Preparation method of carboxylated silk fibroin hydrogel composite nerve conduit

Abstract

The application discloses a preparation method of a carboxylated silk fibroin hydrogel composite nerve conduit. The carboxylated silk fibroin is chemically crosslinked with chitosan amino groups, the crosslinking is carried out in an aqueous solution, and no organic solvent such as ethanol is contained, so that the cell activity is not inhibited, the safety hazard to the human body is avoided, and the temperature and pH do not need to be additionally controlled, which is beneficial to the stability of vascular endothelial growth factor, and the hydrogel can be formed in a very short time, and the VEGF with the function of blood vessel regeneration is rich in the application, so that the neuroangiogenesis is enhanced, and the nerve regeneration is promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of neural tissue engineering and regenerative medicine, and particularly relates to a preparation method of a carboxylated silk fibroin hydrogel composite nerve conduit with proangiogenic function. BACKGROUND

[0002] Peripheral nerve injury is mostly caused by accidental trauma. Partial or complete loss of sensory, motor and autonomic functions or neuropathic pain is the main symptom due to the lack of structure of peripheral nerves. Due to its high incidence and unsatisfactory treatment effect, it brings heavy burden to patients. At present, for short segment nerve injury, regeneration is performed by end-to-end suture; for long segment injury, the most commonly used treatment method is nerve transplantation. Autologous nerve transplantation is considered to be the gold standard for the treatment of peripheral nerve injury. Compared with other treatment methods, autologous nerve transplantation provides the best regeneration effect so far, but it also faces the shortage of donor and the loss of function in the area where the transplanted tissue is obtained. Even the success rate of autologous transplantation is only 50%. At present, tissue engineered nerve conduits made of natural or synthetic biopolymers bring hope to overcome the limitations of nerve transplantation. As a bridge between damaged nerves, nerve conduits provide structural support and nutrient supply for both ends, support the growth of surrounding tissues and the regeneration of axons.

[0003] The design of nerve conduit is divided into five types: hollow, porous, slotted, multi-channel and with filler. Hollow design is the simplest among all designs, but its limitations are also greater, including insufficient nutritional ingredients and poor permeability. In order to overcome these shortcomings, fillers can be added to the conduit, for example, Quigley et al. filled alginate hydrogel rich in neurotrophins into the nerve conduit in order to enhance its neurotrophic effect and material exchange, and proved the improvement of its performance in a four-week transverse sciatic nerve rat model. Hydrogel is a three-dimensional polymer network rich in water, which is usually formed by polymer chemical or physical crosslinking. Hydrogel has many advantages, such as water retention, shape stability and the possibility of functional modification. Therefore, an ideal hydrogel can simulate the three-dimensional microenvironment of the extracellular matrix, and can regulate cell behavior and tissue function. For this reason, hydrogel is widely used in the fields of drug delivery carriers, tissue engineering scaffolds, self-healing materials, tissue adhesives, etc.

[0004] Electrospinning is currently the most popular method for manufacturing nanofiber nerve conduits. This method draws continuous polymer fibers from the nozzle by applying high voltage between the nozzle tip and the receiver. The micropores formed by the structure of nanofibers can simulate the cytoplasmic matrix environment, which is suitable for cell adhesion and growth. At present, electrospinning has been widely used in the production of nerve conduits.

[0005] The basic requirements for nerve conduit and hydrogel material selection are good biocompatibility, appropriate biodegradability and suitable mechanical properties. The most commonly used materials are natural and synthetic polymers. Natural polymers include collagen, gelatin, fibrin and the like based on proteins; cellulose and chitosan and the like based on polysaccharides.

[0006] Chitosan is a polysaccharide obtained by deacetylation of chitin, and has excellent biodegradability, biocompatibility, antibacterial activity and low immunogenicity. These excellent properties make chitosan a viable candidate material for biomedical applications, and it is widely used in drug delivery carriers, surgical sutures, wound healing and tissue engineering. Chitosan is composed of β-(1-4) linked D-glucosamine and randomly distributed N-acetylglucosamine groups. Chitosan chains have amino and hydroxyl functional groups, which enable them to form stable covalent bonds with other functional groups, at the hydroxyl group, etherification or esterification, at the amino group of D-glucosamine, amidation or reaction with aldehyde, through these reactions, various molecules can be grafted and crosslinked with chitosan to prepare its derivatives to improve performance.

