Magnetoelectric hydrogel for treating cervical nerve root injury

Abstract

The invention relates to the technical field of medical materials, in particular to magnetoelectric hydrogel for treating cervical nerve root injury. The magnetoelectric hydrogel is prepared from a combined product and comprises a solution A and a solution B, the solution A comprises 0.1%-2% (w / v) of Fe3O4 (at) BaTiO3 (at) PDA nanoparticles (FBD) and 10%-30% (w / v) of phenylboronic acid modified oxidized dextran (PBA-OD); and the solution B comprises 20-40% (w / v) of a photo-crosslinkable gel matrix. According to the magnetoelectric hydrogel combined product, a bi-crosslinking structure with a first borate network and a second photocuring network is rapidly formed on the surface of a target tissue in the modes of A / B double-liquid split charging, alternate spraying and photocrosslinking, so that the obtained hydrogel has excellent sprayability, rapid gelation ability, strong tissue adhesiveness and magnetic response performance; the effective repair of the neck nerve root injury can be realized under the activation of an external magnetic field.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a magnetoelectric hydrogel for treating cervical nerve root injuries. Background Technology

[0002] Cervical nerve root injury (CNRI) is caused by compression or damage to the cervical nerve roots. Clinically, it often manifests as neck and shoulder pain, muscle weakness, sensory disturbances, and weakened deep tendon reflexes. Intervertebral disc herniation is the most common cause, and it is conservatively estimated that by 2050, the number of people suffering from this condition worldwide will approach 990 million, making it a serious public health problem. Although current surgical interventions can relieve nerve compression, many patients still experience chronic symptoms post-surgery and require long-term rehabilitation. Furthermore, the complex anatomy, deep location, and numerous branches of the cervical nerve roots greatly limit the application of conventional nerve repair materials and methods, necessitating the development of more targeted and adaptive repair strategies.

[0003] Since nerve signals are essentially transmitted in the form of electrical signals, electrical stimulation (ES) has been proven to significantly promote nerve regeneration. However, current percutaneous or implantable ES systems mostly rely on rigid connections or battery power, which are complex to operate and have poor patient compliance. Magnetoelectric nanoparticles (MENPs) offer a potential solution to these problems. When activated by a remote magnetic field, MENPs can generate local electrical pulses at the injury site, eliminating wire dependence and improving stimulation precision. Given that effective stimulation requires proximity to target cells at the micrometer scale, achieving stable anchoring of MENPs is crucial. Therefore, bioactive hydrogel carriers that can mimic the natural microenvironment have become an ideal choice for in-situ delivery and fixation of MENPs. Currently, most methods involve constructing composite hydrogels into conduits or patches and wrapping them around the nerve, using an electric field to reduce nerve inhibition and promote neuroprotection. However, the application of such systems in the deep, narrow, and complex branching structures of the cervical nerve roots is limited, and there is an urgent need for novel material systems with in-situ penetration capabilities and adaptive adhesion properties. Summary of the Invention

[0004] This invention covers the following technical solutions: One aspect of the present invention relates to a magnetoelectric hydrogel combination product, comprising solution A and solution B; Solution A comprises 0.1-2% (w / v) Fe3O4@BaTiO3@PDA nanoparticles (FBD) and 10-30% (w / v) phenylboronic acid-modified dextran oxide (PBA-OD). Solution B contains 20-40% (w / v) of a photocrosslinkable gel matrix.

[0005] Another aspect of the present invention relates to a magnetoelectric hydrogel obtained by mixing the combined products as described above; wherein the PBA-OD and FBD form a first cross-linked network through borate ester bonds, and the gel matrix forms a second cross-linked network under light irradiation, so that the resulting hydrogel has sprayable and tissue adhesion properties.

[0006] Another aspect of the invention relates to the use of the combined products described above, or the magnetoelectric hydrogel described above, in the preparation of medical formulations / repair materials for treating cervical nerve root injuries.

[0007] The magnetoelectric hydrogel combination product of this invention rapidly forms a double-crosslinked structure on the surface of the target tissue through A / B two-liquid dispensing, alternating spraying, and photocrosslinking. This structure combines a first borate ester network and a second photocurable network, resulting in a hydrogel with excellent sprayability, rapid gelation ability, strong tissue adhesion, and magnetic response properties. Under the activation of an external magnetic field, it can enhance nerve regeneration, accelerate myelin formation, and improve the recovery of sensory and motor functions, thereby achieving effective repair of cervical nerve root injuries. It has comprehensive technical effects such as strong structural adaptability, convenient operation, and high safety. Attached Figure Description

[0008] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0009] Figure 1 Synthesis and performance characterization of dopamine-modified Fe3O4@BaTiO3 (FBD) nanoparticles. (A) A schematic diagram of the synthesis process of FBD nanoparticles is shown, including Fe3O4 surface deposition, BaTiO3 conversion, and PDA coating steps; (B) Transmission electron microscopy (TEM) images show that the nanoparticle structure is intact; (C) Energy dispersive X-ray spectroscopy (EDX) shows that the elements are uniformly distributed; (D) Zeta potential results indicate successful PDA functionalization; (E) Fourier transform infrared spectroscopy (FT-IR) confirms the functional group changes; (F) X-ray diffraction (XRD) analysis shows that the crystal structure of Fe3O4 and BaTiO3 is maintained during the synthesis process; (G) Hysteresis loops indicate that the nanoparticles possess superparamagnetic properties, with a magnetic saturation intensity of 18 emu·g. -1(H) Schematic diagram of measuring magnetoelectric effect using atomic force microscopy (AFM); (I–J) Under a magnetic field of 1000 Oe, the nanoparticles exhibit typical piezoelectric amplitude-voltage and phase response curves, indicating that they possess polarization reversal and magnetoelectric coupling capabilities; (K) Finite element analysis shows that under a magnetic field of 25 mT, FBD particles can stably generate potential, electric field, and strain signals, further verifying their magnetoelectric conversion performance.

[0010] Figure 2 Preparation and performance characterization of in-situ sprayable magnetoelectric hydrogel (DG / FBD). (A–G) Performance characterization before photocrosslinking: (A) Schematic diagram of two-step spraying process: Solution A is phenylboronic acid functionalized oligodopamine (PBA-OD) composite with FBD, and solution B is methacrylamide gelatin (MG); (B) Spraying cycle deposition process; (C) Gelation time test; (D) Viscosity change at different shear rates; (E) UV-Vis absorption spectrum at 295 nm; (F) Viscosity stability under different temperature conditions; (G) Adhesion strength on different substrates (pigskin, metal, glass, plastic). (H–O) Performance characterization after photocrosslinking: (H) Infrared spectrum of carbon-carbon covalent bonds formed by photocrosslinking (the above figure is a schematic diagram of UV-induced crosslinking); (I) Rheological analysis of storage modulus changes during crosslinking; (J) Microscopic mechanical modulus distribution obtained by nanoindentation test; (K) Quantitative analysis of elastic modulus; (L) Hysteresis loop measured by vibrating sample magnetometer (VSM), indicating that the hydrogel has superparamagnetic properties; (M) Schematic diagram of shear adhesion test; (N) Quantitative results of shear adhesion strength; (O) Demonstration of adaptive adhesion behavior under coiled pipe and finger deformation. Data are expressed as mean ± standard error (SEM), *p<0.05, **p<0.001 indicate statistical difference.

