Conductive peptide-based multi-modal platform for tissue repair as well as preparation method and application of conductive peptide-based multi-modal platform

Abstract

The invention provides a conductive peptide-based multi-modal platform for tissue repair as well as a preparation method and application thereof, and belongs to the field of biological materials. The Ti3C2 MXene is used as a conductive core, the surface of the Ti3C2 MXene is coated with a polydopamine / cellulose nanocrystal synergistic stable layer, and the Ti3C2 MXene is covalently connected with the anti-inflammatory peptide AF through a phenylboronic acid responsive joint, so that the conductive nano composite material is constructed, and intelligent release triggered by an ROS / acidic microenvironment is realized; the conductive peptide-based multi-modal platform is further prepared from a conductive nano composite material and a VEGFR (vascular endothelial growth factor receptor)-targeted bioactive peptide scaffold, the platform integrates conductivity, inflammation resistance, oxidation resistance and angiogenesis promotion, and through mechanical-electric-biochemical multi-modal synergistic effect, the inflammation microenvironment after stroke is remarkably improved, and neural function recovery is promoted. The platform has important significance in preparation of drugs for preventing and / or treating nerve injury.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to biocompatible, biodegradable, and injectable hydrogel compositions. More specifically, this invention relates to a conductive hydrogel platform comprising surface-engineered MXene nanocomposites and bioactive peptides, and its application in the treatment of nerve injuries, particularly ischemic stroke. Background Technology

[0002] Ischemic stroke (IS) is one of the leading causes of death and long-term disability worldwide. Its pathological feature is obstruction of blood flow to the brain, leading to localized brain tissue hypoxia and insufficient nutrient supply, which in turn causes nerve cell death and irreversible neurological damage. The central nervous system (CNS) has extremely limited endogenous regenerative capacity, making post-stroke functional recovery a significant clinical challenge.

[0003] Current clinical treatment strategies, such as reperfusion therapy (e.g., intravenous thrombolysis and mechanical thrombectomy), while standard treatments in the acute phase, face numerous limitations. First, these therapies have a very narrow therapeutic window (typically 4-6 hours after stroke), limiting their applicability. Second, they are less effective in promoting neurophysiological recovery and angiogenesis. Furthermore, these therapies may cause systemic side effects. Therefore, there is an urgent need to develop novel multimodal treatment strategies that can synergistically modulate multiple pathological processes.

[0004] In recent years, biomaterials, especially hydrogels, have received widespread attention as platforms for tissue repair and regeneration. Hydrogels possess biomimetic three-dimensional (3D) scaffold structures that can provide ideal adhesion sites for cells and alleviate tissue atrophy and promote functional recovery by forming "biobridges" within ischemic lesion cavities. In particular, injectable in situ hydrogels can be delivered via minimally invasive methods and adapt to irregular lesion shapes.

[0005] However, traditional hydrogels used for stroke treatment have several drawbacks: (1) Lack of target specificity: Traditional hydrogels are usually passive scaffolds, lacking the ability to identify and regulate specific pathological targets.

[0006] (2) Limited and single function: Traditional hydrogels cannot achieve multiple therapeutic functions at the same time. For example, they cannot simultaneously provide the ability to reduce oxidative stress, reduce inflammation, promote angiogenesis and restore electrical signal conduction.

[0007] (3) Neglecting electrophysiological recovery: Many treatment strategies neglect the critical importance of electrical signal recovery after stroke.

[0008] (4) Trade-off between stability and responsiveness: There is an inherent contradiction between maintaining the structural stability of the hydrogel (to achieve continuous conductivity or drug release) and the dynamic response to the local ischemic microenvironment (such as inflammation, ROS).

[0009] To address the conductivity issue, two-dimensional (2D) MXenes (such as Ti3C2) and other nanomaterials have attracted attention due to their unique physicochemical properties and biological functions. The conductivity of MXenes is thought to drive axonal regeneration and functional innervation by enhancing cellular mechanotransduction and biochemical signaling pathways. However, the widespread application of MXenes is hindered by a critical bottleneck: pristine MXenes are highly susceptible to oxidation and aggregation under physiological conditions (such as in water and oxygen environments), exhibiting inherent instability. This instability not only causes them to lose their conductivity but may also lead to toxicity problems, impeding their clinical translation.

[0010] Therefore, there is an urgent need in the field for a novel treatment platform that can overcome the shortcomings of existing technologies by providing a physiologically stable conductive material and integrating it into a multifunctional hydrogel scaffold that can actively promote angiogenesis, intelligently respond to the inflammatory microenvironment, and ultimately achieve comprehensive brain tissue repair, including electrophysiological functions. Summary of the Invention

[0011] The purpose of this invention is to provide a novel peptide-based multimodal brain tissue repair (MP-BTR) platform, its preparation method, and its applications, in order to overcome the limitations of the prior art.

[0012] The present invention provides a conductive nanocomposite material, which is prepared from bioactive molecules and conductive nanomaterials coated with a synergistic stabilizing layer as raw materials; wherein the mass ratio of the bioactive molecules to the conductive nanomaterials coated with the synergistic stabilizing layer is 1:(5~7).

[0013] Furthermore, the mass ratio of the bioactive molecules to the conductive nanomaterials coated with the synergistic stabilizing layer is 1:6.

