A magnetically and thermally controlled dynamically powered nanomotor neural graft and its preparation method

Abstract

This invention provides a magnetically and thermally controlled dynamically powered nanomotor neural graft and its preparation method. The neural graft consists of a biodegradable polymer fiber conduit, an internally filled thermosensitive hydrogel, and a nanomotor loaded on the hydrogel. The surface of the nanomotor is loaded with energy molecules such as succinic acid. The nanomotor is prepared using a highly magnetically responsive material and achieves directional guidance under an applied magnetic field. Heat generated under an alternating magnetic field triggers a gel-sol transition in the thermosensitive hydrogel. Energy molecules are continuously released to increase local ATP levels, maintain cellular homeostasis, and support axonal regeneration and synaptic remodeling. This invention combines the advantages of magnetically driven directional navigation and thermosensitive controlled release, overcoming the shortcomings of traditional methods such as insufficient directionality and energy depletion. The graft exhibits good biocompatibility and is biodegradable, can regulate the immune microenvironment of damaged nerves, avoid oxidative stress, and efficiently promote the regeneration and functional recovery of long-segment damaged peripheral nerves.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a magnetic-thermal dual-controlled dynamically powered nanomotor neural graft and its preparation method. Background Technology

[0002] Peripheral nerve injury is the loss of peripheral nerve structure and / or function due to accidents, trauma, and other causes such as diabetes (a specific disease often associated with neuropathy), resulting in partial or complete loss of sensory, motor, and autonomic functions, as well as neuropathic pain. Current traditional surgical treatments, such as autologous and allogeneic nerve transplantation, are limited by donor availability, donor site dysfunction, and adverse immune responses. Drug therapy and structurally unmodified nerve scaffolds have limited efficacy due to low drug half-lives and unsatisfactory traction effects, respectively. Furthermore, the lack of or directional guidance in peripheral nerve regeneration prevents dynamic and precise guidance of axons to overcome the randomness of growth cones, leading to incomplete peripheral nerve regeneration, neuroma formation, or even regeneration failure. Immediately after peripheral nerve injury, metabolic disorders occur, and regenerating tissue suffers oxidative damage. Axonal regeneration requires a large energy supply, and insufficient energy in the local microenvironment (such as ATP) may affect Schwann cell activity and axonal growth rate / distance. Secondly, current regulatory methods are static and limited, making it difficult to achieve complex pathway guidance and continuous regulation.

[0003] Succinate, an energy molecule and an intermediate in the TCA cycle, directly participates in the electron transport chain and promotes ATP synthesis. Following nerve injury, the function of mitochondria in damaged neurons and surrounding glial cells is often impaired. Supplementing with succinate may maintain cellular homeostasis and support axonal regeneration and synaptic remodeling by increasing ATP levels. However, excessive succinate production may affect Cdc42 succinification and activity, thereby inhibiting the proliferation of neural stem cells. Therefore, controlling the duration and pattern of release is crucial for its therapeutic effect.

[0004] Ideally, the intracavitary structure of a neural graft should mimic the natural extracellular matrix, possess an appropriate elastic modulus, and a degradation rate adapted to the neural regeneration process. Currently, injectable thermosensitive hydrogels, by mimicking the natural extracellular matrix, offer multi-dimensional support for neural regeneration through dynamic microenvironment regulation and delivery of bioactive molecules. However, thermosensitive hydrogels lack autonomous movement capabilities: they typically rely on blood circulation or passive diffusion to reach the target area, resulting in low efficiency and difficulty in actively navigating to specific lesions far from the drug delivery point; if the external heat source is not applied precisely enough, it may lead to premature or insufficient drug release in non-target areas. In contrast to drug carriers, magnetically driven micro / nanomotors are micro / nanoscale systems that utilize external magnetic fields for remote, wireless, and precise control. They possess powerful mobility, enabling efficient movement in complex biofluids, overcoming Brownian motion and fluid resistance. The external magnetic field allows for real-time, precise, and non-invasive three-dimensional control of their position, velocity, and direction, enabling them to actively navigate through complex biological barriers and ultimately anchor precisely to the target lesion. Existing magnetic drive systems mostly focus on motion and space navigation, without integrating energy molecule loading and controlled release modules. This makes it difficult to cope with energy metabolism disorders and oxidative stress microenvironment during neural regeneration, thus limiting their role in promoting cell metabolic recovery and axon regeneration. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetic-thermal dual-control dynamically powered nanomotor nerve graft and its preparation method. By combining a magnetic field-driven nanomotor with a temperature-sensitive hydrogel, an intracavitary filling graft with a magnetic-thermal phase transition is constructed. This enables the magnetically driven nanomotor to guide the growth of peripheral nerves in a directional manner, while dynamically and sustainably releasing energy molecules to regulate the immune microenvironment of long-segment damaged peripheral nerves and avoid oxidative stress, providing a large amount of energy to promote the regeneration and functional recovery of damaged nerves.

[0006] The technical solution provided by this invention is as follows: A magnetocalorically controlled dynamically powered nanomotor neural graft, comprising:

[0007] Polymer fiber catheters;

[0008] A thermosensitive hydrogel is filled inside the polymer fiber conduit, and the gel-sol phase transition temperature of the thermosensitive hydrogel is 35~48 ℃.

[0009] The thermosensitive hydrogel contains dispersed nanomotors, which are carried by magnetic nanoparticles with amino-modified surfaces and load energy molecules through amide coupling reactions.

[0010] The magnetocaloric effect generated by the magnetic nanoparticles under the action of a magnetic field triggers the phase transition of the thermosensitive hydrogel, thereby controlling the release of the energy molecules.

