in 0.01 M glacial acetic acid (pH 3.0) at a concentration of 5 mg / mL, slowly stirred at 4 °C for 24 hours to ensure complete dissolution, and finally sterilized through a 0.22 μm filter membrane.

[0066] (3) Preparation of Nb2 Nb@C MXene Functionalized Collagen Vitrified Membrane:

[0067] The Nb 2 C MXene dispersion with a concentration of 0.5 wt% was added dropwise to the 5 mg / mL type I collagen solution under gentle stirring (100 rpm). The final concentration of the mixture was: 2 mg / mL of collagen, and 0.5 wt% of Nb 2 CMXene. The pH value of the mixture was slowly adjusted to 6.8 at a rate of about 0.05 pH units / minute using 1 M NaOH solution to promote collagen self-assembly. The mixture was evenly distributed into a special mold at a volume density of 0.22 mL / cm 2 and incubated in a 37°C constant temperature incubator for 24 hours to allow the collagen to fully self-assemble into a hydrogel. The formed hydrogel was placed in a sterile air dryer, pre-dried at room temperature for 2 hours to slightly shrink the volume of the hydrogel, and then transferred to a 10°C low-temperature sterile incubator at a cooling rate of 2°C / hour and vitrified for 48 hours under a relative humidity of 35% to obtain Nb@CVM with a thickness of 250 μm.

[0068] (4) Preparation of the composite functional membrane:

[0069] CMC (250KDa, Sigma-Aldrich) was dissolved in deionized water at a concentration of 2 wt%, and magnetically stirred (300 rpm) at room temperature for 4 hours to ensure complete dissolution, and then left to stand overnight at 4°C to remove air bubbles. The CMC gel was evenly coated on the surface of the prepared Nb@CVM using a microbrush at a dosage of 0.15 mL / cm 2 . An air stream free of water and oil with a pressure of 0.5 MPa was used to assist the penetration of the CMC gel into the collagen fiber gaps, and the CMC gel was allowed to fully penetrate by standing at room temperature for 1 hour. The constructed bilayer sample was placed in a sterile air dryer and then transferred to a 10°C low-temperature incubator for a second vitrification treatment for 48 hours under a relative humidity of 35%. After vitrification, the prepared composite functional membrane was cut into the required size and sterilized with ultraviolet light for 20 minutes (10 minutes on each side) to finally obtain the Nb0.5@CCJM composite functional membrane.

[0070] The composite functional membrane has excellent biocompatibility with a hemolysis rate of less than 1%; it also has excellent mechanical properties with an elastic modulus of 14.14 MPa and a maximum tensile strength of 1.98 MPa; in addition, the composite functional membrane also exhibits appropriate swelling properties with a swelling rate of 311% and a water retention rate of 245%, which is beneficial for maintaining a moist environment for the wound.

[0071] Due to Nb 2Due to the presence of C MXene, the composite functional membrane has excellent ROS scavenging ability and can effectively scavenge H 2 O 2 , hydroxyl radicals and superoxide radicals, and can effectively protect human umbilical vein endothelial cells (HUVEC) from oxidative stress damage in vitro. The mortality rate of HUVEC treated with H 2 O 2 was reduced from 47.0% to 6.1%. The composite functional membrane also exhibited significant antibacterial activity, which was effective against both Staphylococcus aureus and Pseudomonas aeruginosa, and the antibacterial effect was further enhanced under 808 nm near-infrared light irradiation, with the bacteriostatic rate approaching 100%.

[0072] The composite functional membrane can significantly promote angiogenesis in vitro. The migration and healing rate of HUVEC reached 96.13%, and the number of tube formation nodes increased by about 2 times. Mechanistic studies showed that the composite functional membrane promoted the migration and tube formation of vascular endothelial cells by upregulating the gene expression and protein translation of SDF-1α and CXCR4, activating the SDF-1α / CXCR4 signaling axis and its downstream MAPK and PI3K / AKT signaling pathways. In vivo experiments showed that the composite functional membrane significantly promoted the wound healing of diabetic mice, with the wound healing rate reaching 85.2% on the 7th day and 98.5% on the 14th day.

[0073] Example 2

[0074] Nb 2 A composite functional membrane of Nb 2 C MXene that can promote angiogenesis, with the same composition and preparation method as in Example 1, except that the content of Nb 2 C MXene in the Nb

[0075] When preparing the Nb 2 C MXene-functionalized collagen vitrified membrane, a dispersion of Nb 2 C MXene with a concentration of 0.125 wt% was mixed with the collagen solution, and the remaining steps were the same as in Example 1. The total thickness of the obtained Nb0.125@CCJ M composite functional membrane was 500 μm, of which the thickness of the Nb 2 C MXene-functionalized collagen vitrified membrane layer was 300 μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer was 200 μm. The elastic modulus of this composite functional membrane was 13.75 MPa, and the maximum tensile strength was 1.85 MPa. It had good ROS scavenging ability and could reduce the mortality rate of HUVEC treated with H 2 O 2 from 47.0% to 29.6%.