[0007] Regenerated silk fibroin produced by silkworm cocoon production is widely used to manufacture nerve conduits and hydrogels due to its excellent mechanical properties, slow degradation characteristics and much lower inflammatory response in the human body than common biological materials (collagen or polylactic acid). However, silk fibroin often induces hydrogels by adjusting pH, temperature and using organic solvents, which limits the loading of drugs and the use of hydrogels. For temperature changes, some documents report that the gelation time of 4% silk fibroin solution is 22 days and 13 days at 37 and 60°C, respectively. Obviously, the long gelation time limits its application; for pH adjustment, the isoelectric point of silk fibroin is 3.8-4.0, and lower pH is conducive to accelerating gel formation, but it is also not conducive to the stability of drug loading; for the use of organic solvents, ethanol is usually used to dehydrate the hydrated hydrophobic domains of silk fibroin, making the silk fibroin molecules undergo β-folding to form hydrophobic macromolecular fragments and thus gel, but the introduction of organic solvents is not conducive to cell growth and drug loading, thus also limiting its application.

[0008] Limited to the composition of silk fibroin itself, it contains less active groups such as carboxyl and amino groups, and the silk fibroin hydrogel prepared by chemical crosslinking method is less. At the same time, in the chemical crosslinking hydrogel, the EDC / NHS catalyzed chemical crosslinking method is widely used in the preparation of hydrogel. This is because it can catalyze the formation of amide bond between carboxyl and amino groups in aqueous solution at 37℃, pH=7.4, and it is proved that low concentration (0.5 mol / L) EDC / NHS is safe in biomaterials. Generally, researchers choose polymers containing carboxyl and amino groups to prepare hydrogel, for example, hyaluronic acid and chitosan, hyaluronic acid provides carboxyl, chitosan provides amino, and under the catalysis of EDC / NHS, hydrogel is formed. Because silk fibroin has only 3% carboxyl side groups, it limits its use in chemical crosslinking. But silk fibroin contains 12% hydroxyl side groups, if the hydroxyl is converted to carboxyl, the carboxyl content of silk fibroin can be greatly improved, and it can be applied to the chemical crosslinking which has mild reaction conditions and is suitable for drug loading, making it an ideal material for drug-loaded hydrogel.

[0009] Vascular endothelial growth factor (VEGF) is an effective inducer that can increase vascular permeability and endothelial cell proliferation, migration, survival and angiogenesis. At present, it has been successful to fix VEGF on biomaterials to induce neovascularization and support cell survival and differentiation. After peripheral nerve injury, the construction of local microenvironment suitable for regeneration and the transport of nutrients required for nerve regeneration are closely related to blood supply, so early vascularization of tissue plays an important role in nerve regeneration. However, according to the literature reports, the solubility of vascular endothelial growth factor leads to its rapid diffusion and degradation, and its half-life in vivo is only 50 minutes. SUMMARY

[0010] The purpose of the present application is to provide a preparation method of carboxylated silk fibroin hydrogel composite nerve conduit aiming at the defects existing in the prior art. Firstly, the electrospun silk fibroin nerve conduit has nanofiber structure and a large number of micropores in the microstructure, which can better simulate the environment of extracellular matrix, facilitate the transport of substances, and be suitable for cell adhesion and growth; it has good mechanical properties in macroscopic, which can provide a stable environment during nerve regeneration; secondly, the prepared carboxylated silk fibroin has a higher carboxyl content, which can be used to prepare chemical crosslinking hydrogel. The hydrogel can be formed under mild physiological conditions, and does not depend on pH, temperature and organic solvents, which is suitable for the field of nerve tissue engineering and regenerative medicine; thirdly, the hydrogel can realize slow release of VEGF, effectively solve the problems of low activity and burst release caused by short half-life, rapid diffusion or degradation of VEGF, maintain the activity of VEGF, and better promote nerve regeneration by inducing the formation of blood vessels, so as to achieve the purpose of nerve repair.

