A magnetoelectric heterojunction material for anti-inflammatory and nerve repair promotion and a preparation method and application thereof

Abstract

This invention discloses a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes, its preparation method, and its applications, relating to the field of biomaterials. The invention involves preparing monolayer vanadium carbide (vanadium carbide) Mxene nanosheets via an etching intercalation method, followed by preparing core-shell structured Fe3O4@BaTiO3 magnetoelectric nanoparticles via solvothermal and hydrothermal methods, which are then modified with polyethyleneimine. Finally, the two are mixed and composited through a hydrothermal reaction to form a heterojunction. In this material, the monolayer vanadium carbide (vanadium carbide) Mxene nanosheets can efficiently scavenge reactive oxygen species (ROS) generated by secondary injuries following traumatic brain injury. The Fe3O4@BaTiO3 particles generate piezoelectric signals under the drive of an external alternating magnetic field, which are transmitted to nerve cells via the vanadium carbide (vanadium carbide) Mxene conductive network, wirelessly inducing neural electrical stimulation and promoting the differentiation of neural stem cells into neurons and the directed growth of axons. This invention achieves synergistic treatment of ROS scavenging and radiofrequency stimulation, effectively avoiding the risks of infection and secondary surgery associated with implanted electrodes.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more particularly to the manufacture of biopharmaceuticals, specifically a magnetoelectric heterojunction material for anti-inflammatory and nerve-repairing purposes, its preparation method, and its applications. Background Technology

[0002] Traumatic brain injury (TBI) is the destruction of brain tissue structure and function caused by external mechanical forces. Following TBI, the primary injury leads to the direct death of neurons and glial cells in the lesion area, forming irreversible tissue defects and cavities. Subsequent secondary injuries (such as oxidative stress and neuroinflammation) further deteriorate the microenvironment. Due to the extremely poor intrinsic regenerative capacity of the central nervous system (CNS), damaged brain tissue rarely achieves spontaneous structural repair and functional compensation. Currently, conventional clinical treatments for TBI mainly focus on surgical decompression and conservative drug therapy. These methods aim to save lives and control secondary damage, but they cannot achieve the regeneration of neurons and the remodeling of neural networks in the defect area. Therefore, how to promote in situ regeneration of neural tissue after TBI is a major challenge that urgently needs to be addressed in the field of neuromedicine.

[0003] Nerve tissue possesses natural electrophysiological activity. Numerous studies have shown that endogenous electric fields play a crucial guiding role in neural development and regeneration; while exogenous electrical stimulation has been proven to effectively upregulate the expression of neurotrophic factors, guide the directional growth of nerve axons, and promote synapse formation and neural plasticity.

[0004] However, traditional electrical stimulation therapy typically relies on implanted microelectrodes (such as deep brain stimulation (DBS) devices). This approach has significant limitations. The rigid metal electrodes present a huge mechanical mismatch with the extremely soft brain tissue, easily leading to long-term mechanical friction after implantation. This causes excessive proliferation of glial cells and the formation of glial scars, ultimately encapsulating the electrodes and blocking electrical signal transmission. Furthermore, traditional electrodes require connection to an external power source and wiring, increasing the risk of intracranial infection; and after treatment, a second craniotomy is usually required to remove the device, causing secondary trauma to the patient. Summary of the Invention

[0005] This invention overcomes the shortcomings of existing technologies and provides a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes, along with its preparation method and applications. This material combines monolayer vanadium carbide (Mxene) nanosheets with reactive oxygen species (ROS) scavenging capabilities with Fe3O4@BaTiO3 core-shell nanoparticles with magnetoelectric responsive properties through a hydrothermal reaction to form a heterojunction structure. This eliminates the need for implanted electrodes and wires, avoiding the risks of infection and secondary surgery. Furthermore, its flexible two-dimensional nanosheet structure can closely adhere to nerve cell membranes, exhibiting excellent biocompatibility and mechanical compatibility. This invention aims to regulate the inflammatory microenvironment and in-situ reconstruction of neural networks after traumatic brain injury through a synergistic therapeutic strategy of scavenging ROS and wireless magnetoelectric stimulation, thereby promoting the recovery of neurological function.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Firstly, the present invention provides a method for preparing a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes, comprising the following steps:

[0007] S1. Vanadium-based MAX phase ceramic powder was etched in a mixed solution containing fluoride and inorganic acid. After washing and centrifugation, the resulting precipitate was dispersed in tetramethylammonium hydroxide aqueous solution and stirred. Then, it was sonicated, centrifuged and freeze-dried to obtain monolayer vanadium carbide Mxene nanosheets.

[0008] S2. Fe3O4 nanoparticles were synthesized by a solvothermal method. A TiO2 precursor layer was coated on the surface of the Fe3O4 nanoparticles, and then a hydrothermal reaction was carried out with a barium source under alkaline conditions to generate a BaTiO3 shell. The surface of the obtained Fe3O4@BaTiO3 nanoparticles was modified with a cationic polymer to obtain modified Fe3O4@BaTiO3 magnetoelectric nanoparticles.

[0009] S3. The monolayer vanadium carbide Mxene nanosheets and the modified Fe3O4@BaTiO3 magnetoelectric nanoparticles were mixed and dispersed in water at a mass ratio of 1:2-10. After hydrothermal reaction, the mixture was magnetically separated and freeze-dried to obtain FO@BTO / VC heterojunction.

[0010] In a preferred embodiment of the present invention, in step S1, the vanadium-based MAX phase ceramic powder is V2CT. x The mass-to-volume ratio of MAX to the mixed solution is 1 g: 10-30 mL; the etching conditions are: shaking at 35-45 ℃ for 20-30 h; the ultrasonic and centrifugation steps are repeated 2-4 times.

