Rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances as well as preparation method and application of rigid-flexible composite nerve conduit

Abstract

The invention relates to a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances, which comprises a polymer fiber conduit part and a hydrogel part, and the polymer fiber conduit part is a conduit prepared from a double-layer polymer fiber membrane of which the inner layer is unidirectionally ordered and the outer layer is irregularly arranged; and the hydrogel part is a modified biological macromolecular hydrogel containing anti-inflammatory substance nanoparticles. According to the invention, low-modulus hydrogel with an adjustable degradation rate is used as a carrier of drug-loaded nanoparticles and is combined with an electrostatic spinning nanofiber scaffold which is similar to an extracellular matrix structure and has excellent mechanical properties to form an ideal design of the rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances; due to the existence of the hydrogel, in-situ controllable delivery of the drug-loaded nanoparticles can be realized, the action efficiency of the nanoparticles is improved, the electrostatic spinning nanofiber with an adjustable topological structure can stabilize the form of the hydrogel, and the hydrogel plays a guiding and promoting role in subsequent spinal cord total transection injury repair after being degraded.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances, and a preparation method and application thereof. Background Art

[0002] Complete spinal cord transection injury is a serious clinical disease, and its treatment has always been a difficult problem in the medical community. This injury can lead to loss of sensation and motor ability below the damaged spinal cord segment, as well as sphincter dysfunction, which has a great negative impact on the patient's quality of life and life span. The inflammatory response after spinal cord transection is a key factor leading to secondary damage, and in the subsequent tissue regeneration stage, due to the lack of favorable induction, it is difficult for the regenerated axons to pass through the injured area to reach the descending spinal cord tract and establish neural connections. At present, drug therapy is often used clinically to reduce the inflammatory response, but the presence of the blood-brain-spinal cord barrier limits the effect of drug therapy, and it is difficult for the drug to work for a long time in the injured area. There are also problems such as low utilization rate and serious side effects. In addition, there is currently no effective means to induce directional growth of axons in clinical practice. Therefore, designing a hydrogel loaded with anti-inflammatory substances to provide a fiber conduit with physical induction signals, achieving controlled release of anti-inflammatory substances and promoting directional growth of axons while working for a long time at the injured site has become a good solution to the above problems.

[0003] Anti-inflammatory substances have a short half-life in the body and are quickly inactivated, so they cannot continue to play a role in the early stage of transection. Therefore, they need carrier protection (El-Shishtawy RM, Ahmed NSE, Almulaiky Y Q. Immobilization of catalase on chitosan / ZnO and chitosan / ZnO / Fe2O3 nanocomposites: a comparative study[J]. Catalysts, 2021, 11(7): 820.; Sun X, Yu K, Zhou Y, et al. Self-assembled pH-sensitive polymeric nanoparticles for the inflammation-targeted delivery of Cu / Zn-superoxide dismutase[J]. ACS Applied Materials & Interfaces, 2021, 13(15): 18152-18164.) In the selection of carriers for anti-inflammatory substances, nanoparticles can be a good choice. They can prolong the half-life of drugs and achieve long-term effects of drugs in the damaged area. Nanoparticles can also penetrate the blood-brain-spinal cord barrier to improve the utilization rate of drugs. At present, the size of nanoparticles with three layers of outer, middle and inner structures is generally larger, which is suitable for application scenarios such as patches with large contact areas with tissues and rapid drug release. However, in application scenarios such as catheters with small contact areas with tissues and unable to release drugs quickly, nanoparticles with three-layer structures will be difficult to release on demand due to size factors.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] Based on this, the present invention provides a rigid-flexible composite nerve conduit for controllable delivery of anti-inflammatory substances. By adopting a hydrogel-loaded anti-inflammatory substance nanoparticle delivery system with adjustable degradation rate, controllable delivery of anti-inflammatory substances can be achieved, the inflammatory microenvironment at the early stage of injury can be effectively regulated, and cell survival rate can be improved. At the same time, the rigid-flexible composite design of this conduit enables it to have both strong mechanical properties and a low modulus matrix suitable for cell growth, wherein the degradation rates of hydrogel and nanofiber are quite different. After the hydrogel is delivered in the early stage, the fiber can maintain its morphology and unidirectional ordered topological structure on the surface in vivo, and continuously guide the nerve axons through the injured area in the subsequent regeneration stage, connecting the ascending and descending nerve bundles, and enhancing the repair effect, providing a new approach and method for the design of spinal cord complete transection injury repair materials.

[0006] One of the purposes of the present invention is to provide a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances.