[0014] Furthermore, the bioactive molecule is an anti-inflammatory peptide modified with 4-carboxyphenylboronic acid; wherein the mass ratio of the anti-inflammatory peptide to 4-carboxyphenylboronic acid is 1:(1~5); The conductive nanomaterials coated with the synergistic stabilizing layer are prepared from conductive nanomaterials, dopamine or its salts, and cellulose nanomaterials as raw materials; wherein, the conductive nanomaterials are two-dimensional (2D) transition metal carbides, nitrides, or carbonitrides; the mass ratio of the conductive nanomaterials to dopamine or its salts is 1:(0.2~2); the mass ratio of dopamine or its salts to cellulose nanomaterials is (1~10):1.

[0015] Furthermore, the mass ratio of the anti-inflammatory peptide to 4-carboxyphenylboronic acid is 1:(1.5~2); the mass ratio of the conductive nanomaterial to dopamine or its salt is 1:0.6; and the mass ratio of dopamine or its salt to cellulose nanomaterial is 5:1.

[0016] Furthermore, the amino acid sequence of the anti-inflammatory peptide is shown in SEQ ID No. 1.

[0017] Furthermore, the conductive nanomaterial is Ti3C2MXene nanosheets; the dopamine or its salt is dopamine hydrochloride; and the cellulose nanomaterial is cellulose nanocrystals.

[0018] Furthermore, the preparation of the conductive nanomaterial coated with the synergistic stabilizing layer includes the following steps: mixing and reacting the conductive nanomaterial with dopamine or its salt solution, then adding cellulose nanomaterial to continue the reaction, thereby obtaining the final product; wherein, the temperature of the mixing reaction is 10~40℃ and the time is 0.5~2 hours; the temperature of the continued reaction is 10~40℃ and the time is 2~4 hours. Preferably, the temperature of the mixing reaction is 20~30℃ and the time is 1 hour; the temperature of the continued reaction is 20~30℃ and the time is 3 hours. The preparation of the bioactive molecule includes the following steps: reacting an anti-inflammatory peptide, 4-carboxyphenylboronic acid, a condensing agent, and an organic base to obtain the molecule; wherein the mass ratio of the anti-inflammatory peptide, 4-carboxyphenylboronic acid, condensing agent, and organic base is 1:(1~5):(3~5):(4~6); and the solvent for the reaction is an organic solvent. More preferably, the mass ratio of the anti-inflammatory peptide, 4-carboxyphenylboronic acid, condensing agent, and organic base is 1:(1.5~2):(3.5~4):(4~4.5); the condensing agent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate; the organic base is N,N-diisopropylethylamine; and the solvent for the reaction is N,N-dimethylformamide.

[0019] Furthermore, the preparation of the conductive nanocomposite material includes the following steps: reacting bioactive molecules and conductive nanomaterials coated with a synergistic stabilizing layer to obtain the conductive nanocomposite material.

[0020] Furthermore, the reaction temperature is 10~40℃ and the time is 6~12 hours; preferably, the reaction temperature is 20~30℃ and the time is 3 hours.

[0021] The present invention also provides an angiogenesis-promoting peptide, the amino acid sequence of which is shown in SEQ ID No. 2.

[0022] The present invention also provides a hydrogel composition comprising the above-mentioned conductive nanocomposite material, angiogenic peptide and crosslinking agent; wherein the mass ratio of the conductive nanocomposite material, angiogenic peptide and crosslinking agent is (1~6):(1~5):(3.4~17).

[0023] Furthermore, the mass ratio of the conductive nanocomposite material, the angiogenic peptide, and the crosslinking agent is 4:3:10.

[0024] Furthermore, the amino acid sequence of the pro-angiogenic peptide is shown in SEQ ID No. 2; the crosslinking agent is a multi-arm polyethylene glycol derivative, preferably a 4-arm PEG-maleimide.

[0025] The present invention also provides a multifunctional hydrogel, which is obtained by in-situ reaction of the above-mentioned conductive nanocomposite material solution, angiogenic peptide solution and crosslinking agent solution.

[0026] The present invention also provides the use of the above-mentioned conductive nanocomposite material, the above-mentioned pro-angiogenic peptide, the above-mentioned hydrogel composition, and the above-mentioned multifunctional hydrogel in the preparation of tissue repair materials.

[0027] Furthermore, the tissue repair material is a material for treating ischemic stroke (IS), hemorrhagic stroke, traumatic brain injury (TBI), or spinal cord injury (SCI).

[0028] In one embodiment of the present invention, the treatment includes synergistically promoting tissue repair through a multimodal mechanism, the mechanism including: (a) Provide mechanical support and a conductive microenvironment at the lesion site; (b) Promotes angiogenesis and vascular remodeling; (c) Regulates neuroinflammation in response to the local inflammatory microenvironment; (d) Reduce oxidative stress; (e) Inhibit the formation of glial scars.

[0029] The present invention also provides a pharmaceutical composition, which is a formulation prepared by using the above-mentioned hydrogel composition as the active ingredient and adding pharmaceutically acceptable excipients.