[0011] Figure 3 Precursor solutions A and B are applied sequentially by spraying to form a hydrogel within the cavity.

[0012] Figure 4 Rheological gelation kinetics and gelation points of DG, DG / FB and DG / FBD hydrogels.

[0013] Figure 5 Viscosities of the DG / FBD hydrogel component at different shear rates under different pH conditions.

[0014] Figure 6 Adaptive adhesion of DG / FBD hydrogel under finger deformation.

[0015] Figure 7Magnetoelectric hydrogel promotes cervical stromal stem cell (BMSC) neural differentiation in vitro and enhances CNRI repair in vivo. (A–I) Results of in vitro BMSC neural differentiation regulation experiments. (A–D) Immunofluorescence staining results of neural markers at different time points: Tuj1 on day 7, neurofilament protein (NF) on day 14, and postsynaptic dense protein PSD95 and glial fibrillary acidic protein GFAP on day 21; (E–H) Quantitative analysis of the fluorescence intensity of the corresponding neural markers, showing that magnetoelectric hydrogel combined with magnetic stimulation significantly enhances the expression of neural differentiation markers and inhibits the expression of astrocyte markers. (I–N) Evaluation of in vivo cervical nerve root injury (CNRI) repair effect at week 4 postoperatively. (I) Schematic diagram of CNRI rat model construction; (J) H&E staining of regenerated cervical nerve root tissue; (K) Transmission electron microscopy (TEM) image of mid-segment myelin axons in the regenerated region; (L–M) Statistical analysis of myelin thickness and axon diameter; (N) Immunofluorescence co-staining results of bone morphogenetic protein (BMP, red), NF200 (blue-green), and S100 (yellow) in regenerated tissue, characterizing the degree of nerve repair. Data are expressed as mean ± standard error (mean ± SEM), with **p<0.01, ***p<0.001, and ****p<0.0001 indicating statistical significance.

[0016] Figure 8 After spraying the hydrogel, the DG / FBD spray hydrogel completely covers and adheres to the complex nerve branches and numerous tiny nerve endings at the distal end of the sciatic nerve.

[0017] Figure 9 CNRI Postoperative Functional Recovery Assessment in Rats. (A) Hargreaves Thermal Pain Test; (B) Von Frey Mechanosensitivity Test; (C) Grip Strength Test for Motor Function Assessment; (D) Schematic Diagram of Electrophysiological Analysis Experimental Design; (E–I) Representative Compound Muscle Action Potential (CMAP) Waveforms; (J) Quantitative Analysis of CMAP Amplitude; (K) Nerve Conduction Velocity (NCV) Measurement Results. Each group had a sample size of more than 8 rats, and each experiment was repeated three times, with the average value taken. Data are expressed as mean ± standard error (SEM). **p<0.01, ***p<0.001, and ****p<0.0001 indicate statistical significance.

[0018] Figure 10RNA sequencing analysis 4 weeks after CNRI repair. (A) NMDS (Nonmetric Multidimensional Scale) analysis plot; (B) Volcano plot of differentially expressed genes (DEG) (red: upregulated, blue: downregulated); (C) GO enrichment analysis results; (D–E) GSEA (Gene Set Enrichment Analysis) pathway analysis; (F) Cross-validation of LASSO regression analysis results; (G) Biomarkers screened by LASSO regression; (H) Feature variables screened by Boruta algorithm; (I) Venn plot of overlapping genes; (J) Distribution of differentially expressed genes and biomarkers among different experimental groups.

[0019] Figure 11 Activation of the CXCL12-autophagy signaling axis. (A) Experimental validation strategy; (B–D) qRT-PCR analysis of Nefl, Msr1, and CXCL12; (E) CXCL12 protein expression level; (F) Immunofluorescence co-localization and quantification analysis of CXCL12 (green) and LC3B (yellow); (G) Transmission electron microscopy (TEM) images of autophagosomes; (H) Statistical quantification results of autophagosome counts. Data are expressed as mean ± standard error (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate statistical significance.

[0020] Figure 12 CXCL12 enhances Schwann cell autophagy. (A) Schematic diagram of the proposed mechanism of autophagy-mediated repair in Schwann cells; (B–C) Immunofluorescence staining of S100 (green), CXCL12 (yellow), LC3B (red), and DAPI (blue); (D–E) Quantitative analysis of LC3B and CXCL12 intensities; (F) Correlation analysis between CXCL12 and LC3B. Data are expressed as mean ± standard error (SEM). ns: no significant difference; *p<0.05, **p<0.01, ***p<0.001 indicate statistical significance.

[0021] Figure 13CXCL12 enhances Schwann cell autophagy through the PI3K / AKT / mTOR pathway. (A) Immunofluorescence image of PI3K; (B) Quantitative analysis of PI3K fluorescence intensity; (C) Protein bands of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR (with control: GAPDH); (DF) Relative protein expression levels of p-PI3K / PI3K, p-AKT / AKT, and p-mTOR / mTOR; (G) CXCL12 protein band; (H) Relative protein expression level of CXCL12; (I) Protein bands of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR (with control: GAPDH); (JL) Relative protein expression levels of p-PI3K / PI3K, p-AKT / AKT, and p-mTOR / mTOR; (M) Immunofluorescence image of LC3B; (N) Quantitative analysis of LC3B fluorescence intensity; (O) Schematic diagram of the mechanism of CXCL12-mediated autophagy therapy. Data are expressed as mean ± standard error (SEM), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared with the control group. Detailed Implementation

[0022] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0023] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms materials science, neurobiology, cell and tissue culture, and laboratory procedures used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0024] As used herein, the terms “and / or,” “or / and,” and “and / or” encompass any one of two or more of the relevant listed items, as well as any and all combinations of the relevant listed items, including any two of the relevant listed items, any more of the relevant listed items, or a combination of all the relevant listed items.

[0025] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0026] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0027] As used in this invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5%, of a given value or range. It also includes specific numbers, such as about 20 including 20.

[0028] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0029] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0030] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0031] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0032] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0033] In this invention, X % (w / v) = X grams of solute per 100 mL of solution. This definition applies to all content descriptions involving % (w / v) in this application, including the expression of the amount of each component added in component A and component B.

[0034] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0035] In this invention, "treatment" refers to the act or process of partially or completely alleviating, suppressing, improving, preventing the deterioration of, delaying the progression of, promoting tissue repair, restoring physiological function, reducing symptoms and / or reducing related risks of a disease or pathological state of the object to be treated.

