An artificial hard film based on MXene and a preparation method and application thereof

Abstract

This invention relates to the field of medical materials technology, specifically to an MXene-based artificial dura mater, its preparation method, and its applications. The MXene-based artificial dura mater has a layered structure, comprising an upper membrane and a lower membrane. The upper membrane is composed of amino-modified MXene; the lower membrane uses chitosan / gelatin hydrogel as a matrix, doped with polydopamine-coated MXene. The upper and lower membranes are bonded together by chemical bonds formed by the reaction of amino groups in the upper and lower membranes with a crosslinking agent. The MXene-based artificial dura mater not only achieves the sealing and barrier functions of traditional dura mater repair but also possesses the ability to regulate the injury microenvironment and promote nerve regeneration. Through its unique layered structural design, it can simultaneously regulate inflammatory responses and promote endogenous nerve regeneration during dura mater repair, providing more comprehensive support for the functional recovery of the central nervous system.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to an MXene-based artificial dura mater, its preparation method, and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Trauma and tumor invasion can cause varying degrees of damage and defects to the meninges or spinal cord, leading to cerebrospinal fluid leakage, secondary infection, and even epilepsy due to scarring caused by abnormal meningeal healing. Severe damage and defects can cause substantial and irreversible damage and degeneration of the brain and other central nervous system organs.

[0004] For the treatment of dural defects, different measures are currently adopted clinically depending on the size and degree of the defect. Small dural defects are usually closed with sutures, while large defects require dural patches combined with sutures or sealants for repair. Because dural suturing increases the risk of postoperative infection, biological adhesives such as DuraSeal and CoSeal are often used to supplement the repair and prevent cerebrospinal fluid leakage after simple suturing. However, biological adhesives alone cannot be directly used to repair dural injuries, especially when the dural defect is large. Currently, autologous, allogeneic, or xenogeneic collagen connective tissue transplantation and synthetic connective tissue transplantation are commonly used clinically, but they have drawbacks such as nerve compression, inadequate closure, and unclear toxicity. Furthermore, most artificial dural materials are made of collagen, biodegradable polymers, or synthetic polymers. While they can achieve basic closure and barrier functions, their effects on regulating the microenvironment and promoting nerve regeneration are limited. Summary of the Invention

[0005] To overcome the above problems, the present invention provides an artificial hard membrane based on MXene, its preparation method and application.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an artificial hard membrane based on MXene, which has a layered structure comprising an upper membrane and a lower membrane;

[0008] The upper membrane is amino-modified MXene;

[0009] The lower membrane is based on chitosan / gelatin hydrogel and doped with polydopamine-coated MXene.

[0010] The upper and lower membranes are bonded together by chemical bonds formed by the reaction of amino groups in the upper and lower membranes with the crosslinking agent.

[0011] In one or more embodiments, the crosslinking agent is genipin.

[0012] A second aspect of the present invention provides a method for preparing the MXene-based artificial dura mater described in the first aspect, comprising the following steps:

[0013] (1) MXene is dispersed in an amino modifier to obtain amino-modified MXene;

[0014] (2) MXene was dispersed in a dopamine hydrochloride solution to obtain polydopamine-coated MXene;

[0015] (3) Disperse polydopamine-coated MXene into a chitosan / gelatin mixed solution and add a crosslinking agent for crosslinking; when the mixed solution begins to gel, uniformly coat the surface of the gel with amino-modified MXene; after complete gelation, wash to obtain an artificial hard membrane based on MXene.

[0016] In one or more embodiments, in step (1), the amino modifier includes ammonia or a compound containing an amino group;

[0017] Preferably, the amino-containing compound includes hydrazine compounds or amine compounds;

[0018] More preferably, the hydrazine compounds include hydrazine monohydrate, methylhydrazine, and p-aminohydrazine, etc., and the amine compounds include ethylenediamine, cycloethylenediamine, ethylamine chloride, and methylamine chloride, etc.

[0019] In one or more embodiments, in step (2), the solvent of the dopamine hydrochloride solution is a Tris-HCl solution with a concentration of 0.01-0.05 M and a pH of 8-9, preferably 8.5; the concentration of dopamine hydrochloride is 1-5 g / L.

[0020] In one or more embodiments, the reaction time in step (2) is 4-8 h.

[0021] In one or more embodiments, in step (3), the chitosan / gelatin mixed solution is formed by mixing a chitosan solution and a gelatin solution;

[0022] The chitosan solution has a mass fraction of 1%-3%, and the gelatin solution has a concentration of 2-5 g / L.

[0023] Preferably, the volume ratio of gelatin solution to chitosan solution is (2.5-3.5):2, more preferably 3:2.

[0024] In one or more embodiments, in step (3), the mass of the polydopamine-coated MXene is 1%-3% of the volume of the chitosan / gelatin mixed solution.