[0011] The neural graft provided in this application has a core structure of polymer fiber conduit, thermosensitive hydrogel, and nanomotors. The conduit provides physical bridging support for long-segment nerve injuries and simulates the natural extracellular matrix.

[0012] The thermosensitive aqueous gel has a phase transition temperature of 35~48℃, which is adapted to the internal environment, thus avoiding false triggering by body temperature and precisely regulating the phase transition through magnetothermal control.

[0013] The nanomotor achieves stable loading of energy molecules through amino modification and amide coupling, preventing molecules from falling off. At the same time, the magnetocaloric effect of magnetic nanoparticles triggers the hydrogel phase transition, forming a magnetocaloric dual-control process of magnetic field, magnetocaloric, phase transition and controlled release.

[0014] The overall synergy achieves the effect of directional guidance of magnetic field-driven nanomotors and dynamic energy supply through controllable release of energy molecules, solving the problems of insufficient directionality, energy shortage, and single regulation in existing technologies.

[0015] Preferably, the temperature-sensitive hydrogel has a filling rate of 50-80% in the polymer fiber catheter. Too low a filling rate will result in insufficient load on the nanomotor and unsustainable power supply, while too high a filling rate may damage the catheter structure or affect tissue fluid exchange due to the expansion and solidification of the hydrogel.

[0016] Preferably, the nanomotor has a particle size of 200-400 nm, which can prevent nanomotor aggregation, ensure uniform dispersion in the hydrogel, and adapt to the size of cell migration channels without hindering nerve cell movement; the energy molecule loading rate is 25-60%, which meets the long-term energy requirements of nerve regeneration and prevents excessive release of energy molecules from inhibiting the proliferation of neural stem cells.

[0017] Preferably, the raw material of the polymer fiber conduit is any one or a combination of L-alanine-glycolic acid-lactic acid copolymer, L-alanine-glycolic acid copolymer, L-lysine-glycolic acid-lactic acid copolymer, aspartic acid-glycolic acid-lactic acid copolymer, glycine-glycolic acid-lactic acid copolymer, and glycine-glycolic acid copolymer.

[0018] The raw materials of the thermosensitive hydrogel include a gel substrate and a gel-sol phase transition temperature regulator. The gel substrate and the gel-sol phase transition temperature regulator are mixed to prepare a thermosensitive hydrogel precursor solution. The thermosensitive hydrogel precursor solution is cooled and solidified to form a thermosensitive hydrogel.

[0019] The gel substrate is any one or a combination of poly(N-acryloylglycine), poly(N-acryloylglycine)-polyacrylamide copolymer, polyacrylamide-polyacrylic acid copolymer, gelatin, and gelatin-based polymers; the gel-sol phase transition temperature regulator includes sorbitol solution and PBS buffer, wherein the concentration of sorbitol is 0.2~0.7 g / ml; and the PBS buffer is 1×PBS;

[0020] The magnetic nanoparticles are any one or a combination of magnetite, zinc ferrite, manganese ferrite, nickel ferrite, magnesium ferrite, cobalt ferrite, manganese zinc ferrite, iron-cobalt alloy, and iron oxide-based core-shell composite materials (such as Fe@Fe3O4, Fe3O4@Au). They have high saturation magnetization, strong magnetic responsiveness, and can generate heat under an alternating magnetic field.

[0021] The energy molecule is any one or a combination of isocitrate, α-ketoglutarate, succinyl-CoA, succinic acid, and malic acid.

[0022] Preferably, the raw material for the polymer fiber conduit is an L-alanine-glycolic acid-lactic acid copolymer;

[0023] When the gel substrate of the thermosensitive hydrogel is gelatin, the gel-sol phase transition temperature regulator is sorbitol solution, and the thermosensitive hydrogel precursor solution is prepared by mixing gelatin and sorbitol solution; the mass fraction of gelatin in the thermosensitive hydrogel precursor solution is 10~20wt%.

[0024] When the gel substrate of the thermosensitive hydrogel is polyN-acrylylglycine amide, the gel-sol phase transition temperature regulator is PBS buffer, and the thermosensitive hydrogel precursor solution is prepared by mixing polyN-acrylylglycine amide with PBS buffer; the concentration of polyN-acrylylglycine amide in the thermosensitive hydrogel precursor solution is 60~80 mg / ml.

[0025] The concentration ratios of gelatin and sorbitol, and poly(N-acrylylglycine) amide and PBS buffer, are carefully controlled to ensure the phase transition temperature falls precisely between 35 and 48°C. The magnetic nanoparticles are iron(III) oxide; the energy molecule is succinic acid.

[0026] The method for preparing the magnetically and thermally controlled dynamically powered nanomotor neural implant as described above includes the following steps:

[0027] Step 1) Dissolve the raw materials of the polymer fiber conduit in an organic solvent to prepare a spinning solution, and perform electrospinning to obtain the polymer fiber conduit;

[0028] Step 2) Magnetic nanoparticles are dispersed in deionized water and mixed with polyethyleneimine aqueous solution to obtain surface-modified amino magnetic nanoparticles. The surface-modified amino magnetic nanoparticles are then mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, energy molecules, and deionized water to carry out an amide coupling reaction. After the reaction is completed, a nanomotor is obtained.

[0029] Step 3) Mix the gel substrate of the thermosensitive hydrogel with a gel-sol phase transition temperature regulator to prepare a thermosensitive hydrogel precursor solution. Add a nanomotor to the solution and stir at 60-80 °C for 2-5 h to obtain a mixed solution.

[0030] Step 4) Inject the mixed solution into the polymer fiber conduit and cool it at room temperature for 3-6 hours to form a magnetothermal dual-controlled dynamic power supply nanomotor neural graft.