[0036] In this invention, cervical nerve root injury (CNRI) refers to a type of peripheral nerve injury state in which the spinal nerve roots in the cervical region are compressed, stretched, torn, or contused, resulting in impaired nerve conduction function. This injury typically occurs at the C5–C8 cervical nerve roots, affecting both sensory and motor fibers, leading to symptoms such as radiating pain in the neck and shoulder, numbness, weakness, or diminished reflexes in the upper limbs. Its causes may include herniated discs, osteophyte formation, acute traumatic traction, fractures or dislocations, or iatrogenic manipulations. Essentially, this injury manifests as damage to the structure and function of the nerve root, including axonal rupture, demyelination, local inflammation, and nerve conduction disorders.

[0037] In this invention, a "combination product" refers to a product system consisting of two or more components that are typically kept separate during packaging or storage, but are combined or applied together in a specific manner to achieve the intended function. Such products may be sold or provided as a whole, but their individual components (such as solution A and solution B in this invention) are generally provided in separate packages to ensure stability, portability, or to maintain their chemical properties from reacting before use. The essential characteristic of a combination product is that although the components are stored or transported separately, they need to cooperate to form the target product or achieve the intended technical effect in actual application.

[0038] This invention relates to a magnetoelectric hydrogel combination product, comprising solution A and solution B; Solution A comprises 0.1-2% (w / v) Fe3O4@BaTiO3@PDA nanoparticles (FBD) and 10-30% (w / v) phenylboronic acid-modified dextran oxide (PBA-OD). Solution B contains 20-40% (w / v) of a photocrosslinkable gel matrix.

[0039] The two components are brought into contact through mixing or alternating spraying during use. In solution A, PBA-OD and FBD form a first cross-linked network via borate ester interaction, while the gel matrix in solution B forms a second cross-linked network under light irradiation, resulting in a magnetoelectric hydrogel capable of tissue surface deposition. This combination method aims to achieve stable dispersion and rapid on-site gelation of magnetoelectric nanoparticles, while preventing premature cross-linking or solidification of the two components during storage and transportation. By packaging FBD and PBA-OD separately in solution form and combining them with solution B containing the gel matrix on-site, the combined product of this invention offers significant advantages in storage stability, ease of operation, and on-site gelation efficiency. During use, the two solutions rapidly form a borate ester network upon contact, and further construct a three-dimensional gel framework under subsequent light irradiation, resulting in a magnetoelectric hydrogel with excellent sprayability, adhesion, and tissue compatibility, making it particularly suitable for applications such as nerve tissue surface deposition, encapsulation, and functional repair.

[0040] FBD nanoparticles have a core-shell structure, with Fe3O4 serving as the magnetostrictive core, BaTiO3 as the piezoelectric shell, and PDA as the surface functionalization layer to improve dispersibility and provide borate ester bonding sites. FBD can be synthesized through a multi-step sequential process: First, Fe3O4 nanoparticles are prepared via a solvothermal method; then, TiO2 is deposited on their surface using a sol-gel deposition method to obtain Fe3O4@TiO2; next, a Ba²⁺ hydrothermal conversion forms a BaTiO3 shell, constituting Fe3O4@BaTiO3; finally, dopamine (PDA) is self-polymerized and deposited on the particle surface under weakly alkaline conditions, thus constructing the Fe3O4@BaTiO3@PDA (FBD) core-shell structure. This three-layer structure ensures both the magneto-electric coupling effect and provides reaction sites for the formation of borate ester structures with PBA-OD.

[0041] In this invention, the photocrosslinkable gel matrix is ​​used to construct the second crosslinking network of the magnetoelectric hydrogel. Under light irradiation, it can form a stable three-dimensional network structure, thus providing overall mechanical support and tissue adhesion properties together with the borate ester crosslinking network formed by PBA-OD and FBD nanoparticles. As the main phase of the hydrogel system, the gel matrix's photocurability, hydrophobic / hydrophilic balance, and biocompatibility all significantly influence the gelling effect and clinical applicability after spraying. In some embodiments, the gel matrix is ​​selected from methacrylated gelatin, methacrylated gelatin derivatives, methacrylated hyaluronic acid derivatives, chitosan-methacrylated derivatives, and photocurable natural polymers containing double bonds. All of the above gel matrices can form a three-dimensional network through free radical photopolymerization, meeting the requirements of this invention for photocrosslinking rate, crosslinking density, and biocompatibility.

[0042] In a further embodiment of the present invention, the average particle size of the FBD nanoparticles is 150–500 nm, preferably 250–350 nm. Further observation shows that FBD nanoparticles with an average particle size of approximately 300 nm achieve the best balance between rheological properties, magnetoelectric conversion efficiency, and hydrogel curing uniformity, thereby significantly improving the deposition ability and stimulation output consistency of the resulting magnetoelectric hydrogel on complex neural tissue surfaces. Those skilled in the art will understand that appropriately adjusting the particle size within the above preferred range remains within the scope of the present invention.

[0043] In some embodiments, the magnetoelectric hydrogel composite product may further include a photoinitiator (component C) for triggering a cross-linking reaction in the gel matrix under light irradiation. The photoinitiator can initiate the generation of free radicals when irradiated with light of a specific wavelength, thereby causing an addition reaction of polymerizable double bonds in the gel matrix to form a second cross-linking network, thus improving the mechanical strength and stability of the resulting hydrogel. Photoinitiators that can be used in this invention include, but are not limited to, 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methylpropanone (Irgacure 2959), 2,4,6-trimethylbenzoyl diphenylphosphinate (TPO), diphenyl(2,4,6-trimethylbenzoyl)phosphinate (TPO-L), the methacrylate-type water-soluble photoinitiator LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate), benzoin and its derivatives, acetophenone-based photoinitiators, and other photoinitiating systems capable of generating free radicals in the 320–405 nm wavelength range. Photoinitiators are typically added to the composite product at a ratio of 0.01–1% (w / v) to enable rapid curing during crosslinking while ensuring the biocompatibility of the resulting hydrogel.

[0044] In some embodiments of the present invention, the solvent for the magnetoelectric hydrogel composite product is selected from water or physiological buffer solution. Specifically, solutions A and B can respectively use deionized water, water for injection, or buffer solutions commonly used in biomaterial systems as dispersion media, including but not limited to phosphate-buffered saline (PBS), HEPES buffer, or Tris buffer, so that the FBD nanoparticles, PBA-OD, and gel matrix can maintain good solubility and stability, and ensure suitable rheological properties and biocompatibility when sprayed to form a hydrogel. Those skilled in the art can select different types of aqueous phase systems according to specific application requirements without departing from the spirit of the present invention.

