CN115105621B - Preparation method of silk-spider silk composite silk fibroin nano-microspheres containing chitosan modified graphene oxide - Google Patents
Preparation method of silk-spider silk composite silk fibroin nano-microspheres containing chitosan modified graphene oxide Download PDFInfo
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Abstract
The invention relates to the field of biomedical materials, and discloses a preparation method of silk-spider silk composite silk fibroin nano microspheres containing chitosan modified graphene oxide, which comprises the steps of preparing a spider silk fibroin solution and a silk fibroin solution respectively, then preparing a composite silk fibroin solution, and decomposing silk fibroin into microspheres by heating enzymolysis; preparing chitosan modified graphene oxide particles; and then dispersing the chitosan modified graphene oxide particles and the composite silk fibroin microspheres in water to prepare the composite silk fibroin nanospheres. The composite nano-microsphere has good biocompatibility and biodegradability, can promote the release of loaded drugs so as to sterilize, strength, elasticity and the like, can slowly release graphene oxide to inhibit the breeding of wound bacteria, can promote the healing of wounds by using the spider silk fibroin and the chitosan, and can effectively treat burn and scald wounds if being used as wound dressings and the like.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a preparation method of silk-spider silk composite silk fibroin nano microspheres containing chitosan modified graphene oxide.
Background
Burns and scalds are common accidental injuries in daily life, mild burns can damage the skin of a patient, and severe burns can endanger the life of the patient and cause lifelong injuries. At present, for mild burns and scalds, the traditional method is to disinfect and clean wounds, and then to stick sterile gauze and the like to protect and press the wounds. Of course, this method has a long treatment period and limited effect, and is often used in a dressing based on a biomaterial to promote wound healing and the like, so as to enhance the treatment effect on mild burns and scalds. However, the existing biomaterial dressing mainly keeps the wound moist and breathable, and the biomaterial dressing for active treatment of the wound is less. For burn and scald wounds, bacteria can grow and propagate rapidly due to the change of the microenvironment of the wounds, so that in order to inhibit the mass propagation of the bacteria in the wounds from influencing the healing effect of the wounds, antibacterial drugs and the like are needed to kill the bacteria, protect and promote the change of the microenvironment of the wounds, shorten the healing recovery period of the wounds and achieve the effect of rapid treatment.
Graphene oxide is a biological antibacterial agent with a great application prospect, and when the graphene oxide is used as an antibacterial agent, organisms have excellent compatibility with the antibacterial agent, and the organisms are not easy to generate antibodies, and meanwhile, the graphene oxide has a good antibacterial effect and can kill most bacteria. Therefore, the graphene oxide can help prevent infection of bacteria and the like in a wound healing process when the graphene oxide acts on a burn and scald wound.
Natural macromolecular protein has been widely used in the field of biomedical materials due to its abundant sources, low price and good biocompatibility. A large number of researches show that silk fibroin which is a natural protein product extracted from silk is an extremely ideal drug carrier material, but the application prospect of the silk fibroin serving as a biomedical material is greatly limited due to the fact that the strength is not high enough and the elasticity is limited.
Hydrogel films, fibers and the like are widely applied to materials for treating burns and scalds wound dressings, and the nano microspheres are widely applied to drug carrier materials due to the effects of transdermal absorption, drug loading and the like. It is capable of delivering bioactive components to the deep layers of the skin, releasing the bioactive materials adsorbed by the particles. The nano microsphere transdermal absorption technology breaks the skin absorption barrier, and the skin can actively receive the medicine and generate the effect.