[0031] Preferably, in step 1), the concentration of the polymer fiber conduit raw material in the spinning solution is 0.1~0.4 g / ml, and the electrostatic parameters are set as follows: voltage -2~15 kV, collection distance 10~20 cm, and propulsion rate 0.5~2 ml / h. The organic solvent is one or a mixture of chloroform, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, and hexafluoroisopropanol. The electrospinning process ensures uniform directional arrangement of the conduit fibers. Under these parameters, fiber conduits with uniform diameter and stable mechanical properties can be prepared, providing reliable physical support for nerve regeneration and avoiding repair failure due to conduit structural defects.

[0032] Preferably, in step 2), the mass ratio of magnetic nanoparticles to polyethyleneimine is 1:25-35, and the molar ratio of the surface-modified amino-based magnetic nanoparticles to energy molecules is 1 g:0.05-0.2 mol; the molar ratio of succinic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1-1.5:1-1.5. The 1:25-35 mass ratio of magnetic nanoparticles to polyethyleneimine ensures sufficient amino modification of the particle surface, providing ample reaction sites for the amide coupling reaction and ensuring stable energy molecule loading. The 1 g:0.05-0.2 mol molar ratio avoids insufficient or excessive energy molecule loading, balancing the loading rate and the magnetic response performance of the nanomotor, ensuring that the magnetocaloric conversion efficiency is not affected by the loading process.

[0033] Preferably, in step 3), when the gel substrate of the thermosensitive hydrogel is gelatin, the gel-sol phase transition temperature regulator is a sorbitol solution. The thermosensitive hydrogel precursor solution is prepared by mixing gelatin with a sorbitol solution at a concentration of 0.2~0.7 g / ml, and the mass fraction of gelatin in the thermosensitive hydrogel precursor solution is 10~20 wt%.

[0034] When the gel substrate of the thermosensitive hydrogel is poly-N-acrylylglycine amide, the gel-sol phase transition temperature regulator is PBS buffer, and the thermosensitive hydrogel precursor solution is prepared by mixing poly-N-acrylylglycine amide with PBS buffer; the concentration of poly-N-acrylylglycine amide in the thermosensitive hydrogel precursor solution is 60~80 mg / ml.

[0035] Preferably, in step 3), the content of nanomotors in the mixed solution is 0.1~1 mg / ml. Within this range, the nanomotors are uniformly dispersed and continuously release energy molecules, while maintaining the biocompatibility and phase transition properties of the hydrogel, providing a stable energy microenvironment for nerve regeneration.

[0036] The beneficial effects of this invention are as follows: 1. The neural graft provided by this invention combines magnetic actuation with the properties of reversible thermosensitive hydrogel, possessing the powerful active movement and precise navigation capabilities of magnetically driven nanomotors and the temperature-triggered controllable release capability of thermosensitive hydrogels. This aims to overcome the bottlenecks of single technologies, achieve long-acting controlled drug release, and thus maximize therapeutic efficacy. Utilizing the synergistic effect of targeted therapy by magnetically controlled nanomotors and the gel-sol formation of thermosensitive hydrogels triggered by magnetothermal processes, the material can respond simultaneously or sequentially to two independent stimuli: temperature and magnetic field. This greatly enhances the precise, remote, and non-contact control over the material's state and movement. 2. Secondly, the magnetic field can non-invasively penetrate deep tissues, overcoming the limitation of shallow penetration by traditional heat sources, enabling remote and precise triggering of the phase transition of reversible thermosensitive hydrogels even deep within the body. Using magnetic field control, the clustered motors achieve directional migration within the gel matrix, promoting their active guidance of directional nerve growth. 3. The nanomotor is combined with drug release while moving. Heat triggers a phase change in the locally reversible temperature-sensitive hydrogel material, such as dissolution and flow, which promotes the flow and on-demand release of the material at the site of injury. After cooling, it solidifies and repairs, forming a dynamic and long-lasting controlled release mechanism, creating a good regenerative microenvironment and energy supply for the damaged nerve. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments disclosed in this invention, the accompanying drawings of the embodiments will be briefly described below. These drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0038] Figure 1The image shows the morphology of the nerve graft prepared in Example 1; a is a photograph of the nerve graft, and b is a scanning electron microscope image of the electrospun fiber membrane.

[0039] Figure 2 Transmission electron microscope (TEM) images of the iron oxide nanoparticles and nanomotors prepared in Example 1;

[0040] Figure 3 Magnetization curves of the iron oxide nanoparticles and nanomotor prepared in Example 1;

[0041] Figure 4 The results of hydration particle size analysis of the iron oxide nanoparticles and nanomotors prepared in Example 1 are shown.

[0042] Figure 5 The release curve of succinic acid from the nanomotor prepared in Example 1 under in vitro PBS environment;

[0043] Figure 6 The loading rate of succinic acid changes with different amounts of succinic acid added during the preparation process of the nanomotors in Examples 1-4;

[0044] Figure 7 The image shows a scanning electron microscope (SEM) image of the gelatin-based thermosensitive hydrogel loaded with nanomotors prepared in Example 1; a is an SEM image of the gelatin-based thermosensitive hydrogel loaded with nanomotors, and b is the field of view of the nanomotors loaded in the hydrogel.

[0045] Figure 8 The rheological properties of the gelatin-based thermosensitive hydrogels prepared in Examples 1, 5-7 vary with the amount of sorbitol.

[0046] Figure 9 The gel-sol transition temperature of the gelatin-based thermosensitive hydrogels prepared in Examples 1, 5-7 as a function of sorbitol content is shown in the graph.