[0076] Example 3

[0077] Nb 2 The composite functional membrane of Nb 2 C MXene capable of promoting angiogenesis has the same composition and preparation method as in Example 1, except that the Nb 2 C MXene has a content of 0.25 wt% in the Nb

[0078] When preparing the Nb 2 C MXene-functionalized collagen vitrified membrane, a dispersion of Nb 2 C MXene with a concentration of 0.25 wt% is mixed with the collagen solution, and the remaining steps are the same as in Example 1. The total thickness of the obtained Nb0.25@CCJM composite functional membrane is 450 μm, where the thickness of the Nb 2 C MXene-functionalized collagen vitrified membrane layer is 280 μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer is 170 μm. The elastic modulus of this composite functional membrane is 13.95 MPa, the maximum tensile strength is 1.90 MPa, and it has good ROS scavenging ability, which can reduce the mortality of HUVEC treated with H 2 O 2 from 47.0% to 14.0%.

[0079] Example 4

[0080] Nb 2 The composite functional membrane of Nb 2 C MXene capable of promoting angiogenesis has the same composition and preparation method as in Example 1, except that the Nb 2 C MXene has a content of 1.0 wt% in the Nb

[0081] When preparing the Nb 2 C MXene-functionalized collagen vitrified membrane, a dispersion of Nb 2 C MXene with a concentration of 1.0 wt% is mixed with the collagen solution, and the remaining steps are the same as in Example 1. The total thickness of the obtained Nb1.0@CCJM composite functional membrane is 420 μm, where the thickness of the Nb 2 C MXene-functionalized collagen vitrified membrane layer is 270 μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer is 150 μm. The elastic modulus of this composite functional membrane is 14.35 MPa, the maximum tensile strength is 2.05 MPa, and it has extremely strong ROS scavenging ability and antibacterial properties.

[0082] Example 5

[0083] Nb 2The composite functional membrane of Nb2CT MXene that can promote angiogenesis has the same composition and preparation method as in Example 1, except that the method for preparing Nb2CT MXene nanosheets in step (1) is adjusted: 2 Add 1 g of Nb2AlC MAX phase powder to 20 mL of 50% hydrofluoric acid solution, and magnetically stir at a speed of 600 rpm for 24 hours at 0 °C. After etching is completed, remove the excess HF by centrifugal washing, wash with deionized water 5 times until the pH value of the supernatant reaches 5.5, and finally wash with absolute ethanol 2 times. Disperse the above etching product in 20 mL of 25% tetrapropylammonium hydroxide solution and stir at 37 °C for 3 days. After intercalation is completed, collect the product by centrifugation and wash with deionized water until the pH value of the supernatant is close to neutral. Disperse the dried intercalated material in deionized water, and after ultrasonic treatment, obtain Nb2CT MXene nanosheets with an average lateral size of 150 nm and a thickness of 0.4 nm.

[0084] Add 1 g of Nb 2 AlC MAX phase powder to 20 mL of 50% hydrofluoric acid solution, and magnetically stir at a speed of 600 rpm for 24 hours at 0 °C. After etching is completed, remove the excess HF by centrifugal washing, wash with deionized water 5 times until the pH value of the supernatant reaches 5.5, and finally wash with absolute ethanol 2 times. Disperse the above etching product in 20 mL of 25% tetrapropylammonium hydroxide solution and stir at 37 °C for 3 days. After intercalation is completed, collect the product by centrifugation and wash with deionized water until the pH value of the supernatant is close to neutral. Disperse the dried intercalated material in deionized water, and after ultrasonic treatment, obtain Nb 2 C MXene nanosheets.