[0011] To achieve the above object, the present invention uses the following technical solutions:

[0012] A method for preparing a carboxylated silk fibroin hydrogel composite nerve conduit comprises the following steps:

[0013] 1) dissolving silk fibroin in molten 1-butyl-3-methylimidazolium chloride, adding anhydrous dimethylformamide, and then adding succinic anhydride to react at 80-110° C. to carboxylate hydroxyl groups in the silk fibroin molecular structure. After the reaction, dialyzing the reaction solution with water to obtain insoluble carboxylated silk fibroin, then dissolving the insoluble carboxylated silk fibroin in a calcium alcohol / water ternary solution, dialyzing again with ultrapure water, and freeze-drying the dialyzate to obtain water-soluble carboxylated silk fibroin;

[0014] 2) dissolving the carboxylated silk fibroin prepared in step 1) in hexafluoroisopropanol to prepare a spinning solution, and preparing a hollow nerve conduit by an electrospinning method;

[0015] 3) The carboxylated silk fibroin and chitosan prepared in step 1) are dissolved in water respectively, and then the two aqueous solutions are mixed at a weight ratio of 1-5:1 of carboxylated silk fibroin to chitosan, and then vascular endothelial growth factor is added to a concentration of 0.5-5 ng / ml to obtain a drug-loaded hydrogel precursor sol. EDC and NHS are added to the drug-loaded hydrogel precursor sol to a concentration of 0.05-0.2 mol / L. After mixing evenly, the sol is filled into the hollow nerve conduit of step 2) and placed in a constant temperature incubator at 35-45°C for 5-30 minutes to obtain a hydrogel composite nerve conduit.

[0016] Preferably, the mass ratio of the silk fibroin to succinic anhydride is 1:1-5.

[0017] Preferably, the reaction temperature of the silk fibroin and succinic anhydride is 90°C.

[0018] Preferably, the electrospinning parameters are: spinning solution concentration of 6-15%, outflow speed of 0.1-0.5 mm / min, voltage between the needle tube and the receiving plate of 8-20 KV, receiving distance of 10-25 cm, ambient temperature of 20-25 ° C, and relative humidity of 25-40%.

[0019] Preferably, the weight ratio of the carboxylated silk fibroin to chitosan is 3:1.

[0020] Preferably, the concentration of EDC and NHS is 0.15 mol / L.

[0021] Preferably, the incubation time is 10 min.

[0022] Preferably, the hollow nerve conduit has an inner diameter of 2-3 mm, an outer diameter of 3-5 mm, and a length of 10-20 mm.

[0023] According to one embodiment of the present application, a preferred preparation method is as follows:

[0024] 1) Preparation of water-soluble carboxylated silk fibroin:

[0025] Dissolve 0.5 g of silk fibroin in 3.125 g of molten 1-butyl-3-methylimidazolium chloride, add 10 ml of anhydrous dimethylformamide, and then add 1.5 g of succinic anhydride for reaction. React at 90°C for 2 h to carboxylate the hydroxyl groups in the molecular structure of the silk fibroin. After the reaction is completed, first perform dialysis of the reaction solution with water to obtain insoluble carboxylated silk fibroin. Then dissolve the insoluble carboxylated silk fibroin in a calcium-alcohol-water ternary solution with a molar ratio of CaCl2-EtOH-H2O of 1:2:8, and perform dialysis again with ultrapure water. Freeze-dry the dialyzed solution to obtain water-soluble carboxylated silk fibroin.

[0026] 2) Preparation of a hollow nerve conduit:

[0027] Dissolve the carboxylated silk fibroin prepared in step 1) in hexafluoroisopropanol to prepare a spinning solution. Use an electrospinning method to prepare a hollow nerve conduit. The electrospinning parameters are as follows: the concentration of the spinning solution is 10% (w / v), the flow rate is 0.3 mm / min, the voltage between the needle tube and the receiving plate is 15 KV, the receiving distance is 15 cm, the ambient temperature is 25°C, and the relative humidity is 30%.

[0028] 3) Preparation of a hydrogel composite nerve conduit:

[0029] Dissolve the carboxylated silk fibroin and chitosan prepared in step 1) in water to prepare aqueous solutions with a concentration of 3% (w / v). Then mix the two aqueous solutions in a weight ratio of carboxylated silk fibroin to chitosan of 3:1. Next, add vascular endothelial growth factor to obtain a drug-loaded hydrogel precursor sol with a concentration of 0.5-5 ng / ml. Add EDC and NHS to the drug-loaded hydrogel precursor sol to obtain a concentration of 0.15 mol / L for each. After mixing, fill the sol into the hollow nerve conduit prepared in step 2) and place it in a 37°C constant-temperature incubator for 10 min to obtain a hydrogel composite nerve conduit.