[0047] Figure 10 Phase transition diagrams of polyN-acrylylglycine amide gels of different concentrations prepared in Examples 8-10 in PBS buffer;

[0048] Figure 11 Slow-release curves of succinic acid in the gelatin-based thermosensitive hydrogel loaded with nanomotors prepared in Example 1 at different ambient temperatures.

[0049] Figure 12 Heating curves of the gelatin-based thermosensitive hydrogel loaded with nanomotors prepared in Example 1 under different magnetic field strengths for 10 minutes.

[0050] Figure 13 Heating and cooling temperature distribution of the gelatin-based thermosensitive hydrogel with loaded nanomotor prepared in Example 1 during six repeated magnetothermal on / off cycles.

[0051] Figure 14 The image shows the movement of clustered nanomotors in a gelatin-based thermosensitive hydrogel with nanomotors prepared in Example 1 within a certain magnetothermal cycle under an alternating magnetic field. Detailed Implementation

[0052] The working principle of this invention is as follows: When applying a magnetothermal dual-controlled dynamically powered nanomotor nerve graft to the repair of peripheral nerve injuries, under the action of an external magnetic field, the magnetic nanoparticles in the nanomotor generate heat due to the magnetothermal effect. This causes the thermosensitive hydrogel in the polymer fiber conduit to reach a certain phase transition temperature, resulting in a gel-sol transition. The nanomotor clusters loaded in the hydrogel then migrate directionally along the magnetic field. Simultaneously, since the tissue fluid fills the entire injured area, the hydrogel in the conduit comes into contact with the surrounding tissue fluid, and the nanomotors loaded in the hydrogel release succinic acid in a slow-release manner. This achieves the purpose of simultaneous directional movement and long-term controlled release, thereby playing a role in guiding peripheral nerve regeneration, as well as exerting anti-inflammatory and antioxidant effects, reducing the excessive production of reactive oxygen species, regulating the microenvironment for peripheral nerve regeneration, and avoiding excessive release of succinic acid, which could affect the proliferation of neural stem cells.

[0053] The technical solutions (including preferred technical solutions) of the present invention will be further described in detail below with reference to the accompanying drawings and by way of listing some optional embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0054] Example 1

[0055] This embodiment provides a magnetothermal dual-controlled dynamically powered nanomotor neural graft, including the following steps:

[0056] Step 1): Preparation of polymer fiber conduit: Weigh 1 g of L-alanine-hydroxyacetic acid-lactic acid copolymer with a molecular weight of 100,000.

[0057] The L-alanine-glycolic acid-lactic acid copolymer was prepared in the laboratory using the following method: 1. 50 g (0.56 mol) of L-alanine and 140 mL of 4M sodium hydroxide aqueous solution were mixed and placed in a low-temperature constant temperature reaction bath at -13 ℃. 180 mL of anhydrous dichloromethane (DCM) solution containing chloroacetyl chloride (44.8 mL) and 180 mL of 4M sodium hydroxide aqueous solution were slowly added dropwise, maintaining the pH at approximately 12 during the addition process. After the addition was complete, the mixture was stirred for 2 hours to ensure complete reaction. After the reaction was complete, the pH was adjusted to 1 with concentrated hydrochloric acid to acidify the solution. The reaction solution was extracted three times with 120 mL of ethyl acetate, and the extracted oil phases were combined. The oil phase was washed with saturated sodium chloride aqueous solution and then dried with anhydrous magnesium sulfate. Most of the solvent was removed using a rotary evaporator to concentrate the product, and the resulting colorless crystals were dried under vacuum.

[0058] 2. Weigh 20 g of the above product and dissolve it in 100 mL of anhydrous dimethylformamide (DMF). Transfer this solution to a 250 mL Erlenmeyer flask. Then, add 30 mL of trifluoroacetic acid (TEA) and 250 mL of DMF to a 500 mL three-necked flask. Under nitrogen protection, heat the three-necked flask to 95 °C and use a peristaltic pump to add the former solution dropwise to the flask at a constant rate. After the addition is complete, remove the oil bath and allow the reaction mixture to cool to room temperature. Crystallization and filtration are then performed, followed by repeated washing with dichloromethane. The resulting product is recrystallized from ethyl acetate and dried under vacuum.

[0059] 3. Add 2 g of the above product, 6 g of L-lactide, 2 g of glycolide, and 1.6 mL of a 1% (w / v) Sn(Oct)₂ chloroform solution to a polymerization reaction tube. Freeze the mixture in liquid nitrogen for 20 min, evaporate the chloroform under reduced pressure, and then dry it under vacuum at 50 °C for 1 h. Seal the reaction tube under vacuum. Place it in an oil bath preheated to 140 °C and react for 16 h. After polymerization, dissolve the product in chloroform, and then add the solution dropwise to excess petroleum ether to remove any unreacted monomers and initiators. Repeat this process three times. Finally, dry the product under vacuum at room temperature.

[0060] The obtained product was dissolved in 6.67 ml of hexafluoroisopropanol and mixed evenly using a magnetic stirrer to obtain a spinning solution with a concentration of 0.15 g / ml. The electrospinning process parameters were set as follows: positive voltage of 8 kV, negative voltage of 2 kV, feed rate of 1 ml / h, receiving distance of 15 cm, needle type of 22G, and a roller with a rotation speed of 2400 r / min was selected as the collecting device. The fibers were wound along the surface of the roller to form a fiber membrane. The collected fiber membrane was cut and wound to obtain a polymer fiber guide with a length of 13 mm.