Chitosan, as a natural polymer material, has been widely used in the fields of tissue engineering, biological scaffolds, etc. due to its excellent biosafety and biodegradability. However, the application of the pure chitosan scaffold material in the field of biomedical dressings is limited due to the poor mechanical property of the pure chitosan scaffold material. Besides inhibiting the breeding of burning and scalding wound bacteria, the graphene oxide has larger surface area and good mechanical property and is a good carrier for loading drugs. Researches show that the composite material of chitosan and graphene oxide has good physical and mechanical properties. Therefore, the graphene oxide modified by chitosan is combined with the silk fibroin material, so that the mechanical property of the composite material can be further improved, and the performances of inhibiting the breeding of wound microorganisms, promoting the absorption of loaded drugs, being biodegradable and the like can be realized.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a silk-spider silk composite silk fibroin nano microsphere containing chitosan modified graphene oxide. According to the invention, firstly, spider silk reacts with a sodium carbonate solution and an acetic acid solution respectively to prepare a spider silk fibroin solution, then the sodium carbonate solution is used for degumming silkworm cocoons, and then a lithium bromide solution is used for dissolving to obtain a silk fibroin solution. And then mixing the two silk fibroin solutions, storing the mixture at a low temperature for a period of time, and freeze-drying the mixture to obtain the composite silk fibroin fiber. Dissolving the obtained composite silk fibroin fiber in water, adding a phosphate buffer solution and a protease solution, heating to react to decompose the composite fiber into microspheres, inactivating the protease, and freeze-drying to obtain the silk-spider silk composite silk fibroin microsphere. And secondly, preparing chitosan-modified graphene oxide particles, dispersing the chitosan-modified graphene oxide particles and the composite silk fibroin protein microspheres in a small amount of water respectively, uniformly mixing the particles by ultrasonic waves, centrifuging, and freeze-drying to obtain the silk-spider silk composite silk fibroin protein nanospheres of the chitosan-modified graphene oxide. The composite nano-microsphere has good biocompatibility and biodegradability, can promote the release of a loaded medicament to further sterilize, strength, elasticity and the like, can slowly release graphene oxide to inhibit the breeding of bacteria in a wound, can promote the healing of the wound by using the spider silk fibroin and the chitosan, and can effectively treat the wound of burn and scald if being used as a wound dressing and the like; meanwhile, the method has the advantages of simple operation process, no toxicity, no harm, green and environmental protection.
The specific technical scheme of the invention is as follows: a preparation method of silk-spider silk composite silk fibroin nano-microspheres containing chitosan modified graphene oxide comprises the following steps:
step 1): and (3) putting the cleaned spider silk into a sodium carbonate solution, boiling for degumming, and washing with water to obtain the degummed spider silk.
Step 2): placing degummed spider silk in acetic acid solution for heating reaction, centrifuging the obtained mixed solution after reaction, collecting supernatant to obtain spider silk protein mixed solution, and dialyzing in water to obtain relatively pure spider silk protein solution.
Spider silk fibroin is another natural protein, and has a structure similar to that of silk fibroin and good biocompatibility. The elongation of the spider silk can reach 130% without breaking. Meanwhile, the spider silk fibroin anti-bacterial and anti-mildew fabric has moisture resistance and low temperature resistance, the spider silk can still keep high elasticity and anti-bacterial and anti-mildew properties at low temperature, and the spider silk fibroin has certain relieving and treating effects on burn and scald mouths. The spider silk fibroin obtained in the preparation step has high purity, retains the strength and elasticity of spider silk, can remove substances which enable an organism to generate resistance and low biocompatibility, and is beneficial to the subsequent mixing with the silk fibroin and the formation of nano microspheres.
Therefore, the silk fibroin protein and the spider silk fibroin protein are mixed to obtain the composite silk-spider silk fibroin protein biological material, which is expected to have certain wound treatment effect while improving the elasticity and the strength of the biological medical material, and can greatly enrich the application prospect of the silk fibroin protein as the biological medical material. However, there is currently less research on silk-spider silk compounding as a biomedical material.
Step 3): the degumming silk is obtained by taking silk, cleaning, placing the silk in a sodium carbonate solution, boiling for degumming, and cleaning with water.
Step 4): dissolving degummed silk in a lithium bromide solution to obtain a silk fibroin mixed solution, and dialyzing in water to obtain a relatively pure silk fibroin solution.
The preparation steps are a two-step dissolution method, so that a silk fibroin solution with high purity can be obtained, sericin with immunogenicity and low biocompatibility to biological tissues is removed, and synthesis of composite microspheres containing graphene oxide later is facilitated.
Step 5): uniformly mixing the spidroin solution obtained in the step 2) and the fibroin solution obtained in the step 4), storing at low temperature, and freeze-drying to obtain the silk-spidroin composite fibroin solution.
Step 6): and (3) putting the silk-spider silk composite silk fibroin solution obtained in the step 5) into water, dissolving, putting into a phosphate buffer solution, adding a protease solution, heating the obtained mixed solution in water bath, heating to inactivate the protease, cooling to room temperature, standing, and freeze-drying to obtain the silk-spider silk composite silk fibroin microsphere.