[0007] The catheter includes a polymer fiber catheter portion and a hydrogel portion;

[0008] The polymer fiber conduit portion is a double-layer conduit structure prepared from a polymer fiber membrane; the inner layer of the polymer fiber conduit portion is a unidirectional orderly arranged polymer fiber layer, and the outer layer is a randomly arranged polymer fiber layer;

[0009] The hydrogel part is a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles; the anti-inflammatory substance nanoparticles contain a two-layer structure, wherein the inner layer is the anti-inflammatory substance and the outer layer is the cationic polymer.

[0010] Preferably, the wall thickness of the polymer fiber conduit portion is 50 to 800 μm, preferably 100 to 500 μm;

[0011] The volume of the hydrogel portion is 4 to 12 μL, preferably 6 to 8 μL;

[0012] The particle size of the anti-inflammatory substance nanoparticles is 30 to 200 nm, preferably 50 to 100 nm.

[0013] Preferably, the polymer in the polymer fiber layer is selected from degradable aliphatic polyesters, preferably at least one selected from polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer;

[0014] The modified biomacromolecule in the modified biomacromolecule hydrogel is an acrylated biomacromolecule, preferably at least one selected from methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk fibroin, methacrylated chitosan, and methacrylated sodium alginate;

[0015] The anti-inflammatory substance is selected from at least one of catalase, superoxide dismutase, interleukin, and reducing molecules;

[0016] The cationic polymer is selected from at least one of polyarginine, polylysine, gelatin, polyhistidine, polyethyleneimine, polyacrylamide and polyacrylamine.

[0017] The second object of the present invention is to provide a method for preparing a rigid-flexible composite nerve conduit for controllably delivering anti-inflammatory substances.

[0018] The preparation method comprises electrostatic spinning of a polymer solution to obtain a double-layer nanofiber membrane having an inner layer of unidirectional orderly arranged nanofibers and an outer layer of randomly arranged nanofibers, cross-linking a hydrogel precursor solution containing anti-inflammatory substances by ultraviolet light to obtain a hydrogel containing anti-inflammatory substances, and then wrapping the hydrogel containing anti-inflammatory substances with a polymer fiber membrane having an inner layer of unidirectional order and an outer layer of randomly arranged nanofibers, thereby obtaining the rigid-flexible composite nerve conduit for controllably delivering anti-inflammatory substances.

[0019] Preferably, the preparation method specifically comprises the following steps:

[0020] Step (1): electrospinning the polymer solution A to prepare a double-layer polymer fiber layer with an inner layer having unidirectional order and an outer layer having random arrangement;

[0021] Step (2): mixing the anti-inflammatory substance solution B with the cationic polymer solution C to obtain an anti-inflammatory substance nanoparticle suspension D;

[0022] Step (3): adding an initiator to the anti-inflammatory substance nanoparticle suspension D to obtain an initiator solution E, and then adding the modified biomacromolecule to the initiator solution E to obtain a hydrogel precursor solution F;

[0023] Step (4): injecting the hydrogel precursor solution F into the mold, and crosslinking it under ultraviolet light to obtain a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles;

[0024] Step (5): The inner layer of the polymer fiber membrane obtained in step (1) with unidirectional order and the outer layer with random arrangement is contacted with the modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles obtained in step (4), and wrapped into a tube, and finally bonded with polymer solution A to obtain the rigid-flexible composite nerve conduit for controllable delivery of anti-inflammatory substances.

[0025] Preferably, in step (1):

[0026] The solvent in the polymer solution A is selected from at least one of hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane, and N,N'-dimethylformamide; the mass percentage concentration of the polymer solution A is 5 to 25%, preferably 8 to 15%;

[0027] The electrospinning comprises: performing uniaxial electrospinning on a polymer solution A, using a roller or a U-shaped groove as a receiver, the spinning solution flow rate is 0.1-3 mL / h, the voltage is 10-20 kV, and the receiving distance is 10-20 cm; preferably, when the electrospinning uses a roller as a receiver, first spinning for 2-10 hours with a high-speed rotating roller as a receiver, and then spinning for 2-10 hours by reducing the roller speed; or, when the electrospinning uses a U-shaped groove as a receiver, spinning for 2-10 hours with the U-shaped groove as a receiver, and then peeling the collected fiber membrane from the U-shaped groove, removing the U-shaped groove, and placing the obtained fiber membrane on tin foil as a receiver for spinning for 2-10 hours.