[0030] The present application has the following advantages:

[0031] 1) The hollow drug-loaded nerve conduit provided by the present invention has a nanofiber structure and a large number of micropores in its microstructure, which can better simulate the environment of the extracellular matrix, facilitate the transport of substances, and is suitable for cell adhesion and growth; on a macroscopic level, the prepared conduit has good mechanical properties and can provide a stable environment during nerve regeneration.

[0032] 2) Although silk fibroin is widely used to prepare hydrogels, the preparation conditions of traditional silk fibroin gels are not suitable for preparation under physiological conditions. The carboxylated silk fibroin in the present invention can form hydrogels with chitosan under physiological conditions and is suitable for the fields of neural tissue engineering and regenerative medicine;

[0033] 3) Protein drugs such as VEGF have high biological activity, strong solubility, and low toxicity, but they are easily degraded by proteases in the body, and their short half-life in the body limits their use. Therefore, improving drug stability, increasing bioavailability, and extending their half-life in plasma are the main challenges facing such drugs. The carboxylated silk fibroin-chitosan hydrogel of the present invention can load VEGF and achieve its slow release;

[0034] 4) Single hollow nerve conduits have the disadvantages of insufficient nutrients and poor permeability. The main problem currently faced is the production of multi-porous, highly permeable nerve conduits rich in various nutritional factors. The hydrogel composite nerve conduit of the present invention has excellent permeability and is rich in VEGF, which has angiogenesis function. It can enhance angiogenesis within the nerve and promote nerve regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the reaction principle of silk fibroin carboxylation.

[0036] Figure 2 This is the H NMR spectrum of carboxylated silk fibroin.

[0037] Figure 3 This is a scanning electron micrograph of electrospun fibers.

[0038] Figure 4 This is a physical picture of the hydrogel composite nerve conduit.

[0039] Figure 5 This is the release curve of VEGF in the hydrogel composite nerve conduit. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to specific embodiments.

[0041] Example 1

[0042] 1) Preparation of carboxylated silk protein:

[0043] 0.5 g of silk fibroin was dissolved in 3.125 g of melted 1-butyl-3-methylimidazolium chloride, the obtained solution was added into 10 ml of anhydrous dimethylformamide (DMF) and mixed until uniform; then 1.5 g of succinic anhydride (dissolved in 5 ml of DMF) was added into the solution and reacted at 90°C for 2 h. After the reaction was completed, a small amount of ultrapure water was first added into the reaction system to hydrolyze the succinic anhydride that did not participate in the reaction, then the reaction solvent and succinic anhydride were dialyzed with ultrapure water, and the obtained solid precipitate after dialysis was the insoluble carboxylated silk fibroin. Finally, the insoluble carboxylated silk fibroin was dissolved in a calcium alcohol ternary solution (CaCl2-EtOH-H2O molar ratio 1:2:8), and dialyzed with ultrapure water again. After dialysis was completed, the solution was freeze-dried to obtain the water-soluble carboxylated silk fibroin.

[0044] From Figure 2 It can be seen from the hydrogen nuclear magnetic resonance spectrum that the hydrogen atoms with chemical shift values of 2.50 and 2.63 prove that succinic anhydride is successfully grafted onto the silk fibroin molecular chain. By integrating the valine γ-CH3 protons (0.95 ppm) and setting the integral to 6 protons, and integrating the new peak (2.44-2.40 ppm) in the carboxylated sample caused by the methylene protons of succinic anhydride, the carboxylation rate of silk fibroin is calculated and analyzed. The results show that 87% of the silk fibroin in this embodiment is successfully carboxylated. The reaction temperature and time have important influences on the carboxylation of the protein, and in addition, the amount of succinic anhydride also has an influence on the carboxylation rate. The higher the amount of succinic anhydride, the higher the carboxylation rate of the protein, but it will reach saturation after a certain range.

[0045] 2) Preparation of a hollow nerve conduit:

[0046] The carboxylated silk fibroin was dissolved in hexafluoroisopropanol to obtain an electrospinning solution, and the concentration of the carboxylated silk fibroin was 10% (w / v). The electrospinning parameters were as follows: the flow rate was 0.3 mm / min, the voltage between the needle tube and the receiving plate was 15 KV, the receiving distance was 15 cm, the environmental temperature was 25°C, and the relative humidity was 30%.