The preparation method comprises the step of mixing silk and spider silk fibroin to obtain a composite silk fibroin solution. Through enzymolysis of a protease solution, silk fibroin and spider silk fibroin are mixed more uniformly, the molecular weight of the mixed silk fibroin is reduced, and the obtained silk-spider silk composite silk fibroin microspheres with a certain particle size provide a good basis for subsequent compounding with the chitosan-modified graphene oxide microspheres.
Step 7): mixing a graphene oxide solution, a chitosan solution, a cross-linking agent and a catalyst, and then stirring for reaction to covalently bond graphene oxide and chitosan to obtain a graphene oxide hydrogel modified by chitosan; and (4) after low-temperature storage, freezing and drying to obtain the chitosan modified graphene oxide particles.
Step 8): dispersing the chitosan modified graphene oxide particles and the silk-spider silk composite fibroin microspheres in water to form a suspension, performing ultrasonic treatment to uniformly mix the suspension, centrifuging the obtained mixed solution, taking down the precipitate of the lower layer, and performing freeze drying to obtain the silk-spider silk composite fibroin nanospheres containing the chitosan modified graphene oxide.
The preparation method comprises the steps of dispersing chitosan modified graphene oxide particles and silk-spider silk composite silk fibroin microspheres in water, wherein active amino acid residues exist on the surfaces of the composite silk fibroin microspheres, active residues also exist on the surfaces of the chitosan modified graphene oxide particles, and the active residues on the surfaces of the chitosan modified graphene oxide particles and the silk-spider silk composite silk fibroin microspheres are subjected to conjugate adsorption by mixing and ultrasonic. The stability of the composite material structure is enhanced through conjugated adsorption among residues, and the silk-spider silk composite silk fibroin nano-microsphere containing the chitosan modified graphene oxide is obtained.
Preferably, in step 1), the concentration of sodium carbonate is 0.3 to 3.0wt%, and the ratio of the spider silk to the sodium carbonate solution is 1g: (100-300) mL, and the boiling degumming time is 20-50 minutes.
Preferably, in step 2), the concentration of the acetic acid solution is 70-90wt%, and the dosage ratio of the spider silk to the acetic acid solution is 1g: (30-70) mL, the heating reaction temperature is 35-72 ℃, the reaction time is 4-12 hours, the centrifugal speed is 8500-14000 r/min, the centrifugal time is 10-20 minutes, and the dialysis time is 30-50 hours.
Preferably, in the step 3), the concentration of sodium carbonate is 0.04-0.1wt%, and the dosage ratio of silk to sodium carbonate solution is 1g: (200-800) mL, and the boiling degumming time is 20-50 minutes.
Preferably, in the step 4), the concentration of lithium bromide is 7-10M, and the dosage ratio of the silk to the lithium bromide solution is 1g: (20-150) mL, and the dialysis time is 45-85 hours.
Preferably, in the step 5), the mass ratio of the spider silk protein solution to the silk protein solution is 1: (5-20), the low-temperature storage temperature is (-60) - (20 ℃), the storage time is 8-20 hours, and the freeze-drying time is 16-30 hours.
Preferably, in step 6), the concentration of the silk-spider silk composite fibroin solution is 10-15wt%, the pH of the phosphate buffer is =7.5-8.0, and the mixing volume ratio of the phosphate buffer to the silk-spider silk composite fibroin solution is 1: (2-5), the mixing volume ratio of the protease solution to the silk-spider silk composite silk fibroin solution is 1: (30-80), heating in water bath at 40-70 deg.C for 2-5 hr, heating to 105 deg.C for 5-15 min, cooling to room temperature, standing for 9-20 hr, and freeze drying for 16-30 hr.
Preferably, in the step 7), the concentration of the graphene oxide solution is 2-9g/L, the mass concentration of the chitosan solution is 1-10%, the cross-linking agent is a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the mass concentration of 90-98%, the catalyst is an N-hydroxysuccinimide solution with the mass concentration of 95-100%, and the mass ratio of the components of the mixed solution is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride: n-hydroxysuccinimide: chitosan =1: (3-8); the mixed solution is stirred for reaction time of 12-24 hours. The freeze-drying time is 24-48 hours.