[0085] In the obtained Nb0.5@CCJM composite functional membrane, the Nb 2 C MXene content in the functionalized collagen vitrified membrane layer of C MXene is 0.5 wt%, and the total thickness is 465 μm. Its performance is similar to that of Example 1. 2 In the obtained Nb0.5@CCJM composite functional membrane, the Nb

[0086] Example 6

[0087] Nb 2 The composite functional membrane of Nb2CT MXene that can promote angiogenesis has the same composition and preparation method as in Example 1, except that the method for preparing Nb2CT MXene nanosheets in step (1) is adjusted: 2 Add 1 g of Nb2AlC MAX phase powder to 20 mL of 50% hydrofluoric acid solution, and magnetically stir at a speed of 600 rpm for 24 hours at 4 °C. After etching is completed, remove the excess HF by centrifugal washing, wash with deionized water 5 times until the pH value of the supernatant reaches 6.5, and finally wash with absolute ethanol 2 times. Disperse the above etching product in 20 mL of 25% tetrapropylammonium hydroxide solution and stir at 37 °C for 3 days. After intercalation is completed, collect the product by centrifugation and wash with deionized water until the pH value of the supernatant is close to neutral. Disperse the dried intercalated material in deionized water, and after ultrasonic treatment, obtain Nb2CT MXene nanosheets with an average lateral size of 200 nm and a thickness of 0.6 nm.

[0088] Add 1 g of Nb 2 AlC MAX phase powder to 20 mL of 50% hydrofluoric acid solution, and magnetically stir at a speed of 600 rpm for 24 hours at 4 °C. After etching is completed, remove the excess HF by centrifugal washing, wash with deionized water 5 times until the pH value of the supernatant reaches 6.5, and finally wash with absolute ethanol 2 times. Disperse the above etching product in 20 mL of 25% tetrapropylammonium hydroxide solution and stir at 37 °C for 3 days. After intercalation is completed, collect the product by centrifugation and wash with deionized water until the pH value of the supernatant is close to neutral. Disperse the dried intercalated material in deionized water, and after ultrasonic treatment, obtain Nb 2 C MXene nanosheets.

[0089] Nb in the obtained Nb0.5@CCJM composite functional membrane 2 Nb in the NbC MXene-functionalized collagen vitrified membrane layer 2 The content of NbC MXene is 0.5 wt%, the total thickness is 445 μm, and its performance is similar to that of Example 1.

[0090] Example 7

[0091] Nb 2 A composite functional membrane of NbC MXene that can promote angiogenesis, with the same composition and preparation method as in Example 1, except that the glass transition temperature in steps (3) and (5) is 9.5 °C and the relative humidity is 30%.

[0092] The total thickness of the obtained Nb0.5@CCJM composite functional membrane is 468 μm, where the thickness of the NbC MXene-functionalized collagen vitrified membrane layer 2 is 270 μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer is 198 μm. The elastic modulus of this composite functional membrane is 14.05 MPa, and the maximum tensile strength is 1.95 MPa. Other properties are similar to those of Example 1.

[0093] Example 8

[0094] Nb 2 A composite functional membrane of NbC MXene that can promote angiogenesis, with the same composition and preparation method as in Example 1, except that the glass transition temperature in steps (3) and (5) is 10.5 °C and the relative humidity is 40%.

[0095] The total thickness of the obtained Nb0.5@CCJM composite functional membrane is 460 μm, where the thickness of the NbC MXene-functionalized collagen vitrified membrane layer 2 is 260 μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer is 200 μm. The elastic modulus of this composite functional membrane is 14.20 MPa, and the maximum tensile strength is 2.00 MPa. Other properties are similar to those of Example 1.

[0096] Example 9

[0097] Nb 2 A composite functional membrane of NbC MXene that can promote angiogenesis, with the same composition and preparation method as in Example 1, except that the final concentration of the mixed collagen in step (2) is 1.8 mg / mL, and the concentration of the carboxymethyl chitosan solution in step (4) is 1.8 wt%.

[0098] The total thickness of the obtained Nb0.5@CCJM composite functional membrane is 430 μm, where the Nb 2The thickness of the C MXene-functionalized collagen vitrified film layer is 270 μm, and the thickness of the carboxymethyl chitosan vitrified film layer is 160 μm. The elastic modulus of this composite functional film is 13.85 MPa, and the maximum tensile strength is 1.90 MPa. Other properties are similar to those of Example 1.

[0099] Example 10

[0100] Nb 2 A composite functional film of Nb0.5@CCJM that can promote angiogenesis, with the same composition and preparation method as in Example 1, except that the final concentration of mixed collagen in step (2) is 2.2 mg / mL, and the concentration of the carboxymethyl chitosan solution in step (4) is 2.2 wt%.

[0101] The total thickness of the obtained Nb0.5@CCJM composite functional film is 470 μm, in which Nb 2 The thickness of the C MXene-functionalized collagen vitrified film layer is 290 μm, and the thickness of the carboxymethyl chitosan vitrified film layer is 180 μm. The elastic modulus of this composite functional film is 14.30 MPa, and the maximum tensile strength is 2.05 MPa. Other properties are similar to those of Example 1.