[0047] From Figure 3 It can be seen from the scanning electron microscope image of the electrospun fiber that the electrospun fiber has a relatively uniform diameter of about 720 nm. From the nerve conduit real object image of Figure 4 It can be seen from the nerve conduit real object image of

[0048] 3) Preparation of a hydrogel composite nerve conduit:

[0049] The prepared part of carboxylated silk fibroin is dissolved in ultrapure water to prepare a 3% (w / v) aqueous solution and placed at 4°C for standby, and chitosan is dissolved in ultrapure water to prepare a 3% (w / v) chitosan solution and placed at 4°C for standby. According to the weight ratio of carboxylated silk fibroin to chitosan of 3:1, the two solutions are mixed, then vascular endothelial growth factor is added to a concentration of 4 ng / ml to obtain a drug-loaded hydrogel precursor sol, and EDC and NHS are added to the solution system to a concentration of 0.15 mol / L, and the sol is uniformly mixed and filled into the hollow nerve conduit of step 2) to form a hydrogel in a 37°C constant temperature incubator for 10 min, thereby obtaining a hydrogel composite nerve conduit.

[0050] In this embodiment, the carboxylated silk fibroin is chemically crosslinked with the amino groups of chitosan, and the crosslinking is carried out in an aqueous solution without any organic solvent residues such as ethanol, so it does not inhibit cell activity and does not pose a safety hazard to the human body. Moreover, this method does not require additional temperature and pH control, which is not only conducive to the stability of the vascular endothelial growth factor, but also enables the formation of a hydrogel in a very short time. Among them, part of the amino groups on the chitosan are crosslinked with the carboxylated silk fibroin in the precursor sol to form a hydrogel, and the other part of the amino groups are crosslinked with the carboxylated silk fibroin on the hollow nerve conduit, increasing the structural stability, and the vascular endothelial growth factor is loaded inside the hydrogel and released slowly. Therefore, the ratio of carboxylated silk fibroin to chitosan plays an important role in the molding speed, structural stability, and drug release of the conduit, and the preferred ratio is 3:1.

[0051] Release detection of the hydrogel composite nerve conduit:

[0052] Under a clean bench, the hydrogel composite nerve conduit prepared in Example 1 is placed in sterile phosphate buffer solution (PBS) with a pH value of 7.4, and then placed in a constant temperature shaking incubator at 37°C and 60 r / min. The release medium is collected at a specific time, and fresh PBS is used to compensate for the test tube, and the extraction solution is uniformly shaken to ensure sterility during the production process. Then, the amount of dissolved VEGF in the sample solution is determined by the Elisa method.

[0053] The experimental results are shown in Figure 5 VEGF is released at about 38% in the first day, and then continuously released, with a cumulative release rate of 75% in 20 days.

[0054] Example 2

[0055] 1) Preparation of carboxylated silk fibroin:

[0056] 0.5 g of silk fibroin was dissolved in 3.125 g of melted 1-butyl-3-methylimidazolium chloride, the obtained solution was added into 10 ml of anhydrous dimethylformamide (DMF) and mixed until uniform; 2.5 g of succinic anhydride (5 ml of DMF solution) was added into the solution, and reacted at 80℃ for 3 h. After the reaction was completed, a small amount of ultrapure water was first added into the reaction system to hydrolyze the succinic anhydride which did not participate in the reaction, then the reaction solvent and succinic anhydride were dialyzed with ultrapure water, and the obtained solid precipitate after dialysis was the insoluble carboxylated silk fibroin. Finally, the insoluble carboxylated silk fibroin was dissolved in a calcium alcohol ternary solution, and dialyzed with ultrapure water again. After dialysis was completed, the solution was freeze-dried to obtain the water-soluble carboxylated silk fibroin.

[0057] In this embodiment, about 80% of the silk fibroin was carboxylated.

[0058] 2) Preparation of a hollow nerve conduit:

[0059] The carboxylated silk fibroin was dissolved in hexafluoroisopropanol to obtain an electrospinning solution, the concentration of the carboxylated silk fibroin was 10% (w / v), and the electrospinning parameters were as follows: the flow rate was 0.3 mm / min, the voltage between the needle tube and the receiving plate was 15 KV, the receiving distance was 15 cm, the environmental temperature was 25℃, and the relative humidity was 30%.

