Preparation method of bio-based neural restoration cannula material

Abstract

The invention discloses a preparation method of a bio-based neural restoration cannula material, and relates to the field of biological materials. The preparation method comprises the following steps: firstly, enabling chitosan, 3-carboxyphenylboronic acid and maleic anhydride to react with nanogold to prepare modified chitosan; acrylic acid, acrylic acid-N-hydroxysuccinimide, alpha-ketoglutaric acid, modified chitosan and glutaraldehyde are subjected to a reaction, hydrogel is prepared, insulin-like growth factor-1 is added, and the bio-based neural restoration cannula is prepared. The prepared bio-based nerve repair sleeve has excellent biocompatibility, antibacterial property and mechanical property, nerves can be effectively repaired without suture, and secondary damage to nerve broken ends is avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological materials, in particular to a preparation method of a bio-based nerve repair sleeve material. BACKGROUND

[0002] Nerve injury is a common clinical disease, which can cause sensory and motor dysfunction and seriously affect the quality of life of patients. At present, for the repair of nerve injury, the traditional treatment methods mainly include direct suture, autologous nerve transplantation, allogeneic nerve transplantation and the like, but the above treatment methods not only have limited application range, but also have limitations in nerve regeneration effect.

[0003] Most of the existing nerve repair sleeve materials are synthetic polymer materials, such as polylactic acid, polycaprolactone and the like. Although they have good mechanical properties and processing properties, the biocompatibility and biodegradability often cannot fully meet the ideal nerve repair requirements. During in vivo degradation, acidic degradation products may be produced, causing local inflammatory reactions and affecting nerve regeneration. Bio-based materials have the advantages of wide source and biodegradability, and good biocompatibility avoids causing immune rejection reactions, ensuring the stability of the nerve regeneration microenvironment, and gradually becoming a research hotspot of nerve repair sleeve materials. SUMMARY

[0004] The purpose of the present application is to provide a preparation method of a bio-based nerve repair sleeve material to solve the problems in the prior art.

[0005] In order to solve the above technical problems, the present application provides the following technical scheme: the bio-based nerve repair sleeve material is a hydrogel prepared by reacting acrylic acid, acrylic acid-N-hydroxysuccinimide, alpha-ketoglutaric acid, modified chitosan and glutaraldehyde, and then reacting with insulin-like nerve factor-1;The modified chitosan is prepared by reacting chitosan, 3-carboxyphenylboronic acid, maleic anhydride and nano-gold

[0006] Further, a preparation method of a bio-based nerve repair sleeve material includes the following preparation steps:

[0007] (1) mixing 3-carboxyphenylboronic acid, maleic anhydride, 1-(3-dimethylamine propyl)-3-ethyl carbodiimide hydrochloride, N-hydroxysuccinimide and N,N-dimethylformamide in a mass ratio of (0.5-0.6):1:(1.1-1.2):(1.1-1.2):(10-15) to obtain a mixed solution, stirring at 20-30℃ and 300-500r / min for 30-40min, mixing chitosan, 10% dilute hydrochloric acid and deionized water in a mass ratio of 1:(5-7):(15-20) to prepare a chitosan solution, adding the chitosan solution into the mixed solution dropwise at a speed of 20mL / min, and continuing to stir for 2-4h, mixing chloroauric acid and deionized water in a ratio of 1:(80-90) to obtain a chloroauric acid solution, adding the chloroauric acid solution into the mixed solution dropwise at a speed of 15mL / min, heating to 40-50℃, stirring at 300-500r / min for 6-8h, filtering, washing the filter cake with deionized water for 3-5 times, and drying in a drying box at 50-60℃ and 5-10Pa for 10-12h to obtain modified chitosan;

[0008] (2) mixing modified chitosan, acrylic acid, acrylic acid-N-hydroxysuccinimide, alpha-ketoglutaric acid, benzophenone and deionized water in a mass ratio of 1:(10-15):(0.4-0.7):(0.1-0.2):(0.01-0.02):(50-60) to obtain a mixed solution, irradiating the mixed solution with a UV lamp as a light source for 20-30min, filtering to obtain a filtrate, adding 0.05-0.1 times of glutaraldehyde of the modified chitosan into the filtrate, stirring at 20-30℃ and 200-300r / min for 10-20min, and drying in a drying box at 5-10Pa and 30-40℃ for 6-8h to obtain a hydrogel;

[0009] (3) mixing the hydrogel, insulin-like growth factor-1 and 10% phosphate buffer solution in a mass ratio of 1:(0.0001-0.0002):(30-40), stirring at 0-5℃ and 200-300r / min for 3-5h to obtain a biological nerve repair cannula material.