[0028] Preferably, in step (2):

[0029] The solvent of the anti-inflammatory substance solution B is selected from at least one of phosphate buffered saline, water, chloroform, glycerol, trifluoroethanol, and hexafluoroisopropanol; the concentration of the anti-inflammatory substance solution B is 1 μg / mL to 5 mg / mL, preferably 0.1 to 2 mg / mL;

[0030] The solvent of the cationic polymer solution C is at least one of phosphate buffered saline, water, ethanol, polyvinyl alcohol, polyacrylamide, and polyacrylic acid; the concentration of the cationic polymer solution C is 1 μg / mL to 5 mg / mL, preferably 0.05 to 2 mg / mL;

[0031] In the suspension D, the volume ratio of the anti-inflammatory substance solution B to the cationic polymer solution C is 1:(0.1-10), preferably 1:(0.5-3).

[0032] Preferably, in step (3):

[0033] The initiator is selected from at least one of 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate; the mass percentage of the initiator in the initiator solution E is 0.1 to 0.5%, preferably 0.15 to 0.3%;

[0034] The mass percentage of the modified biomacromolecules in the hydrogel precursor solution F is 5 to 15%, preferably 8 to 12%;

[0035] In step (3), the initiator is added to the anti-inflammatory substance nanoparticle suspension D, and the mixture is carried out at 37°C in the dark, and the initiator solution E is obtained after sufficient shaking and mixing. When preparing the hydrogel precursor solution F, the initiator solution E is first placed in a constant temperature water bath in the dark and heated to 37°C, and then the modified biomacromolecule is added to the initiator solution E, and the mixture is fully shaken and mixed at 37°C to obtain the hydrogel precursor solution F.

[0036] Preferably, in step (4):

[0037] The mold is a tubular mold; the conditions for UV cross-linking are: the cross-linking temperature is 18 to 37° C., preferably 20 to 30° C.; the cross-linking time is 4 to 60 seconds, preferably 4 to 30 seconds;

[0038] UV crosslinking includes: firstly, heating the hydrogel precursor solution F to 37°C in a constant temperature water bath in the dark, then adding the hydrogel precursor solution F into a syringe and rapidly injecting it into a tubular mold through a 0.22 μm filter, crosslinking the hydrogel precursor solution in the mold under light, and the light crosslinking time is 4 to 60 seconds. Hydrogels with different crosslinking degrees can be obtained by adjusting the crosslinking time.

[0039] The third purpose of the present invention is to provide a rigid-flexible composite nerve conduit with controllable delivery of anti-inflammatory substances as described above, or a rigid-flexible composite nerve conduit with controllable delivery of anti-inflammatory substances obtained by the preparation method described above, and use it in the repair of complete spinal cord transection injury.

[0040] When the rigid-flexible composite nerve conduit with controllable delivery of anti-inflammatory substances described in the present invention is used as a spinal cord total transection injury repair material, the controlled delivery of nanoparticles containing anti-inflammatory substances is achieved by regulating the degradation rate of the hydrogel, which can inhibit the severe inflammatory response in the early stage of the injury and inhibit cell apoptosis. Among them, the protection of the cationic polymer on the outer layer of the nanoparticles can achieve its long-term effect at the lesion site, avoiding the problem of rapid inactivation of anti-inflammatory substances in the body. In addition, while regulating the inflammatory response, the migration of cells and the extension of nerve axons are promoted by constructing nanofibers with a unidirectional ordered topological structure. The rigid-flexible composite nerve conduit with controlled delivery of anti-inflammatory substances can improve the microenvironment of the spinal cord total transection injury, create conditions for the regulation of the inflammatory response and subsequent injury repair, and after the spinal cord total transection injury repair process is completed, the conduit can be degraded and metabolized by the body.

[0041] The present invention combines a low-modulus hydrogel with adjustable degradation rate as a carrier of drug-loaded nanoparticles and an electrospun nanofiber scaffold with excellent mechanical properties similar to the extracellular matrix structure to form an ideal design for a rigid-flexible composite nerve conduit for controllable delivery of anti-inflammatory substances. The presence of the hydrogel can achieve in situ controllable delivery of drug-loaded nanoparticles and improve the action efficiency of the nanoparticles. The electrospun nanofibers with adjustable topological structure can stabilize the morphology of the hydrogel and guide and promote the subsequent repair of complete spinal cord transection injury after the hydrogel is degraded.