[0060] 3) Preparation of a hydrogel composite nerve conduit:

[0061] A part of the prepared carboxylated silk fibroin was dissolved in ultrapure water to prepare a 3% (w / v) aqueous solution and placed at 4℃ for standby, and chitosan was dissolved in ultrapure water to prepare a 3% (w / v) chitosan solution and placed at 4℃ for standby. According to the weight ratio of 5:1 of the carboxylated silk fibroin to chitosan, the two solutions were mixed, then vascular endothelial growth factor was added to make the concentration be 4 ng / ml, and the obtained drug-loaded hydrogel sol was added with EDC and NHS to make the concentration of each be 0.1 mol / L. After uniform mixing, the sol was filled into the hollow nerve conduit of step 2), and placed in a 37℃ constant temperature incubator for 30 min to form a hydrogel, and thus the hydrogel composite nerve conduit was obtained.

[0062] It was detected that about 45% of the VEGF in the hydrogel composite nerve conduit of the application was released within the first day, and then continuously released, and the cumulative release rate reached 77% within 20 days.

[0063] Example 3

[0064] 1) Preparation of carboxylated silk fibroin:

[0065] 0.5 g of silk fibroin was dissolved in 3.125 g of melted 1-butyl-3-methylimidazolium chloride, the obtained solution was added into 10 ml of anhydrous dimethylformamide (DMF) and mixed until uniform; 1 g of succinic anhydride (5 ml dissolved in DMF) was added into the solution, and reacted at 110℃ for 1 h. After the reaction was completed, a small amount of ultrapure water was first added into the reaction system to hydrolyze the succinic anhydride which did not participate in the reaction, then the reaction solvent and succinic anhydride were dialyzed with ultrapure water, and the obtained solid precipitate after dialysis was the insoluble carboxylated silk fibroin. Finally, the insoluble carboxylated silk fibroin was dissolved in a calcium alcohol water ternary solution, and dialyzed with ultrapure water again. After dialysis was completed, the solution was freeze-dried to obtain the water-soluble carboxylated silk fibroin.

[0066] In this embodiment, about 72% of the silk fibroin was carboxylated.

[0067] 2) Preparation of a hollow nerve conduit:

[0068] The carboxylated silk fibroin was dissolved in hexafluoroisopropanol to obtain an electrospinning solution, the concentration of the carboxylated silk fibroin was 10% (w / v), and the electrospinning parameters were as follows: the flow rate was 0.5 mm / min, the voltage between the needle tube and the receiving plate was 12 KV, the receiving distance was 15 cm, the environmental temperature was 25℃, and the relative humidity was 30%.

[0069] 3) Preparation of a hydrogel composite nerve conduit:

[0070] A part of the prepared carboxylated silk fibroin was dissolved in ultrapure water to prepare a 3% (w / v) aqueous solution and placed at 4℃ for standby, and chitosan was dissolved in ultrapure water to prepare a 3% (w / v) chitosan solution and placed at 4℃ for standby. According to the weight ratio of 2:1 of the carboxylated silk fibroin to chitosan, the two solutions were mixed, then vascular endothelial growth factor was added to make the concentration be 4 ng / ml, and the obtained drug-loaded hydrogel sol was added with EDC and NHS to make the concentration of each be 0.2 mol / L. After uniform mixing, the sol was filled into the hollow nerve conduit of step 2), and placed in a 37℃ constant temperature incubator for 15 min to form a hydrogel, and thus a hydrogel composite nerve conduit was obtained.

[0071] It was detected that about 37% of the VEGF in the hydrogel composite nerve conduit of the application was released within the first day, and then continuously released, and the cumulative release rate reached 65% within 20 days.

[0072] Example 4

[0073] 1) Preparation of carboxylated silk fibroin:

[0074] 0.5 g of silk fibroin was dissolved in 3.125 g of melted 1-butyl-3-methylimidazolium chloride, the obtained solution was added into 10 ml of anhydrous dimethylformamide (DMF) and mixed until uniform; 2 g of succinic anhydride (5 ml dissolved in DMF) was added into the solution, and reacted at 100 ℃ for 1.5 h. After the reaction was completed, a small amount of ultrapure water was first added into the reaction system to hydrolyze succinic anhydride which did not participate in the reaction, then the reaction solvent and succinic anhydride were dialyzed with ultrapure water, and the obtained solid precipitate after dialysis was insoluble carboxylated silk fibroin. Finally, the insoluble carboxylated silk fibroin was dissolved in a calcium alcohol ternary solution, and dialyzed with ultrapure water again. After dialysis was completed, the solution was freeze-dried to obtain water-soluble carboxylated silk fibroin.