[0061] Step 2): Preparation of nanomotors: Weigh 2 mmol of ferric chloride hexahydrate, 4 mmol of trisodium citrate, 6 mmol of urea, and polyacrylamide and add them to 40 ml of deionized water. Stir until completely dissolved and react at 200 °C for 12 h. After washing and drying, iron oxide particles are obtained. Disperse 10 mg of iron oxide particles in 5 ml of deionized water, add polyethyleneimine aqueous solution (2%, 15 ml), and mechanically stir for 3 h. After treatment, surface-modified amino magnetic nanoparticles (surface-modified amino iron oxide particles) are obtained.

[0062] 10 mg of amino-modified iron oxide particles were weighed and reacted with 2 mmol of succinic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) (wherein the molar ratio of succinic acid, EDC, and NHS was 1:1:1.5) and 25 ml of deionized water at room temperature in the dark for 12 h. After washing and drying, succinic acid-loaded iron oxide particles (i.e., nanomotors) were obtained.

[0063] The particle size of the nanomotor was determined to be 334.5 nm using a Malvern particle size analyzer, based on its hydration kinetic diameter. The concentration of succinic acid was measured by detecting a characteristic peak at 213 nm using high-performance liquid chromatography (HPLC), yielding a succinic acid loading rate of 54.71% for the nanomotor and a succinic acid release test period of 14 days. The loading rate, MS, and MC represent the mass of the loaded succinic acid and the mass of the amino-terminated magnetic nanoparticles used, respectively.

[0064] Step 3): Preparation of the thermosensitive gel precursor solution for dispersing nanomotors: 0.45 g sorbitol, 1 ml deionized water, and gelatin (Gelatin from porcine skin, type A, ~300 g Bloom) were dissolved by stirring at 60 °C for 3 h to form a homogeneous solution, wherein the gelatin content in the homogeneous solution was 18 wt%. 0.5 mg of the nanomotors prepared in Step 2 were added to the solution, and the mixture was homogenized by vortex mixing to obtain the thermosensitive gel precursor solution for dispersing nanomotors.

[0065] Step 4): Using a syringe, draw up the thermosensitive gel precursor solution of the dispersed nanomotor prepared in Step 3 and inject it into the polymer fiber conduit prepared in Step 1. The filling amount is 60% of the volume of the polymer fiber conduit. Place it at room temperature to cool for 3 hours to form a magnetothermal dual-controlled dynamic power supply nanomotor neural graft.

[0066] The gel-sol transition temperature of the thermosensitive hydrogel was determined to be 43.4 ℃ by performing a dynamic temperature scan from 20 ℃ to 50 ℃ using an MCR 302e rotational rheometer at a constant frequency of 1 Hz, a constant strain of 1% and a programmed temperature rise rate of 1 ℃ / min.

[0067] Example 2

[0068] This embodiment provides a magnetic-thermal dual-controlled dynamically powered nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 2, the amount of succinic acid added is 1.5 mmol.

[0069] The hydrated particle size of the nanomotor prepared in step 2 was 350 nm, the loading rate of succinic acid was 49.59%, and the succinic acid release test period was 14 days.

[0070] Example 3

[0071] This embodiment provides a magnetic-thermal dual-controlled dynamically powered nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 2, the amount of succinic acid added is 1 mmol.

[0072] The hydrated particle size of the nanomotor prepared in step 2 was 342.6 nm, the loading rate of succinic acid was 41.27%, and the succinic acid release test period was 14 days.

[0073] Example 4

[0074] This embodiment provides a magnetic-thermal dual-controlled dynamically powered nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 2, the amount of succinic acid added is 0.5 mmol.

[0075] The hydrated particle size of the nanomotor prepared in step 2 was 310.3 nm, the loading rate of succinic acid was 25.7%, and the succinic acid release test period was 14 days.

[0076] Example 5

[0077] This embodiment provides a magnetic-thermal dual-controlled dynamic power supply nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 3, the amount of sorbitol added is 0.225 g.

[0078] The gel-sol transition temperature of the thermosensitive hydrogel in the magnetothermal dual-controlled dynamic power supply nanomotor neural graft is 41.5 ℃.

[0079] Example 6

[0080] This embodiment provides a magnetic-thermal dual-controlled dynamic power supply nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 3, the amount of sorbitol added is 0.05 g.

[0081] The gel-sol transition temperature of the thermosensitive hydrogel in the magnetothermal dual-controlled dynamic power supply nanomotor neural graft is 37.8 ℃.

[0082] Example 7

[0083] This embodiment provides a magnetic-thermal dual-controlled dynamic power supply nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 3, the amount of sorbitol added is 0.675 g.

[0084] The gel-sol transition temperature of the thermosensitive hydrogel in the magnetothermal dual-controlled dynamic power supply nanomotor neural graft is 45.8 ℃.

[0085] Example 8

[0086] This embodiment provides a magnetothermal dual-controlled dynamically powered nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except for step 3, where N-acryloylglycine amide (3.9 mmol) is dissolved in dimethyl sulfoxide (DMSO, 18 ml), and then 3.5 mg of AIBN is added, followed by degassing with nitrogen for 40 min. The solution is then polymerized in a 70°C oil bath for 2 hours, cooled in an ice bath to stop the reaction, and precipitated in 200 mL of methanol. The precipitate is centrifuged and washed three times with methanol, then vacuum dried at 80°C for 72 hours to obtain poly(N-acryloylglycine amide) in powder form. 60 mg / ml of poly(N-acryloylglycine amide) and 1×PBS buffer are dissolved by stirring at 60°C to form a homogeneous solution. 0.5 mg of succinic acid-loaded iron oxide particles obtained in step 2 are added to this solution, and the mixture is vortexed to obtain a thermosensitive gel precursor solution loaded with nanomotors.

[0087] The gel-sol transition temperature of the thermosensitive hydrogel in the magnetothermal dual-controlled dynamic power supply nanomotor neural graft is 35 ℃.