[0102] As can be seen from the above examples, the Nb 2 Composite functional film of C MXene that can promote angiogenesis, through the bilayer asymmetric structure design, combined with the multifunctional characteristics of Nb 2 C MXene realizes the synergistic effects of antioxidant, antibacterial and angiogenesis promotion, providing a safe, economical and effective solution for diabetic wound healing. The preparation process of this composite functional film is simple and safe, without using toxic cross-linking agents, and at the same time has excellent mechanical properties and appropriate degradation behavior, showing good application prospects.

[0103] To further verify the superior performance of the Nb 2 Composite functional film of C MXene that can promote angiogenesis of the present invention, we designed the following comparative examples and carried out systematic comparative tests.

[0104] Comparative Example 1

[0105] A bilayer composite film (CCJM) without Nb 2 C MXene, with the same preparation method as in Example 1, but in step (2), no Nb 2 C MXene nanosheets are added, and only collagen solution is used to prepare the collagen vitrified film (CVM), and the remaining steps remain unchanged.

[0106] Comparative Example 2

[0107] A Nb-containing one prepared by replacing the vitrification process with the glutaraldehyde cross-linking method2 Composite film of NbC MXene (Nb0.5-GTA), and the specific preparation method is as follows: Disperse NbC MXene with a concentration of 0.5 wt% in a collagen solution, and the final concentration of the mixture is 2 mg / mL of collagen and 0.5 wt% of NbC MXene. Use 1 M NaOH solution to adjust the pH value of the mixture to 6.8. After incubating at 37 °C to form a hydrogel, immerse the hydrogel in 0.5% glutaraldehyde PBS solution for 4 hours for crosslinking. The crosslinked hydrogel is thoroughly washed with deionized water to remove residual glutaraldehyde, and then immersed in 0.1 M glycine solution for 2 hours to block unreacted aldehyde groups. After washing and air drying, coat a 2 wt% carboxymethyl chitosan solution on its surface, and spray it with 0.5% glutaraldehyde solution for crosslinking for 2 hours. Finally, wash and block to obtain the composite film. 2 C MXene dispersion is mixed with the collagen solution, and the final concentration of the mixture is 2 mg / mL of collagen and 0.5 wt% of Nb 2 C MXene. Use 1 M NaOH solution to adjust the pH value of the mixture to 6.8. After incubating at 37 °C to form a hydrogel, immerse the hydrogel in 0.5% glutaraldehyde PBS solution for 4 hours for crosslinking. The crosslinked hydrogel is thoroughly washed with deionized water to remove residual glutaraldehyde, and then immersed in 0.1 M glycine solution for 2 hours to block unreacted aldehyde groups. After washing and air drying, coat a 2 wt% carboxymethyl chitosan solution on its surface, and spray it with 0.5% glutaraldehyde solution for crosslinking for 2 hours. Finally, wash and block to obtain the composite film.

[0108] Comparative Example 3

[0109] Commercially available polyurethane foam wound dressing (3M TM Tegaderm TM Foam Adhesive Dressing), with a specification of 8 cm × 8 cm, is sampled and used according to the instructions.

[0110] Comparative Example 4

[0111] A single-layer Nb 2 C MXene-functionalized collagen film (Nb0.5@CVM), the preparation method of which is the same as that of Example 1, but only steps (1) to (3) are completed, and the carboxymethyl chitosan vitrification film layer is not added.

[0112] Performance testing methods and results

[0113] 1. Mechanical property testing

[0114] Testing method: Use an electronic universal material testing machine (Instron 5943, USA) for tensile testing. Cut the sample into a dumbbell shape (length 30 mm, middle width 4 mm), and measure the thickness with a micrometer. Set the tensile rate to 10 mm / min and conduct the test at room temperature. Each group of samples is tested 5 times, and the average value is taken. Calculate the elastic modulus, maximum tensile strength, and elongation at break through the stress-strain curve.

[0115] 2. Swelling property and degradation characteristic testing

[0116] Swelling ratio testing: Weigh the dry sample (W 0 ), and then immerse it in PBS solution (pH 7.4) for 24 hours. Take out the sample, gently wipe off the excess liquid on the surface, and quickly weigh (W 1)。Swelling rate calculation formula: Swelling rate (%) = (W 1 - W 0 ) / W 0 × 100%.

[0117] Water retention rate test: Centrifuge the swollen sample at 3500 rpm for 5 minutes and immediately weigh (W 2 ). Water retention rate calculation formula: Water retention rate (%) = (W 2 - W 0 ) / W 0 × 100%.