[0011] In a preferred embodiment of the present invention, in step S1, the mass-to-volume ratio of the fluoride to the inorganic acid in the mixed solution is 1 g: 5-15 mL; the inorganic acid is hydrochloric acid with a concentration of 8-10 mol / L; the fluoride is lithium fluoride; the concentration of the tetramethylammonium hydroxide aqueous solution is 10-25 wt%, and the mass-to-volume ratio of the tetramethylammonium hydroxide aqueous solution to the vanadium-based MAX phase ceramic powder is 10-30 mL: 1 g.

[0012] In a preferred embodiment of the present invention, in step S2, the specific conditions for the solvothermal synthesis of Fe3O4 nanoparticles are as follows: ferric chloride hexahydrate, sodium citrate and ammonium acetate are dissolved in ethylene glycol, reacted at 190-210 °C for 8-12 h, and washed after magnetic separation;

[0013] The mass-to-volume ratio of ferric chloride hexahydrate to ethylene glycol is 1 g: 40-60 mL, the mass ratio of sodium citrate to ferric chloride hexahydrate is 1: 2-5, and the mass ratio of ammonium acetate to ferric chloride hexahydrate is 2-4: 1.

[0014] In a preferred embodiment of the present invention, in step S2, the specific method for coating the TiO2 precursor layer is as follows: Fe3O4 nanoparticles are dispersed in a mixed solvent of ethanol and acetonitrile, tetrabutyl titanate and ammonia are added under stirring conditions, the reaction is carried out for 1-2 hours, and the mixture is washed after magnetic separation.

[0015] The volume ratio of ethanol to acetonitrile in the mixed solvent is 2-4:1, the mass-volume ratio of Fe3O4 nanoparticles to the mixed solvent is 1 g:100-300 mL, the mass ratio of tetrabutyl titanate to Fe3O4 nanoparticles is 1-3:1, and the volume ratio of ammonia to tetrabutyl titanate is 1-2:2-1.

[0016] In a preferred embodiment of the present invention, in step S2, the specific conditions for the hydrothermal reaction with the barium source are as follows: Fe3O4 nanoparticles coated with TiO2 precursor layer at a mass ratio of 1:2-5 are mixed with barium hydroxide in water, ammonia is added to adjust the pH value to 9-11, and the reaction is carried out at 190-210 °C for 6-10 h. The product is washed sequentially with formic acid aqueous solution, water and ethanol.

[0017] The volume-to-mass ratio of water to Fe3O4 nanoparticles is 20-50 mL:1 g, and the concentration of formic acid aqueous solution is 0.5-2 wt%.

[0018] In a preferred embodiment of the present invention, in step S2, the cationic polymer is polyethyleneimine; the specific method of surface modification is as follows: Fe3O4@BaTiO3 nanoparticles are dispersed in a polyethyleneimine aqueous solution with a mass fraction of 4-6%, stirred for 2-4 h, magnetically separated, and then dried;

[0019] The mass-to-volume ratio of Fe3O4@BaTiO3 nanoparticles to polyethyleneimine aqueous solution was 1 g: 20-35 mL.

[0020] In a preferred embodiment of the present invention, in step S3, the ratio of the volume of water to the total mass of the two solids is 10-50 mL:1 g; the temperature of the hydrothermal reaction is 90-110 °C, and the reaction time is 2-4 h.

[0021] Secondly, the present invention provides a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes, which is prepared by any one of the above-mentioned methods.

[0022] Thirdly, the present invention provides an application of the magnetoelectric heterojunction material for anti-inflammatory and neuroreparative purposes as described above, wherein the magnetoelectric heterojunction material for anti-inflammatory and neuroreparative purposes is used in the preparation of drugs or medical devices for treating traumatic brain injury.

[0023] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0024] (1) This invention provides a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes, its preparation method and application. By combining monolayer vanadium carbide Mxene nanosheets with Fe3O4@BaTiO3 magnetoelectric nanoparticles to form a heterojunction structure, the surface of the monolayer vanadium carbide Mxene nanosheets is rich in hydroxyl and oxygen functional groups, which can act as electron donors to undergo redox reactions with reactive oxygen free radicals, converting superoxide anions, hydrogen peroxide and hydroxyl free radicals into water molecules and oxygen, thereby reducing the oxidative stress level in the lesion area. This process reduces the generation of lipid peroxidation products and DNA oxidative damage markers, and can protect the membrane integrity of neurons and glial cells. Compared with traditional small molecule antioxidant drugs such as edaravone, the material of this invention does not require frequent administration and can be easily recovered from the body through magnetic separation, effectively avoiding the accumulation toxicity of drugs in non-target organs, thereby solving the problem of excessive accumulation of free radicals leading to continuous death of nerve cells in secondary injuries caused by traumatic brain injury.

[0025] (2) This invention utilizes the magnetoelectric coupling effect of Fe3O4@BaTiO3 core-shell nanoparticles. The Fe3O4 core has superparamagnetism and generates magnetostrictive stress in an applied magnetic field, which can be transferred to the piezoelectric BaTiO3 shell, causing non-centrosymmetric distortion of the titanium octahedron, thereby generating a local potential on the particle surface. This potential is efficiently transmitted to the attached nerve cell membrane through a conductive network composed of a single layer of vanadium carbide Mxene nanosheets, activating voltage-gated calcium ion channels, which can cause an increase in intracellular calcium ion concentration, thereby initiating calcium regulation. Protein kinases and cyclic adenosine monophosphate (cAMP) response elements bind to protein signaling pathways, upregulating the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Compared with traditional deep brain stimulation (DBS) devices, the material of this invention does not require the implantation of rigid metal electrodes and percutaneous leads, avoiding the risks of glial scarring and intracranial infection caused by mechanical mismatch. It also eliminates the need for secondary surgery to remove the device, enabling directional guidance of neural stem cell differentiation into neurons and effectively promoting the directional extension of axons and synapse formation. This solves the problems of existing electrical stimulation treatments being highly invasive, risky, and unsuitable for long-term use.