[0030] This invention overcomes the shortcomings of the prior art and achieves the following beneficial effects: (1) The present invention adopts a synergistic stabilization strategy of polydopamine (PDA) and cellulose nanocrystals (CNC) to significantly improve the structural stability of MXene in physiological environment and ensure its long-term conductivity.

[0031] (2) This invention constructs a multimodal active repair system, rather than a traditional passive stent: (i) The VP peptide in the scaffold component has a pro-angiogenic function that targets VEGFR; (ii) The MPA nanocomposite material loaded with the material has anti-inflammatory (AF peptide), antioxidant (MP) and conductive (MXene) functions; (iii) Intelligent responsive release of anti-inflammatory peptides via phenylboronic acid (PBA) linkers.

[0032] (3) Through the synergistic effect of the mechanical-electrical-biochemical multimodal regulation platform, this invention ultimately achieves comprehensive intervention and functional recovery of the complex pathological process after stroke.

[0033] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0034] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0035] Figure 1 Computer-aided design, structural characterization, and angiogenesis potential of VP. (A) Screening flowchart for VEGFR-targeting peptide discovery (including the entire process from peptide library screening to ADMET analysis and molecular dynamics simulation). (B) Root mean square deviation (RMSD) stability analysis of the modified Top1-VEGFR complex, showing that the complex remains stable during the simulation time. (C) Root mean square fluctuation (RMSF) analysis of key binding site residues, verifying the structural rigidity of the binding region. (D) Two-dimensional interaction network diagram between modified Top1 and VEGFR. (E) Visualization of B-factor analysis of binding site rigidity. (F) Interaction frequency analysis of modified Top1 with key residues. (G, H) Comparison of binding mode structures of Top1 and modified Top1 at the VEGFR binding site. (I) Comparison of binding free energy of Top1 and modified Top1 calculated based on molecular mechanics / generalized Born surface area (MM / GBSA) method. (J) Circular dichroism (CD) spectrum of VP, showing its characteristic beta-fold conformation. (K) Analysis of the secondary structure composition of VP. (L) Mass spectrometry analysis confirms the successful synthesis of VP ([M+H]+ = 1490.86). (M) Dose-dependent effect of different concentrations of VP on VEGF-A expression levels in HUVEC cells (n=4).

[0036] Figure 2 : Molecular interaction analysis, peptide engineering (AF-PBA), and development of MXene-based composite materials (MPA). (A) 3D conformational simulation of the binding of anti-inflammatory peptide (AF) to TNF-α. (B) 2D interaction map of the AF-TNF-α hydrogen bond network. (C) Analysis of hydrophobic contact residues at the AF-TNF-α interface. (D) Schematic diagram of AF-PBA synthesis for controlled release (showing the reaction of the anti-inflammatory peptide with 4-carboxyphenylboronic acid). (E) Circular dichroism (CD) spectra of the secondary structures of AF and AF-PBA. (F) Quantitative analysis of the changes in the secondary structure composition before and after PBA modification. (G, H) Mass spectra validating the molecular weights of AF and AF-PBA. (I) In vitro anti-inflammatory efficacy evaluation of AF-PBA (n=4). (J) Schematic diagram of the synthesis process of MXene@PDA nanosheets. (K) Transmission electron microscopy (TEM) image and elemental distribution map of MXene@PDA. (L) UV-Vis spectroscopy validation of conductive materials. (M) Comparison of Raman spectra of fresh and aged conductive materials. (N) Comparison of colloidal stability of conductive materials and control group (after 2 months of storage). (O) Verification of successful synthesis of MPA by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). (P, Q) Atomic force microscopy (AFM) 3D surface morphology, planar morphology, and height profile analysis of MXene and MPA (n=3).

[0037] Figure 3 Construction and functional validation of the MP-BTR platform (PEV / MPA hydrogel). (A, B) Preparation process, injectability demonstration, and conductivity demonstration of PEV / MPA hydrogel. (C) Statistical analysis of the conductivity of hydrogels at different MPA concentrations (1-6 mg / mL). (D, E) Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) curve analysis of different hydrogel groups. (F) Comparative analysis of the microstructure of different hydrogels using scanning electron microscopy (SEM). (GI) Rheological property analysis of different hydrogels: frequency-dependent viscoelasticity (storage modulus G' / loss modulus G''), strain amplitude-dependent shear thinning behavior, and self-healing properties. (J) Swelling rate of each hydrogel group in physiological buffer solution. (K) AF-PBA controlled release kinetic curves under simulated physiological (PBS pH 7.4) and pathological (PBS pH 6.5, H2O2) conditions. Statistical significance was determined using two-way ANOVA. p<0.0001. (L, M) Cell viability thermograms of HT22 neurons and BV2 microglia after 48 hours of co-culture with hydrogel (n = 6). (NP) Representative immunofluorescence images and quantitative analysis of TNF-α and IL-1β expression in BV2 microglia after 48 hours of hydrogel treatment following glucose-oxygen deprivation (OGD). (QS) Tubular structures of HUVEC vascular endothelial cells after 48 hours of hydrogel treatment following OGD, as well as quantitative analysis of tube length and node number, revealed by FDA staining. (T) Semi-quantitative analysis of HUVEC cell migration rate in scratch assays after OGD treatment and different treatments.