[0010] Further, the reaction process of the modified chitosan in step (1) is as follows:

[0011]

[0012] Further, the volume ratio of the mixed solution and the chitosan solution in step (1) is 1:(20-30).

[0013] Further, the volume ratio of the mixed solution and the chloroauric acid solution in step (1) is 1:(0.03-0.04).

[0014] Further, the chitosan type in the step (1) is R21.

[0015] Further, the reaction process of the hydrogel in the step (2) is as follows:

[0016]

[0017] Further, the parameters of the ultraviolet lamp in the step (2) are as follows: the wavelength is 365 nm, the power is 10-15 W, and the light intensity is 45-50 mW / cm 2 .

[0018] Further, the insulin-like growth factor-1 type in the step (3) is XFCZ0012.

[0019] Further, the configuration process of the 10% mass fraction of the phosphate buffer solution in the step (3) is as follows: the sodium chloride, potassium chloride, sodium phosphate dodecahydrate, potassium phosphate and deionized water are uniformly mixed according to the mass ratio (40-45) : 1 : (14-16) : (1.05-1.1) : (500-700), and the pH is 7.0-7.5.

[0020] Compared with the prior art, the beneficial effects achieved by the present application are:

[0021] (1) The present application prepares a nerve repair sleeve that can respond to stimulation, has self-coiling and adjustable diameter, and does not need to be sutured, thereby avoiding secondary puncture site damage and suture-related inflammation. The hydroxyl groups on the surface of boric acid and insulin-like growth factor-1 can form reversible borate ester bonds. The dissociation of the borate ester bonds releases insulin growth factor, effectively promotes peripheral nerve regeneration, and has good biological activity. The NHS group in the acrylic acid-N-hydroxysuccinimide can not only make it tightly combined with the nerve stump through covalent cross-linking to form a dense gap, but also can avoid the dissociation of the carrier and the insulin-like growth factor-1 in the physiological environment, which is conducive to the efficient local delivery and long-acting repair of the insulin-like growth factor.

[0022] (2) The nano-gold is loaded on the chitosan through the adsorption of the carboxyl group, which can not only scavenge active oxygen and reduce neuronal oxidative damage, but also can make the cells maintain a higher proliferation and axon growth rate, and improve the neural differentiation due to its conductivity. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0024] Example 1; (1) 3-carboxyphenylboronic acid, maleic anhydride, 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N, N-dimethylformamide were mixed in a mass ratio of 0.5:1:1.1:1.1:10 to obtain a mixed solution, which was stirred at 20°C and 300 r / min for 30-40 min. Chitosan, 10% dilute hydrochloric acid and deionized water were mixed in a mass ratio of 1:(5-7):(15-20) to prepare a chitosan solution. The chitosan solution was added dropwise to the mixed solution at a rate of 20 mL / min, and the stirring was continued for 2 h. Chloroauric acid and deionized water were mixed in a ratio of 1:80 to prepare a chloroauric acid solution. The chloroauric acid solution was added dropwise to the mixed solution at a rate of 15 mL / min, and the temperature was raised to 40°C. The stirring was continued at 300 r / min for 6 h. The filter cake was washed with deionized water for 5 times, and was placed in a drying box. The drying was carried out at 50°C and 5 Pa for 10 h to obtain modified chitosan;

[0025] (2) The modified chitosan, acrylic acid, acrylic acid-N-hydroxysuccinimide, α-ketoglutaric acid, benzophenone and deionized water were mixed in a mass ratio of 1:10:0.4:0.1:0.01:50. A UV lamp with a wavelength of 365 nm, a power of 10 W and an illumination intensity of 45 mW / cm 2 was used as a light source for irradiation for 20 min. The filtrate was obtained by filtration. Modified chitosan was added to the filtrate in an amount of 0.05 times the amount of glutaraldehyde. The stirring was carried out at 20°C and 200 r / min for 10 min. The drying was carried out in a drying box at 5 Pa and 30°C for 6 h to obtain a hydrogel;

[0026] (3) Sodium chloride, potassium chloride, sodium phosphate dodecahydrate, potassium phosphate and deionized water were mixed in a mass ratio of 40:1:14:1.05:500 to prepare a 10% phosphate buffer solution. The hydrogel, insulin-like growth factor-1 and the 10% phosphate buffer solution were mixed in a mass ratio of 1:0.0001:30. The stirring was carried out at 0°C and 200 r / min for 3 h to obtain a biological nerve repair sleeve material.

