CN107118361B - Silk fibroin/carboxymethyl chitosan composite gel and preparation method thereof - Google Patents

Silk fibroin/carboxymethyl chitosan composite gel and preparation method thereof Download PDF

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CN107118361B
CN107118361B CN201710273386.7A CN201710273386A CN107118361B CN 107118361 B CN107118361 B CN 107118361B CN 201710273386 A CN201710273386 A CN 201710273386A CN 107118361 B CN107118361 B CN 107118361B
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solution
silk fibroin
carboxymethyl chitosan
composite gel
fibroin
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CN107118361A (en
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朱良均
刘迪
许宗溥
胡豆豆
杨明英
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Zhejiang University ZJU
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    • C08JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00Processes of treating or compounding macromolecular substances
    • C08J3/02Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/09Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in organic liquids
    • C08J3/091Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in organic liquids characterised by the chemical constitution of the organic liquid
    • C08J3/095Oxygen containing compounds
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    • C08J3/24Crosslinking, e.g. vulcanising, of macromolecules
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    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08L5/00Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
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    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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    • C08J2305/00Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
    • C08J2305/08Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
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    • C08JWORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
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    • C08J2489/00Characterised by the use of proteins; Derivatives thereof

Abstract

The invention discloses silk fibroin/carboxymethyl chitosan composite gel and a preparation method thereof. The composite gel is mainly composed of silk fibroin and carboxymethyl chitosan, and the mass ratio is (0.05-2): 1; the method comprises the steps of firstly processing a cocoon layer to obtain silk fibroin fibers, then preparing a silk fibroin solution to obtain a carboxymethyl chitosan solution, and uniformly mixing the silk fibroin solution and the carboxymethyl chitosan solution to obtain a mixed solution; adding a certain amount of cross-linking agent, stirring the solution on a magnetic stirrer for a short time, and immediately pouring the solution into a mold for standing; then immersing into ethanol water solution to finally obtain the fibroin/carboxymethyl chitosan composite gel. The invention prepares the gel with stronger mechanical property and better stability by a simpler process and shorter time, provides a new thought for improving the preparation method of the silk fibroin composite gel, and further expands the application approach of the silk fibroin.

Description

Silk fibroin/carboxymethyl chitosan composite gel and preparation method thereof
Technical Field
The invention belongs to a processing technology of a regenerated silk fibroin solution, and particularly relates to a silk fibroin/carboxymethyl chitosan composite gel and a preparation method thereof.
Background
In recent years, the gel material is distinguished in the biomedical field, is used in the fields of tissue engineering scaffolds, drug delivery carriers, wound dressings and the like, is favored by researchers, and many researchers lock the gel raw material into a natural polymer biomaterial which is cheap and easily available, good in biocompatibility and high in safety performance.
1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCL) has the characteristic of activating carboxyl, and is generally applied to polymer modification as a condensing agent and a crosslinking agent due to the characteristics of mild reaction conditions, high yield, good selectivity, environmental friendliness and the like ([ J ] desalinization, 2008, 234: 134-.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide the silk fibroin/carboxymethyl chitosan composite gel and the preparation method thereof, the process is simple, the time consumption is short, and the composite gel has excellent mechanical properties.
The technical scheme adopted by the invention comprises the following steps:
a silk fibroin/carboxymethyl chitosan composite gel:
the composite gel is mainly composed of silk fibroin and carboxymethyl chitosan.
In the composite gel, the mass ratio of the silk fibroin to the carboxymethyl chitosan is (0.05-2): 1, preferably (0.5-2): 1.
secondly, a preparation method of the silk fibroin/carboxymethyl chitosan composite gel comprises the following steps:
dissolving the degummed silk fibroin fiber in LiBr solution, dialyzing to obtain regenerated silk fibroin solution, mixing the regenerated silk fibroin solution with carboxymethyl chitosan solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride serving as a cross-linking agent, and finally preparing the silk fibroin/carboxymethyl chitosan composite hydrogel under the action of ethanol.