[0045] Optionally, to further improve the efficiency of borate bond formation between PBA-OD and the polyphenol groups on the FBD surface, and to enhance the initial crosslinking rate of the combined product during use, the pH of the solvent can be controlled between 7.4 and 8.0; in some preferred embodiments, the pH of the solvent is controlled between 7.0 and 7.8. In this slightly alkaline environment, the phenylboronic acid groups are in a dissociated state more conducive to the reversible formation of borate esters with the vicinal diol structure on the FBD surface, which helps to rapidly build the first crosslinking network during the spraying stage, improving the interfacial stability and tissue adhesion of the initial gel. Furthermore, this pH range remains within a physiologically acceptable range, avoiding tissue irritation and making it suitable for in-situ application to nerve tissue surfaces.

[0046] According to another aspect of the invention, there is a magnetoelectric hydrogel obtained by mixing the combined products as described above; wherein the PBA-OD and FBD form a first cross-linked network through borate ester bonds, and the gel matrix forms a second cross-linked network under light irradiation, so that the resulting hydrogel has sprayable and tissue-adhesive properties.

[0047] In some embodiments, the first crosslinking network can be further enhanced by increasing the PBA-OD content or increasing the exposure of hydroxyl / dopamine on the FBD surface; the second crosslinking network can be formed using methacrylamide gelatin (GelMA), hyaluronic acid methacrylamide derivatives, chitosan-methacrylamide derivatives, etc., and a photoinitiator can be added if necessary to improve the photocuring efficiency.

[0048] The magnetoelectric hydrogel obtained in this invention possesses both excellent biocompatibility and materials engineering properties. First, the hydrogel matrix is ​​composed of natural polymer derivatives, exhibiting a soft, skin-friendly feel and good interfacial compatibility with nerve tissue and soft tissue, allowing for close adhesion to curved or irregular surfaces without triggering significant irritation. Second, the hydrogel rapidly solidifies within 1–3 seconds after spraying; its borate ester dynamic bonds endow the material with instantaneous adaptive curing ability, forming a stable covering layer in the surgical area and helping to prevent material erosion. Furthermore, the photocrosslinked second network structure significantly improves the gel's shape retention and durability, enabling it to maintain necessary mechanical support under physiological conditions while retaining flexibility to avoid compressing nerve roots. In addition, the hydrogel system of this invention can achieve biodegradation through stepwise hydrolysis. The degradation rate can be controlled by adjusting the PBA-OD content and the crosslinking density of the gel matrix, allowing the material to maintain structural integrity within the nerve repair window and be gradually eliminated from the body after completing the critical stage of tissue regeneration, without producing residual foreign body reactions. In summary, this magnetoelectric hydrogel exhibits excellent performance in terms of sprayability, interfacial stability, biocompatibility, and controllable degradation, making it suitable for precise application and long-term functional recovery in neurological injury environments.

[0049] According to another aspect of the present invention, a method for preparing the magnetoelectric hydrogel as described above is provided, comprising: Solution A and solution B are applied to the surface of the target substrate by alternating spraying to obtain a primary gel, wherein PBA-OD and FBD form borate ester bonds and construct a first crosslinking network in the primary gel; The resulting primary gel is exposed to light to form a second cross-linked network in the gel matrix, thereby obtaining an adhesive magnetoelectric hydrogel.

[0050] The above-mentioned alternating spraying can be completed using handheld spraying equipment, dual-channel spray guns, or medical atomizing nozzles to ensure that the coating uniformly covers complex anatomical structures.

[0051] The two-component alternating spraying method of this invention exhibits excellent anti-nozzle clogging ability in clinical or animal model environments: because solutions A and B do not premix at the nozzle, they do not gel prematurely, thus avoiding nozzle clogging; and the system has moderate viscosity and shear-thinning rheological behavior, which significantly reduces spraying resistance and improves operability in narrow or branching nerve regions. Through the rapid formation of the first cross-linking network and the light-triggered construction of the second cross-linking network, the resulting hydrogel can achieve rapid curing, firm adhesion, and precise shaping on complex nerve surfaces, significantly enhancing the clinical usability and operational stability of the magnetoelectric hydrogel. This method not only achieves precise coverage in cervical nerve root regions where surgical space is limited, but also ensures that FBD nanoparticles are uniformly distributed in the hydrogel network, generating a stable magnetoelectric response when activated by an external magnetic field, thereby improving nerve repair efficacy.

[0052] In some embodiments, the wavelength of the illumination is 320–405 nm, preferably 365 nm.

[0053] In some embodiments, the intensity of the light is 0.5–5 W / cm², and the irradiation time is 5–60 s.

[0054] In some embodiments, solution A and solution B can be sprayed alternately at a volume ratio of 1:(0.5-1.5), more preferably 1:(0.7-1.3), and even more preferably 1:(0.9-1.1). This ratio can maintain the content of the gel matrix within a suitable range while ensuring the dynamic crosslinking efficiency of the borate ester, avoiding local over-dilution due to excessive B solution or the formation of a discontinuous gel layer due to insufficient B solution.

[0055] In another embodiment, the total amount of each alternating spray can be controlled within 0.04–0.3 mL, for example 0.06 mL, 0.08 mL, 0.1 mL, 0.15 mL, 0.2 mL, or 0.25 mL. Depending on the size, curvature, and desired coverage thickness of the target area, 2–20 cycles (e.g., 5, 10, or 15 cycles) of deposition are selected to achieve a final hydrogel layer thickness of 0.4–2 mm (e.g., 0.5 mm, 0.7 mm, 1.0 mm, or 1.5 mm). The aforementioned deposition thickness has been shown to provide sufficient three-dimensional network support for the magnetoelectric nanoparticles without affecting the activity of surrounding tissues, thereby generating a stable and uniform magnetoelectric stimulation output under subsequent magnetic field action.

[0056] During alternating spraying, the time interval between two sprays can optionally be set to 0.5–5 seconds, preferably 0.5–2 seconds. This time range ensures sufficient contact between solution A and solution B, allowing for in-situ gelation and initial coagulation to rapidly form a borate ester network, while avoiding localized accumulation or sagging caused by repeated nozzle triggering. Experimental results of this invention show that the above spraying parameters can significantly improve the uniformity of coverage and the stability of the initial coagulated structure on the surface of narrow nerve channels or multi-branched nerves.

[0057] According to another aspect of the invention, there is a connection to the use of the combined product as described above, or the magnetoelectric hydrogel as described above, in the preparation of a medical formulation / repair material for treating cervical nerve root injury.

[0058] In some embodiments, the medical preparation / repair material is used in conjunction with magnetic field activation.

[0059] The magnetoelectric hydrogel of this invention possesses sprayable, rapid-solidifying, strong adhesive, and strong magnetoelectric response properties, enabling it to firmly adhere to the complex surfaces of nerve roots or surrounding tissues, forming a stable local microenvironment at the injury site. This microenvironment constructed by the hydrogel supports nerve cell attachment, growth, and migration, and generates localized weak electrical stimulation under subsequent magnetic field action, which is beneficial for promoting axonal regeneration, myelin formation, inflammation relief, and nerve function recovery. Those skilled in the art can further prepare this hydrogel into different forms of medical preparations or repair materials, including injectable solutions, sprayable formulations, cross-linkable in-situ formed hydrogels, tissue adhesion films, and local filling materials, to meet the treatment needs of different types of cervical nerve root injuries.