[0042] Compared with the prior art, the present invention has the following advantages:

[0043] 1. The nanoparticle drug delivery system used in the present invention avoids direct exposure of the drug in the body, prolongs the half-life of the drug, and enables the nanoparticles to act in the damaged area for a long time;

[0044] 2. The present invention uses hydrogels with adjustable degradation rates to wrap drug-loaded nanoparticles, which can achieve in-situ controllable delivery of drug-loaded nanoparticles and improve the efficiency of the nanoparticles. The controllable delivery system of anti-inflammatory substances used in the present invention can effectively inhibit the severe inflammatory response and cell death in the early stage of spinal cord transection injury, so that the inflammatory microenvironment in the early stage of spinal cord transection is regulated in a long-term and stable manner, and provide a suitable microenvironment for subsequent axon regeneration.

[0045] 3. The present invention adopts an electrospun nanofiber and hydrogel rigid-flexible composite design, which significantly improves the mechanical properties of the hydrogel. The presence of nanofibers is beneficial to stabilizing the morphology of the hydrogel. The nanofibers prepared by electrospinning can imitate the function of the extracellular matrix (ECM) as much as possible, so that the material as a whole has a bionic structure of the spinal cord, and the unidirectional and orderly arranged fiber structure of the inner layer of the nanofibers enables it to promote the regeneration and migration of axons in the damaged area for a long time after the hydrogel is degraded.

[0046] 4. The randomly arranged fibers of the outer layer of the degradable aliphatic polyester fiber of the present invention provide sufficient elasticity and toughness to meet the needs of spinal movement during and after surgery;

[0047] 5. The rigid-flexible composite nerve conduit of the present invention has excellent biocompatibility and degradation performance, and can realize the controllable delivery of anti-inflammatory substances to regulate the microenvironment after complete transection of the spinal cord injury, promote the subsequent repair process and improve the repair effect.

[0048] 6. The nanoparticles of the present invention have a double-layer structure, and can still be quickly released to achieve a good repair effect in the case of a catheter with a small contact area with the tissue.

[0049] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0051] Figure 1This is a scanning electron microscope image of the polymer fiber membrane, hydrogel and nanoparticles in the rigid-flexible composite nerve conduit obtained in Example 1.

[0052] Figure 2 The modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles obtained in Example 1 affects the intracellular reactive oxygen content level of two cell models, SHSY-5Y and astrocytes, under oxidative stress conditions.

[0053] Figure 3 The modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles obtained in Example 1 has an effect on the cell survival rate of two cell models, SHSY-5Y and astrocytes, under oxidative stress conditions.

[0054] Figure 4 This is the surgical procedure of using the rigid-flexible composite nerve conduit with controllable release of anti-inflammatory substances obtained in Example 1 in a 2 mm rat spinal cord complete transection model. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present invention.

[0056] The test instruments and test conditions used in the examples are as follows:

[0057] Electrospinning equipment: Model: TEADFS-103, Beijing Xinrui Baina Technology Co., Ltd.; (roller as receiver)

[0058] Electrospinning equipment: Model: NANO 01, MECC Co., Ltd., Japan; (U-shaped groove as receiver)

[0059] Field emission scanning electron microscope: Model: S-4800, Hitachi, Japan;

[0060] Transmission electron microscope: Model: HT7700, Hitachi, Japan;

[0061] Inverted fluorescence microscope: Model: LSM900, Carl Zeiss GmbH.

[0062] The raw materials and sources used in the examples are as follows:

[0063] The degradable aliphatic polyester raw materials used in the examples were purchased from Sigma Reagent Company, organic solvents were purchased from Aladdin Company, and growth factors were purchased from Thermo Fisher Scientific Company. Modified biomacromolecules and initiators were purchased from EFL Company, and anti-inflammatory substances and cationic polymers were purchased from Yuanye Company.

[0064] Unless otherwise specified, other raw materials used in the embodiments and comparative examples are disclosed in the prior art, for example, they can be directly purchased or prepared according to the preparation method disclosed in the prior art.

[0065] Embodiment 1:

[0066] Step (1) preparing a polylactic acid-glycolic acid copolymer (PLGA) solution with a volume ratio of dichloromethane: N,N-dimethylformamide = 4:1, and magnetically stirring at room temperature for 24 hours to obtain a polymer solution A with a mass concentration of 10%; using solution A for uniaxial electrospinning, the flow rate of solution A is 1 mL / h, the voltage is 16 kV, the receiving distance is 15 cm, and a high-speed rotating roller is used as a receiver. The spinning is performed for 5 hours to obtain unidirectional ordered fibers, and then the roller speed is gradually reduced, and the randomly arranged fibers are continuously collected for 5 hours to obtain a polymer fiber membrane with an inner layer unidirectionally ordered and an outer layer randomly arranged; the polymer fiber membrane is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate;