[0027] Example 2; (1) 3-carboxyphenylboronic acid, maleic anhydride, 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N, N-dimethylformamide were mixed in a mass ratio of 0.54:1:1.13:1.13:12 to obtain a mixed solution, and stirred at 25°C and 400 r / min for 35 min. Chitosan, 10% dilute hydrochloric acid and deionized water were mixed in a mass ratio of 1:5.6:17 to prepare a chitosan solution. The chitosan solution was added dropwise to the mixed solution at a rate of 20 mL / min, and the stirring was continued for 3 h. Chloroauric acid and deionized water were mixed in a ratio of 1:86 to prepare a chloroauric acid solution. The chloroauric acid solution was added dropwise to the mixed solution at a rate of 15 mL / min, and the temperature was raised to 45°C. The stirring was continued at 400 r / min for 7 h. The filter cake was washed with deionized water for 5 times, and placed in a drying box. The drying was carried out at 55°C and 7 Pa for 11 h to obtain the modified chitosan.

[0028] (2) The modified chitosan, acrylic acid, acrylic acid-N-hydroxysuccinimide, alpha-ketoglutaric acid, benzophenone and deionized water were mixed in a mass ratio of 1:12:0.6:0.15:0.013:55. A UV lamp with a wavelength of 365 nm, a power of 12 W and an illumination intensity of 48 mW / cm 2 was used as a light source and irradiated for 25 min. The filtrate was obtained by filtration. The modified chitosan was added to the filtrate at a ratio of 0.08 times the amount of glutaraldehyde. The stirring was carried out at 25°C and 300 r / min for 16 min. The drying was carried out in a drying box at 7 Pa and 35°C for 7 h to obtain the hydrogel.

[0029] (3) Sodium chloride, potassium chloride, sodium phosphate dodecahydrate, potassium phosphate and deionized water were mixed in a mass ratio of 42:1:15:1.08:600 to prepare a 10% phosphate buffer solution. The hydrogel, insulin-like growth factor-1 and the 10% phosphate buffer solution were mixed in a mass ratio of 1:0.0002:35. The stirring was carried out at 3°C and 300 r / min for 4 h to obtain the bio-based nerve repair cannula material.

[0030] Example 3; (1) 3-carboxyphenyl boronic acid, maleic anhydride, 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N, N-dimethylformamide were mixed in a mass ratio of 0.6:1:1.1:1.2:15 to obtain a mixed solution, which was stirred at 30°C and 500 r / min for 40 min. Chitosan, 10% dilute hydrochloric acid and deionized water were mixed in a mass ratio of 1:7:20 to prepare a chitosan solution. The chitosan solution was added dropwise into the mixed solution at a rate of 20 mL / min, and the stirring was continued for 4 h. Chloroauric acid and deionized water were mixed in a ratio of 1:90 to prepare a chloroauric acid solution. The chloroauric acid solution was added dropwise into the mixed solution at a rate of 15 mL / min, and the temperature was raised to 50°C. The stirring was continued at 500 r / min for 8 h. The filter cake was washed with deionized water for 5 times, and was placed in a drying box. The drying was carried out at 60°C and 10 Pa for 12 h to obtain modified chitosan;

[0031] (2) The modified chitosan, acrylic acid, acrylic acid-N-hydroxysuccinimide, alpha-ketoglutaric acid, benzophenone and deionized water were mixed in a mass ratio of 1:15:0.7:0.2:0.02:60. An ultraviolet lamp with a wavelength of 365 nm, a power of 15 W and an illumination intensity of 50 mW / cm 2 was used as a light source and irradiated for 30 min. The filtrate was obtained by filtration. The modified chitosan was added to the filtrate in an amount of 0.1 times the amount of glutaraldehyde. The stirring was carried out at 30°C and 300 r / min for 20 min. The drying was carried out in a drying box at 10 Pa and 40°C for 8 h to obtain a hydrogel;

[0032] (3) Sodium chloride, potassium chloride, sodium phosphate dodecahydrate, potassium phosphate and deionized water were mixed in a mass ratio of 45:1:16:1.1:700 to prepare a 10% phosphate buffer solution. The hydrogel, insulin-like growth factor-1 and the phosphate buffer solution were mixed in a mass ratio of 1:0.0002:40. The stirring was carried out at 5°C and 300 r / min for 5 h to obtain a bio-based nerve repair cannula material;