The method comprises the following specific processes:
(1) silk fibroin is obtained by processing a cocoon layer, and then silk fibroin solution is prepared;
(2) dissolving carboxymethyl chitosan in water to prepare carboxymethyl chitosan solution;
(3) uniformly mixing the fibroin solution obtained in the step (1) and the carboxymethyl chitosan solution obtained in the step (2) to obtain a mixed solution;
(4) adding a certain amount of cross-linking agent into the mixed solution obtained in the step (3), stirring the solution on a magnetic stirrer for a short time, and immediately pouring the solution into a mold for standing;
(5) then immersing into ethanol water solution to finally obtain the fibroin/carboxymethyl chitosan composite gel.
The step (1) is specifically as follows:
(1) placing the cocoon shell in 0.5 percent of Na by mass fraction2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) placing the silk fibroin fibers obtained in the step (1) in 9M LiBr solution, dissolving for 4h at 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag, dialyzing in deionized water for 4d, and continuously changing water during the dialysis, thus obtaining the silk fibroin solution.
The concentration of the silk fibroin solution obtained in the step (1) is 0.5-10% w/v, the concentration of the carboxymethyl chitosan solution obtained in the step (2) is 1-5% w/v, and the mixing volume ratio of the silk fibroin solution and the carboxymethyl chitosan solution in the step (3) is 1: 1.
Finally, the mass ratio of mixed solute silk fibroin and carboxymethyl chitosan is (0.05-2): 1, in the range of. Preferably, the concentration ratio of the silk fibroin solution to the carboxymethyl chitosan solution is (0.1-2): 1.
the degree of deacetylation of the carboxymethyl chitosan is 55-99%, and the degree of substitution is 60-99%.
Preferably, the concentration of the silk fibroin solution in the step (1) is 0.5-8% w/v, the concentration of the carboxymethyl chitosan solution in the step (2) is 1-4% w/v, the deacetylation degree of the carboxymethyl chitosan is 90%, and the substitution degree is 90%.
More preferably, the concentration of the silk fibroin solution in the step (1) is 0.5-4% w/v and the concentration of the carboxymethyl chitosan solution in the step (2) is 4% w/v.
Most preferably, the concentration of the silk fibroin solution is 4% w/v, the concentration of the carboxymethyl chitosan solution is 4% w/v, and the mixing volume ratio is 1:1, so that the silk fibroin and carboxymethyl chitosan solution in the invention is simpler to prepare, easy to control, more uniform to mix, better in molding of the obtained composite gel, stronger in mechanical property and excellent in water absorption stability of the composite gel.
The molecular weight of the dialysis bag in the step (1) is 8000-14000.
In the step (4), solid powder of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCL) is used as the crosslinking agent, and the addition amount of the crosslinking agent is 0.3-0.75% w/v of the system.
The system refers to the mixed solution obtained in the step (4).
Preferably, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCL) is added in an amount of 0.45-0.6% w/v of the fibroin/carboxymethyl chitosan solution system.
More preferably, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCL) is added at 0.6% w/v of the fibroin/carboxymethyl chitosan solution system. The composite gel thus obtained is excellent in crosslinking effect and does not cause an excessive amount of the crosslinking agent.
The addition mode of the cross-linking agent is divided into two modes: one is a system of adding solid powder directly and the other is a system of adding solid powder dissolved in water, so long as the crosslinking agent powder can be dissolved and the volume of added water must be as small as possible.
Preferably, in the step (5) of the present invention, the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC. HCL) is added by dissolving the solid powder in a small amount of water and adding the solution, so that the distribution of the cross-linking agent in the mixed solution is more uniform, the reaction is faster and more efficient, the obtained composite gel has a tighter structure, and the performance is better.
In the step (4), the stirring time is 20s, and the standing time is 6-12 h; in the step (5), the concentration of ethanol in the ethanol aqueous solution is 60-90% v/v, and the time for immersing the ethanol aqueous solution is 12 hours.
Preferably, the standing time for pouring the solution system into the mold in the step (4) is 12 h.