[0067] Step (2) adding catalase and superoxide dismutase to water, mixing them thoroughly to obtain an anti-inflammatory substance solution B with a concentration of 1 mg / mL; adding polylysine to water, mixing them thoroughly to obtain a cationic polymer solution C with a concentration of 50 μg / mL; mixing the two solutions in a volume ratio of 1:1, and magnetically stirring at room temperature for 30 minutes to obtain an anti-inflammatory substance nanoparticle suspension D;

[0068] Step (3) adding LAP initiator to the anti-inflammatory substance nanoparticle suspension D obtained in step (2), shaking and mixing at 37°C in the dark, to obtain an initiator solution E with a mass fraction of 0.25%; adding methacrylated gelatin to the initiator solution E, shaking and mixing at 37°C in the dark, to obtain a hydrogel precursor solution F with a mass fraction of 10%;

[0069] Step (4) firstly heat the hydrogel precursor solution F obtained in step (3) to 37° C. in a constant temperature water bath in the dark, then add the hydrogel precursor solution F into a syringe and quickly inject 8 μL of the hydrogel precursor solution F into a tubular mold through a 0.22 μm filter, crosslink the hydrogel precursor solution F in the mold under 405 nm blue light irradiation, the light crosslinking time is 8 s, and demold to obtain a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles;

[0070] In step (5), the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles obtained in step (4) is used as a mold, and the inner layer of the polymer fiber membrane with a unidirectional ordered inner layer and a randomly arranged outer layer obtained in step (1) is contacted with the hydrogel and wrapped into a tube, and polymer solution A is applied to the adhesion at room temperature. After adhesion, it is dried in an ultra-clean bench, and finally a rigid-flexible composite nerve conduit with controllable delivery of anti-inflammatory substances is obtained.

[0071] Embodiment 2:

[0072] Step (1) adding polycaprolactone to N,N-dimethylformamide, and magnetically stirring at room temperature for 24 hours to obtain a polymer solution A with a mass concentration of 10%; performing uniaxial electrospinning with solution A, the flow rate of solution A being 1 mL / h, the voltage being 16 kV, the receiving distance being 15 cm, and a high-speed rotating roller being used as a receiver, spinning for 5 hours to obtain unidirectional ordered fibers, then gradually reducing the roller speed, and continuing to collect randomly arranged fibers for 5 hours to obtain a polymer fiber membrane with an inner layer having unidirectional order and an outer layer having random arrangement; placing the polymer fiber membrane in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate;

[0073] Step (2) adding curcumin and resveratrol to water and mixing them thoroughly to obtain an anti-inflammatory substance solution B with a concentration of 1 mg / mL; adding polylysine to water and mixing them thoroughly to obtain a cationic polymer solution C with a concentration of 50 μg / mL; mixing the two solutions in a volume ratio of 1:1, and magnetically stirring at room temperature for 30 minutes to obtain an anti-inflammatory substance nanoparticle suspension D;

[0074] Step (3) adding the I2929 initiator to the anti-inflammatory substance nanoparticle suspension D obtained in step (2), shaking and mixing at 37° C. in the dark, to obtain an initiator solution E with a mass fraction of 0.25%; adding methacrylated gelatin to the initiator solution E, shaking and mixing at 37° C. in the dark, to obtain a hydrogel precursor solution F with a mass fraction of 10%;

[0075] Step (4) firstly heat the hydrogel precursor solution F obtained in step (3) to 37° C. in a constant temperature water bath in the dark, then add the hydrogel precursor solution F into a syringe and quickly inject 8 μL of the hydrogel precursor solution F into a tubular mold through a 0.22 μm filter, crosslink the hydrogel precursor solution F in the mold under 405 nm blue light irradiation, the light crosslinking time is 8 s, and demold to obtain a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles;

[0076] In step (5), the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles obtained in step (4) is used as a mold, and the inner layer of the polymer fiber membrane with a unidirectional ordered inner layer and a randomly arranged outer layer obtained in step (1) is contacted with the hydrogel and wrapped into a tube, and polymer solution A is applied to the adhesion at room temperature. After adhesion, it is dried in an ultra-clean bench, and finally a rigid-flexible composite nerve conduit with controllable delivery of anti-inflammatory substances is obtained.