[0033] Comparative Example 1; Comparative Example 1 differs from Example 2 in that step (1) is modified as follows: 3-carboxyphenylboronic acid, maleic anhydride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N,N-dimethylformamide are mixed in a mass ratio of 0.54:1:1.13:1.13:12 to obtain a mixed solution, which is stirred at 25°C and 400 r / min for 35 min, chitosan, 10% dilute hydrochloric acid and deionized water are mixed in a mass ratio of 1:5.6:17 to prepare a chitosan solution, 23 times the volume of the chitosan solution is added dropwise into the mixed solution at a rate of 20 mL / min, and stirring is continued for 3 h, then the product is filtered, the filter cake is washed with deionized water for 5 times, and the filter cake is placed in a drying box and dried at 55°C and 7 Pa for 11 h to obtain the modified chitosan; the remaining steps are the same as those in Example 2.

[0034] Comparative Example 2; Comparative Example 2 differs from Example 2 in that step (1) is modified as follows: maleic anhydride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N,N-dimethylformamide are mixed in a mass ratio of 1:1.13:1.13:12 to obtain a mixed solution, which is stirred at 25°C and 400 r / min for 35 min, chitosan, 10% dilute hydrochloric acid and deionized water are mixed in a mass ratio of 1:5.6:17 to prepare a chitosan solution, 23 times the volume of the chitosan solution is added dropwise into the mixed solution at a rate of 20 mL / min, and stirring is continued for 3 h, then chloroauric acid and deionized water are mixed in a mass ratio of 1:86 to obtain a chloroauric acid solution, 0.035 times the volume of the chloroauric acid solution is added dropwise into the mixed solution at a rate of 15 mL / min, the temperature is raised to 45°C, and stirring is continued at 400 r / min for 7 h, then the product is filtered, the filter cake is washed with deionized water for 5 times, and the filter cake is placed in a drying box and dried at 55°C and 7 Pa for 11 h to obtain the modified chitosan; the remaining steps are the same as those in Example 2.

[0035] Comparative Example 3; Comparative Example 3 differs from Example 2 in that step (2) is modified as follows: the modified chitosan, acrylic acid, α-ketoglutaric acid, benzophenone and deionized water are mixed in a mass ratio of 1:12:0.15:0.013:55, a UV lamp with a wavelength of 365 nm, a power of 12 W and an illumination intensity of 48 mW / cm 2 is used as a light source, and the mixture is irradiated for 25 min, then the filtrate is obtained by filtration, 0.08 times the volume of glutaraldehyde is added into the filtrate, the mixture is stirred at 25°C and 300 r / min for 16 min, and the mixture is placed in a drying box and dried at 7 Pa and 35°C for 7 h to obtain the hydrogel; the remaining steps are the same as those in Example 2.

[0036] Effect Example

[0037] The performance analysis results of a bio-based nerve repair sleeve material using the present embodiments 1 to 3 and comparative examples 1 to 3 are shown in the following table.

[0038] The test method of biocompatibility: according to the GB / T16886 standard test, 2 mL of adult rabbit blood was collected in a centrifuge tube containing 15 mL of edetic acid at a speed of 1000 rpm for 2 minutes, washed with 15 mL of 10% by mass fraction of phosphate buffer solution to remove serum, and finally diluted with 10% by mass fraction of phosphate buffer solution to a concentration of 1 x 10 -8 mL of red blood cell suspension, then the comparative examples and embodiments were added to the red blood cell suspension to prepare samples, 10% by mass fraction of triton X-100 was used as a positive control, after incubation at 37°C for 60 minutes, the upper suspension was collected and centrifuged at 1000 rpm for 5 minutes, the supernatant was taken and the absorbance value at 540 nm wavelength was determined using an enzyme marker, hemolysis rate = sample absorbance - negative control absorbance / positive control absorbance x 100%. The results are shown in Table 1. Rat Schwann cells were inoculated in DMEM high-sugar medium containing 10% of 10 μL CCK-8 reagent and cultured at 37°C in a 5% carbon dioxide incubator for 24 hours as experimental group 1, the culture dish was added with the embodiments and comparative examples as experimental group 2, and the medium without rat Schwann cells was set as a blank control group, the absorbance value at 490 nm wavelength was determined using an enzyme marker. Cell survival rate = (experimental group 2 absorbance - blank control group absorbance) / (experimental group 1 absorbance - blank control group absorbance) x 100%. The results are shown in Table 1.