[0025] In one or more embodiments, in step (3), the crosslinking agent is genipin.

[0026] Preferably, the concentration of genipin is 20-100 mM;

[0027] More preferably, the volume ratio of the genipin solution to the chitosan / gelatin mixed solution is 1:(80-120), more preferably 1:100.

[0028] A third aspect of the present invention provides the use of the MXene-based artificial dura mater described in the first aspect or the MXene-based artificial dura mater prepared by the preparation method described in the second aspect in the preparation of medical materials for treating dural injuries.

[0029] The beneficial effects of this invention are as follows:

[0030] (1) The MXene-based artificial dura mater provided by this invention not only achieves the sealing and barrier functions of traditional dura mater repair, but also has the ability to regulate the injury microenvironment and promote nerve regeneration. Through its unique layered structure design, it can regulate the inflammatory response and promote endogenous nerve regeneration while repairing the dura mater, providing more comprehensive support for the functional recovery of the central nervous system. Specifically, amino-modified MXene can react with reactive oxygen species in the microenvironment of the injury site to reduce oxidative stress; polydopamine-coated MXene can respond to external changing magnetic fields to generate electrical stimulation signals, thereby regulating the proliferation, migration and differentiation of endogenous neural stem cells; in addition, MXene can also regulate the fate of vascular endothelial cells and promote angiogenesis at the injury site.

[0031] (2) The MXene-based artificial dura mater prepared in this invention has good biocompatibility and biodegradability. MXene contains elements with human affinity such as carbon, nitrogen, oxygen, and titanium. Dopamine is a natural substance in the human body. Collagen and chitosan are commonly used medical biomaterials. Genipin, as a crosslinking agent with excellent biocompatibility, makes the dura mater material have good biocompatibility and can be widely used in the treatment of central nervous system diseases such as traumatic brain injury, spinal cord injury, and brain tumors. It provides a new solution for the recovery of nerve function and has high clinical application value. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 The images show the transmission electron microscope (TEM) image and X-ray diffraction (XRD) pattern of the raw material MXene; where A and B are TEM images and C is an X-ray diffraction pattern.

[0034] Figure 2 The images shown are transmission electron microscope (TEM) images, elemental analysis diagrams, and infrared spectra of the polydopamine-coated MXene prepared in Example 1; where A is a transmission electron microscope (TEM) image, B is an elemental analysis diagram, and C is an infrared spectrum.

[0035] Figure 3 Transmission electron microscopy (TEM) image and elemental analysis of the amino-modified MXene prepared in Example 1. Figure X X-ray photoelectron spectroscopy (XPS) and degradation rate test results; where A is a transmission electron microscope (TEM) image, B is an elemental analysis diagram, C is an X-ray photoelectron spectroscopy image, and D is a degradation rate test result diagram.

[0036] Figure 4 Example 1 introduces scanning electron microscope (SEM) images, infrared spectra, adhesion demonstration images, conductivity and electrical conductivity demonstration images, and degradation rate test results of chitosan / gelatin hydrogels coated with MXene with different contents of polydopamine. In this example, AD is a scanning electron microscope (SEM) image, E is an infrared spectrum, F is an adhesion demonstration image, G is an electrical conductivity image, H is an electrical conductivity demonstration image, and I is a degradation rate test result.

[0037] Figure 5 Scanning electron microscopy (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra of chitosan / gelatin hydrogel before and after coating with amino-modified MXene; where A is the SEM image of chitosan / gelatin hydrogel before coating with amino-modified MXene, B is the SEM image of chitosan / gelatin hydrogel after coating with amino-modified MXene, C is the XPS spectrum of chitosan / gelatin hydrogel before coating with amino-modified MXene, and D is the XPS spectrum of chitosan / gelatin hydrogel after coating with amino-modified MXene.

[0038] Figure 6 Biocompatibility diagrams of chitosan / gelatin hydrogels containing amino-modified MXene and MXenes coated with different polydopamines are shown. In the diagrams, A and B represent the cell viability and inactivation staining results of amino-modified MXene, and C represents the quantification of A and B. DG represents the cell viability and inactivation staining results of chitosan / gelatin hydrogels containing MXenes coated with different polydopamines, and H represents the quantification of DG.

[0039] Figure 7The figures represent the levels of amino-modified MXene reactive oxygen species scavenging and pro-inflammatory factor secretion. In the figures, A represents the fluorescence staining results, B represents the quantification of A, C and D represent the Western blotting images of pro-inflammatory factor secretion, and E and F represent the quantifications of C and D, respectively.