[0077] Example 3

[0078] Step (1) preparing a polylactic acid-glycolic acid copolymer (PLGA) solution in a volume ratio of dichloromethane: N,N-dimethylformamide = 4:1, and magnetically stirring at room temperature for 24 hours to obtain a polymer solution A with a mass concentration of 12%; using solution A for uniaxial electrospinning, the flow rate of solution A is 0.8 mL / h, the voltage is 15 kV, the receiving distance is 15 cm, and the U-shaped groove is used as a receiver. The spinning is performed for 6 hours to obtain a unidirectional ordered fiber membrane, and then the U-shaped groove is removed, and the unidirectional ordered fiber membrane obtained is used as a collector to collect randomly arranged fibers. The collection is performed for 6 hours to obtain a polymer fiber membrane with a unidirectional ordered inner layer and a randomly arranged outer layer; the polymer fiber membrane is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate;

[0079] Step (2) adding IL-10 and IL-4 to PBS and mixing them thoroughly to obtain an anti-inflammatory substance solution B with a concentration of 500 μg / mL; adding polylysine to water and mixing them thoroughly to obtain a cationic polymer solution C with a concentration of 100 μg / mL; mixing the two solutions at a volume ratio of 1:0.5, and magnetically stirring at room temperature for 30 minutes to obtain an anti-inflammatory substance nanoparticle suspension D;

[0080] Step (3) adding Irgacure 2959 initiator to the anti-inflammatory substance nanoparticle suspension N obtained in step (2), shaking and mixing at 37° C. in the dark, to obtain an initiator solution E with a mass fraction of 0.25%; adding methacrylylated silk fibroin to the initiator solution E, shaking and mixing at 37° C. in the dark, to obtain a hydrogel precursor solution F with a mass fraction of 8%;

[0081] Step (4) firstly heat the hydrogel precursor solution F obtained in step (3) to 37° C. in a constant temperature water bath in the dark, then add the hydrogel precursor solution F into a syringe and quickly inject 8 μL of the hydrogel precursor solution F into a tubular mold through a 0.22 μm filter, crosslink the hydrogel precursor solution F in the mold under 405 nm blue light irradiation, the light crosslinking time is 8 s, and demold to obtain a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles;

[0082] In step (5), the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles obtained in step (4) is used as a mold, and the inner layer of the polymer fiber membrane with a unidirectional ordered inner layer and a randomly arranged outer layer obtained in step (1) is contacted with the hydrogel and wrapped into a tube, and polymer solution A is applied to the adhesion at room temperature. After adhesion, it is dried in an ultra-clean bench, and finally a rigid-flexible composite nerve conduit with controllable delivery of anti-inflammatory substances is obtained.

[0083] Comparative Example 1:

[0084] Step (1) preparing a PLGA solution with a volume ratio of dichloromethane: N,N-dimethylformamide = 4:1, and magnetically stirring at room temperature for 24 hours to obtain a polymer solution A with a mass concentration of 10%; using solution A for uniaxial electrospinning, the flow rate of solution A is 1 mL / h, the voltage is 16 kV, the receiving distance is 15 cm, and a high-speed rotating roller is used as a receiver. The spinning is performed for 5 hours to obtain unidirectional ordered fibers, and then the roller speed is gradually reduced, and the randomly arranged fibers are continuously collected for 5 hours to obtain a polymer fiber membrane with an inner layer unidirectionally ordered and an outer layer randomly arranged; the polymer fiber membrane is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate;

[0085] Step (2) adding catalase and superoxide dismutase to water, mixing them thoroughly to obtain an anti-inflammatory substance solution B with a concentration of 1 mg / mL; adding polylysine to water, mixing them thoroughly to obtain a cationic polymer solution C with a concentration of 50 μg / mL; mixing the two solutions in a volume ratio of 1:1, and magnetically stirring at room temperature for 30 minutes to obtain an anti-inflammatory substance nanoparticle suspension D;

[0086] Step (3) adding LAP initiator to the anti-inflammatory substance nanoparticle suspension D obtained in step (2), shaking and mixing at 37°C in the dark, to obtain an initiator solution E with a mass fraction of 0.25%; adding methacrylated gelatin to the initiator solution E, shaking and mixing at 37°C in the dark, to obtain a hydrogel precursor solution F with a mass fraction of 10%;

[0087] Step (4) firstly heat the hydrogel precursor solution F obtained in step (3) to 37° C. in a constant temperature water bath in the dark, then add the hydrogel precursor solution F into a syringe and quickly inject 8 μL of the solution into a tubular mold through a 0.22 μm filter, crosslink the hydrogel precursor solution F in the mold under 405 nm blue light irradiation, the light crosslinking time is 4 s, and demold to obtain a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles;

[0088] Step (5) uses the modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles obtained in step (4) as a mold, and the inner layer of the polymer fiber membrane obtained in step (1) with unidirectional order and the outer layer randomly arranged is contacted with the hydrogel and wrapped into a tube, and the polymer solution A is applied to the adhesion at room temperature, and after adhesion, it is dried in a clean bench, and finally a rigid-flexible composite nerve conduit that can controllably deliver anti-inflammatory substances is obtained.