[0038] Figure 4 : The regulatory effect of the MP-BTR platform on cell function in vitro. (A, B) JC-1 confocal microscopy images used to assess mitochondrial membrane potential (MMP), and semi-quantitative analysis of MMP (red / green fluorescence intensity ratio) in HT22 neurons after treatment with oxygen deprivation (OGD) and co-culture with hydrogel for 48 hours. (CF) Oxidative stress markers (4HNE, Nrf2) and inflammatory markers (IL-1β, NLRP3) in BV2 cells. (GH) Angiogenic markers (CD31, VEGF-A) in HUVEC cells. (IK) Quantitative analysis of VEGF-A and CD31 expression levels (I, J) and representative immunofluorescence images (K) in HUVEC cells after OGD treatment and co-culture with hydrogel for 48 hours. (L) Inhibition of apoptosis and restoration of MMP by regulating oxidative stress (ROS / Nrf2) and inflammation (TNF-α / IL-1β / NLRP3). (M) promotes angiogenesis by upregulating CD31 and VEGF-A.

[0039] Figure 5The MP-BTR platform alleviates ischemic stroke injury by modulating the oxidative-inflammatory cascade. (A) Experimental timeline design for in vivo treatment evaluation. (B) Cerebral edema (brain water content) measured by gravity method (wet-dry weight method) on days 3 and 7 post-treatment (3 dpi and 7 dpi). (C) Hematoxylin-eosin (H&E) stained brain tissue sections of each group at 3 dpi, showing the infarct area. (D, H) Biodegradation fluorescence photographs (D) and degradation efficiency curves (H) of MP-BTR hydrogel in vivo (n=3). (EG) Representative immunofluorescence images of key markers in brain tissue at 3 dpi: (E) lipid peroxidation marker 4HNE (red) and antioxidant factor Nrf2 (green); (F) microglial marker IBA1 (green) and inflammasome NLRP3 (red); (G) pro-inflammatory factor IL-1β (green). Quantitative analysis of the expression levels of 4HNE, Nrf2, IBA1, NLRP3 and IL-1β in brain tissue at 3 dpi (IM).

[0040] Figure 6 : The hydrogen bond network between the Top1 compound and key residues of VEGFR. Detailed Implementation

[0041] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0042] Unless otherwise stated, the technical and scientific terms used in this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] "MXene" refers to a two-dimensional (2D) transition metal carbide, nitride, or carbonitride. In a preferred embodiment of the invention, MXene specifically refers to Ti3C2 or a derivative thereof.

[0044] "Conductive nanocomposite material (MPA)" refers to the surface-engineered MXene nanocomposite material developed in this invention. Its core structure is MXene, and its surface is modified with a synergistic stabilizing layer (such as PDA and CNC), and further grafted with bioactive molecules (such as AF-PBA peptide).

[0045] "Angiogenic peptide (VP)" refers to a peptide that has been designed or screened to promote the proliferation, migration, or formation of tubular structures of vascular endothelial cells, or to activate angiogenic signaling pathways (such as the VEGFR pathway). In a preferred embodiment of the present invention, VP refers to a peptide with the amino acid sequence CLEYKLHDFGYC.

[0046] "Anti-inflammatory peptide (AF)" refers to a peptide that can reduce or inhibit inflammatory responses, for example, by binding to pro-inflammatory cytokines such as TNF-α. In a preferred embodiment of the present invention, AF refers to a peptide with the amino acid sequence MQMKKVLDS.

[0047] "Hydrogel": refers to a three-dimensional network of hydrophilic polymers capable of absorbing and retaining large amounts of water. In this article, "hydrogel" specifically refers to a type of hydrogel that can be rapidly (e.g., under physiological conditions) formed by mixing two or more precursor solutions and then chemically cross-linking them (e.g., "click chemistry"). in situ Injectable hydrogels that form a gel.

[0048] "Treatment" or "treatment" refers to the application of the composition of the present invention to an individual suffering from a target disease or condition (such as ischemic stroke) with the aim of relieving symptoms, slowing disease progression, repairing damaged tissues, or improving biological functions (such as motor or electrophysiological functions).

[0049] Example 1: Preparation of Conductive Nanocomposite Material (MPA) The preparation process of MPA nanocomposites includes: (1) Preparation of MXene@PDA: Ultrathin Ti3C2MXene nanosheets (Beijing Beike New Material Technology Co., Ltd.) were dispersed in a buffer solution containing dopamine hydrochloride (10 mM Tris-HCl buffer, pH 8.5), where the concentration of dopamine hydrochloride was 2 mg / mL and the mass ratio of Ti3C2MXene nanosheets to dopamine was 1:0.6. Under magnetic stirring, the reaction was carried out at room temperature (25℃) for 1 h (to allow dopamine to polymerize in situ on the Xene surface to form a PDA coating); after the reaction, the precipitate was collected by centrifugation and washed three times with deionized water to remove unreacted dopamine and free polydopamine particles, thus obtaining MXene@PDA.

[0050] (2) Preparation of MP nanosheets (PDA / CNC synergistic stabilization): According to the mass ratio of dopamine hydrochloride to cellulose nanocrystals (CNC) of 5:1, an aqueous dispersion of cellulose nanocrystals (CNC) with a concentration of 1 mg / mL (purchased from Sigma-Aldrich) was added to the MXene@PDA obtained in the above steps. The mixture was magnetically stirred at room temperature (25℃) and the reaction was continued for 3 hours. After the reaction was completed, the precipitate was collected by centrifugation and washed with water to form more stable and better dispersible MP (MXene@CNC@PDA) nanosheets.