[0118] Degradability test: Immerse the sample in PBS solution (pH 7.4) containing 1 mg / mL collagenase and incubate at 37°C. Take out the samples on the 1st, 3rd, 7th, 10th, and 14th days respectively, wash with deionized water, freeze-dry and weigh (W_t). Degradation rate calculation formula: Degradation rate (%) = (W 0 - W_t) / W 0 × 100%.

[0119] 3. ROS scavenging ability test

[0120] H 2 O 2 Scavenging test: Immerse the sample (2 cm × 2 cm) in 5 mL of 0.5 mM H 2 O 2 solution and incubate at 37°C for 30 minutes. Monitor the change in absorbance at 240 nm using a UV-Vis spectrophotometer (Shimadzu UV-2600, Japan) and calculate the scavenging rate of H 2 O 2 .

[0121] Cell protection experiment: Seed HUVEC cells at a density of 2 × 10 5 cells / well in a 24-well plate and culture for 24 hours. Co-treat the cells with the sample extract and 500 μM H 2 O 2 for 6 hours and evaluate cell viability using a Calcein-AM / PI double staining kit (Invitrogen). Observe and take pictures through a fluorescence microscope (Olympus IX71, Japan) and calculate the cell mortality rate using ImageJ software.

[0122] Intracellular ROS test: Stain the treated HUVEC cells with a DCFH-DA probe (10 μM) for 20 minutes, wash 3 times with PBS, observe the intracellular ROS level through a fluorescence microscope, and quantitatively measure the fluorescence intensity using a fluorescence microplate reader (Synergy H1, BioTek).

[0123] 4. Antibacterial Performance Test

[0124] Plate Count Method: Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) were cultured to the logarithmic phase, and the concentration was adjusted to 1×10 6 CFU / mL. 100 μL of the bacterial suspension was evenly spread on the surface of the sample (1 cm × 1 cm) and incubated at 37 °C for 24 hours. For the near-infrared light irradiation group, an 808 nm laser (power density 1.0 W / cm2) was used to irradiate for 10 minutes. After incubation, the sample was placed in 10 mL of sterile PBS, vortexed for 10 minutes to extract bacteria, serially diluted, and plate counted on LB agar plates. Calculation formula: Inhibitory rate (%) = (number of bacteria in the control group - number of bacteria in the experimental group) / number of bacteria in the control group × 100%.

[0125] Live / Dead Bacteria Staining: The bacteria after treatment were stained using a SYTO 9 / PI Bacterial Live / Dead Staining Kit (Invitrogen), observed and photographed by a confocal laser scanning microscope (Leica TCS SP8, Germany), and the bacterial mortality rate was calculated using ImageJ software.

[0126] 5. Pro-Angiogenesis Ability Test

[0127] Cell Migration Assay: A uniform scratch was made on the confluent HUVEC monolayer cells using a 200 μL pipette tip. After washing with PBS, serum-free medium containing the sample extract was added. Photographs were taken at 0 h and 24 h, and the scratch healing area was calculated using ImageJ software. Calculation formula: Migration rate (%) = (scratch area at 0 h - scratch area at 24 h) / scratch area at 0 h × 100%.

[0128] Transwell Migration Assay: HUVEC cells (5×10 4 cells / well) suspended in serum-free medium containing the sample extract were added to the upper chamber of the Transwell, and complete medium containing 10% FBS was added to the lower chamber. After incubation at 37 °C for 6 hours, the non-migrated cells in the upper chamber were wiped off with a cotton swab, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. Five fields of view were randomly selected and photographed and counted under an optical microscope.

[0129] Tube Formation Assay: 100 μL of Matrigel (BD Biosciences) was added to a pre-cooled 48-well plate and incubated at 37 °C for 30 minutes to solidify. HUVEC cells were seeded at 2×10 4Cells were seeded at a density of [cells / well] on Matrigel, and a medium containing the sample extract was added. After incubation for 6 hours, observations and photographs were taken under an optical microscope. The network structure, number of branches, and length of tubular structures were analyzed using the Angiogenesis Analyzer plugin of ImageJ software.

[0130] Immunofluorescence staining: HUVEC cells were seeded at a density of 1×10 5 cells / well in a 24-well plate pre-placed with cover slips and cultured until 60 - 70% confluence. They were then treated with the sample extract for 24 hours. Fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, blocked with 5% BSA for 1 hour, incubated with primary antibodies (anti-CD31, 1:200; anti-F-actin, 1:100) overnight at 4°C, incubated with secondary antibodies (Alexa Fluor 488 / 594-labeled, 1:500) for 1 hour at room temperature, stained with DAPI for cell nuclei, and observations and photographs were taken under a fluorescence microscope. The fluorescence intensity was analyzed using ImageJ software.