[0088] Example 9

[0089] This embodiment provides a magnetothermal dual-controlled dynamic power supply nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 3, the concentration of polyN-acrylylglycine amide is 70 mg / ml.

[0090] The gel-sol transition temperature of the thermosensitive hydrogel in the magnetothermal dual-controlled dynamic power supply nanomotor neural graft is 42 ℃.

[0091] Example 10

[0092] This embodiment provides a magnetothermal dual-controlled dynamic power supply nanomotor neural graft, the preparation steps of which are basically the same as those in Example 1, except that in step 3, the concentration of polyN-acrylylglycine amide is 80 mg / ml.

[0093] The gel-sol transition temperature of the thermosensitive hydrogel in the magnetothermal dual-controlled dynamic power supply nanomotor neural graft is 48 ℃.

[0094] like Figure 1 As shown, the magnetothermal dual-controlled dynamic power supply nanomotor nerve graft prepared in Example 1 exhibits no obvious deformation, cracking, or delamination in its macroscopic morphology, indicating that the fiber conduit rolled after electrospinning has good formability and can serve as a physical bridging scaffold for nerve injury sites, adapting to the repair needs of long-segment peripheral nerve injuries. Microscopically, the polymer fiber conduit is formed by the continuous and uniform directional arrangement of nanoscale fibers with uniform fiber diameter, proving that the electrospinning process parameters are conducive to the stable preparation of conduits with consistent mechanical properties, avoiding support failure caused by fiber breakage and aggregation.

[0095] like Figure 2 As shown, the iron oxide particles prepared in Example 1 are spherical or near-spherical, with no obvious agglomeration, and the particles are independent of each other, ensuring high saturation magnetization and magnetic responsiveness. The surface of the nanomotor (FNSA) particles loaded with succinic acid becomes rough and blurred, but still maintains a spherical structure, proving that succinic acid was successfully loaded onto the surface of iron oxide through polyethyleneimine adsorption and amide coupling reaction.

[0096] like Figure 3 As shown, the nanomotor prepared in Example 1 exhibits a saturation magnetization value of 63.8 emu g due to the loading of succinic acid. -1 Reduced to 52.4 emu g -1 However, the nanomotor (FNSA) still exhibits superparamagnetism. This strong magnetic response characteristic makes it possible to apply an external magnetic field for targeted action in vivo. Figure 4As shown, both the iron oxide particles prepared in Example 1 and the nanomotor particles loaded with succinic acid exhibit single, sharp particle size distribution peaks, without obvious broad peaks or overlapping peaks. The hydrated particle size of FNSA is concentrated in the range of 200–400 nm (the horizontal axis in the figure is presented in logarithmic form). This indicates that the amide coupling reaction did not induce particle aggregation, and FNSA can be independently dispersed in aqueous solution, laying the foundation for subsequent uniform mixing into the thermosensitive hydrogel. The size difference between the hydrated particle size measured by Malvern particle size analyzer and the TEM results can be attributed to the presence of the hydrated shell of the particles in the solution during the hydrodynamic size measurement.

[0097] like Figure 5 As shown, the succinic acid-loaded nanomotor prepared in Example 1 showed a high rate of succinic acid release in the first two days under PBS conditions, followed by a stable release phase, and continued to release succinic acid until the 14th day.

[0098] like Figure 6 As shown in Examples 1-4, the loading rate of succinic acid by the nanomotors increased with the increase of succinic acid addition, but the rate of increase in loading rate decreased. In particular, the growth rate of loading rate was the largest when the amount of succinic acid added increased from 0.5 mmol to 1 mmol, indicating that in this range, the amount added is the most important factor affecting the loading rate of succinic acid.

[0099] like Figure 7 As shown, the gelatin-based thermosensitive hydrogel loaded with nanomotors prepared in Example 1 exhibits a porous structure with nanomotor particles uniformly dispersed within it. The porous structure of the hydrogel provides a hydrated three-dimensional environment, more suitable for cell migration and nutrient exchange, and allows for easy doping with growth factors or other synthetic materials to promote nerve regeneration. Furthermore, the integration of nanomotors offers an innovative approach for precisely controlling drug delivery systems.

[0100] like Figures 8-9 As shown in Examples 1, 5-7, the phase transition temperature of the hydrogel changes with the amount of sorbitol added. The temperature phase transition point of the hydrogel is tested using a rotational rheometer, where G' represents the storage modulus of the hydrogel and G'' represents the loss modulus. During the dynamic temperature scan, the intersection of G' and G'' represents the collapse point of the three-dimensional network of the hydrogel, that is, the transition of the hydrogel from the gel state to the sol state, also known as the phase transition point of the thermosensitive hydrogel.

[0101] With increasing sorbitol content, the phase transition temperature increases. During cooling, due to the addition of sorbitol, some β-turns of the amorphous gelatin molecular chains transform into short-range β-sheets. The appearance of short-range β-sheets makes the three-dimensional network structure of the hydrogel more compact and stable. The properties of the hydrogel are modulated to ensure it can provide mechanical support for nerve regeneration. Simultaneously, the behavior of the hydrogel—maintaining a gel state below the phase transition temperature and a sol state above it—was verified using a more intuitive vial inversion method. This thermal reversibility also plays a regulatory role in the subsequent release of succinic acid from the nanomotor. This temperature-sensitive behavior has wide applications as a drug and gene carrier, a temperature-targeted therapeutic material, and a tissue engineering material.