[0060] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0061] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0062] The chemical reagents used in the examples are as follows: FeCl3·6H2O, Ba(OH)2·8H2O, tetrabutyltitanium (TBOT), 3-aminophenylboronic acid, and dopamine hydrochloride (DA-HCl) were all sourced from Shanghai Aladdin Reagent Co., Ltd. (China). Dextran (molecular weight: 70,000), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (I2959), methacrylic anhydride (MA, >94%), and type A porcine gelatin were purchased from Sigma-Aldrich (USA). Sodium picolinate (NaIO4, >99%) was provided by Adamas Pharmaceuticals (China). Other reagents, such as ethylene glycol (EG), sodium citrate (Na3CT), anhydrous ethanol, acetonitrile (ACN), ammonium acetate (NH4OAc), sodium picolinate (NaIO4), and 30% ammonia, were provided by Chengdu Kelon Reagent Co., Ltd. (China). All chemical reagents used were of analytical grade and were not further purified.

[0063] Example 1: Preparation and Characterization of FBD Nanoparticles This invention prepares core-shell structured Fe3O4@BaTiO3@PDA nanoparticles (FBD, particle size approximately 300 nm) via a multi-step sequential synthesis method. The Fe3O4 (FO) nanoparticles are synthesized via a solvothermal method. The specific process includes: first, dissolving FeCl3·6H2O (1.350 g), Na3CT (0.4 g), and NH4Ac (3.854 g) in 70 mL of ethylene glycol, stirring at 25°C for 1 hour, and reacting under high pressure at 200°C for 12 hours. The product is then magnetically separated, washed, and dispersed in ethanol. Subsequently, the FO solution (5 mL) is mixed with ethanol (85 mL) / acetonitrile (30 mL), sonicated, and reacted with tetrabutyltitanium (TBOT) for 1.5 hours. FO@TO (FT) is then obtained by magnetic separation and washing. The FT solution was further ultrasonically dispersed, mixed with Ba(OH)₂·8H₂O solution (0.015 g / mL) and stirred for 15 minutes. Ammonia (2 ml) was added, and the mixture was hydrothermally reacted at 200°C for 8 hours. The product was washed with formic acid / water and magnetically separated to obtain FO@BTO (FB). Finally, FB was mixed with Tris-HCl buffer (10 mM, pH 8.5), stirred for 10 minutes, and dopamine (DA, 1.2 g) was added. The mixture was continuously stirred at 25°C for 24 hours. The product was washed three times with distilled water and collected using a neodymium magnet to obtain FO@BTO@DA (FBD) (see...). Figure 1(Figure A). Transmission electron microscopy (TEM, Figure B) and field emission scanning electron microscopy results showed that a uniform PDA layer of approximately 25 nm was successfully coated on the surface of the nanoparticles. Energy dispersive X-ray spectroscopy (EDX, Figure C) further confirmed the uniform distribution of elements. The successful functionalization of PDA was also reflected in the significant negative change in zeta potential (Figure D) and the shift in the characteristic peak position of Fourier transform infrared spectroscopy (FT-IR) (Figure E), proving that the nanostructure possesses the key hydrogel binding ability. X-ray diffraction (XRD) analysis showed that the FBD nanoparticles simultaneously retained the cubic phase of Fe3O4 (PDF#97-015-8505) and the tetragonal phase of BaTiO3 (PDF#97-008-6286) (Figure F), indicating that the structure was not affected by the coating process. Subsequently, we evaluated its functional properties. Vibrating sample magnetometer (VSM) tests showed that the nanoparticles retained superparamagnetism even after PDA coating, with a magnetic saturation strength (Ms) of 18 emu·g. -1 (Figure 1, G). Piezoelectric force microscopy (PFM) further verified the integrity of the material's magnetoelectric function (Figure 1, H). The 180° phase reversal behavior (Figure 1, I) reflects the material's polarization reversal capability; while the "butterfly-shaped" strain curve (Figure 1, J) confirms its reversible polarization behavior, which is fundamental for achieving magnetoelectric conversion. Under a magnetic field of 1000 Oe, the voltage coercivity of BaTiO3 changed from -0.59 / 0.86 V to -0.51 / 1.02 V, further demonstrating the effective magnetostrictive-piezoelectric coupling characteristics of this structure. Furthermore, finite element analysis results (Figure 1, K) show that PDA coating does not significantly impair the magnetoelectric properties, and the voltage generated by the FBD under a 25 mT magnetic field (approximately 2.5 μV) is comparable to that of the uncoated FB. In summary, these results indicate that, while preserving magnetoelectric conversion efficiency, PDA functionalization not only provides an interface site for subsequent binding with hydrogels but also provides functional support for wireless neural modulation.

[0064] Example 2: Preparation and property characterization of DG / FBD magnetoelectric hydrogel To address the multiple challenges in the repair of cervical nerve root injury (CNRI), this invention constructs an in-situ sprayable magnetoelectric adhesive hydrogel (DG / FBD), which is synergistically assembled from phenylboronic acid-functionalized oligodopamine (PBA-OD), methacrylamide gelatin (MG), and FBD nanoparticles (Figure 2A).

[0065] The synthesis of phenylboronic acid-modified oxidized dextran (PBA-OD) and gelatin acrylamide (MG) followed a previously established method. First, oxidized dextran (OD) was obtained by a redox reaction using a sodium desired acid. Subsequently, PBA-OD was synthesized via a Schiff base reaction. Furthermore, MG was synthesized by grafting methacrylic anhydride (MA) onto the gelatin molecular chain via esterification modification. The successful synthesis of PBA-OD and MG was confirmed by ¹H NMR (AV II-600 MHz, Bruker) and FTIR spectroscopy (Thermo Fisher Nicolet Is10).

[0066] The DG / FBD hydrogel was prepared using the following formulation: FBD (0.5% w / v), PBA-OD (20% w / v), and MG (30% w / v). To meet clinical application needs, we developed an optimized two-step spraying process that effectively avoids nozzle clogging and achieves uniform, layered adhesion deposition through alternating spraying cycles (Figure 2B). Figure 3 The hydrogel gels rapidly in less than 2 seconds, enabling immediate tissue fixation (Figure 2, C). Figure 4 Solution A (containing PBA-OD and FBD) and solution B (containing MG), both with pH 7.0–7.8, were transferred to two separate spray bottles. The solutions were applied in an alternating sequence: solution A was sprayed onto the target surface first, followed immediately by solution B. This alternating spraying sequence was repeated to obtain a sprayable DG / FBD hydrogel. The prepared DG / FBD hydrogel was placed under UV radiation (wavelength: 365 nm, intensity: 3 W / cm²) for 20 seconds, and crosslinking of the DG / FBD hydrogel was completed using an OmniCure® S1500 EXPO system; the volume ratio of solution A to solution B was 1:1; the total amount of solution A and solution B sprayed each time was 0.04–0.3 mL, with 15 cycles, resulting in a final thickness of 1.5 mm; each cycle was spaced 2 seconds apart.