[0026] (3) In this invention, Fe3O4@BaTiO3 magnetoelectric nanoparticles are surface modified with polyethyleneimine to impart a positive charge to the surface of the material. This allows the material to electrostatically adsorb onto the negatively charged proteoglycans on the surface of nerve cell membranes, making the heterojunction material adhere tightly to the cell membrane surface. At the same time, the positively charged surface can also have a strong electrostatic interaction with negatively charged hydrogel matrices such as hyaluronic acid or chondroitin sulfate, which anchors the material stably in the injectable hydrogel three-dimensional network and serves as a physical cross-linking point to enhance the mechanical strength of the hydrogel. This shortens the transmission distance of the magnetoelectric signal from the material to the cell membrane, reduces signal attenuation, and improves the efficiency of electrical stimulation. This reduces the potential thermal and mechanical effects of the magnetic field on surrounding normal tissues, effectively improving the safety and effectiveness of radiofrequency stimulation therapy.

[0027] (4) The present invention uses hydrothermal reaction to form a heterojunction interface. During the formation process, electrons on the surface of vanadium carbide Mxene are transferred to Fe3O4@BaTiO3 particles, making vanadium carbide Mxene positively charged. Its charge separation effect can enhance the antioxidant activity of vanadium carbide Mxene because the positively charged surface is more likely to adsorb negatively charged superoxide anions and hydroxyl radicals. At the same time, electron transfer can improve the overall conductivity of the composite material, so that the weak piezoelectric signal generated by the magnetoelectric particles can propagate along the vanadium carbide nanosheets with lower impedance, avoiding the accumulation and dissipation of charge at the particle interface. This makes the actual electrical signal intensity transmitted to the nerve cell membrane higher under the same external magnetic field strength, thereby more effectively activating the intracellular calcium signaling pathway. Compared with Fe3O4@BaTiO3 particles alone, the heterojunction material of the present invention can synergistically enhance the ability to remove hydrogen peroxide and generate oxygen, and also significantly improve the efficiency of promoting the differentiation of neural stem cells into neurons, thus taking into account the dual functions of antioxidant and electrical stimulation, and achieving synergistic effect of the two functional modules. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 These are the characterization data of the material prepared in Example 1 of the present invention, wherein Figure 1 A and 1B are FO@BTO and FO@BTO / VCSEM and TEM images, respectively. Figure 1 C is a TEM image of the heterojunction material FO@BTO / VC. Figure 1 D is the elemental mapping diagram of the composite material FO@BTO / VC;

[0030] Figure 2 This is a characterization of the physicochemical properties of the material prepared in Example 1 of the present invention, wherein... Figure 2 A, 2B, and 2C are particle size distribution diagrams for materials FO, FO@BTO, and FO@BTO / VC, respectively. Figure 2 D represents the Zeta potential values ​​of FO, FO@BTO, and FO@BTO / VC. Figure 2 E and 2F are the powder XRD and XPS patterns of the material. Figure 2 G, 2H, and 2I are the XPS elemental fine spectra of the material, representing the electron transfer that occurs after the reaction forms a heterojunction.

[0031] Figure 3This is a characterization of the magnetoelectric properties of the material prepared in Example 1 of the present invention, wherein... Figure 3 A represents the hysteresis curves of VC, FO@BTO, and FO@BTO / VC. Figure 3 B represents the change in the photoresponse surface potential of FO@BTO / VC. Figure 3 C and 3D are the piezoelectric response phase curve and amplitude curve of FO@BTO / VC, respectively;

[0032] Figure 4 This is a characterization of the antioxidant properties of the material prepared in Example 1 of the present invention, wherein... Figure 4 A and 4B represent the material's ability to scavenge ABTS free radicals. Figure 4 C and 4D represent the material's ability to scavenge DPPH free radicals. Figure 4 E and 4F represent the material's ability to scavenge hydroxyl radicals. Figure 4 G, 4H, and 4I are the properties of the material to remove hydrogen peroxide and generate oxygen;

[0033] Figure 5 This is a characterization of the in vitro ROS scavenging and neuroinflammatory inhibition properties of the material prepared in Example 1 of the present invention, wherein... Figure 5 A and 5B represent the material's ROS removal performance. Figure 5 C represents the anti-inflammatory phenotypic transformation performance. Figure 5 D represents the performance of the MitoSox probe in detecting mitochondrial ROS levels. Figure 5 E represents the performance of ATP synthase immunofluorescence in assessing mitochondrial functional status.

[0034] Figure 6 This is an experimental result showing that the material prepared in Example 1 of this invention promotes the differentiation of NSCs into neurons through remote magnetic field-mediated radio stimulation. Figure 6 A is a schematic diagram of a bio-heterostructure nanopatch constructed by functionalizing laminin to anchor onto the surface of NSCs membranes. Figure 6 Image B shows a SEM image revealing that the nanopatch tightly wraps around the cell membrane, forming a patch-cell complex. Figure 6 C represents the results of immunofluorescence staining showing the differentiation of NSCs into Tuj-1+ functional neurons; Figure 6 D represents the result of a calcium imaging experiment confirming that radio stimulation activates intracellular calcium signaling. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0037] It should be noted that the raw materials, equipment and reagents used in this invention can all be purchased from the market or obtained through existing preparation methods.

[0038] Example 1:

[0039] This embodiment provides a method for preparing a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes, comprising the following steps:

[0040] S1, 1 g V2CT x MAX powder was added to 20 mL of hydrochloric acid solution (9 mol / L) containing 1 g of lithium fluoride (LiF) and etched at 40 °C with constant temperature shaking at 200 rpm for 24 h. After the reaction, the resulting mixture was washed repeatedly with water and centrifuged at 3500 rpm for 5 min until the pH of the supernatant was greater than 6. The precipitate was dispersed in 20 mL of 20 wt% tetramethylammonium hydroxide aqueous solution and stirred at room temperature for 12 h for intercalation. Subsequently, the intercalated dispersion was sonicated in an ice-water bath at 300 W for 1 h and then centrifuged at 3500 rpm for 1 h. The supernatant was collected, which was the monolayer VC nanosheet dispersion. Finally, the dispersion was freeze-dried at -50 °C for 48 h to obtain monolayer VC nanosheet powder.