[0040] Figure 8 To introduce 2% polydopamine-coated MXene into chitosan / gelatin hydrogels, different electrical stimulations were generated under different frequency magnetic fields to regulate neural stem cell differentiation. Real-time quantitative polymerase chain reaction and related marker protein immunofluorescence staining images were obtained. In the images, AD is the real-time quantitative polymerase chain reaction image, E is the related marker protein immunofluorescence staining image, and F is the quantification of E.

[0041] Figure 9 Fluorescent staining images showing how amino-modified MXene and polydopamine-coated MXene promote angiogenesis.

[0042] Figure 10 The images show the dura mater repair in mice with spinal cord injury using an MXene-based artificial dura mater; in A, chitosan / gelatin hydrogel was used, and in B, an MXene-based artificial dura mater was used.

[0043] Figure 11 The image shows the results of MXene-based artificial dura mater inhibiting inflammation in mice with spinal cord injury; A represents the staining results of spinal cord tissue, and B and C represent the quantification of A.

[0044] Figure 12 BMS score map (A) and gait analysis map (B) for the recovery of motor function in mice with spinal cord injury using MXene-based artificial dura mater.

[0045] Figure 13 The images show somatosensory evoked potentials (SEP) and motor evoked potentials (MEP) in mice with spinal cord injury, where MXene-based artificial dura mater promotes the recovery of electrophysiological activity. A represents the SEP image, B represents the quantification of A, C represents the MEP image, and D represents the quantification of C.

[0046] Figure 14 The images show tissue staining of MXene-based artificial dura mater in mice with spinal cord injury, promoting nerve regeneration. In the images, A shows the staining of neurons and glial cells at the injury site, and B shows the staining of mature neurons and their axons at the injury site.

[0047] Figure 15 This diagram illustrates the degradation rate and biosafety of MXene-based artificial dura mater in vivo. A represents the experimental results, B represents the quantification of A, and C represents HE staining of heart, lung, liver, spleen, and kidney tissues. Detailed Implementation

[0048] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0050] Glossary: ​​NSCs are neural stem cells; BV2 cells are mouse microglia; MXN is amino-modified MXene; CG is chitosan / collagen hydrogel; MXP is polydopamine-coated MXene; CGMXP is CG hydrogel doped with MXP; MXN-CGMXP is an MXene-based artificial dura mater; DA is dopamine hydrochloride; PDA is polydopamine; Tris-HCl solution is tris(hydroxymethyl)aminomethane hydrochloride solution; MF is time-varying magnetic field; SCI is spinal cord injury; H2O2 is hydrogen peroxide.

[0051] For the treatment of dural defects, different measures are currently adopted clinically depending on the size and degree of the defect. Small dural defects are usually closed with sutures, while large defects require dural patches combined with sutures or sealants for repair. Because dural suturing increases the risk of postoperative infection, biological adhesives such as DuraSeal and CoSeal are often used to supplement the repair and prevent cerebrospinal fluid leakage after simple suturing. However, biological adhesives alone cannot be directly used to repair dural injuries, especially when the dural defect is large. Currently, autologous, allogeneic, or xenogeneic collagen connective tissue transplantation and synthetic connective tissue transplantation are commonly used clinically, but they have drawbacks such as nerve compression, inadequate closure, and unclear toxicity. Furthermore, most artificial dural materials are made of collagen, biodegradable polymers, or synthetic polymers. While they can achieve basic closure and barrier functions, their effects on regulating the microenvironment and promoting nerve regeneration are limited.

[0052] This invention provides an artificial hard membrane based on MXene, which has a layered structure including an upper membrane and a lower membrane; the upper membrane is amino-modified MXene; the lower membrane is based on chitosan / gelatin hydrogel and doped with polydopamine-coated MXene; the upper membrane and the lower membrane are bonded by chemical bonds formed by the reaction of amino groups in the upper and lower membranes with a crosslinking agent.

[0053] The MXene-based artificial dura mater provided by this invention not only achieves the sealing and barrier functions of traditional dura mater repair, but also possesses the ability to regulate the injury microenvironment and promote nerve regeneration. Through its unique layered structural design, it can simultaneously regulate inflammatory responses and promote endogenous nerve regeneration during dura mater repair, providing more comprehensive support for the functional recovery of the central nervous system. Specifically, amino-modified MXene can react with reactive oxygen species in the injury site microenvironment, reducing oxidative stress; polydopamine-coated MXene can respond to external changing magnetic fields, generating electrical stimulation signals, thereby regulating the proliferation, migration, and differentiation of endogenous neural stem cells; furthermore, MXene can also regulate the fate of vascular endothelial cells and promote angiogenesis at the injury site. Subsequent experiments using a spinal cord injury model further validated that the amount of pro-inflammatory factors secreted by microglia in the spinal cord tissue of mice treated with MXene-based artificial dura mater was effectively reduced, and the inflammatory microenvironment was effectively controlled. Mice treated with MXene-based artificial dura mater showed the most significant recovery in motor function, with coherent gait and continuous and regular hind limb footprints, although the walking speed was slower. Mice treated with MXene-based artificial dura mater were able to induce higher amplitude SEP and MEP, exhibiting stronger electrophysiological activity. Mice treated with MXene-based artificial dura mater showed more neurons at the injury site and demonstrated better effects in neural regeneration and neural circuit reconstruction.