[0089] This comparative example produces a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles obtained at different cross-linking times corresponding to Example 1.

[0090] Figure 1 This is a scanning electron microscope image of the polymer fiber membrane, hydrogel and nanoparticles in the rigid-flexible composite nerve conduit obtained in Example 1. It can be seen that the fiber membrane has a unidirectional orderly arrangement in the inner layer (upper left) and a random arrangement structure in the outer layer (lower left). The hydrogel (upper right) has a porous structure. The size of the nanoparticles (lower right) is less than 100nm and the particle size is relatively uniform. Structurally, Example 1 successfully prepared a rigid-flexible composite nerve conduit with controllable release of anti-inflammatory substances.

[0091] Figure 2 This is the effect of the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles obtained in Example 1 on the intracellular reactive oxygen content levels of two cell models, SHSY-5Y and astrocytes, under oxidative stress conditions. It can be seen that the modified biomacromolecule hydrogel group containing anti-inflammatory nanoparticles (the bottom row) has the lowest fluorescence intensity, that is, the lowest reactive oxygen content, indicating that the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles can inhibit oxidative stress.

[0092] Figure 3 This is the effect of the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles obtained in Example 1 on the cell survival rate of two cell models, SHSY-5Y and astrocytes, under oxidative stress conditions. It can be seen that the number of live cells in the modified biomacromolecule hydrogel group containing anti-inflammatory nanoparticles (the rightmost column) is the highest, indicating that the modified biomacromolecule hydrogel containing anti-inflammatory nanoparticles can achieve cell protection under oxidative stress conditions.

[0093] Figure 4This is the surgical procedure of using the rigid-flexible composite nerve conduit with controllable release of anti-inflammatory substances obtained in Example 1 in a 2 mm rat spinal cord complete transection model.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

Claims

1. A rigid-flexible composite nerve conduit for controllable delivery of anti-inflammatory substances, characterized in that: comprising a polymer fiber conduit portion and a hydrogel portion; The polymer fiber conduit portion is a double-layer conduit structure prepared from a polymer fiber membrane; the inner layer of the polymer fiber conduit portion is a unidirectional orderly arranged polymer fiber layer, and the outer layer is a randomly arranged polymer fiber layer; The hydrogel part is a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles; the anti-inflammatory substance nanoparticles contain a two-layer structure, wherein the inner layer is the anti-inflammatory substance and the outer layer is the cationic polymer.

2. A rigid-flexible composite nerve conduit for controllably delivering anti-inflammatory substances according to claim 1, characterized in that: The wall thickness of the polymer fiber conduit portion is 50 to 800 μm, preferably 100 to 500 μm; The volume of the hydrogel portion is 4 to 12 μL, preferably 6 to 8 μL; The particle size of the anti-inflammatory substance nanoparticles is 30 to 200 nm, preferably 50 to 100 nm.

3. A rigid-flexible composite nerve conduit for controllably delivering anti-inflammatory substances according to claim 2, characterized in that: The polymer in the polymer fiber layer is selected from degradable aliphatic polyesters, preferably at least one selected from polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer; The modified biomacromolecule in the modified biomacromolecule hydrogel is an acrylated biomacromolecule, preferably at least one selected from methacrylated gelatin, methacrylated hyaluronic acid, methacrylated silk fibroin, methacrylated chitosan, and methacrylated sodium alginate; The anti-inflammatory substance is selected from at least one of catalase, superoxide dismutase, interleukin, and reducing molecules; The cationic polymer is selected from at least one of polyarginine, polylysine, gelatin, polyhistidine, polyethyleneimine, polyacrylamide and polyacrylamine.

4. A method for preparing a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances, characterized in that: The polymer solution is electrospun to obtain a double-layer nanofiber membrane with an inner layer of unidirectionally ordered nanofibers and an outer layer of randomly arranged nanofibers. The hydrogel precursor solution containing anti-inflammatory substances is cross-linked by ultraviolet light to obtain a hydrogel containing anti-inflammatory substances. The hydrogel containing anti-inflammatory substances is then wrapped with a polymer fiber membrane with an inner layer of unidirectional order and an outer layer of randomly arranged nanofibers to obtain the rigid-flexible composite nerve conduit for controllable delivery of anti-inflammatory substances.