[0041] Preparation and modification of S2, Fe3O4@BaTiO3 magnetoelectric nanoparticles:

[0042] S21. Dissolve 1.350 g ferric chloride hexahydrate, 0.4 g sodium citrate and 3.854 g ammonium acetate in 70 mL ethylene glycol and stir magnetically until completely dissolved. Transfer the yellow transparent solution to a 100 mL polytetrafluoroethylene-lined high-pressure reactor, seal it, and heat it to 200 °C at a rate of 5 °C / min for 10 h. After naturally cooling to room temperature, separate the black precipitate with a neodymium magnet and wash it three times each with water and anhydrous ethanol. Finally, disperse the obtained FO nanoparticles in 20 mL anhydrous ethanol and store them at 4 °C for later use.

[0043] S22. Take 5 mL of the above FO suspension and mix it with 85 mL of anhydrous ethanol and 30 mL of acetonitrile. Disperse the mixture by sonication for 15 min. Under vigorous mechanical stirring, add 1 mL of tetrabutyl titanate and 0.3 mL of ammonia water in sequence. React at room temperature for 1.5 h. After the reaction is complete, collect the product with a magnet and wash it 3 times each with anhydrous ethanol and water. Disperse the product in 20 mL of anhydrous ethanol to obtain FO@TO suspension.

[0044] S23. Dissolve 0.315 g of barium hydroxide in 20 mL of water. After ultrasonically dispersing the entire FO@TO suspension for 15 min, add the above barium hydroxide solution. Under vigorous stirring, add 2 mL of ammonia water to adjust the pH value to 10. Transfer the mixture to a 100 mL high-pressure reactor lined with polytetrafluoroethylene and react at 200 ℃ for 8 h. After natural cooling, wash the product three times each with 1 wt% formic acid aqueous solution, water, and anhydrous ethanol. Separate with a magnet and dry the product in a vacuum drying oven at 60 ℃ for 12 h to obtain FO@BTO nanoparticles.

[0045] S24. Disperse 500 mg of the above FO@BTO nanoparticles in 50 mL of 5% (w / w) polyethyleneimine (PEI) aqueous solution, and mechanically stir at room temperature for 3 h. After the reaction is completed, separate and collect the product with a magnet, wash it three times with water to remove unbound PEI, and finally dry it in a vacuum drying oven at 60 °C for 12 h to obtain PEI-modified FO@BTO-PEI nanoparticles.

[0046] S3. Weigh 20 mg of monolayer VC nanosheet powder, disperse it in 30 mL of water, and ultrasonically disperse it for 10 min to obtain a VC dispersion. Separately weigh 100 mg of FO@BTO-PEI nanoparticles, disperse them in 30 mL of water, and ultrasonically disperse them for 10 min to obtain an FO@BTO dispersion. Under continuous mechanical stirring, slowly add the FO@BTO dispersion to the VC dispersion, and continue stirring for 30 min. Transfer the mixture to a 100 mL high-pressure reactor lined with polytetrafluoroethylene, and hydrothermally react at 100 ℃ for 3 h. After the reaction, allow it to cool naturally to room temperature, separate and collect the black product using a magnet, wash it three times with water, and finally freeze-dry it at -50 ℃ for 48 h to obtain the FO@BTO / VC heterojunction material. In this example, the mass ratio of VC to FO@BTO is 1:5.

[0047] To verify the successful acquisition of the material in Example 1, the physicochemical properties of the material in Example 1 were characterized. The morphology and corresponding elements of the material were characterized by SEM and TEM, such as... Figure 1 As shown, in the FO@BTO / VC heterojunction prepared in Example 1, the core-shell structured FO@BTO particles are uniformly distributed on the surface of the monolayer V2C. Figure 1 A-1C), the elemental mapping diagram confirms the coexistence and uniform distribution of Fe, O, Ti, Ba, V, and C elements. Figure 1 D). The particle size and zeta potential of the material were analyzed using a nanoparticle size analyzer, such as... Figure 2 As shown in AD; XRD analysis of the material phases revealed characteristic peaks for each phase, such as... Figure 2 As shown in Figure E; the electronic structure of the material was analyzed by XPS, revealing the transfer of electrons from FO@BTO to V2C and the formation of surface heterostructures, as shown in Figure E. Figure 2 FI. The magnetoelectric properties of the material were characterized using a magnetometer and piezoelectric microscopy. Compared to V2C, the FO@BTO / VC heterojunction exhibits enhanced magnetic field strength and magnetoelectric properties, such as... Figure 3 As shown.

[0048] Free radical scavenging performance experiment:

[0049] Experiment 1: The clearance experiments for ABTS and DPPH were performed according to the instructions provided with their respective clearance kits. The results are as follows: Figure 4 As shown in Figures A, B, C, and D, after FO@BTO / VC removal, the absorbance of ABTS at 740 nm decreased from 1.225 to 0.009, a significant improvement compared to 0.862 for FO@BTO. DPPH showed a similar trend, with its absorbance at 520 nm decreasing from 1.433 to 1.116. However, the change after FO@BTO treatment was minimal. Furthermore, the removal effect increased significantly with further increases in the concentration of FO@BTO / VC. When the concentration reached 200 ppm, both free radicals were nearly completely removed, with an ABTS removal rate of 99.3% and a DPPH removal rate of 98.1%.

[0050] Experiment 2: The FO@BTO / VC heterojunction material prepared in Example 1 was subjected to an ∙OH scavenging performance experiment. The ∙OH scavenging effect was evaluated using TMB. The specific procedure was as follows: ∙OH was generated by reacting 200 µL of ferrous sulfate heptahydrate (9 mM) with 200 µL of hydrogen peroxide (8.8 mM); after reacting for 5 min, 200 µL of an aqueous dispersion of the FO@BTO / VC material was added and reacted for 10 min; after centrifugation, 200 µL of TMB (2 mM) was added to the supernatant, and its absorbance was then measured. Figure 4 As can be seen from E, after FO@BTO / VC removal, the absorbance of TMB oxidized by ∙OH at 654 nm decreased from 1.881 to 0.916, which is a significant improvement compared to 1.444 in the FO@BTO treated group. Figure 4As can be seen from F, the scavenging effect significantly improved with the increase of FO@BTO / VC concentration, reaching 98.5%.