Preferably, the ethanol concentration of the aqueous ethanol solution is 75% v/v.
The product of the invention obtains higher mechanical property and stable water retention property through the example test, the elastic modulus reaches (363.94 +/-45.57) kPa, the compression recovery rate reaches 100%, the volume is relatively stable in a phosphate buffer solution with the pH value of 7.4, and the swelling rate is (9.32 +/-0.09) multiplied by 100%.
The invention promotes the silk fibroin/carboxymethyl chitosan to form the composite gel by the crosslinking effect of EDC & HCL and the characteristic that ethanol promotes the silk fibroin to generate β transformation, prepares the gel with stronger mechanical property and better stability by a simpler process and shorter time, provides a new idea for improving the preparation method of the silk fibroin composite gel, and further expands the application approach of the silk fibroin.
Compared with the prior art, the invention has the following advantages and effects:
1. the process is simple: in the prior art, methods such as organic solvent, ultrasonic treatment, optical crosslinking and the like are usually needed for preparing the silk fibroin composite gel, but the preparation can be completed in a room temperature environment;
2. the gel time is short: the preparation of the silk fibroin composite gel in the prior art generally needs longer time, but the preparation method only needs about 1-2 days, so that the efficiency is greatly improved;
3. the cross-linking agent required by the invention is taken as an active agent, can be easily removed by a water washing method, and has high biological safety;
4. the composite gel prepared by the invention has stronger mechanical property, good water absorption stability and moisture retention, and is superior to silk fibroin composite gels prepared by other methods. The composite gel prepared by the invention can be used in the fields of medicine and health, tissue engineering, environmental protection and the like, expands the application approaches of the silk fibroin and also provides a new idea and a new method for preparing the high-strength composite gel.
Detailed Description
The present invention is further illustrated by the following examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
Example 1
The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in the embodiment sequentially comprises the following steps.
(1) Weighing 10g of cut cocoon shells in 0.5% by mass of Na2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) putting the silk fibroin fibers obtained in the step (1) into 9M LiBr solution, dissolving for 4 hours at the temperature of 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag (molecular weight of 8000-14000), dissolving a proper amount of carboxymethyl chitosan (degree of deacetylation is 90%, degree of substitution is 90%) in deionized water (3), and dissolving in water to obtain a carboxymethyl chitosan solution with the concentration of 4% w/v.
(4) Mixing the fibroin solution with the concentration of 0.5% w/v obtained in the step (2) and the carboxymethyl chitosan solution with the concentration of 4% w/v obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing to obtain a mixed solution.
(5) Adding a certain amount of a cross-linking agent 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC & HCL) into the mixed solution obtained in the step (4), wherein the adding modes are divided into two modes: one is to add the solid powder directly into the system, and the other is to add the solid powder into the system by dissolving the solid powder into a small amount of water. The addition amount of the cross-linking agent is 0.3-0.75% w/v of the system, preferably 0.45-0.6% w/v, and most preferably 0.6% w/v. The best mode of addition of the cross-linking agent is the second.
(6) And adding EDC & HCL into the fibroin/carboxymethyl chitosan composite solution under the continuous stirring of a magnetic stirrer, pouring the solution into a mould after 20s, and standing for 6-12h, preferably 12 h.
(7) And immersing the silk fibroin/carboxymethyl chitosan composite gel in 60-90% v/v ethanol water solution for 12h, wherein the optimal concentration of ethanol is 75% v/v, and finally obtaining the silk fibroin/carboxymethyl chitosan composite gel.