[0051] (3) Synthesis of AF-PBA: 100 mg of anti-inflammatory peptide AF (amino acid sequence: MQMKKVLDS, SEQ ID No. 1) was dissolved in 8 mL of DMF, and 183 mg of 4-carboxyphenylboronic acid (PBA), 380 mg of HBTU and 435 µL of DIPEA were added. The reaction promoted the formation of amide bonds between the peptide and PBA to obtain AF-PBA.

[0052] (4) Preparation of MPA (AF-PBA grafting): The synthesized AF-PBA solution was added to the aqueous dispersion of MP nanosheets, and the mass ratio of AF-PBA to MP was controlled to be 1:6. The reaction was carried out at room temperature (25℃) with magnetic stirring for 8 hours. After the reaction was completed, the mixture was freeze-dried to obtain the final MPA nanocomposite material.

[0053] Example 2: Preparation and Characterization of PEV / MPA Hydrogel 1. Preparation of precursor solution: Solution A: Dissolve the angiogenic peptide VP (amino acid sequence: CLEYKLHDFGYC, SEQ ID No. 2) in PBS buffer to obtain a VP solution of 2 mg / mL.

[0054] Solution B: Dissolve 4-arm PEG-maleimide (4-Arm PEG-Mal) in PBS buffer to obtain a 1 mg / mL 4-Arm PEG-Mal solution.

[0055] Solution C: The MPA nanocomposite material prepared in Example 1 was ultrasonically dispersed in PBS buffer to form a uniform suspension with a concentration of 4 mg / mL.

[0056] 2. Hydrogel formation: Solutions A, B, and C (with a mass ratio of 4:3:10 for the pro-angiogenic peptide VP, 4-Arm PEG-Mal, and MPA nanocomposite material) were rapidly mixed to obtain a PEV / MPA hydrogel (also known as the MP-BTR platform) with a final MPA concentration of 4 mg / mL.

[0057] The following experimental examples demonstrate the beneficial effects of the present invention.

[0058] Unless otherwise specified, all experimental examples of this invention use the hydrogel prepared in Example 1 (i.e., the final MPA concentration is 4 mg / mL) for characterization and performance testing.

[0059] Example 1: Preparation, Screening and Characterization of PEV / MPA Hydrogels 1. Optimized screening of pro-angiogenic peptides (VP) The present invention uses a computer-designed angiogenesis-promoting peptide (VP). Figure 1 J~L), this VP screened nearly a million candidate molecules through a complex computer-aided screening process (including ADMET analysis, high-throughput screening, molecular docking, and molecular dynamics simulation). Figure 1 A) and obtained through optimization.

[0060] This sequence (originating from the hydrogen bond network between the Top1 compound and key residues of VEGFR, such as...) Figure 6 The modified (shown) was designed to specifically bind to and activate the vascular endothelial growth factor receptor (VEGFR). Molecular dynamics simulations ( Figure 1 BI confirmed that VP can form a stable hydrogen bond and hydrophobic interaction network with key residues (such as Asn897 and Glu1003) at the active site of VEGFR, and its binding affinity is even better than that of the original Top1 compound.

[0061] In vitro functional experiments ( Figure 1 M) confirmed that VP can effectively upregulate the expression of VEGF-A in human umbilical vein endothelial cells (HUVECs), demonstrating its strong pro-angiogenic potential.

[0062] 2. Characterization of PEV / MPA hydrogel The preparation method of Example 2 is referenced, except that the final concentration of MPA in the hydrogel is controlled to be 1, 2, 3, 4, 5 or 6 mg / mL to prepare different hydrogels.

[0063] Molecular interaction analysis, development of AF-PBA and MPA, etc. Figure 2 As shown in A~Q. Physicochemical properties are characterized as follows: Figure 3 As shown: (1) Injectability and mechanical compatibility: The platform prepared by this invention using in-situ click chemistry has good injectability. Figure 3 (A, B), can fill irregular ischemic lesion cavities. Its rheological properties ( Figure 3 The GI shows that the storage modulus (G') is always higher than the loss modulus (G''), forming a stable elastic network with shear thinning and self-healing properties.

[0064] (2) Brain tissue compatibility: This hydrogel has a low swelling ratio (<10%). Figure 3 J), which is crucial in the limited space of the cranial cavity to avoid additional compression of surrounding brain tissue.

[0065] (3) Stable conductivity: The PEV / MPA hydrogel exhibits significant and stable conductivity (the conductivity is highest at 4 mg / mL MPA, reaching 1.2 mS / cm). Figure 3C), as well as lower impedance (EIS) and enhanced redox activity (CV) Figure 3 D, E).

[0066] (4) Inflammatory response and anti-inflammatory effects: Under simulated inflammatory conditions (pH 6.5 + H2O2), the cumulative release rate of AF-PBA (approximately 56.75%) was significantly higher than that under physiological conditions (pH 7.4, approximately 36.24%). Figure 3 K).