[0102] like Figure 10 As shown, different concentrations of poly(N-acrylylglycine amide) gels in Examples 8-10 exhibited different phase transition temperatures. The gel-sol state transition behavior was verified using the inverted test tube method. Gelatin-based gels are naturally derived, bio-based protein hydrogels whose properties depend on the collagen source, extraction process, and degree of modification. Poly(N-acrylylglycine amide) gel (PNAGA) is a fully synthetic hydrogel formed by the free radical polymerization of monomers to create a polymer network. Its properties can be controlled by monomer concentration, polymerization conditions, etc. Both are temperature-sensitive. Gelatin gel is softer, more biomimetic, has better biocompatibility, promotes cell adhesion, and is biodegradable, making it widely used in tissue engineering scaffolds and drug delivery. PNAGA has high strength and toughness, long-term stability, and high reproducibility, but its degradability is somewhat worse than that of natural gelatin.

[0103] like Figure 11 As shown, the succinic acid release from the gelatin-based thermosensitive hydrogel loaded with nanomotors prepared in Example 1 differed at three different ambient temperatures. 1) Below the phase transition temperature (37 °C), the succinic acid release rate was the slowest, while the release amount gradually increased; however, compared to the succinic acid release from the nanomotors... Figure 5 The overall release rate and amount are reduced. This is because the hydrogel is in a gel state at this time, which restricts the diffusion of nanomotors, and succinic acid also needs time to be released from the nanomotors and pass through the gel network. This release behavior is relatively stable and controllable, and is suitable for scenarios requiring long-term, low-dose continuous drug release.

[0104] 2) Above the phase transition temperature (45 °C), succinic acid exhibits the fastest release rate and the largest release volume, similar to the release of succinic acid by nanomotors. This is because the hydrogel is in a sol state at this temperature, allowing the nanomotors to diffuse freely in the low-viscosity liquid with minimal hindrance; simultaneously, succinic acid released from the nanomotors also diffuses rapidly due to the low viscosity of the environment. This high and rapid release behavior is suitable for scenarios requiring rapid and large-volume drug delivery (such as local administration in response to inflammation and fever, or chemotherapy triggered by thermotherapy).

[0105] 3) When magnetothermal application is intermittent, the temperature fluctuates around the phase transition temperature (37 / 45 ℃). In this state, magnetothermal application leads to the dominant release of succinic acid, similar to 2 (high temperature sustained), with a rapid release rate. Without magnetothermal application, release slows drastically or even stops, similar to 1 (low temperature sustained). This is because intermittent magnetothermal application causes the hydrogel to repeatedly transition between gel and sol states. During the high-temperature (sol) phase, nanomotors gain freedom and begin to diffuse and release succinic acid. When the temperature decreases and the hydrogel returns to its gel state, it may be captured or blocked by the reformed network. The recaptured nanomotors and succinic acid molecules are slowly released at low temperatures. This highly controllable, on-demand release is suitable for scenarios requiring timed, quantitative, and on-demand drug delivery (such as precision chemotherapy in response to external thermal stimuli).

[0106] like Figure 12 As shown, the magnetocaloric properties of the gelatin-based thermosensitive hydrogel loaded with nanomotors prepared in Example 1 were evaluated. The temperature change of the 0.5 mg / ml nanomotor-loaded hydrogel under magnetocaloric heating for 10 min at different magnetic field strengths (12, 18, 24, 36 mT) was observed using a high-frequency magnetic induction heater. The temperature showed a gradual increasing trend with increasing magnetic field strength. This demonstrates that magnetocaloric heating can reach the phase transition temperature of the hydrogel, thereby triggering the gel-sol transition.

[0107] like Figure 13 As shown, in six cycles of magnetic field on and off at room temperature (0.5 mg / ml, 24 mT, on for 10 minutes, then off for 15 minutes), the gelatin-based thermosensitive hydrogel with nanomotor loaded in Example 1 exhibited a similar maximum temperature without decay in its magnetocaloric effect, demonstrating a durable and temperature-dependent magnetocaloric response. Increased temperature reduces magnetic anisotropy, leading to easy reversal of particle magnetic moments and weakening targeting ability. Highly stable particles maintain superparamagnetism at body temperature, avoiding aggregation.

[0108] like Figure 14As shown, the directional movement effect of the magnetothermal dual-controlled dynamically powered nanomotor neural graft prepared in Example 1 is demonstrated. Under an alternating magnetic field, the magnetic field (24 mT) is activated every 10 minutes to reach the phase transition temperature of the hydrogel. From the first to the 21st activation, the clustered nanomotors exhibit significant directional displacement. The nanomotors, precisely controlled by an external magnetic field, can overcome the limitations of non-directional diffusion and achieve active movement, exhibiting a cluster synergistic effect. Through the integrated function of precise navigation, dynamic scaffolding, intelligent drug release, and energy supply, the clustered nanomotors provide a new approach to transforming neural regeneration from passive waiting to active reconstruction.

[0109] It will be readily understood by those skilled in the art that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, combinations, substitutions, improvements, etc., made under the spirit and principles of the present invention are included within the protection scope of the present invention.

Claims

1. A magnetothermal dual-controlled dynamically powered nanomotor neural graft, characterized in that, include: Polymer fiber catheters; A thermosensitive hydrogel is filled inside the polymer fiber conduit, and the gel-sol phase transition temperature of the thermosensitive hydrogel is 35~48 ℃. The thermosensitive hydrogel contains dispersed nanomotors, which are carried by magnetic nanoparticles with amino-modified surfaces and load energy molecules through amide coupling reactions. The magnetocaloric effect generated by the magnetic nanoparticles under the action of a magnetic field triggers the phase transition of the thermosensitive hydrogel, thereby controlling the release of the energy molecules; The energy molecule is any one or a combination of isocitrate, α-ketoglutarate, succinyl-CoA, succinic acid, and malic acid.