[0054] The MXene-based artificial dura mater prepared in this invention exhibits good biocompatibility and biodegradability. MXene contains elements with human affinity such as carbon, nitrogen, oxygen, and titanium; dopamine is a natural human substance; collagen and chitosan are commonly used medical biomaterials; and genipin, as a cross-linking agent with excellent biocompatibility, ensures that the dura mater material has good biocompatibility and can be widely used in the treatment of central nervous system diseases such as traumatic brain injury, spinal cord injury, and brain tumors, providing a new solution for nerve function recovery and possessing high clinical application value.

[0055] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0056] Example 1

[0057] (1) Disperse 0.1 g MXene in 200 mL of ammonia solution (pH=9), vortex for 10 min, and then sonicate for 1 h; centrifuge (3500 rpm, 10 min) and collect the supernatant; shake the lower layer solution again and sonicate, then centrifuge and collect the supernatant; repeat the operation 2-3 times for the lower layer solution to collect the supernatant. Under nitrogen protection, sonicate the supernatant for 5 min, then transfer it to a high-pressure reactor and carry out a hydrothermal reaction at 70 ℃ for 4 h. After the reaction, centrifuge the suspension (1000 rpm, 5 min), collect the precipitate, wash with deionized water until pH 7, and finally collect the precipitate and freeze-dry it to obtain amino-modified MXene, denoted as MXN.

[0058] (2) Add 0.5 g MXene to 200 mL of 0.01 M Tris-HCl solution (pH 8.5), add 400 mg dopamine hydrochloride, and stir at room temperature for 8 h; after the reaction is complete, centrifuge to collect the precipitate, and wash with deionized water until the pH of the supernatant is 7. After collecting the precipitate, transfer it to an oven for drying to obtain polydopamine-coated MXene, denoted as MXP;

[0059] (3) Prepare a 3 g / L gelatin solution, prepare a 2% (w / v) chitosan solution using 0.2% (v / v) acetic acid, and neutralize the pH to 7.0 using a 1.0 mol / L sodium hydroxide standard solution; mix the gelatin solution and chitosan solution at a ratio of 3:2, then add 1%, 2%, and 3% (v / v) of MXP to the chitosan / gelatin mixture respectively; and add a 25 mM genipin solution as a crosslinking agent at a volume ratio of 1:100, mix thoroughly, and crosslink at room temperature. When the mixture begins to gel, MXN is coated onto the surface. After complete gelation, rinse with deionized water to remove uncrosslinked MXN, obtaining an MXene-based artificial hard membrane.

[0060] Example 2

[0061] (1) Disperse 0.1 g MXene in 200 mL of ammonia solution (pH=9), vortex for 10 min, and then sonicate for 1 h; centrifuge (3500 rpm, 10 min) and collect the supernatant; shake the lower layer solution again and sonicate, then centrifuge and collect the supernatant; repeat the operation 2-3 times for the lower layer solution to collect the supernatant. Under nitrogen protection, sonicate the supernatant for 5 min, then transfer it to a high-pressure reactor and carry out a hydrothermal reaction at 80 ℃ for 5 h. After the reaction, centrifuge the suspension (1000 rpm, 5 min), collect the precipitate, wash with deionized water until pH 7, and finally collect the precipitate and freeze-dry it to obtain amino-modified MXene, denoted as MXN.

[0062] (2) Add 0.5 g MXene to 200 mL of 0.01 M Tris-HCl solution (pH 8.8), add 600 mg dopamine hydrochloride, and stir at room temperature for 6 h; after the reaction is complete, centrifuge to collect the precipitate, and wash with deionized water until the pH of the supernatant is 7. After collecting the precipitate, transfer it to an oven for drying to obtain polydopamine-coated MXene, denoted as MXP;

[0063] (3) Prepare a 3 g / L gelatin solution, prepare a 2.5% (w / v) chitosan solution using 0.2% (v / v) acetic acid, and neutralize the pH to 7.0 using a 1.0 mol / L sodium hydroxide standard solution; mix the gelatin solution and chitosan solution at a ratio of 3:2, then add 1% (v / v) of MXP to the chitosan / gelatin mixture; and add 50 mM genipin solution as a crosslinking agent at a volume ratio of 1:100. After thorough mixing, crosslink at room temperature. When the mixture begins to gel, MXN is coated onto the surface. After complete gelation, rinse with deionized water to remove uncrosslinked MXN, obtaining an MXene-based artificial hard membrane.