[0067] (5) Antioxidant properties: PEV / MPA hydrogel showed a clearance rate of approximately 82.9% as measured by the DPPH method.

[0068] (6) Multiple bioactivities: This platform simultaneously promotes angiogenesis and exhibits excellent cell compatibility (>95% viability in HT22 and BV2 cells). Figure 3 L, M).

[0069] (7) Microscopic morphology characterization: Scanning electron microscope (SEM) images ( Figure 3 F) shows that, compared with pure PEV hydrogel, PEV / MPA hydrogel exhibits a denser and more uniform network structure.

[0070] (8) Anti-neuroinflammatory assessment: Immunofluorescence images ( Figure 3 N) and quantitative analysis ( Figure 3 (O, 3P) indicated that oxygen-glucose deprivation (OGD) treatment significantly upregulated the expression levels of pro-inflammatory factors TNF-α and IL-1beta in BV2 microglia (p<0.0001). However, treatment with PEV / MPA hydrogel significantly suppressed the levels of these pro-inflammatory factors (p<0.0001).

[0071] (9) Angiogenesis-tube formation experiment ( Figure 3 Q~S): Tube formation experiments of HUVEC cells showed that after hydrogel treatment, the cells were able to form a denser vascular network. Quantitative analysis showed that, compared with the control group, the hydrogel group (containing the pro-angiogenic peptide VP) had significantly increased total tube length and number of branch nodes.

[0072] (10) Angiogenesis-migration experiment ( Figure 3 T): Semi-quantitative analysis of the scratch assay showed that, compared with the control group and the OGD group, the PEV composite hydrogel significantly improved the migration rate of HUVEC cells.

[0073] Experimental Example 2: In vitro cell function regulation using PEV / MPA hydrogel 1. Experimental Methods Construction of an oxygen-glucose deprivation (OGD) cell model (simulating an ischemic microenvironment): The culture medium for conventionally cultured cells (HT22 hippocampal neurons, BV2 microglia, or HUVECs vascular endothelial cells) was replaced with DMEM medium without D-glucose. Cells were placed in a hypoxic incubator at 37°C, 1% O2, 94% N2, and 5% CO2 for 2 hours to simulate ischemic hypoxia injury. After OGD treatment, cells were returned to standard culture conditions and reoxygenated and reglucose-replenished culture in complete medium or medium containing hydrogel samples.

[0074] Treatment method: Cells were subjected to the above-mentioned 2-hour OGD treatment 24 hours after seeding and culture. After treatment, the old culture medium was removed and fresh culture medium containing different groups of hydrogels (PEV, PEV / MXene, PEV / MP, PEV / MPA) was added. After culturing for 48 hours (ROS detection was performed after 24 hours), various indicators were measured.

[0075] Effect detection method: Cell viability and toxicity assessment: CCK-8 method: After co-culturing for 48 hours, cell viability was detected using the CellCounting Kit-8 (CCK-8) kit (n=6).

[0076] Live / dead cell staining: HT22 cells were stained with a double staining method using fluorescein diacetate (FDA, which stains live cells green) and propidium iodide (PI, which stains dead cells red). Cell viability was observed and calculated using a fluorescence microscope.

[0077] Oxidative stress level detection: ROS detection: 24 hours after OGD treatment and hydrogel therapy, cells were incubated with a 10 uM DCFH-DA fluorescent probe, and the intracellular reactive oxygen species (ROS) level (green fluorescence intensity) was detected by fluorescence microscopy.

[0078] Mitochondrial membrane potential (MMP) detection: HT22 cells were stained with JC-1 fluorescent probe and changes in mitochondrial membrane potential were observed using laser confocal scanning microscopy (CLSM) (the red / green fluorescence ratio reflects the health of mitochondria).

[0079] Inflammation and angiogenesis factor detection: ELISA detection: Cell culture supernatant of BV2 microglia (detecting inflammatory factors: TNF-alpha, IL-1beta, NLRP3) and HUVECs (detecting angiogenesis factors: VEGF-A, CD31) was collected and quantitatively analyzed using an enzyme-linked immunosorbent assay (ELISA) kit.

[0080] Immunofluorescence staining: Cells were fixed and labeled with specific antibodies (such as TNF-α, IL-1beta, VEGF-A, CD31), and the expression levels of related proteins were observed and quantitatively analyzed by CLSM.

[0081] 2. Experimental Results Experimental results are as follows Figure 4 As shown.

[0082] (1) Neuroprotection and mitochondrial function: In a simulated ischemic oxygen-glucose deprivation (OGD) model, JC-1 staining ( Figure 4 A, B) show that PEV / MPA can reverse the OGD-induced decrease in mitochondrial membrane potential (MMP), restore mitochondrial function, and thus inhibit early apoptosis.

[0083] (2) Anti-inflammatory and antioxidant effects: ELISA and immunofluorescence analysis ( Figure 4 CF, Figure 3 NP) showed that PEV / MPA hydrogel significantly downregulated the expression of pro-inflammatory factors (TNF-α, IL-1β) and inflammasome components (NLRP3) in BV2 microglia, while downregulating the oxidative stress marker (4HNE) and upregulating the endogenous antioxidant factor (Nrf2).