2. The magnetothermal dual-controlled dynamically powered nanomotor neural graft according to claim 1, characterized in that, The thermosensitive hydrogel has a filling rate of 50-80% in the polymer fiber conduit.

3. The magnetothermal dual-controlled dynamically powered nanomotor neural graft according to claim 1, characterized in that, The nanomotor has a particle size of 200-400 nm; the energy molecule loading rate is 25-60%.

4. The magnetothermal dual-controlled dynamically powered nanomotor neural graft according to claim 1, characterized in that, The raw material of the polymer fiber duct is any one or a combination of L-alanine-glycolic acid-lactic acid copolymer, L-alanine-glycolic acid copolymer, L-lysine-glycolic acid-lactic acid copolymer, aspartic acid-glycolic acid-lactic acid copolymer, glycine-glycolic acid-lactic acid copolymer, and glycine-glycolic acid copolymer. The raw materials of the thermosensitive hydrogel include a gel substrate and a gel-sol phase transition temperature regulator. The gel substrate and the gel-sol phase transition temperature regulator are mixed to prepare a thermosensitive hydrogel precursor solution. The thermosensitive hydrogel precursor solution is cooled and solidified to form a thermosensitive hydrogel. The gel substrate is any one or a combination of poly(N-acryloylglycine), poly(N-acryloylglycine)-polyacrylamide copolymer, polyacrylamide-polyacrylic acid copolymer, gelatin, and gelatin-based polymers; the gel-sol phase transition temperature regulator includes sorbitol solution and PBS buffer, wherein the concentration of sorbitol is 0.2~0.7 g / ml; and the PBS buffer is 1×PBS; The magnetic nanoparticles are any one or more combinations of iron oxide, zinc ferrite, manganese ferrite, nickel ferrite, magnesium ferrite, cobalt ferrite, manganese zinc ferrite, iron-cobalt alloy, and iron oxide-based core-shell composite materials.

5. The magnetocalorically controlled dynamically powered nanomotor neural graft according to claim 4, characterized in that, The raw material for the polymer fiber duct is an L-alanine-hydroxyacetic acid-lactic acid copolymer; When the gel substrate of the thermosensitive hydrogel is gelatin, the gel-sol phase transition temperature regulator is sorbitol solution, and the thermosensitive hydrogel precursor solution is prepared by mixing gelatin and sorbitol solution; the mass fraction of gelatin in the thermosensitive hydrogel precursor solution is 10~20wt%. When the gel substrate of the thermosensitive hydrogel is polyN-acrylylglycine amide, the gel-sol phase transition temperature regulator is PBS buffer, and the thermosensitive hydrogel precursor solution is prepared by mixing polyN-acrylylglycine amide with PBS buffer; the concentration of polyN-acrylylglycine amide in the thermosensitive hydrogel precursor solution is 60~80 mg / ml. The magnetic nanoparticles are iron(III) oxide; the energy molecules are succinic acid.

6. A method for preparing a magnetically and thermally controlled dynamically powered nanomotor neural implant according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1) Dissolve the raw materials of the polymer fiber conduit in an organic solvent to prepare a spinning solution, and perform electrospinning to obtain the polymer fiber conduit; Step 2) Magnetic nanoparticles are dispersed in deionized water and mixed with polyethyleneimine aqueous solution to obtain surface-modified amino magnetic nanoparticles. The surface-modified amino magnetic nanoparticles are then mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, energy molecules, and deionized water to carry out an amide coupling reaction. After the reaction is completed, a nanomotor is obtained. Step 3) Mix the gel substrate of the thermosensitive hydrogel with a gel-sol phase transition temperature regulator to prepare a thermosensitive hydrogel precursor solution, and add nanomotors to it to obtain a mixed solution. Step 4) The mixed solution is injected into the polymer fiber conduit and cooled and shaped at room temperature to obtain a magnetothermal dual-controlled dynamic power supply nanomotor neural graft.

7. The method for preparing a magnetically and thermally controlled dynamically powered nanomotor neural implant according to claim 6, characterized in that, In step 1), the concentration of the raw material for the polymer fiber conduit in the spinning solution is 0.1~0.4 g / ml, and the electrospinning parameters are set as follows: voltage is -2~15 kV, collection distance is 10~20 cm, and propulsion rate is 0.5~2 ml / h.

8. The method for preparing a magnetically and thermally controlled dynamically powered nanomotor neural implant according to claim 6, characterized in that, In step 2), the mass ratio of magnetic nanoparticles to polyethyleneimine is 1:25~35, and the mass ratio of the surface-modified amino magnetic nanoparticles to the molar amount of energy molecules is 1 g:0.05~0.2 mol.

9. The method for preparing a magnetically and thermally controlled dynamically powered nanomotor neural implant according to claim 6, characterized in that, In step 3), when the gel substrate of the thermosensitive hydrogel is gelatin, the gel-sol phase transition temperature regulator is sorbitol solution. The thermosensitive hydrogel precursor solution is prepared by mixing gelatin with a sorbitol solution at a concentration of 0.2~0.7 g / ml, and the mass fraction of gelatin in the thermosensitive hydrogel precursor solution is 10~20 wt%. When the gel substrate of the thermosensitive hydrogel is poly-N-acrylylglycine amide, the gel-sol phase transition temperature regulator is PBS buffer, and the thermosensitive hydrogel precursor solution is prepared by mixing poly-N-acrylylglycine amide with PBS buffer; the concentration of poly-N-acrylylglycine amide in the thermosensitive hydrogel precursor solution is 60~80 mg / ml.

10. The method for preparing a magnetically and thermally controlled dynamically powered nanomotor neural implant according to claim 6, characterized in that, In step 3), the content of nanomotors in the mixed solution is 0.1~1 mg / ml.

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