[0051] Experiment 3: The FO@BTO / VC heterojunction material prepared in Example 1 was used to decompose hydrogen peroxide to produce oxygen. The oxygen yield was evaluated using [Ru(dpp)3]Cl2. The specific procedure was as follows: 200 µL of hydrogen peroxide (1 mM), 200 µL of [Ru(dpp)3]Cl2 (2 mM dissolved in DMSO), and 200 µL of FO@BTO / VC material aqueous dispersion were added sequentially. After reacting for 5 min, the fluorescence intensity was measured. The lower the fluorescence intensity, the more oxygen was produced, indicating a stronger H2O2 decomposition ability. The results are as follows: Figure 4 As shown in G, H, and I, the fluorescence intensity of the FO@BTO / VC heterojunction material in Example 1 decreased to 125.3.

[0052] The results show that the introduction of V2C into the FO@BTO / VC bioheterojunction system can significantly enhance the material's ability to scavenge various free radicals and ROS, confirming that FO@BTO / VC has a stronger broad-spectrum antioxidant capacity.

[0053] In vitro ROS scavenging and neuroinflammation inhibition experiments: The biological functions of FO@BTO and FO@BTO / VC were systematically evaluated, focusing on their synergistic effects in scavenging ROS and inhibiting neuroinflammation. A series of assessments were performed by cell immunofluorescence staining, and fluorescence intensity was quantitatively analyzed using ImageJ software.

[0054] First, in an H2O2-induced oxidative stress model of HT-22 neurons, intracellular ROS levels were detected using DCFH-DA and DHE fluorescent probes. Figure 5 In A, the fluorescence intensities fitted for the FO@BTO and FO@BTO / VC treatment groups were 110.056 and 51.334, respectively. Figure 5 In B, the fluorescence intensities fitted by the FO@BTO and FO@BTO / VC treatment groups were 50.999 and 25.533, respectively, confirming that the intracellular ROS content in the FO@BTO / VC treatment group was significantly reduced, indicating a stronger broad-spectrum antioxidant capacity. Secondly, in the LPS-induced BV2 inflammation model, CD206 immunofluorescence detection of the M2 anti-inflammatory phenotype (M2 type) showed that CD206 expression was significantly upregulated in the FO@BTO / VC treatment group, with a fluorescence intensity of 39.876, much greater than the 12.662 in the FO@BTO treatment group. This suggests that it can effectively promote the polarization of BV2 towards the anti-inflammatory phenotype, inhibit the continuous amplification of neuroinflammation, and thus sever the downstream inflammatory cascade of the pathological amplification axis, such as... Figure 5 As shown in C.

[0055] MitoSox probes were used to detect mitochondrial ROS levels. Figure 5 Results showed that the red fluorescence intensity of the FO@BTO / VC treatment group was significantly lower than that of the FO@BTO treatment group, with mitochondrial ROS fluorescence intensities of 47.28 and 25.152, respectively, indicating that FO@BTO / VC can significantly reduce mitochondrial oxidative stress damage. Further analysis using ATP synthase immunofluorescence was used to assess mitochondrial functional status. Figure 5 The results showed that the green fluorescence of the FO@BTO / VC treatment group was significantly higher than that of the FO@BTO treatment group, and the mitochondrial ATP fluorescence intensities were 29.646 and 21.207, respectively. This indicates that FO@BTO / VC can maintain the stability of mitochondrial structure and function, and confirms that it has a direct protective effect against mitochondrial dysfunction.

[0056] In summary, FO@BTO / VC exhibits synergistic multi-target advantages in clearing ROS bursts, inhibiting neuroinflammation, and protecting mitochondrial function. It can effectively block the key pathological amplification axis of secondary damage after TBI, laying a solid experimental foundation for its further research in nerve injury repair.

[0057] Remote magnetic field-mediated radio stimulation promoting NSC differentiation into neurons: This experiment aims to verify the ability of FO@BTO / VC heterostructures to promote neural stem cell (NSC) differentiation under the drive of an external magnetic field. Membrane-anchored electrical stimulation was achieved using the two-dimensional structure of V2C MXene. Preliminary experiments were conducted to further verify the feasibility of membrane-anchored electrical stimulation. Through laminin functionalization, a bio-heterostructure nanopatch that can stably anchor to the NSC membrane surface was successfully constructed, such as… Figure 6 As shown in Figure A; SEM results show that the nanopatch can tightly and uniformly encapsulate the cell membrane, forming a stable patch-cell complex, effectively preventing endocytosis. Figure 6 As shown in Figure B, based on this cell model, applying an external alternating magnetic field allows nanopatches anchored to the membrane to generate local electrical signals in situ and wirelessly. Immunofluorescence staining and calcium imaging experiments confirmed that this radio stimulation significantly activated intracellular calcium signaling and efficiently drove the differentiation of NSCs into Tuj-1+ functional neurons, demonstrating the effectiveness of membrane-anchored electrical signals in regulating NSC fate determination. Figure 6 As shown in C and D, these results provide direct and strong preliminary experimental support for the design concept of introducing V2C as a nanopatch in the FO@BTO / VC bioheterojunction system to enhance magnetoelectric conversion and achieve sustained and precise electrical stimulation, significantly reducing the implementation risk of the core technology of this project.

[0058] To further simplify and make the present invention achieve its objectives and effects, the present invention will be further illustrated in conjunction with the following specific embodiments and comparative examples. The preferred embodiment 1 is used as the basis for the illustration and comparative examples, but the present invention is not limited to the scope of the embodiments described herein.

[0059] Example 2:

[0060] This embodiment is basically the same as embodiment 1, except that the mass ratio of VC to FO@BTO is 1:2.