[0064] Example 3

[0065] (1) Disperse 0.2 g MXene in 200 mL of ammonia solution (pH=9), vortex for 10 min, and then sonicate for 1 h; centrifuge (3500 rpm, 10 min) and collect the supernatant; shake the lower layer solution again and sonicate, then centrifuge and collect the supernatant; repeat the operation 2-3 times for the lower layer solution to collect the supernatant. Under nitrogen protection, sonicate the supernatant for 5 min, then transfer it to a high-pressure reactor and carry out a hydrothermal reaction at 70 ℃ for 6 h. After the reaction, centrifuge the suspension (1000 rpm, 5 min), collect the precipitate, wash with deionized water until pH 7, and finally collect the precipitate and freeze-dry it to obtain amino-modified MXene, denoted as MXN.

[0066] (2) 0.2 g MXene was added to 200 mL of 0.01 M Tris-HCl solution (pH 8.8), and 300 mg of dopamine hydrochloride was added. The mixture was stirred at room temperature for 8 h. After the reaction was completed, the precipitate was collected by centrifugation and washed with deionized water until the pH of the supernatant was 7. The precipitate was collected and transferred to an oven for drying to obtain polydopamine-coated MXene, which was denoted as MXP.

[0067] (3) Prepare a 4 g / L gelatin solution, prepare a 2% (w / v) chitosan solution using 0.2% (v / v) acetic acid, and neutralize the pH to 7.0 using a 1.0 mol / L sodium hydroxide standard solution; mix the gelatin solution and chitosan solution at a ratio of 3:2, then add 1% (v / v) MXP to the chitosan / gelatin mixture; and add 100 mM genipin solution as a crosslinking agent at a volume ratio of 1:100. After thorough mixing, crosslink at room temperature. When the mixture begins to gel, MXN is coated onto the surface. After complete gelation, rinse with deionized water to remove uncrosslinked MXN, obtaining an MXene-based artificial hard membrane.

[0068] Example 4

[0069] (1) 0.2 g MXene was dispersed in 30 mL ethylenediamine (EDA) and vortexed for 10 min. The mixture was then transferred to a high-pressure reactor and reacted at 40 °C for 24 h with stirring at 600 rpm. After the reaction was complete, the precipitate was collected by centrifugation and washed with ethanol and deionized water, respectively. After washing, the precipitate was dispersed in 30 mL deionized water, stirred for 5 min, transferred to an ice bath for sonication for 30 min, and then vacuum filtered. Finally, the filtered solution was vacuum dried at 40 °C to obtain amino-modified MXene, denoted as MXN.

[0070] (2) Add 0.5 g MXene to 200 mL of 0.01 M Tris-HCl solution (pH 8.8), add 400 mg dopamine hydrochloride, and stir at room temperature for 8 h; after the reaction is complete, centrifuge to collect the precipitate, and wash with deionized water until the pH of the supernatant is 7. After collecting the precipitate, transfer it to an oven for drying to obtain polydopamine-coated MXene, denoted as MXP;

[0071] (3) Prepare a 3 g / L gelatin solution, prepare a 2% (w / v) chitosan solution using 0.2% (v / v) acetic acid, and neutralize the pH to 7.0 using a 1.0 mol / L sodium hydroxide standard solution; mix the gelatin solution and chitosan solution at a ratio of 3:2, then add 1% (v / v) MXP to the chitosan / gelatin mixture; and add 25 mM genipin solution as a crosslinking agent at a volume ratio of 1:100. After thorough mixing, crosslink at room temperature. When the mixture begins to gel, MXN is coated onto the surface. After complete gelation, rinse with deionized water to remove uncrosslinked MXN, obtaining an MXene-based artificial hard membrane.

[0072] Example 5

[0073] (1) 0.1 g MXene was dispersed in 30 mL of hydrazine monohydrate (HM) and vortexed for 10 min. The mixture was then transferred to a high-pressure reactor and reacted at 40 °C for 24 h with stirring at 600 rpm. After the reaction was complete, the precipitate was collected by centrifugation and washed with ethanol and deionized water, respectively. After washing, the precipitate was dispersed in 30 mL of deionized water, stirred for 5 min, transferred to an ice bath for sonication for 30 min, and then vacuum filtered. Finally, the filtered solution was vacuum dried at 40 °C to obtain amino-modified MXene, denoted as MXN.