(8) In this example, the mechanical properties of the gel were measured by uniaxial compression (compression rate: 1mm/min) using a mechanical universal tester (Shimadzu, AGS-J series). The swelling performance of the gel is tested by adopting a mass weighing method: firstly, soaking the composite gel material in deionized water or phosphate buffer solution for 24h to make the composite gel material reach swelling equilibrium, then absorbing surface moisture by using filter paper, and weighing the mass of the composite gel material as M1(g) Then placing the composite gel in a 60 ℃ oven for drying, and measuring the mass of the composite gel as M2(g) In that respect The calculation formula is as follows: swelling ratio ═ M1-M2)/M2×100%。
The elastic modulus of the composite gel reaches (29.46 +/-16.70) kPa, and the compression recovery rate reaches 100%. The gel had a water swelling rate of (103.63 ± 4.91) × 100% in deionized water, and was relatively stable in volume in a phosphate buffer at pH 7.4, with a swelling rate of (20.79 ± 2.64) × 100%.
Example 2
The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in the embodiment sequentially comprises the following steps.
(1) Weighing 10g of cut cocoon shells in 0.5% by mass of Na2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) placing the silk fibroin fibers obtained in the step (1) into 9M LiBr solution, dissolving for 4h at 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag (with molecular weight of 8000-14000), and dialyzing in deionized water for 4d, wherein water is continuously changed in the dialysis bag, and the concentration of the finally obtained silk fibroin solution is 1.0% w/v.
(3) An appropriate amount of carboxymethyl chitosan (degree of deacetylation 90%, degree of substitution 90%) was dissolved in water to make a 4% w/v carboxymethyl chitosan solution.
(4) Mixing the fibroin solution with the concentration of 1.0% w/v obtained in the step (2) and the carboxymethyl chitosan solution with the concentration of 4% w/v obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing to obtain a mixed solution.
(5) And (3) adding a certain amount of a cross-linking agent 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC & HCL) into the mixed solution obtained in the step (4), wherein the adding mode is that the solid powder is firstly dissolved in a small amount of water and then added into the system. The crosslinker was added in an amount of 0.6% w/v of the system.
(6) Adding EDC & HCL into the fibroin/carboxymethyl chitosan composite solution under the continuous stirring of a magnetic stirrer, pouring the solution into a mould after 20s, and standing for 12 h.
(7) And soaking the silk fibroin/carboxymethyl chitosan composite gel in 75% v/v ethanol water solution for 12h to finally obtain the silk fibroin/carboxymethyl chitosan composite gel.
(8) The composite gel was tested in the same manner as in example 1 to obtain an elastic modulus (78.89 ± 5.67) kPa, a compression recovery rate of 100%, a water absorption swelling rate (56.95 ± 6.34) × 100% in deionized water, and a volume stability in a phosphate buffer solution at pH 7.4 of (19.17 ± 4.30) × 100%.
Example 3
The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in the embodiment sequentially comprises the following steps.
(1) Weighing 10g of cut cocoon shells in 0.5% by mass of Na2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) placing the silk fibroin fibers obtained in the step (1) into 9M LiBr solution, dissolving for 4h at 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag (with molecular weight of 8000-14000), and dialyzing in deionized water for 4d, wherein water is continuously changed in the dialysis bag, and the concentration of the finally obtained silk fibroin solution is 1.5% w/v.
(3) An appropriate amount of carboxymethyl chitosan (degree of deacetylation 90%, degree of substitution 90%) was dissolved in water to make a 4% w/v carboxymethyl chitosan solution.
(4) Mixing the fibroin solution with the concentration of 1.5% w/v obtained in the step (2) and the carboxymethyl chitosan solution with the concentration of 4% w/v obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing to obtain a mixed solution.
(5) And (3) adding a certain amount of a cross-linking agent 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC & HCL) into the mixed solution obtained in the step (4), wherein the adding mode is that the solid powder is firstly dissolved in a small amount of water and then added into the system. The crosslinker was added in an amount of 0.6% w/v of the system.
(6) Adding EDC & HCL into the fibroin/carboxymethyl chitosan composite solution under the continuous stirring of a magnetic stirrer, pouring the solution into a mould after 20s, and standing for 12 h.
(7) And soaking the silk fibroin/carboxymethyl chitosan composite gel in 75% v/v ethanol water solution for 12h to finally obtain the silk fibroin/carboxymethyl chitosan composite gel.