[0131] 6. In vivo wound healing effect test

[0132] Animal model: Male C57BL / 6 mice (6 - 8 weeks old, body weight 20 - 25 g) were induced to establish a diabetic model by streptozotocin (50 mg / kg, intraperitoneal injection for 5 consecutive days). The successful establishment of the diabetic model was confirmed when the fasting blood glucose level was ≥16.8 mmol / L for 2 weeks. After anesthetizing the mice, the hair on the back was shaved and disinfected, and a full-thickness wound was created using an 8-mm skin punch. The mice were randomly divided into 5 groups (n = 6 / group): control group (without dressing), comparative example 1 group, comparative example 2 group, comparative example 3 group, and example 1 group. The dressing was changed every 3 days until day 14.

[0133] Wound healing rate determination: Wound photographs were taken on days 0, 3, 7, 10, and 14, and the wound area was measured using ImageJ software. The formula for the healing rate: Healing rate (%) = (Initial wound area - Current wound area) / Initial wound area × 100%.

[0134] Histological analysis: On days 7 and 14, some mice in each group were euthanized, the wound tissues were collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (5-μm thickness). Hematoxylin-eosin (H&E) staining was used to evaluate tissue morphology and re-epithelialization; Masson's trichrome staining was used to evaluate collagen deposition; immunohistochemical staining (anti-CD31 antibody, 1:100) was used to evaluate neovascularization. Observations and photographs were taken using an optical microscope, and quantitative analysis was performed using ImageJ software.

[0135] DHE staining: Fresh wound tissues were embedded with OCT, cryosectioned (8 μm thickness), stained with 5 μM dihydroethidium (DHE) in the dark at 37 °C for 30 minutes, washed three times with PBS, observed and photographed under a fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ software.

[0136] All experimental results and data were statistically analyzed using GraphPad Prism 8.0 software. The results were expressed as mean ± standard deviation. One-way ANOVA and Tukey's post hoc test were used for comparison among multiple groups. A p value < 0.05 indicated a statistically significant difference.

[0137] The following table summarizes the main performance parameters of each group of samples:

[0138] Table 1 Comparison of mechanical properties and physical characteristics of each group of samples

[0139] Performance parameters Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Elastic modulus (MPa) 14.14±1.01 11.61±0.45 8.35±0.72 3.21±0.35 10.25±0.68 Maximum tensile strength (MPa) 1.98±0.17 1.65±0.12 1.25±0.15 0.92±0.10 1.70±0.14 Elongation at break (%) 14.5±1.2 15.2±1.3 11.8±1.0 125.5±15.6 13.2±1.1 Swelling rate (%) 311±15 375±18 450±25 580±30 335±20 Water retention rate (%) 245±10 205±12 185±15 410±25 190±15 Degradation rate in 14 days (%) 52±3 55±4 30±5 - 62±5

[0140] Table 2 Comparison of ROS scavenging ability and antibacterial properties of each group of samples

[0141] Performance parameters Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 <![CDATA[H 2 O 2 Clearance rate (%, 30 min)]]> 82.5±3.6 10.3±1.5 75.8±4.2 5.2±1.0 84.1±3.8 <![CDATA[HUVEC mortality (%, H 2 O 2 treatment)]]> 6.1±1.0 48.5±2.5 12.3±1.8 45.2±2.8 5.8±0.9 Intracellular ROS level (relative fluorescence intensity) 1.5±0.2 9.8±0.6 2.5±0.3 9.5±0.7 1.4±0.2 Inhibitory rate against Staphylococcus aureus (%) 85.6±3.2 5.3±1.0 60.2±3.5 45.5±3.0 87.2±3.5 Inhibitory rate against Pseudomonas aeruginosa (%) 84.1±2.6 4.5±0.8 56.8±3.0 42.3±2.8 85.5±3.2 Inhibitory rate under NIR assistance (%) (Staphylococcus aureus) 96.2±0.5 6.2±1.2 70.5±3.2 48.2±3.1 97.5±0.4

[0142] Table 3 Comparison of angiogenesis-promoting ability of each group of samples

[0143]

[0144]

[0145] Table 4 Comparison of in vivo wound healing effects of each group of samples

[0146] Performance parameters Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Control group Wound healing rate (%, on the 7th day) 85.2±0.6 61.5±1.4 70.3±1.2 50.2±2.4 48.0±3.0 Wound healing rate (%, on the 14th day) 98.5±0.6 90.2±1.0 93.5±0.9 88.5±1.2 81.7±1.6 Granulation tissue thickness (μm, on the 7th day) 650±35 420±30 510±32 380±28 360±25 Collagen deposition amount (relative intensity, on the 14th day) 4.5±0.3 2.2±0.2 3.0±0.2 1.8±0.2 1.0±0.1 <![CDATA[Number of CD31-positive blood vessels (per mm 2 , day 7)]]> 185±15 85±10 130±12 75±8 60±7 Wound ROS level (relative fluorescence intensity) 25±5 95±10 45±8 90±10 100±10