[0067] Rheological analysis revealed that the DG / FBD hydrogel exhibited shear-thinning non-Newtonian fluid properties and maintained a moderate viscosity (87 mPa·s) across the entire shear rate range, ensuring good sprayability (Figure 2, D). In contrast, the control group hydrogels DG and DG / FB, lacking sufficient borate ester crosslinking, exhibited lower viscosities (17 mPa·s and 37 mPa·s, respectively) and slower gel times (3.3 seconds and 2.44 seconds, respectively), thus affecting post-spraying stability (Figure 2, D). Under mild alkaline conditions, the crucial role of borate ester bonds was further validated by the increased viscosity observed in the shear rate-dependent curve. Figure 5 Meanwhile, UV-Vis absorption analysis showed that the absorption intensity of DG / FBD at 295 nm (0.58) was significantly lower than that of DG (1.21) and DG / FB (1.08), further demonstrating the improved bonding efficiency (E in Figure 2). Furthermore, the DG / FBD hydrogel exhibited excellent thermal stability and tissue adhesion properties during dynamic deformation (F in Figure 2). Figure 6 It is worth noting that the hydrogel exhibits strong interfacial adhesion to skin tissue (22.11 kPa), and its adhesive properties may be due to the synergistic effect of hydrogen bonding, electrostatic interaction and cation-π interaction (G in Figure 2).

[0068] The UV crosslinking process following spraying further strengthens the hydrogel matrix through the formation of carbon-carbon (C–C) bonds (FTIR absorption peak 1322 cm⁻¹, H in Figure 2), increasing the storage modulus to a level matching that of neural tissue (approximately 100 Pa). Figure 2 While maintaining good self-healing ability, the DG / FBD also exhibits excellent adhesion and branch penetration in complex neck anatomy. Nanoindentation testing further evaluated its micromechanical homogeneity, showing an elastic modulus of 0.21 kPa, which falls between that of DG (0.08 kPa) and DG / FB (0.41 kPa) (J, K in Figure 2). Functionally, vibrational sample magnetometer (VSM) confirmed that the DG / FBD maintains good superparamagnetic properties (L in Figure 2) and magnetic saturation, ensuring its non-invasive magnetic response characteristics in neuromodulation. In summary, this system achieves rapid adhesion and branch penetration in complex neck anatomy, possesses good spatiotemporal control capabilities, and provides an adaptive platform for CNRI repair (M–O in Figure 2).

[0069] Example 3: DG / FBD hydrogel in vitro regulation of neural differentiation and maturation of BMSCs To ensure that the hydrogel spray possesses both excellent biocompatibility and effective magnetoelectric stimulation, we first screened for suitable FBD nanoparticle (NP) doping concentrations through co-culture experiments with bone marrow mesenchymal stem cells (BMSCs) and PC12 cells. Cell viability assays showed that NP concentrations below 0.5% had no significant inhibitory effect on BMSC proliferation, while a significant inhibitory effect was observed at a concentration of 1%. Further PC12 cell spreading experiments demonstrated that axonal extension capacity was significantly enhanced under magnetic field conditions, and cell extension length was positively correlated with NP concentration. Based on these results, and considering both biocompatibility and magnetoelectric stimulation efficiency, a 0.5% NP concentration was selected as the standard ratio in subsequent experiments.

[0070] Subsequently, we evaluated the effects of DG / FBD hydrogel on the neural differentiation and maturation of BMSCs under magnetoelectric stimulation. On day 7, the expression of the early neuronal marker Tuj1 was significantly higher in the DG / FBD + magnetic stimulation (MS) group than in the DG, DG + MS, and DG / FBD groups, indicating that this condition helps promote early neural differentiation. Figure 7 (A, E). Similarly, on day 21, neurofilament (NF, Figure 7 B, F) and postsynaptic dense protein PSD95 ( Figure 7 The expression of C and G was also significantly upregulated in the DG / FBD+MS group, reflecting enhanced neuronal maturation and synaptic remodeling, respectively. Conversely, the expression of the astrocyte differentiation marker GFAP was significantly inhibited in the DG / FBD+MS group. Figure 7 The results (D and H) suggest that magnetoelectric stimulation can effectively inhibit glial cell activation and glial scar formation. In summary, DG / FBD hydrogel-mediated magnetoelectric stimulation can not only effectively induce BMSCs to differentiate into neuronal lineages and promote their maturation, but also simultaneously inhibit their differentiation into glial cells, demonstrating dual regulatory potential in the regulation of the neural regeneration microenvironment.

[0071] Example 4: DG / FBD hydrogel spray accelerates nerve regeneration in CNRI rats To further verify the positive effects of DG / FBD hydrogel on cervical nerve root structural repair and pathological improvement, we performed in-situ spraying treatment in a CNRI rat model (Figure 2, I), followed by external magnetic field stimulation. Multiple experimental groups were established using standard surgical procedures, including CNRI, DG+magnetic stimulation (DG+MS), DG / FBD, DG / FBD+magnetic stimulation (DG / FBD+MS), and a sham-operated group (Sham). Due to the narrow anatomical location of the cervical nerve roots, it was difficult to visually demonstrate the coating effect of hydrogel spraying in this area; therefore, we selected the distal branches of the sciatic nerve for demonstration. Figure 8 As shown, DG / FBD hydrogel spray can form a uniform coverage and tight adhesion on the surface of complex nerve branches. After 4 weeks of treatment, H&E staining results showed that the nerve axons in the CNRI, DG+MS, and DG / FBD groups were sparse and structurally hollow, while the DG / FBD+MS group formed dense, orderly arranged nerve fibers, with a structure similar to the Sham group (J in Figure 7). Transmission electron microscopy (TEM) observation results and quantitative analysis of myelin regeneration further confirmed the above conclusions. The DG / FBD+MS group showed a thick, structurally complete myelin sheath layer surrounding the regularly arranged axons, while the control group had a thin and sparse myelin sheath. Figure 7(Middle K). Statistical data show that the myelin sheath thickness was significantly increased in the DG / FBD+MS group ( Figure 7 (Middle L), axon diameter close to normal level ( Figure 7 The presence of hydrogels in the middle M (middle M) reinforced their role in promoting nerve regeneration. G ratio analysis (reflecting myelination efficiency) further confirmed that this group exhibited good axonal function recovery and structural integrity. Further longitudinal immunofluorescence staining ( Figure 7 The results showed that multiple neural repair-related structural proteins were significantly upregulated in the DG / FBD+MS group, including the axonal marker NF200, the myelin marker MBP, and the Schwann cell marker S100. These results collectively indicate that DG / FBD hydrogel spray under magnetoelectric stimulation can effectively accelerate structural neural regeneration in CNRI rats.