[0061] Example 3:

[0062] This embodiment is basically the same as embodiment 1, except that the mass ratio of VC to FO@BTO is 1:10.

[0063] Example 4:

[0064] This embodiment is basically the same as Example 1, except that the amount of polyethyleneimine aqueous solution used is different. Specifically, the mass-volume ratio of Fe3O4@BaTiO3 nanoparticles to polyethyleneimine aqueous solution is 1 g: 20 mL.

[0065] Example 5:

[0066] This embodiment is basically the same as Example 1, except that the amount of polyethyleneimine aqueous solution used is different. Specifically, the mass-volume ratio of Fe3O4@BaTiO3 nanoparticles to polyethyleneimine aqueous solution is 1 g: 35 mL.

[0067] Comparative Example 1:

[0068] This comparative example is basically the same as Example 1, except that the mass ratio of VC to FO@BTO is 1:1.

[0069] Comparative Example 2:

[0070] This comparative example is basically the same as Example 1, except that the mass ratio of VC to FO@BTO is 1:11.

[0071] Comparative Example 3:

[0072] This comparative example is basically the same as Example 1, except that the FO@BTO nanoparticles were not modified with PEI.

[0073] Comparative Example 4:

[0074] This comparative example is basically the same as Example 1, except that the amount of polyethyleneimine aqueous solution used is different. Specifically, the mass-volume ratio of Fe3O4@BaTiO3 nanoparticles to polyethyleneimine aqueous solution is 1 g: 15 mL.

[0075] Comparative Example 5:

[0076] This comparative example is basically the same as Example 1, except that the amount of polyethyleneimine aqueous solution used is different. Specifically, the mass-volume ratio of Fe3O4@BaTiO3 nanoparticles to polyethyleneimine aqueous solution is 1 g: 40 mL.

[0077] Comparative Example 6:

[0078] This comparative example is basically the same as Example 1, except that the FO@BTO / VC heterojunction material was not prepared by hydrothermal reaction. The specific steps of S3 are as follows: 20 mg of monolayer VC nanosheet powder and 100 mg of FO@BTO-PEI nanoparticles were weighed and ultrasonically dispersed in 30 mL of water. The FO@BTO dispersion was slowly added dropwise to the VC dispersion. After stirring for 30 min, the product was directly separated and collected by magnet. The product was then freeze-dried at -50 °C for 48 h to obtain the FO@BTO / VC heterojunction material.

[0079] Performance testing: The FO@BTO / VC heterojunction materials obtained in Examples 2-5 and Comparative Examples 1-6 were subjected to in vitro free radical scavenging performance tests and in vitro ROS scavenging and neuroinflammation inhibition tests, respectively, and compared with those in Example 1. The results are shown in Table 1 and Table 2, respectively.

[0080] Table 1: Free radical scavenging performance at a concentration of 200 ppm

[0081] project ABTS clearance rate (%) DPPH removal rate (%) OH removal rate (%) <![CDATA[H2O2 decomposition (relative fluorescence intensity)]]> Example 1 99.3 98.1 98.5 125.3 Example 2 97.5 95.6 96.2 145.6 Example 3 96.8 94.2 95.1 168.2 Example 4 94.2 91.5 92.8 178.5 Example 5 95.5 93.0 94.0 162.3 Comparative Example 1 93.4 92.1 92.5 184.6 Comparative Example 2 87.9 85.8 96.7 204.2 Comparative Example 3 91.6 89.2 89.8 195.1 Comparative Example 4 86.5 83.2 84.1 235.6 Comparative Example 5 89.3 86.7 87.5 212.4 Comparative Example 6 38.5 30.1 42.5 485.5

[0082] Table 2: In vitro cell protection and anti-inflammatory properties

[0083] project DCFH-DA fluorescence intensity (relative value) DHE fluorescence intensity (relative value) CD206 fluorescence intensity (relative value) MitoSOX fluorescence intensity (relative value) ATP synthase fluorescence intensity (relative value) Example 1 1.8 1.6 3.2 1.9 2.8 Example 2 2.2 2.0 2.8 2.3 2.4 Example 3 2.6 2.3 2.4 2.7 2.1 Example 4 2.5 2.2 2.5 2.6 2.2 Example 5 2.4 2.1 2.6 2.5 2.3 Comparative Example 1 2.3 2.1 2.5 2.4 2.3 Comparative Example 2 2.9 2.7 1.9 3.0 1.8 Comparative Example 3 2.7 2.5 2.1 2.8 2.0 Comparative Example 4 3.2 2.9 1.7 3.3 1.5 Comparative Example 5 3.0 2.8 1.8 3.1 1.7 Comparative Example 6 3.1 2.8 2.0 2.9 1.9

[0084] As shown in Tables 1 and 2:

[0085] A comparison of Examples 1-3 and Comparative Examples 1-2 reveals that with appropriate amounts of VC and FO@BTO, monolayer VC nanosheets, acting as highly conductive two-dimensional carriers, possess a surface rich in functional groups such as hydroxyl and epoxy groups. These functional groups can form a tight heterojunction interface with the titanium and iron sites on the surface of FO@BTO particles through hydrogen bonding and electrostatic adsorption. During the hydrothermal reaction, directional electron transfer occurs at the interface, making the VC surface positively charged. The positively charged VC efficiently captures negatively charged superoxide anions and hydroxyl radicals through electrostatic attraction. Simultaneously, the functional groups on the VC surface act as electron donors, reducing these free radicals to water molecules or oxygen, thereby significantly enhancing the broad-spectrum antioxidant capacity. Furthermore, electron transfer improves the overall conductivity of the composite system, enabling the local piezoelectric signal generated by FO@BTO in an alternating magnetic field to be transmitted with low impedance along the two-dimensional conductive channels of VC to the attached nerve cell membrane, activating voltage-gated calcium channels and promoting the differentiation of neural stem cells into neurons.