[0075] In the embodiment, about 77% of the silk fibroin was carboxylated.

[0076] 2) Preparation of a hollow nerve conduit:

[0077] The carboxylated silk fibroin was dissolved in hexafluoroisopropanol to obtain an electrospinning solution, the concentration of the carboxylated silk fibroin was 10% (w / v), and the electrospinning parameters were as follows: the flow rate was 0.1 mm / min, the voltage between the needle tube and the receiving plate was 15 KV, the receiving distance was 10 cm, the environmental temperature was 25 ℃, and the relative humidity was 30%.

[0078] 3) Preparation of a hydrogel composite nerve conduit:

[0079] A part of the prepared carboxylated silk fibroin was dissolved in ultrapure water to prepare a 3% (w / v) aqueous solution and placed at 4 ℃ for standby, and chitosan was dissolved in ultrapure water to prepare a 3% (w / v) chitosan solution and placed at 4 ℃ for standby. The two solutions were mixed according to the weight ratio of 1:1 of the carboxylated silk fibroin and the chitosan, then vascular endothelial growth factor was added to make the concentration be 4 ng / ml, and the obtained drug-loaded hydrogel sol was added into the hollow nerve conduit of step 2) after EDC and NHS were added into the solution system to make the concentration of each be 0.08 mol / L. After the sol was mixed uniformly, the hydrogel was formed by filling the sol into the hollow nerve conduit of step 2) and placing in a 37 ℃ constant temperature incubator for 5 min, and the hydrogel composite nerve conduit was obtained.

[0080] It was detected that the VEGF in the hydrogel composite nerve conduit of the application was released by 52% in the first day, and then continuously released, and the cumulative release rate reached 71% within 20 days.

Claims

1. A method for preparing a carboxylated silk fibroin hydrogel composite nerve conduit, characterized by The method comprises the following steps: 1) 0.5 g of silk fibroin is dissolved in 3.125 g of melted 1-butyl-3-methylimidazolium chloride, 10 ml of anhydrous dimethylformamide is added, and 1.5 g of succinic anhydride is added for reaction, and the reaction is carried out at 90°C for 2 h, so that the hydroxyl groups in the molecular structure of the silk fibroin are carboxylated; after the reaction is completed, the reaction solution is first dialyzed with water to obtain insoluble carboxylated silk fibroin, and then the insoluble carboxylated silk fibroin is dissolved in a calcium-alcohol-water ternary solution with a molar ratio of CaCl2-EtOH-H2O of 1:2:8, and is dialyzed again with ultrapure water, and the dialyzed solution is freeze-dried to obtain water-soluble carboxylated silk fibroin; 2) The carboxylated silk fibroin prepared in step 1) is dissolved in hexafluoroisopropanol to prepare a spinning solution, and a hollow nerve conduit is prepared by using an electrospinning method, wherein the parameters of the electrospinning are as follows: the concentration of the spinning solution is 10%, w / v, the flow rate is 0.3 mm / min, the voltage between the needle tube and the receiving plate is 15 KV, the receiving distance is 15 cm, the ambient temperature is 25°C, and the relative humidity is 30%; 3) The carboxylated silk fibroin and chitosan prepared in step 1) are respectively dissolved in water to prepare a 3%, w / v water-soluble carboxylated silk fibroin solution and a 3%, w / v chitosan solution, then the two kinds of water solutions are mixed in a weight ratio of carboxylated silk fibroin to chitosan of 3:1, and then vascular endothelial growth factor is added to make the concentration of the vascular endothelial growth factor be 0.5-5 ng / ml, so as to obtain a drug-loaded water hydrogel precursor sol, EDC and NHS are added to the drug-loaded water hydrogel precursor sol to make the concentrations of EDC and NHS both be 0.15 mol / L, and after being uniformly mixed, the sol is filled in the hollow nerve conduit of step 2), and is placed in a 37°C constant-temperature box for culture for 10 min, so as to obtain a hydrogel composite nerve conduit.

2. The method for preparing the carboxylated silk fibroin hydrogel composite nerve conduit according to claim 1, wherein: The hollow nerve conduit has an inner diameter of 2-3 mm, an outer diameter of 3-5 mm, and a length of 10-20 mm.

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