(8) The composite gel was tested in the same manner as in example 1 to obtain an elastic modulus of (99.03 ± 9.72) KPa, a compression recovery of 100%, a water absorption swelling rate of (51.74 ± 10.03) × 100% in deionized water, and a volume stability of (16.60 ± 2.11) × 100% in a phosphate buffer at pH 7.4.
Example 4
The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in the embodiment sequentially comprises the following steps.
(1) Weighing 10g of cut cocoon shells in 0.5% by mass of Na2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) placing the silk fibroin fibers obtained in the step (1) into 9M LiBr solution, dissolving for 4h at 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag (with molecular weight of 8000-14000), and dialyzing in deionized water for 4d, wherein water is continuously changed in the dialysis bag, and the concentration of the finally obtained silk fibroin solution is 2.0% w/v.
(3) An appropriate amount of carboxymethyl chitosan (degree of deacetylation 90%, degree of substitution 90%) was dissolved in water to make a 4% w/v carboxymethyl chitosan solution.
(4) Mixing the fibroin solution with the concentration of 2.0% w/v obtained in the step (2) and the carboxymethyl chitosan solution with the concentration of 4% w/v obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing to obtain a mixed solution.
(5) And (3) adding a certain amount of a cross-linking agent 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC & HCL) into the mixed solution obtained in the step (4), and dissolving the solid powder into a small amount of water to be added into the system. The crosslinker was added in an amount of 0.6% w/v of the system.
(6) Adding EDC & HCL into the fibroin/carboxymethyl chitosan composite solution under the continuous stirring of a magnetic stirrer, pouring the solution into a mould after 20s, and standing for 12 h.
(7) And soaking the silk fibroin/carboxymethyl chitosan composite gel in 75% v/v ethanol water solution for 12h to finally obtain the silk fibroin/carboxymethyl chitosan composite gel.
(8) The composite gel was tested in the same manner as in example 1 to obtain an elastic modulus of (248.60 ± 34.47) kPa, a compression recovery of 100%, a water absorption swelling rate of (35.78 ± 10.75) × 100% in deionized water, and a volume stability of (13.18 ± 0.53) × 100% in a phosphate buffer at pH 7.4.
Example 5
The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in the embodiment sequentially comprises the following steps.
(1) Weighing 10g of cut cocoon shells in 4.0% by mass of Na2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) placing the silk fibroin fibers obtained in the step (1) into 9M LiBr solution, dissolving for 4h at 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag (with molecular weight of 8000-14000), and dialyzing in deionized water for 4d, wherein water is continuously changed in the dialysis bag, and the concentration of the finally obtained silk fibroin solution is 0.5% w/v.
(3) An appropriate amount of carboxymethyl chitosan (degree of deacetylation 90%, degree of substitution 90%) was dissolved in water to make a 4% w/v carboxymethyl chitosan solution.
(4) Mixing the fibroin solution with the concentration of 4.0% w/v obtained in the step (2) and the carboxymethyl chitosan solution with the concentration of 4% w/v obtained in the step (3) according to the volume ratio of 1:1, uniformly mixing to obtain a mixed solution.
(5) And (3) adding a certain amount of a cross-linking agent 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC & HCL) into the mixed solution obtained in the step (4), wherein the adding mode is that the solid powder is firstly dissolved in a small amount of water and then added into the system. The crosslinker was added in an amount of 0.6% w/v of the system.
(6) Adding EDC & HCL into the fibroin/carboxymethyl chitosan composite solution under the continuous stirring of a magnetic stirrer, pouring the solution into a mould after 20s, and standing for 12 h.
(7) And soaking the silk fibroin/carboxymethyl chitosan composite gel in 75% v/v ethanol water solution for 12h to finally obtain the silk fibroin/carboxymethyl chitosan composite gel.
(8) The composite gel was tested in the same manner as in example 1 to obtain an elastic modulus (363.99 ± 45.57) kPa, a compression recovery rate of 100%, a water absorption swelling rate (20.79 ± 3.14) × 100% in deionized water, and a volume stability in a phosphate buffer solution at pH 7.4 of (9.32 ± 0.09) × 100%.