[0074] (2) Add 0.5 g MXene to 200 mL of 0.01 M Tris-HCl solution (pH 8.8), add 400 mg dopamine hydrochloride, and stir at room temperature for 8 h; after the reaction is complete, centrifuge to collect the precipitate, and wash with deionized water until the pH of the supernatant is 7. After collecting the precipitate, transfer it to an oven for drying to obtain polydopamine-coated MXene, denoted as MXP;

[0075] (3) Prepare a 3 g / L gelatin solution, prepare a 2% (w / v) chitosan solution using 0.2% (v / v) acetic acid, and neutralize the pH to 7.0 using a 1.0 mol / L sodium hydroxide standard solution; mix the gelatin solution and chitosan solution at a ratio of 3:2, then add 1% (v / v) MXP to the chitosan / gelatin mixture; and add 25 mM genipin solution as a crosslinking agent at a volume ratio of 1:100. After thorough mixing, crosslink at room temperature. When the mixture begins to gel, MXN is coated onto the surface. After complete gelation, rinse with deionized water to remove uncrosslinked MXN, obtaining an MXene-based artificial hard membrane.

[0076] Example 6

[0077] Figure 1 The images show transmission electron microscopy (TEM) and X-ray diffraction (XRD) patterns of the raw material MXene; such as... Figure 1 As shown in Figures A and B, TEM results indicate that MXene exhibits a plate-like structure and good crystallinity; Figure 1 As shown in Figure C, the XRD results show that the characteristic peak at 7.6° corresponds to the (002) crystal plane of MXene, indicating that MXene has a layered structure and good crystallinity.

[0078] Figure 2 The transmission electron microscope (TEM) image, elemental analysis diagram, and infrared spectrum of the MXP prepared in Example 1 are shown below; Figure 2 As shown in Figure A, TEM results show that MXP also exhibits a nanosheet-like morphology with uniform coating effect; Figure 2 As shown in Figure B, elemental analysis results indicate that, in addition to the C and Ti elements present in MXene itself, N elements were also detected in MXP; simultaneously, as shown in Figure B... Figure 2 As shown in Figure C, the infrared spectroscopy results show that, compared with MXene, MXP detected characteristic absorption peaks of CO, CN and NH bonds, further verifying that polydopamine was successfully coated on the MXene surface.

[0079] Figure 3 Transmission electron microscopy (TEM) image and elemental analysis of MXN prepared in Example 1. Figure X X-ray photoelectron spectroscopy (XPS) and degradation rate test results; such as Figure 3 As shown in Figure A, TEM results show that MXN also exhibits a nanosheet-like structure; as Figure 3 As shown in Figure B, elemental analysis results indicate that, in addition to the C and Ti elements inherent in MXene, MXN also contains N elements; simultaneously, as shown in Figure B... Figure 3As shown in Figure C, the XPS results indicate that MXN detected the characteristic signal of nitrogen compared to MXene, further verifying the successful amino modification of the MXene surface. Finally, as... Figure 3 As shown in Figure D, MXN can be degraded under different conditions, especially in the presence of reactive oxygen species, where the degradation rate is significantly accelerated.

[0080] Figure 4 The images show scanning electron microscopy (SEM) images, infrared spectra, adhesion demonstration images, electrical conductivity and conductivity demonstration images, and degradation rate test results of the chitosan / gelatin hydrogels with different MXP contents introduced in Example 1. 1%, 2%, and 3% MXP were added to the chitosan / gelatin (CG) hydrogels prepared in Example 1, respectively, to obtain CGMXP-1%, CGMXP-2%, and CGMXP-3%. Figure 4 As shown in Figures A through D, SEM results indicate that the hydrogels maintained good porosity even after introducing different amounts of MXP; Figure 4 As shown in Figure E, the infrared spectroscopy results indicate that MXP was successfully incorporated into the CG hydrogel. Figure 4 As shown in Figure F, the CGMXP hydrogel, rich in amino groups, exhibits excellent adhesion properties and can connect well with spinal cord tissue. Figure 4 As shown in Figures G~H, the conductivity results indicate that the conductivity of CGMXP gradually increases with increasing MXP content, and it can successfully light a bulb by utilizing its excellent conductivity to transport current. Figure 4 As shown in Figure I, the degradation rate results show that the CGMXP hydrogel has good degradability, achieving nearly 50% degradation within about 4 weeks, and the degradation rate decreases slightly with the increase of MXP content.

[0081] Figure 5 Scanning electron microscopy (SEM) images and X-ray photoelectron spectroscopy (XPS) images before and after CG coating with MXN. Figure 5 As shown in Figures A and B, SEM results indicate that a coating composed of numerous sheet-like MXN layers formed on the smooth CG hydrogel surface; as shown in Figures A and B. Figure 5 As shown in C and D, XPS results indicate that MXN and CG are mainly bonded through chemical bonds, thus ensuring the stability of the coating.