[0072] Example 5: DG / FBD hydrogel spray enhances the recovery of sensory, motor, and nerve conduction functions in CNRI rats. CNRI is often accompanied by sensory impairments such as pain and temperature perception disturbances; therefore, sensory recovery is one of the key indicators for assessing neurological function recovery. To evaluate the potential of magnetically driven DG / FBD hydrogel spray for sensory recovery, we used Hargreaves and von Frey experiments. Hargreaves results showed that the thermal pain threshold in the DG / FBD+MS group was significantly reduced in the second week after surgery, indicating faster recovery of temperature sensitivity; by the fourth week, the pain threshold in this group was close to that of the Sham group, suggesting good recovery of thermal perception function. Figure 9 (A). In the von Frey experiment, the DG / FBD+MS group also showed significant improvement, with the mechanical pain threshold approaching normal levels by week 6, indicating a significant recovery in pain perception function. Figure 9 (Middle B). To further assess the recovery of motor function, we conducted a grip strength test. The results showed that the grip strength of rats in the DG / FBD+MS group gradually recovered during the 6-week observation period, reaching a level similar to that of the Sham group by week 4. Figure 9 (C) According to Figure 9 As shown in the schematic diagram (D), we also performed electrophysiological analysis to assess the recovery of nerve conduction. Specific procedures included exposing the cervical nerve roots and applying a single electrical stimulation, recording the compound muscle action potentials (CMAP). Figure 9 (E–I). The results showed that the CMAP amplitude on the injured side of the DG / FBD+MS group was significantly higher than that of the unstimulated DG / FBD group and the DG+MS group, and approached the level of the Sham group; while the CMAP amplitude of the CNRI group remained at a low level due to limited functional recovery. Figure 9Further analysis showed that the neural conduction velocity (NCV) in this group increased from 13.69 ± 6.84 m / s in the CNRI group to 41.78 ± 8.29 m / s, indicating the most significant recovery effect among all groups. Figure 9 (K). In summary, the DG / FBD+MS group showed significant improvements in sensation, motor function, and neural electrical signal transmission. The enhancement of CMAP and NCV reflects the recovery of nerve fiber regeneration and motor response. The innovation of this magnetoelectric hydrogel spray system lies in its excellent biocompatibility and magnetoelectric responsiveness, as well as its significant advantages in anatomical adaptability. It can better conform to tissue morphology and achieve effective stimulation, thus providing a multi-level, systematic solution for the functional recovery of CNRI.

[0073] Example 6: Magnetoelectric stimulation enhances autophagy activity and upregulates CXCL12 expression To elucidate the mechanism by which magnetically driven DG / FBD hydrogel promotes nerve regeneration, we performed RNA transcriptome sequencing analysis on tissue samples collected from the CNRI regeneration region 4 weeks post-surgery. The results showed significant clustering differences in gene expression profiles among different groups. Figure 10 (A) verified the reliability of the sequencing data. Comparing the Sham group with the DG / FBD+MS group, a total of 126 genes were upregulated and 212 genes were downregulated ( ). Figure 10 (B). Gene ontology (GO) enrichment analysis revealed several autophagy-related terms, including autophagosome assembly, nuclear fragmentation macroautophagy, macroautophagy, and pre-autophagosome membrane structure. Figure 10 In addition, gene set enrichment analysis (GSEA) showed significant enrichment of autophagy-related pathways, such as autophagy-animal, mitophagy-animal, and PI3K-Akt signaling pathways. Figure 10 (D and E). These findings suggest that autophagy may play a key role in the promotion of CNRI repair by magnetoelectric stimulation-mediated DG / FBD hydrogels. Next, we applied the LASSO and Boruta algorithms to identify key regulatory genes by selecting 15 and 25 feature variables, respectively. Figure 10 (FH). Nefl, Msr1, and CXCL12 were identified as overlapping genes. Figure 10 (I). Relative expression level analysis showed that CXCL12 expression was significantly upregulated in the DG / FBD+MS group, consistent with the improvement results of CNRI repair. In contrast, Nefl and Msr1 did not show a similar upregulation trend. Figure 10(J). These multi-omics analyses showed that magnetoelectric stimulation-mediated DG / FBD hydrogels not only enhanced autophagy activity but also upregulated CXCL12 expression. Previous studies have shown that after peripheral nerve injury, neurons use autophagy to clear myelin debris from Waller's degeneration zone, thereby providing space for nerve regeneration. Furthermore, the CXCL12-CXCR4 axis is also associated with the repair of injured sciatic nerves. However, this study, by demonstrating that magnetoelectric stimulation not only enhances autophagy activity but also upregulates CXCL12 expression, provides new insights into the field of neuroelectric modulation.

[0074] To verify these findings, we subsequently conducted related experiments ( Figure 11 (A). Quantitative PCR (qPCR), Figure 11 (BD) and enzyme-linked immunosorbent assay (ELISA) Figure 11 Consistent results from the studies (E) indicated that CXCL12 expression was significantly higher in the DG / FBD+MS group than in other experimental groups. CXCL12, also known as stromal cell-derived factor 1A (SDF-1A), is a chemokine crucial for neural migration and tissue repair. Following nerve injury, CXCL12 is typically upregulated and promotes repair by enhancing mesenchymal stem cell homing, myelin regeneration, and modulating neuropathic pain through the CXCR4 signaling pathway. The upregulation of CXCL12 in the DG / FBD+MS group further supports its role as a key mediator in CNRI repair.

[0075] Immunofluorescence staining results at 4 weeks post-surgery showed that CXCL12 expression was significantly higher in the DG / FBD+MS group than in other groups. Figure 11 This result is consistent with the results of qPCR and ELISA. Meanwhile, the autophagy marker LC3B (a widely used marker for autophagosome formation) was upregulated in the DG / FBD+MS group. Figure 11 While LC3B was upregulated, P62 (a key indicator of autophagic flux) was significantly downregulated. Previous studies have shown that upregulation of LC3B and downregulation of P62 indicate enhanced autophagic activity. This differential expression pattern suggests an increased autophagic flux in the DG / FBD+MS group.

[0076] Transmission electron microscopy (TEM) further confirmed this observation, showing a significant increase in the abundance of autophagosomes in the DG / FBD+MS group. Figure 11(G and H). In summary, these results indicate that magnetically driven hydrogel spraying simultaneously upregulates CXCL12 expression and promotes autophagic flux. The potential association between these two factors warrants further investigation, but these findings highlight the dual role of hydrogels in enhancing both CXCL12 expression and autophagic activity.