Therefore, the novel method for preparing the fibroin composite gel provided by the invention has the advantages of simple preparation process, mild condition and short gel time, and the obtained composite gel has better mechanical property and water absorption stability: the elastic modulus reaches (363.94 +/-45.57) kPa, the compression recovery rate reaches 100 percent, namely when certain pressure is applied on the composite gel, the composite gel has strong bearing capacity and is not crushed, and when the pressure is removed, the gel is recovered; the volume was relatively stable in phosphate buffer at pH 7.4 with a swelling ratio of (9.32 ± 0.09) × 100%, and the dried sample was swollen by rapidly absorbing a large amount of water in water, but still maintained a good form without being broken loose.
While only a few specific embodiments of the present invention have been shown and described, it will be obvious that the invention is not limited thereto, but may be embodied in many different forms and that all changes and modifications that can be derived from the disclosure set forth herein by one of ordinary skill in the art are deemed to be within the scope of the present invention.

Claims (5)

1. A preparation method of silk fibroin/carboxymethyl chitosan composite gel is characterized by comprising the following steps:
dissolving the degummed silk fibroin in LiBr solution, dialyzing to obtain regenerated silk fibroin solution, mixing the regenerated silk fibroin solution with carboxymethyl chitosan solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride serving as a cross-linking agent, and finally preparing the silk fibroin/carboxymethyl chitosan composite hydrogel under the action of ethanol;
the method comprises the following specific processes:
(1) silk fibroin is obtained by processing a cocoon layer, and then silk fibroin solution is prepared;
(2) dissolving carboxymethyl chitosan in water to prepare carboxymethyl chitosan solution; the concentration of the silk fibroin solution in the step (1) is 0.5-4% w/v, and the concentration of the carboxymethyl chitosan solution in the step (2) is 4% w/v;
(3) uniformly mixing the fibroin solution obtained in the step (1) and the carboxymethyl chitosan solution obtained in the step (2) to obtain a mixed solution;
(4) adding a certain amount of cross-linking agent into the mixed solution obtained in the step (3), stirring the solution on a magnetic stirrer for a short time, and immediately pouring the solution into a mold for standing; the cross-linking agent is solid powder of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDC & HCL), and the addition amount of the cross-linking agent is 0.3-0.75% w/v of the system;
(5) then immersing into ethanol water solution to finally obtain the fibroin/carboxymethyl chitosan composite gel.
2. The method for preparing silk fibroin/carboxymethyl chitosan composite gel according to claim 1, characterized in that: the step (1) is specifically as follows:
(1) placing the cocoon shell in 0.5 percent of Na by mass fraction2CO3Degumming twice in boiling water solution for 30min each time, washing the obtained silk fibroin fiber with deionized water, wringing, and drying in an oven;
(2) and (2) placing the silk fibroin fibers obtained in the step (1) in 9M LiBr solution, dissolving for 4h at 60 ℃, filtering the obtained liquid, putting the filtered liquid into a dialysis bag, dialyzing in deionized water for 4d, and continuously changing water during the dialysis, thus obtaining the silk fibroin solution.
3. The method for preparing silk fibroin/carboxymethyl chitosan composite gel according to claim 1, characterized in that: the mixing volume ratio of the silk fibroin solution and the carboxymethyl chitosan solution in the step (3) is 1: 1.
4. The method for preparing silk fibroin/carboxymethyl chitosan composite gel according to claim 1, characterized in that: the degree of deacetylation of the carboxymethyl chitosan is 55-99%, and the degree of substitution is 60-99%.
5. The method for preparing silk fibroin/carboxymethyl chitosan composite gel according to claim 1, characterized in that: in the step (4), the stirring time is 20s, and the standing time is 6-12 h; in the step (5), the concentration of ethanol in the ethanol aqueous solution is 60-90% v/v, and the time for immersing the ethanol aqueous solution is 12 hours.
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