[0084] (3) Promoting angiogenesis: In the HUVEC model ( Figure 4 GK, Figure 3 QS), PEV / MPA hydrogel (and its VP component) significantly upregulated the expression of angiogenesis markers (VEGF-A, CD31) and significantly increased the formation, total length and number of branch points of tubular structures.

[0085] (4) Synergistic treatment mechanism: Figure 4 L and 4M revealed that the PEV / MPA hydrogel alleviates OGD-induced cell damage through a triple synergistic mechanism of "antioxidant-anti-inflammatory-pro-angiogenesis": On the one hand, the hydrogel activates the endogenous antioxidant defense system by downregulating lipid peroxides (4HNE) and upregulating the antioxidant transcription factor (Nrf2), while improving the inflammatory microenvironment by inhibiting the expression of pro-inflammatory factors (TNF-α, IL-1β) and the NLRP3 inflammasome, thereby effectively inhibiting neuronal apoptosis and restoring mitochondrial function; on the other hand, the platform successfully constructs a microenvironment conducive to angiogenesis by upregulating the expression of key angiogenic factors (VEGF-A) and endothelial cell markers (CD31), thereby achieving comprehensive repair of damaged tissues through multi-target regulation.

[0086] Experimental Example 3: Early in vivo protection and inflammation / oxidation regulation of PEV / MPA hydrogel 1. Experimental Methods Establishment of a photothrombotic (PT) ischemic stroke mouse model: Male C57 BL / 6 mice were selected and anesthetized with isoflurane (induction concentration 5%, maintenance concentration 2%). Rose Bengal (concentration 1%) was injected intraperitoneally. A bone window with a diameter of approximately 3 mm was drilled 2 mm to the right of the anterior fontanelle, ensuring the integrity of the dura mater. The bone window area was continuously irradiated for 7 minutes using a 2 mm diameter laser (power 100 mW, wavelength 560 nm) to induce thrombus formation. After irradiation, the incision was sutured and disinfected.

[0087] Experimental grouping and treatment: Mice after modeling were randomly divided into 6 groups: PT model group, Sham group, PEV hydrogel group, PEV / MXene hydrogel group, PEV / MP hydrogel group, and PEV / MPA hydrogel group. On the 3rd day after modeling (3 days post-IS), the corresponding hydrogel was injected into the center of the infarct area, 2.0 mm below the dura mater, using a microinjector for in situ treatment.

[0088] Cerebral edema assessment: On day 3 (3 dpi) and day 7 (7 dpi) after treatment, brain water content was measured using the gravity method (wet and dry weight method) to assess the relief of cerebral edema.

[0089] Histopathological evaluation: On day 3 post-treatment (3 dpi), brain tissue was harvested and prepared into sections for hematoxylin and eosin (H&E) staining to observe infarct volume, vacuolation, and tissue necrosis, and to assess the neuroprotective effect. Simultaneously, the heart, liver, spleen, and kidneys were stained with H&E to assess the biosafety of the materials.

[0090] Oxidative stress level detection: The expression levels of lipid peroxidation marker 4HNE and antioxidant transcription factor Nrf2 in brain tissue sections on day 3 after treatment were detected by immunofluorescence double labeling method to assess the activation of the endogenous antioxidant defense system.

[0091] Neuroinflammatory level detection: Immunofluorescence staining was used to analyze the expression and distribution of microglia markers IBA1, inflammasome NLRP3, and pro-inflammatory cytokine IL-1beta on day 3 after treatment, and to evaluate the inhibitory effect of hydrogel on microglia polarization and inflammatory cascade.

[0092] Hydrogel degradation monitoring: Cy7 fluorescently labeled PEV / MPA hydrogels were injected into the infarct cavity, and fluorescence signals were monitored at different time points (e.g., days 1, 3, 7, 14, 21, and 35) using a small animal in vivo imaging system (IVIS) to assess the retention and degradation of the material in vivo.

[0093] 2. Experimental Results Experimental results are as follows Figure 5 As shown.

[0094] Injecting PEV / MPA hydrogel into a PT stroke mouse model ( Figure 5 A): (1) Reduction of cerebral edema and infarction: On days 3 and 7 after injection, the brain water content (cerebral edema index) in the PEV / MPA group was significantly lower than that in the PT control group ( Figure 5 B). H&E staining ( Figure 5 C) The infarct volume was significantly reduced, and the degree of necrosis was alleviated.

[0095] (2) Monitoring of hydrogel degradation: in vivo fluorescence imaging ( Figure 5 D) and quantitative analysis of fluorescence intensity ( Figure 5 The results showed that the PEV / MPA hydrogel gradually biodegraded over time after injection and was completely degraded by day 35 after injection.

[0096] (3) Inhibition of oxidative stress: At 3 dpi, the PT group showed strong oxidative damage (high expression of 4HNE) and depleted antioxidant defenses (Nrf2). + (Low expression). PEV / MPA treatment significantly reversed this phenomenon, 4HNE + The level was close to that of the sham surgery group, while Nrf2 + The expression was strongly upregulated. Figure 5 E, I, J).