[0147] Based on the above comparative test results, the following analysis conclusions can be drawn:

[0148] 1. Mechanical properties and physical characteristics: Nb0.5@CCJM (Example 1) of the present invention exhibited the optimal mechanical properties, with the elastic modulus and maximum tensile strength significantly higher than those of other control groups. This is mainly attributed to the formation of a double-layer vitrified structure and a nano-interlocked interface, which provided better interlayer bonding force and overall strength. In contrast, Nb0.5-GTA (Comparative Example 2) crosslinked with glutaraldehyde, although containing the same concentration of Nb 2 C MXene, had significantly poorer mechanical properties, which may be due to the fact that the crosslinking process damaged the microstructure of the material. In addition, the swelling ratio and water retention rate of the present invention also showed a better balance, being able to maintain a moist environment for the wound without excessive swelling, which is beneficial for stably covering the wound.

[0149] 2. ROS Scavenging Ability: Both Example 1 and Comparative Example 4 exhibited excellent ROS scavenging ability, which demonstrated that Nb 2 CMXene was the main source of antioxidant activity. Comparative Example 1 (without Nb 2 CMXene) had almost no ROS scavenging ability, while Comparative Example 2, although containing Nb 2 CMXene, had a lower scavenging activity, which might be due to the partial blocking of the active sites of Nb 2 CMXene during the glutaraldehyde crosslinking process. In the cell protection experiment, Example 1 could significantly reduce the mortality of H 2 O 2 2-induced HUVECs from 47.0% to 6.1%, and this protective effect was far better than that of the control group.

[0150] 3. Antibacterial Performance: Both Example 1 and Comparative Example 4 showed significant antibacterial activity, and the antibacterial rates against Staphylococcus aureus and Pseudomonas aeruginosa reached about 85%, and under near-infrared light irradiation, the antibacterial rate was further increased to over 96%. In contrast, Comparative Example 1 had almost no antibacterial activity, and the antibacterial effects of Comparative Example 2 and Comparative Example 3 were also significantly weaker. This result further confirmed the photothermal bactericidal effect of Nb 2 CMXene and the antibacterial adhesion effect of the outer layer CMC in the bilayer structure.

[0151] 4. Angiogenesis-Promoting Ability: In terms of promoting angiogenesis, Example 1 showed the strongest activity, with the HUVEC migration rate reaching 96.1%, and the number of tube formation nodes and the total length being 1.96 times and 1.98 times that of the control group, respectively. Through immunofluorescence staining analysis, Example 1 also significantly upregulated the expressions of CD31 and F-actin, which are closely related to the migration and tube formation of vascular endothelial cells. In contrast, Comparative Example 1 and Comparative Example 3 had almost no angiogenesis-promoting activity, and Comparative Example 2 only showed moderate angiogenesis-promoting activity.

[0152] 5. In Vivo Wound Healing Effect: In the full-thickness skin wound model of diabetic mice, Example 1 showed the best wound healing effect, with the wound healing rate reaching 85.2% on the 7th day and 98.5% on the 14th day, significantly higher than other control groups. Histological analysis showed that the wound treated with Example 1 had the thickest granulation tissue layer, the most new blood vessels, and the highest collagen deposition, and these indicators are all important signs of wound healing quality. In addition, Example 1 significantly reduced the ROS level at the wound site, which helped to reduce oxidative stress and provide a more favorable survival environment for cells.

[0153] In summary, through multi-dimensional comparative tests, Nb of the present invention 2The composite functional membrane (Example 1) of C MXene that can promote angiogenesis shows significant advantages in terms of mechanical properties, ROS scavenging ability, antibacterial property, angiogenesis promotion ability, and in vivo wound healing effect. This advantage mainly stems from Nb 2 The synergistic effect of the multifunctional properties (antioxidant, antibacterial, angiogenesis promotion) of C MXene and the bilayer asymmetric structure (the inner layer adheres to the wound, and the outer layer prevents bacterial and tissue adhesion), as well as the excellent mechanical properties and appropriate degradation behavior conferred by the dual vitrification process adopted. These results fully demonstrate the innovation and effectiveness of the present invention in the application of diabetic wound healing.