[0086] In Comparative Example 1, the VC ratio was too high. Excessive VC nanosheets would stack up between layers, partially covering the surface of FO@BTO particles. This hindered the effective electron transfer interface between FO@BTO and VC. The stacked VC layers increased the interfacial contact resistance, causing dissipation of the magnetoelectric signal during transmission. At the same time, although excessive VC itself has antioxidant capacity, due to the lack of synergistic electronic coupling with FO@BTO, its free radical scavenging efficiency per unit mass did not improve further. Instead, the overall scavenging rate decreased slightly due to interface degradation. In Comparative Example 2, the VC ratio was too low, resulting in insufficient VC nanosheets to form a continuous conductive network between FO@BTO particles. Some FO@BTO particles were isolated from each other, and the piezoelectric signals they generated could not be effectively transmitted to the cells. In addition, the insufficient number of VC as antioxidant active centers meant that they could not adequately remove the large amount of ROS generated by secondary damage, leading to a decrease in ABTS scavenging rate to 87.9% and an increase in H2O2 decomposition fluorescence intensity to 204.2 (higher fluorescence intensity indicates weaker decomposition ability). More importantly, when the VC ratio was too low, the total amount of electrons transferred from FO@BTO to VC decreased, the charge separation effect of the heterojunction was weakened, the positive charge density on the VC surface was insufficient, and the electrostatic capture ability of negatively charged free radicals decreased, ultimately leading to a comprehensive deterioration of antioxidant and neuroprotective functions.

[0087] A comparison of Example 1 and Comparative Example 3 reveals that PEI is a cationic polymer rich in amine groups. The primary, secondary, and tertiary amine groups on its molecular chain, after protonation in aqueous solution, impart a dense positive charge to the FO@BTO surface. When FO@BTO is not modified with PEI, its surface typically carries a weak negative charge in aqueous solution at around pH 7 (due to the deprotonation of surface hydroxyl groups). Therefore, the interaction between FO@BTO and VC mainly relies on weak van der Waals forces and hydrophobic interactions, making it difficult to form a tight heterojunction interface. During the hydrothermal reaction, it is difficult for the two to undergo a significant interaction. The effective electron transfer was significantly reduced, and the migration of electrons from FO@BTO to VC observed in XPS was significantly weakened. This resulted in insufficient positive charge on the VC surface, decreased electrostatic capture ability of ROS, and reduced free radical scavenging rate. Furthermore, the heterojunction interface resistance increased, and the piezoelectric signal generated by FO@BTO was severely attenuated when transmitted to VC, failing to effectively stimulate nerve cells. Consequently, the fluorescence intensity of CD206 (anti-inflammatory phenotype marker) decreased from 3.2 in Example 1 to 2.1, and the fluorescence intensity of ATP synthase (mitochondrial functional marker) decreased from 2.8 to 2.0.

[0088] By comparing Examples 1 and 4-5 with Comparative Examples 4-5, it can be seen that in this invention, the optimization of PEI dosage needs to ensure that the FO@BTO surface is fully and uniformly coated. When the PEI dosage is appropriate, the PEI molecular chains form a dense monolayer or multilayer on the FO@BTO surface, the surface zeta potential changes from negative to positive and reaches more than +30 mV, generating sufficient electrostatic repulsion to prevent particle agglomeration, while providing sufficient positive charge sites to bind with VC.

[0089] In Comparative Example 4, the amount of PEI was too low, resulting in only a small amount of PEI molecules adsorbing on the FO@BTO surface. This prevented charge reversal, and the particle surface remained weakly negatively charged or nearly neutral. Consequently, there was a lack of electrostatic attraction between FO@BTO and VC, making it difficult for them to form a uniform composite during the hydrothermal reaction. Most FO@BTO particles remained independent and failed to anchor to the VC surface. Furthermore, the insufficiently coated FO@BTO particles were prone to agglomeration in aqueous solution, forming aggregates with a size of hundreds of nanometers. This reduced the effective specific surface area and magnetoelectric response efficiency. Therefore, the ABTS scavenging rate of Comparative Example 4 decreased to 86.5%, the CD206 fluorescence intensity decreased to 1.7, and the MitoSOX fluorescence intensity increased to 3.3 (due to increased mitochondrial ROS levels). In Comparative Example 5, when the PEI dosage was too high, although the FO@BTO surface was covered by PEI and the zeta potential might be close to zero or slightly positive, a complete protective layer could not be formed. Local aggregation could still occur on the exposed FO@BTO surface, and the binding force with VC was uneven, resulting in high interfacial resistance and low electron transfer efficiency in some areas of the heterojunction. Compared to Example 1, the ·OH scavenging rate and CD206 fluorescence intensity of Comparative Example 5 were significantly reduced, indicating that when the PEI dosage deviates from the optimal range, even slight changes in the ratio can affect the synergistic effect of the heterojunction due to insufficient surface charge density or incomplete coating. It should be noted that more PEI is not necessarily better; excessive PEI will form an excessively thick polymer layer on the particle surface. This insulating layer will hinder the tunneling transfer of electrons from FO@BTO to VC, and excessive positive charge may cause non-specific cytotoxicity.

[0090] A comparison between Example 1 and Comparative Example 6 reveals that in Comparative Example 6, FO@BTO and VC were only physically mixed without undergoing a hydrothermal reaction. Under hydrothermal conditions, the high temperature and pressure environment promoted the chemical bonding between the functional groups on the surface of VC nanosheets and the titanium or iron atoms on the surface of FO@BTO particles, forming covalent bonding interfaces such as Ti-OC, Fe-OC, or VO-Ti, which is the structural basis for achieving efficient electron transfer. XPS characterization ( Figure 2 In F-2I, after the formation of the heterojunction, the V 2p peak of VC shifts to a higher binding energy, while the Fe 2p and Ti 2p peaks of FO@BTO shift to a lower binding energy, confirming the directional migration of electrons from FO@BTO to VC.