[0082] The upper and lower membranes of the MXene-based artificial hard membrane prepared in Example 1 were subjected to biocompatibility tests, and the results are as follows: Figure 6 As shown, where Figure 6 Cell viability staining results from MXN showed no significant difference in cell viability between cells seeded on MXN and those cultured under normal conditions. Figure 6Cell viability staining results in D~G showed that, compared with normal cell culture conditions, no significant change in cell viability was observed in CG hydrogels with the introduction of 1% and 2% MXP; however, cell viability decreased slightly when CG hydrogels with the introduction of 3% MXP were used.

[0083] Example 7

[0084] BV2 cell polarization state regulation experiment, neural stem cell trilineage differentiation experiment, and angiogenesis experiment:

[0085] After co-incubating the MXN prepared in Example 1 with BV2 cells, the cells were stimulated with H2O2 to induce the production of reactive oxygen species. Figure 7 As shown in Figures A and B, fluorescence staining results indicated that H2O2 treatment significantly increased intracellular reactive oxygen species (ROS) levels, while MXN intervention significantly reduced ROS levels in cells. Subsequently, MXN prepared in Example 1 was co-incubated with BV2 cells, and lipopolysaccharide (LPS) treatment was used to stimulate the cells, inducing a cellular inflammation model. Figure 7 As shown in Figures C-F, Western blotting results indicated that LPS treatment significantly increased the secretion of pro-inflammatory factors in BV2 cells, while MXN intervention significantly reduced the secretion of pro-inflammatory factors in BV2 cells. These two experimental results suggest that MXN can react with reactive oxygen species, regulate oxidative stress in inflammatory cells, and reduce the secretion of pro-inflammatory factors.

[0086] The CGMXP-2% prepared in Example 1 was co-incubated with mouse-derived neural stem cells (NSCs) for 10 days. During co-culture, the cells were treated twice daily (10 minutes each time) with a magnetic field of different rotation speeds. The expression levels of relevant neural markers (Nestin, a neural stem cell marker; Tuj1 and MAP2, neuronal markers; and GFAP, astrocyte marker) were detected using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. Figure 8 As shown in Figures A through D, the RT-qPCR results indicate that as the rotation speed of the magnetic field increases, the stronger electrical stimulation accelerates the differentiation of neural stem cells and promotes their differentiation towards neurons, while inhibiting their differentiation towards astrocytes. Figure 8 The immunofluorescence staining results shown in E~F are consistent with the RT-qPCR results, further verifying that CGMXP hydrogel can regulate the differentiation of neural stem cells and generate more neurons under the action of a time-varying magnetic field.

[0087] The MXP and MXN prepared in Example 1 were co-incubated with vascular endothelial cells seeded on matrix gel for 8 hours. The cells were stained with calcein to observe lumen formation. Figure 9As shown, the fluorescence staining results indicate that treatment with MXN and MXP can accelerate the formation of new blood vessels.

[0088] Example 8

[0089] Based on the experimental results in Examples 6 and 7, there is sufficient evidence to show that MXene-based artificial dura maters can effectively adhere spinal cord tissue, regulate inflammatory responses, promote angiogenesis, and induce neural stem cell differentiation. Therefore, this example further provides experiments to verify that MXene-based artificial dura maters promote neural regeneration after spinal cord injury.

[0090] A spinal cord injury model was established in 6-8 week old female C57BL6J mice using a typical method. The MXene-based artificial dura mater prepared in Example 1 was applied to the injured site for structural repair. Figure 10 As shown, MXene-based artificial dura maters can fulfill the sealing and barrier functions of traditional dura mater repair.

[0091] On day 7, perfusion samples were collected from mice with spinal cord injuries treated with different interventions, and immunofluorescence staining was performed on the spinal cord injury sites. Figure 11 As shown, the spinal cord tissue staining results indicate that the amount of pro-inflammatory factors secreted by microglia in the spinal cord tissue of mice treated with MXene-based artificial dura mater was effectively reduced, and the inflammatory microenvironment was effectively controlled.

[0092] During weeks 1 to 8, mice with spinal cord injuries treated with different interventions were scored weekly using the BMS system to observe the functional recovery of their hind limbs. Figure 12 As shown in Figure A, the BMS scoring results indicate that hindlimb motor function gradually improved in mice with spinal cord injuries after different interventions over time. Mice receiving MXene-based artificial dura mater showed the best motor function recovery, with an average score of 5, demonstrating the ability to stand and walk, although with an unsteady and uncoordinated gait. Simultaneously, at week 8, Catwalk gait analysis was performed on mice with spinal cord injuries receiving different interventions to evaluate their motor function recovery. Figure 12 As shown in Figure B, Catwalk gait analysis results showed that mice treated with MXene-based artificial dura mater experienced the most significant recovery in motor function. Their gait was coherent, and although their walking speed was slow, their hind limb footprints were continuous and regular.