[0039] The test method of antibacterial performance: the embodiments and comparative examples were added to the culture dish to prepare nutrient broth, inoculated with Staphylococcus aureus and Escherichia coli, diluted with phosphate buffer solution, and cultured on plate counting agar medium for 24 h, and the number of colonies was counted; the control group of nutrient broth and phosphate buffer solution was inoculated with Staphylococcus aureus and Escherichia coli, and the antibacterial rate = control group of viable bacteria - experimental group of viable bacteria / control group of viable bacteria x 100%. The results are shown in Table 1.

[0040] The test method of tensile properties: the embodiments and comparative examples were cut into samples with a length of 10 mm, a width of 10 mm, and a thickness of 1 mm, and a universal tensile testing machine was used for tensile testing until fracture, and the tensile strength and elongation of the sample were calculated. The results are shown in Table 1.

[0041] Table 1

[0042]

[0043] It will be apparent to those skilled in the art that the application is not limited to the details of the above-exemplified embodiments and that the present application can be implemented in other particular forms without departing from the spirit or essential characteristics of the present application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the above description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. No reference herein to any prior patents or applications shall be taken as an admission that the application is not entitled to antedate such prior patents or applications by virtue of prior application. Any reference to the use of a term in the singular herein shall be taken as an acknowledgment of the existence of the term in the plural and vice versa.

Claims

1. A method for preparing a bio-based neural repair cannula material, characterized in that, The bio-based nerve repair cannula material is prepared by reacting acrylic acid, acrylate-N-hydroxysuccinimide, α-ketoglutaric acid, modified chitosan, and glutaraldehyde to obtain a hydrogel, which is then reacted with insulin-like neurotrophic factor-1. The modified chitosan is prepared by reacting chitosan, 3-carboxyphenylboronic acid, maleic anhydride, and gold nanoparticles.

2. The method for preparing a bio-based neural repair cannula material according to claim 1, characterized in that, The preparation steps include the following: (1) 3-Carboxyphenylboronic acid, maleic anhydride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N,N-dimethylformamide are mixed and stirred evenly in a mass ratio of (0.5-0.6):1:(1.1-1.2):(1.1-1.2):(10-15) to obtain a mixed solution. Stir at 20-30℃ for 30-40 min. Chitosan, 10% dilute hydrochloric acid and deionized water are mixed in a mass ratio of 1:(5-7):(15-20) to prepare a chitosan solution. Chitosan solution is added dropwise to the mixed solution and stirred for 2-4 h. Chloroauric acid and deionized water are mixed in a mass ratio of 1:(80-90) to prepare a chloroauric acid solution. Chloroauric acid solution is added dropwise to the mixed solution. The temperature is raised to 40-50℃ and stirred for 6-8 h to obtain modified chitosan. (2) Modified chitosan, acrylic acid, acrylate-N-hydroxysuccinimide, α-ketoglutaric acid, benzophenone and deionized water are mixed evenly in a mass ratio of 1:(10~15):(0.4~0.7):(0.1~0.2):(0.01~0.02):(50~60). The mixture is irradiated with an ultraviolet lamp for 20~30 min, filtered, and 0.05~0.1 times the amount of modified chitosan in glutaraldehyde is added to the filtrate. The mixture is stirred at 20~30℃ for 10~20 min to obtain a hydrogel. (3) The hydrogel, insulin-like growth factor-1 and 10% phosphate buffer solution were mixed at a mass ratio of 1:(0.0001~0.0002):(30~40) and stirred at 0~5℃ and 200~300r / min for 3~5h to obtain the bio-based nerve repair cannula material.

3. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The reaction process of modified chitosan in step (1) is as follows:

4. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The volume ratio of the mixed solution and the chitosan solution in step (1) is 1:(20-30).

5. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The volume ratio of the mixed solution and the chloroauric acid solution in step (1) is 1:(0.03-0.04).

6. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The chitosan type in step (1) is R21.

7. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The reaction process of the hydrogel in step (2) is as follows:

8. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The parameters of the ultraviolet lamp in step (2) are: wavelength 365nm, power 10-15W, and light intensity 45-50mW / cm². 2 .

9. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The insulin-like growth factor-1 in step (3) is of model number XFCZ0012.

10. The method for preparing a bio-based neural repair cannula material according to claim 2, characterized in that, The preparation process of the 10% phosphate buffer solution in step (3) is as follows: Sodium chloride, potassium chloride, sodium dodecahydrate, potassium phosphate and deionized water are mixed evenly in a mass ratio of (40-45):1:(14-16):(1.05-1.1):(500-700) and the pH is 7.0-7.5.

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