[0091] Comparative Example 6 employed physical mixing. Only weak van der Waals forces and electrostatic adsorption existed between FO@BTO and VC, preventing the formation of a chemically bonded heterojunction interface. Therefore, a high-impedance interfacial barrier existed in the magnetoelectric signal transmission path. The weak piezoelectric signal generated by FO@BTO in the magnetic field was almost completely dissipated when crossing this physical interface, failing to reach the VC conductive network and thus unable to stimulate nerve cells. Simultaneously, due to the lack of interfacial electron transfer, the VC surface failed to acquire a positive charge. Its antioxidant capacity relied solely on the direct redox reaction of its own functional groups, lacking synergistic enhancement with FO@BTO. Consequently, Comparative Example 6 exhibited an ABTS scavenging rate of only 38.5%, and a H2O2 decomposition fluorescence intensity as high as 485.5 (almost no decomposition ability). In cell experiments, the fluorescence intensities of CD206 and ATP synthase were significantly lower than in Example 1.

[0092] The above description is based on the preferred embodiments of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0093] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes, characterized in that, Includes the following steps: S1. Vanadium-based MAX phase ceramic powder was etched in a mixed solution containing fluoride and inorganic acid. After washing and centrifugation, the resulting precipitate was dispersed in tetramethylammonium hydroxide aqueous solution and stirred. Then, it was sonicated, centrifuged and freeze-dried to obtain monolayer vanadium carbide Mxene nanosheets. S2. Fe3O4 nanoparticles were synthesized by a solvothermal method. A TiO2 precursor layer was coated on the surface of the Fe3O4 nanoparticles, and then a hydrothermal reaction was carried out with a barium source under alkaline conditions to generate a BaTiO3 shell. The surface of the obtained Fe3O4@BaTiO3 nanoparticles was modified with a cationic polymer to obtain modified Fe3O4@BaTiO3 magnetoelectric nanoparticles. S3. The monolayer vanadium carbide Mxene nanosheets and the modified Fe3O4@BaTiO3 magnetoelectric nanoparticles were mixed and dispersed in water at a mass ratio of 1:2-10. After hydrothermal reaction, the mixture was magnetically separated and freeze-dried to obtain FO@BTO / VC heterojunction.

2. The method for preparing a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S1, the vanadium-based MAX phase ceramic powder is V2CT. x The mass-to-volume ratio of MAX to the mixed solution is 1 g: 10-30 mL; the etching conditions are: shaking at 35-45 ℃ for 20-30 h; the ultrasonic and centrifugation steps are repeated 2-4 times.

3. The method for preparing a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S1, the mass-to-volume ratio of the fluoride to the inorganic acid in the mixed solution is 1 g: 5-15 mL; the inorganic acid is hydrochloric acid with a concentration of 8-10 mol / L; the fluoride is lithium fluoride; the concentration of the tetramethylammonium hydroxide aqueous solution is 10-25 wt%, and the mass-to-volume ratio of the tetramethylammonium hydroxide aqueous solution to the vanadium-based MAX phase ceramic powder is 10-30 mL: 1 g.

4. The method for preparing a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S2, the specific conditions for the solvothermal synthesis of Fe3O4 nanoparticles are as follows: ferric chloride hexahydrate, sodium citrate and ammonium acetate are dissolved in ethylene glycol, reacted at 190-210 °C for 8-12 h, and washed after magnetic separation; The mass-to-volume ratio of ferric chloride hexahydrate to ethylene glycol is 1 g: 40-60 mL, the mass ratio of sodium citrate to ferric chloride hexahydrate is 1: 2-5, and the mass ratio of ammonium acetate to ferric chloride hexahydrate is 2-4:

1.

5. The method for preparing a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S2, the specific method for coating the TiO2 precursor layer is as follows: Fe3O4 nanoparticles are dispersed in a mixed solvent of ethanol and acetonitrile, tetrabutyl titanate and ammonia are added under stirring conditions, the reaction is carried out for 1-2 hours, and the mixture is washed after magnetic separation. The volume ratio of ethanol to acetonitrile in the mixed solvent is 2-4:1, the mass-volume ratio of Fe3O4 nanoparticles to the mixed solvent is 1 g:100-300 mL, the mass ratio of tetrabutyl titanate to Fe3O4 nanoparticles is 1-3:1, and the volume ratio of ammonia to tetrabutyl titanate is 1-2:2-1.

6. The method for preparing a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S2, the specific conditions for the hydrothermal reaction with the barium source are as follows: Fe3O4 nanoparticles coated with TiO2 precursor layer with a mass ratio of 1:2-5 are mixed with barium hydroxide in water, ammonia is added to adjust the pH value to 9-11, and the reaction is carried out at 190-210 °C for 6-10 h. The product is washed sequentially with formic acid aqueous solution, water and ethanol. The volume-to-mass ratio of water to Fe3O4 nanoparticles is 20-50 mL:1 g, and the concentration of formic acid aqueous solution is 0.5-2 wt%.

7. The method for preparing a magnetoelectric heterostructure material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S2, the cationic polymer is polyethyleneimine; the specific method of surface modification is as follows: Fe3O4@BaTiO3 nanoparticles are dispersed in a polyethyleneimine aqueous solution with a mass fraction of 4-6%, stirred for 2-4 h, magnetically separated, and then dried. The mass-to-volume ratio of Fe3O4@BaTiO3 nanoparticles to polyethyleneimine aqueous solution was 1 g: 20-35 mL.

8. The method for preparing a magnetoelectric heterojunction material for anti-inflammatory and nerve repair purposes according to claim 1, characterized in that, In step S3, the ratio of the volume of water to the total mass of the two solids is 10-50 mL:1 g; the temperature of the hydrothermal reaction is 90-110 °C, and the reaction time is 2-4 h.

9. A magnetoelectric heterostructure material for anti-inflammatory and nerve-repairing purposes, characterized in that, It is prepared by any one of claims 1-8.

10. An application of the magnetoelectric heterostructure material according to claim 9 for anti-inflammatory and neurorepair-promoting purposes, characterized in that, The magnetoelectric heterojunction material used for anti-inflammatory and neuroreparative purposes is used in the preparation of drugs or medical devices for treating traumatic brain injury.

Images