[0154] It should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composite functional membrane of Nb2C MXene that can promote angiogenesis, characterized in that: The composite functional film comprises: Nb2C MXene functionalized collagen vitrified membrane layer; and Carboxymethyl chitosan vitrified membrane layer; Wherein, a nano interlocking interface is formed between the Nb2C MXene functionalized collagen vitrified membrane layer and the carboxymethyl chitosan vitrified membrane layer; The Nb2C MXene functionalized collagen vitrified membrane layer is hydrophobic, and the carboxymethyl chitosan vitrified membrane layer is hydrophilic.

2. The composite functional film of Nb2C MXene capable of promoting angiogenesis according to claim 1, characterized in that: The content of Nb2C MXene in the Nb2C MXene functionalized collagen vitrified membrane layer is 0.125 wt% to 1.0 wt%.

3. The composite functional film of Nb2C MXene capable of promoting angiogenesis according to claim 1, characterized in that: The water contact angle of the Nb2C MXene functionalized collagen vitrified membrane layer is 80° to 90°, and the water contact angle of the carboxymethyl chitosan vitrified membrane layer is 20° to 30°.

4. The composite functional film of Nb2C MXene capable of promoting angiogenesis according to claim 1, characterized in that: The thickness of the composite functional membrane is 400um to 500um, wherein the thickness of the Nb2C MXe ne functionalized collagen vitrified membrane layer is 250μm to 300μm, and the thickness of the carboxymethyl chitosan vitrified membrane layer is 150μm to 200μm.

5. A method for preparing a composite functional film of Nb2C MXene capable of promoting angiogenesis according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Preparation of Nb2C MXene nanosheets; (2) mixing the Nb2C MXene nanosheets with a collagen solution, adjusting the pH value to 6.8±0.1, and allowing the collagen to self-assemble to form a hydrogel; (3) vitrifying the hydrogel at 10±0.5°C for 48 hours to form a Nb2C MXene functionalized collagen vitrified membrane; (4) uniformly coating a carboxymethyl chitosan solution on the Nb2C MXene functionalized collagen vitrified membrane; (5) The coated structure is vitrified for a second time at 10±0.5° C. for 48 hours to form a composite functional film of Nb2CMXene that can promote angiogenesis.

6. The method according to claim 5, characterized in that The method for preparing the Nb2C MXene nanosheet in step (1) comprises: (a) adding Nb2AlC MAX phase powder into a 50% hydrofluoric acid solution and stirring the mixture at 0-4°C for 24 hours; (b) washing the obtained product by centrifugation until the pH value of the supernatant reaches about 6; (c) dispersing the product obtained in step (b) in a 25% concentration of tetrapropylammonium hydroxide solution and stirring at 37° C. for 3 days; (d) washing the obtained product by centrifugation until the pH value of the supernatant is close to neutral; (e) dispersing the product obtained in step (d) in deionized water and subjecting it to ultrasonic treatment to obtain Nb2C MXene nanosheets with an average lateral size of 150-200 nm and a thickness of 0.4-0.6 nm.

7. The method according to claim 5, characterized in that The collagen solution in step (2) is a type I collagen solution prepared by the following method: (a) Rat tail tendons were immersed in 0.5 M glacial acetic acid solution and extracted with stirring at 4 °C for 48 h. (b) filtering and centrifuging the extract, and adding 5 M sodium chloride solution to the supernatant to a final concentration of 0.9 M; (c) After standing at 4°C overnight, the precipitate was collected by centrifugation and redissolved in 0.5 M glacial acetic acid; (d) dialyzing and freeze-drying the solution to obtain purified type I collagen; (e) The purified type I collagen was dissolved in 0.01 M glacial acetic acid (pH 3.0) at a concentration of 5 mg / mL.

8. The method according to claim 5, characterized in that The final concentration of collagen in the mixed solution in step (2) is 2 mg / mL, and the mixture is heated at 0.22 mL / cm 2 The volume density of 1000 μg / ml was evenly distributed into the mold and incubated at 37 °C for 24 h to allow the collagen to fully self-assemble.

9. The method according to claim 5, characterized in that The concentration of the carboxymethyl chitosan solution in step (4) is 2 wt % and the dosage is 0.15 mL / cm 2 , use a water-free and oil-free air flow with a pressure of 0.5 MPa to assist it in penetrating into the gaps between collagen fibers and let it stand at room temperature for 1 hour.

10. The method according to claim 5, characterized in that The vitrification process in step (3) and step (5) is carried out under a relative humidity of 30-40%, wherein the first vitrification is preceded by pre-drying at room temperature for 2 hours, and after the second vitrification is completed, the Nb2C MXene composite functional film that can promote angiogenesis is sterilized by ultraviolet light for 20 minutes.

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