[0093] At week 8, electrophysiological experiments were performed on mice with spinal cord injuries treated with different interventions. The recovery of neural conduction function was evaluated by detecting somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs). Figure 13As shown, electrophysiological experiments indicate that mice treated with MXene-based artificial dura mater can induce higher amplitude SEP and MEP, exhibiting stronger electrophysiological activity.

[0094] In week 8, perfusion samples were collected from mice with spinal cord injuries treated with different interventions, and immunofluorescence staining was performed on the spinal cord injury sites. Figure 14 As shown, immunofluorescence staining results indicate that mice treated with MXene-based artificial dura mater exhibited more neurons at the site of injury and showed better results in neurogenesis and neural circuit reconstruction.

[0095] The degradation of the MXene-based artificial dura mater in mice was evaluated over a period of 1 to 4 weeks. Degradation results are shown below. Figure 15 As shown in Figures A-B, the MXene-based artificial dura mater achieved a degradation rate of nearly 75% within 4 weeks. Furthermore, perfusion sampling was performed on mice 4 weeks after treatment, and HE staining was performed on their heart, lung, liver, spleen, and kidney tissues, as shown... Figure 15 As shown in Figure C. Staining results showed that, compared with normal mice, the major organs of mice receiving MXene-based artificial dura mater transplants were intact in morphology and normal in color, with no swelling, necrosis or other pathological degeneration observed, indicating that the material has good biocompatibility.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An MXene-based artificial hard coating, characterized by, It is a layered structure, including an upper layer film and a lower layer film; The upper layer film is an amino-modified MXene; The lower layer film is based on a chitosan / gelatin hydrogel doped with polydopamine-coated MXene; The upper layer film and the lower layer film are combined by a chemical bond formed by the reaction of the amino group in the upper layer film and the crosslinking agent in the lower layer film.

2. The MXene-based artificial hardmask of claim 1, wherein, The crosslinking agent is genipin.

3. The method of claim 1 or 2, wherein the MXene-based artificial hard coating is prepared by the steps of: It comprises the following steps: (1) dispersing MXene into an amino-modifying agent to obtain an amino-modified MXene; (2) dispersing MXene into a dopamine hydrochloride solution to obtain polydopamine-coated MXene; (3) dispersing the polydopamine-coated MXene into a chitosan / gelatin mixed solution, adding a crosslinking agent for crosslinking; when the mixed solution begins to gel, uniformly coat the amino-modified MXene on the surface of the gel; after complete gelation, wash to obtain an MXene-based artificial dura mater.

4. The production method according to claim 3, wherein In step (1), the amino-modifying agent includes ammonia or an amino-containing compound.

5. The production method according to claim 4, wherein The amino-containing compound includes a hydrazine compound or an amine compound.

6. The production method according to claim 5, wherein The hydrazine compound includes monohydrate hydrazine, methyl hydrazine, and p-amino hydrazine, and the amine compound includes ethylenediamine, cycloethylenediamine, ethylamine chloride, and methylamine chloride.

7. The production method according to claim 3, wherein In step (2), the solvent of the dopamine hydrochloride solution is a Tris-HCl solution, the concentration of the Tris-HCl solution is 0.01-0.05 M, and the pH is 8-9; the concentration of dopamine hydrochloride is 1-5 g / L; Alternatively, in step (2), the reaction time is 4-8 h.

8. The production method according to claim 7, wherein In step (2), the pH of the Tris-HCl solution is 8.

5.

9. The production method according to claim 3, wherein In step (3), the chitosan / gelatin mixed solution is obtained by mixing a chitosan solution and a gelatin solution; Among them, the mass fraction of the chitosan solution is 1%-3%, and the concentration of the gelatin solution is 2-5 g / L.

10. The production method according to claim 9, wherein The volume ratio of the gelatin solution to the chitosan solution is (2.5-3.5):

2.

11. The production method according to claim 9, wherein The volume ratio of the gelatin solution to the chitosan solution is 3:

2.

12. The production method according to claim 3, wherein In step (3), the mass of the polydopamine-coated MXene is 1%-3% of the volume of the chitosan / gelatin mixed solution.

13. The production method according to claim 3, wherein In step (3), the crosslinking agent is genipin.

14. The production method according to claim 13, wherein The concentration of genipin is 20-100 mM.

15. The production method according to claim 14, wherein The volume ratio of the genipin solution to the chitosan / gelatin mixed solution is 1:(80-120).

16. The production method according to claim 15, wherein The volume ratio of the genipin solution to the chitosan / gelatin mixed solution is 1:

100.

17. The MXene-based artificial dura mater of claim 1 or 2 or prepared by the preparation method of any one of claims 3-16 for use in the preparation of a medical material for treating dura mater injury.

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