5. The method for preparing a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances according to claim 4, characterized in that: The steps include: Step (1): electrospinning the polymer solution A to prepare a double-layer polymer fiber layer with an inner layer having unidirectional order and an outer layer having random arrangement; Step (2): mixing the anti-inflammatory substance solution B with the cationic polymer solution C to obtain an anti-inflammatory substance nanoparticle suspension D; Step (3): adding an initiator to the anti-inflammatory substance nanoparticle suspension D to obtain an initiator solution E, and then adding the modified biomacromolecule to the initiator solution E to obtain a hydrogel precursor solution F; Step (4): injecting the hydrogel precursor solution F into the mold, and cross-linking it under ultraviolet light to obtain a modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles; Step (5): The inner layer of the polymer fiber membrane obtained in step (1) with unidirectional order and the outer layer with random arrangement is contacted with the modified biomacromolecule hydrogel containing anti-inflammatory substance nanoparticles obtained in step (4), and wrapped into a tube, and finally bonded with polymer solution A to obtain the rigid-flexible composite nerve conduit for controllable delivery of anti-inflammatory substances.

6. The method for preparing a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances according to claim 5, characterized in that: In the step (1): The solvent in the polymer solution A is selected from at least one of hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane, and N,N'-dimethylformamide; the mass percentage concentration of the polymer solution A is 5 to 25%, preferably 8 to 15%; The electrospinning comprises: performing uniaxial electrospinning on a polymer solution A, using a roller or a U-shaped groove as a receiver, the spinning solution flow rate is 0.1-3 mL / h, the voltage is 10-20 kV, and the receiving distance is 10-20 cm; preferably, when the electrospinning uses a roller as a receiver, first spinning for 2-10 hours with a high-speed rotating roller as a receiver, and then spinning for 2-10 hours by reducing the roller speed; or, when the electrospinning uses a U-shaped groove as a receiver, spinning for 2-10 hours with the U-shaped groove as a receiver, and then peeling the collected fiber membrane from the U-shaped groove, removing the U-shaped groove, and placing the obtained fiber membrane on tin foil as a receiver for spinning for 2-10 hours.

7. The method for preparing a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances according to claim 6, characterized in that: In step (2): The solvent of the anti-inflammatory substance solution B is selected from at least one of phosphate buffered saline, water, chloroform, glycerol, trifluoroethanol, and hexafluoroisopropanol; the concentration of the anti-inflammatory substance solution B is 1 μg / mL to 5 mg / mL, preferably 0.1 to 2 mg / mL; The solvent of the cationic polymer solution C is at least one of phosphate buffered saline, water, ethanol, polyvinyl alcohol, polyacrylamide, and polyacrylic acid; the concentration of the cationic polymer solution C is 1 μg / mL to 5 mg / mL, preferably 0.05 to 2 mg / mL; In the suspension D, the volume ratio of the anti-inflammatory substance solution B to the cationic polymer solution C is 1:(0.1-10), preferably 1:(0.5-3).

8. The method for preparing a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances according to claim 7, characterized in that: In step (3): The initiator is selected from at least one of 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate; the mass percentage of the initiator in the initiator solution E is 0.1 to 0.5%, preferably 0.15 to 0.3%; The mass percentage of the modified biomacromolecules in the hydrogel precursor solution F is 5 to 15%, preferably 8 to 12%; In step (3), the initiator is added to the anti-inflammatory substance nanoparticle suspension D, and the mixture is carried out at 37°C in the dark, and the initiator solution E is obtained after sufficient shaking and mixing. When preparing the hydrogel precursor solution F, the initiator solution E is first placed in a constant temperature water bath in the dark and heated to 37°C, and then the modified biomacromolecule is added to the initiator solution E, and the mixture is fully shaken and mixed at 37°C to obtain the hydrogel precursor solution F.

9. The method for preparing a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances according to claim 8, characterized in that: In step (4): The mold is a tubular mold; the conditions for UV cross-linking are: the cross-linking temperature is 18 to 37° C., preferably 20 to 30° C.; the cross-linking time is 4 to 60 seconds, preferably 4 to 30 seconds; UV crosslinking includes: firstly, heating the hydrogel precursor solution F to 37°C in a constant temperature water bath in the dark, then adding the hydrogel precursor solution F into a syringe and rapidly injecting it into a tubular mold through a 0.22 μm filter, crosslinking the hydrogel precursor solution in the mold under light, and the light crosslinking time is 4 to 60 seconds. Hydrogels with different crosslinking degrees can be obtained by adjusting the crosslinking time.

10. Use of a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances as claimed in any one of claims 1 to 3 or a rigid-flexible composite nerve conduit capable of controllably delivering anti-inflammatory substances obtained by the preparation method as claimed in any one of claims 4 to 9 in the repair of complete transection of the spinal cord.

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