[0097] (4) Inhibition of neuroinflammation: The PT group showed a large number of activated microglia (IBA1) + ) and strong NLRP3 + Inflammatory body activation ( Figure 5 F, K, L). PEV / MPA treatment significantly reduced IBA1. + The cell count was reduced, microglia morphology returned to a resting state, and NLRP3 expression was almost completely suppressed. Simultaneously, the expression of the pro-inflammatory factor IL-1β was also significantly inhibited. Figure 5 G, M).

[0098] In summary, this invention provides a conductive peptide-based multimodal platform for tissue repair, its preparation method, and its applications. This invention uses Ti3C2MXene as a conductive core, coated with a polydopamine / cellulose nanocrystal synergistic stabilizing layer, and covalently links the anti-inflammatory peptide AF via a phenylboronic acid responsive linker to construct a conductive nanocomposite material, achieving intelligent release triggered by ROS / acidic microenvironment. Furthermore, a conductive peptide-based multimodal platform is prepared using the conductive nanocomposite material and a VEGFR-targeting bioactive peptide scaffold. This platform integrates conductivity, anti-inflammation, antioxidant properties, and angiogenesis promotion, significantly improving the inflammatory microenvironment after stroke and promoting neurological function recovery through mechano-electro-biochemical multimodal synergistic effects. This platform has significant implications for the preparation of drugs for the prevention and / or treatment of neurological injury.

Claims

1. An electrically conductive nanocomposite, characterized by, The conductive nanocomposite is prepared from a bioactive molecule and a synergistically stable layer coated conductive nanomaterial; wherein the mass ratio of the bioactive molecule and the synergistically stable layer coated conductive nanomaterial is 1: (5-7).

2. The electrically conductive nanocomposite of claim 1, wherein, The bioactive molecule is an anti-inflammatory peptide modified by 4-carboxyphenylboronic acid; wherein the mass ratio of the anti-inflammatory peptide and the 4-carboxyphenylboronic acid is 1: (1-5). The synergistically stable layer coated conductive nanomaterial is prepared from a conductive nanomaterial, dopamine or a salt thereof, and a cellulose nanomaterial; wherein the conductive nanomaterial is a two-dimensional transition metal carbide, nitride, or carbonitride; the mass ratio of the conductive nanomaterial and the dopamine or a salt thereof is 1: (0.2-2); and the mass ratio of the dopamine or a salt thereof and the cellulose nanomaterial is (1-10):

1.

3. The electrically conductive nanocomposite of claim 1, wherein, The preparation of the synergistically stable layer coated conductive nanomaterial comprises the following steps: mixing and reacting the conductive nanomaterial with a dopamine or a salt thereof solution, and then adding a cellulose nanomaterial to continue the reaction, to obtain the product; wherein the mixing and reacting is performed at a temperature of 10-40°C for 0.5-2 hours; and the continuing reaction is performed at a temperature of 10-40°C for 2-4 hours. Preferably, the mixing and reacting is performed at a temperature of 20-30°C for 1 hour; and the continuing reaction is performed at a temperature of 20-30°C for 3 hours. The preparation of the bioactive molecule comprises the following steps: reacting an anti-inflammatory peptide, 4-carboxyphenylboronic acid, a condensing agent, and an organic base, to obtain the product; wherein the mass ratio of the anti-inflammatory peptide, 4-carboxyphenylboronic acid, condensing agent, and organic base is 1: (1-5): (3-5): (4-6); and the reaction is performed in an organic solvent. More preferably, the mass ratio of the anti-inflammatory peptide, 4-carboxyphenylboronic acid, condensing agent, and organic base is 1: (1.5-2): (3.5-4): (4-4.5); the condensing agent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; the organic base is N,N-diisopropylethylamine; and the reaction is performed in N,N-dimethylformamide.

4. A method of preparing the electrically conductive nanocomposite of any one of claims 1 to 3, characterized in that, The method comprises the following step: reacting a bioactive molecule and a synergistically stable layer coated conductive nanomaterial, to obtain a conductive nanocomposite.

5. An angiogenic peptide, characterized in that, The amino acid sequence of the pro-angiogenic peptide is shown in SEQ ID No.

2.

6. A hydrogel composition, characterized by, The hydrogel composition comprises the conductive nanocomposite of any one of claims 1-3, a pro-angiogenic peptide, and a crosslinking agent; wherein the mass ratio of the conductive nanocomposite, pro-angiogenic peptide, and crosslinking agent is (1-6): (1-5): (3.4-17).

7. The hydrogel composition of claim 6, wherein, The amino acid sequence of the pro-angiogenic peptide is shown in SEQ ID No. 2; and the crosslinking agent is a multi-armed polyethylene glycol derivative, preferably a 4-armed PEG-maleimide.

8. A multifunctional hydrogel, characterized by, The multifunctional hydrogel is obtained by mixing the conductive nanocomposite solution, pro-angiogenic peptide solution, and crosslinking agent solution of claims 6 or 7, and then reacting in situ.

9. Use of the electrically conductive nanocomposite according to any one of claims 1 to 3, the pro-angiogenic peptide according to claim 5, the hydrogel composition according to claim 6 or 7, or the multifunctional hydrogel according to claim 8 for the preparation of a tissue repair material.

10. Use according to claim 9, characterized in that, The tissue repair material is a material for treating ischemic stroke, hemorrhagic stroke, traumatic brain injury, or spinal cord injury.

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