[0077] Notably, previous studies have shown that magnetoelectric stimulation promotes neural repair through multiple mechanisms, including modulating the MAPK / ERK / PI3K pathway, influencing intracellular ion channel activity, and enhancing cell migration. However, our study differs in that we innovatively discover that magnetoelectric stimulation not only upregulates CXCL12 expression but also promotes autophagic flux. This new finding provides new insights into how magnetoelectric stimulation simultaneously modulates chemokine signaling and autophagy activity to enhance neural repair.

[0078] Example 7: CXCL12 regulates enhanced autophagy in Schwann cells via the PI3K / AKT / mTOR signaling pathway Next, we explored the potential association between CXCL12 and autophagy. Schwann cells (SCs) play a crucial role in peripheral nerve injury repair, creating a favorable environment for nerve regeneration by migrating to the injury site, clearing myelin debris, and promoting autophagosome formation. Figure 12 (A). Against this backdrop, we selected Schwann cells as a model to investigate whether CXCL12 directly enhances autophagy under the action of magnetically driven DG / FBD hydrogel.

[0079] The specific experimental procedure involved treating Schwann cells with exogenous CXCL12 overexpression, the autophagy inhibitor 3-MA, and the autophagy agonist rapamycin, respectively, for in vitro experiments. Immunofluorescence images ( Figure 12 (B and C) and quantitative analysis ( Figure 12 The results (D and E) showed significant differences among the Blank, rapamycin, and 3-MA groups, demonstrating that autophagy agonists and inhibitors effectively regulated Schwann cell activity and validating the effectiveness of the experimental conditions.

[0080] Compared with the Blank group and the control group, magnetically driven DG / FBD hydrogel significantly increased CXCL12 expression and autophagy. Figure 12 (D and E). Notably, the CXCL12 treatment group showed enhanced autophagic flux compared to the Blank group, while the autophagic activity of the CXCL12 + rapamycin group was significantly higher than that of the rapamycin alone group. Simultaneously, the CXCL12 + 3-MA group partially reversed the 3-MA-induced autophagy inhibition.

[0081] Correlation analysis using the Schwann cell marker S100 showed that upregulation of CXCL12 expression was associated with an increase in the autophagy marker LC3B. Figure 12 The presence of F indicates that CXCL12 promotes autophagy.

[0082] Currently, detailed research on the regulatory mechanisms of autophagy in magnetoelectric neural modulation is insufficient. Our gene set enrichment analysis (GSEA) indicates that this signaling pathway is mediated by magnetically driven DG / FBD hydrogels in Schwann cells (SCs). Immunofluorescence staining showed that p-PI3K levels were significantly reduced in Schwann cells exposed to DG / FBD hydrogels. Figure 13 (A and B in the middle). Based on these findings, we hypothesize that the hydrogel enhances Schwann cell autophagy by modulating the CXCL12-mediated PI3K / AKT / mTOR signaling pathway.

[0083] To verify this hypothesis, we performed Western blot analysis to evaluate key signaling proteins such as PI3K, AKT, mTOR, p-PI3K, p-AKT, and p-mTOR. Figure 13 (C). The results showed that the expression of p-PI3K, p-AKT and p-mTOR in Schwann cells treated with DG / FBD hydrogel was significantly reduced (C). Figure 13 The results (DF) indicate that the hydrogel inhibited the PI3K / AKT / mTOR signaling pathway.

[0084] Next, we evaluated whether autophagy affected CXCL12 expression. Western blot analysis showed that autophagy induction did not lead to a significant change in CXCL12 expression levels. Figure 13 In addition, the DG / FBD hydrogel modulates the PI3K / AKT / mTOR signaling pathway by regulating CXCL12 ( GH). Figure 13 (IL).

[0085] The application of the PI3K inhibitor 740Y-P further confirmed that the hydrogel promotes autophagy by inhibiting the PI3K / AKT / mTOR signaling pathway (Figure 8M-N). These findings support our hypothesis that CXCL12 enhances Schwann cell autophagy by inactivating the PI3K / AKT / mTOR signaling pathway. Figure 13 (O). Based on the above, our work innovatively confirms the role of CXCL12 in promoting Schwann cell migration and autophagy through the PI3K / AKT / mTOR signaling pathway in magnetoelectric hydrogel-mediated neurogenesis. These findings expand our understanding of Schwann cell-mediated repair mechanisms and open new therapeutic avenues for improving post-injury functional recovery.

[0086] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A magnetoelectric hydrogel combination product, comprising solution A and solution B; Solution A comprises 0.1-2% (w / v) Fe3O4@BaTiO3@PDA nanoparticles (FBD) and 10-30% (w / v) phenylboronic acid-modified dextran oxide (PBA-OD). Solution B contains 20-40% (w / v) of a photocrosslinkable gel matrix.

2. The combined product according to claim 1, wherein the gel matrix is ​​selected from methacrylated gelatin, methacrylated gelatin derivatives, methacrylated hyaluronic acid derivatives, chitosan-methacrylated derivatives, and photocurable natural polymers containing double bonds.

3. The combined product according to claim 1, wherein the average particle size of the FBD nanoparticles is 150–500 nm, preferably 250–350 nm.

4. The combined product according to any one of claims 1-3, further comprising component C: a photoinitiator.

5. The combined product according to any one of claims 1-3, wherein the solvent is water or a physiological buffer solution; optionally, the pH of the solvent is 7.4–8.0; preferably, the pH is 7.0–7.

8.

6. A magnetoelectric hydrogel, obtained by mixing the combined products according to any one of claims 1-5; wherein, The PBA-OD and FBD form a first cross-linked network through borate ester bonds, and the gel matrix forms a second cross-linked network under light irradiation, giving the resulting hydrogel sprayable and tissue-adhesive properties.

7. A method for preparing the magnetoelectric hydrogel according to claim 6, comprising: Solution A and solution B are applied to the surface of the target substrate by alternating spraying to obtain a primary gel, wherein PBA-OD and FBD form borate ester bonds and construct a first crosslinking network in the primary gel; The resulting primary gel is exposed to light to form a second cross-linked network in the gel matrix, thereby obtaining an adhesive magnetoelectric hydrogel.

8. The preparation method according to claim 7, wherein the wavelength of the light irradiation is 320–405 nm, preferably 365 nm; preferably the intensity of the light irradiation is 0.5–5 W / cm², and the irradiation time is 5–60 s; Optionally, the volume ratio of the sprayed amount of solution A to solution B is 1:(0.5-1.5); Optionally, the total amount of solution A and solution B sprayed each time is 0.04-0.3 mL, and the cycle is repeated 2-20 times, with a final thickness of 0.4-2 mm; Optionally, the interval between each cycle is 0.5-5 seconds, preferably 0.5-2 seconds.

9. Use of the combined product according to any one of claims 1-5, or the magnetoelectric hydrogel according to claim 6, in the preparation of a medical preparation / repair material for treating cervical nerve root injury.

10. The use according to claim 9, wherein the medical preparation / repair material is used in conjunction with magnetic field activation.

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