Preferably, in the step 8), the concentration of the chitosan-modified graphene oxide solution is 1-20g/L, and the mixing volume ratio of the chitosan-modified graphene oxide solution to the silk-spider silk composite fibroin solution is 1: (10-80), the ultrasonic time is 20-50 minutes, the centrifugal speed is 8000-15000 r/min, the centrifugal time is 20-40 minutes, and the freeze-drying time is 24-48 hours.
Compared with the prior art, the invention has the beneficial effects that:
(1) The silk-spider silk composite silk fibroin nanospheres containing chitosan modified graphene oxide can be used as a carrier for loading a graphene oxide antibacterial agent, can effectively inhibit bacterial growth and reproduction of burn and scald wounds, protects the wounds and provides a good healing microenvironment for the wounds.
(2) According to the silk-spider silk composite silk fibroin nano-microsphere containing chitosan modified graphene oxide, spider silk fibroin and chitosan can effectively improve elasticity and strength of the material, meanwhile, the spider silk fibroin and chitosan can effectively promote wound healing, if the microsphere is further made into a dressing for treating a wound and the like, the wound can be quickly healed while a better dressing material is obtained, and the treatment period and pain of a patient are effectively shortened.
(3) The silk-spider silk composite silk fibroin nanospheres containing the chitosan modified graphene oxide have good biocompatibility, no obvious toxic or side effect and anaphylactic reaction, and the preparation materials are easy to obtain, simple to operate and low in cost.
(4) The silk-spider silk composite silk fibroin nanoparticle particles of the chitosan-modified graphene oxide are uniformly dispersed, stable and efficient, and the particle sizes are uniform.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
3g of washed spider silk was taken, and boiled in 500 mL of 1.0wt% sodium carbonate solution for 30 minutes, and washed with deionized water, and the above procedure was repeated once. And (2) placing the spider silk obtained in the step (a) into 150 mL of 70wt% acetic acid solution, heating and reacting for 6 hours at 60 ℃, centrifuging the mixed solution at 10000 r/min for 12 minutes after reaction, collecting supernatant to obtain spider silk protein mixed solution, and dialyzing the solution in deionized water for 30 hours to obtain a purer spider silk protein solution.
50g of silk is washed and then boiled in 1.5L of 0.05wt% sodium carbonate solution for 30 minutes, and after washing with deionized water, the above steps are repeated once. And dissolving the obtained silk in 1.5L of 8M lithium bromide solution to obtain silk fibroin mixed solution, and dialyzing the solution in deionized water for 60 hours to obtain pure silk fibroin solution.
And (3) uniformly mixing the obtained spidroin solution and fibroin solution according to the proportion of 1. And (2) dissolving the obtained silk-spider silk composite silk fibroin fibers in deionized water to enable the concentration of the silk-spider silk composite silk fibroin fibers to be 10wt%, placing the dissolved silk-spider silk composite silk fibroin fibers in a phosphate buffer solution with the pH =7.9 according to the proportion of 1.
After 8g/L of a graphene oxide solution, a 10% by mass chitosan solution, 98% by mass of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 98% by mass of N-hydroxysuccinimide were mixed in a ratio of 5. Obtaining the graphene oxide hydrogel modified by chitosan. After low-temperature storage, the graphene oxide particles modified by chitosan are obtained after freeze drying for 36 hours.
Dispersing the obtained silk-spider silk composite silk fibroin microspheres in a small amount of deionized water to form a suspension, adding 5g/L of chitosan modified graphene oxide solution according to a ratio of 1.
The silk fibroin used in the method is more, the spider silk fibroin is less, the mechanical property of the obtained composite nano-microsphere is slightly poor, the tensile strength is 1.6MPa, and the elongation rate reaches 121.68%. The dosage of the chitosan modified graphene oxide is slightly higher, and the chitosan modified graphene oxide respectively releases about 13.7%, 36.8%, 65.1% and 80.3% in an in vitro release test through the antibacterial agent loaded tetracycline at 1, 3, 7 and 12 days, and the cumulative release content at 24 days is about 88.6%. The composite nanometer particle is prepared into dressing to cover the burned rat epidermal wound, the wound closure rate reaches 79.1 percent after 14 days, and no obvious wound exists in the 4 th week.
Example 2
3g of washed spider silk was taken, boiled in 600 mL of 2wt% sodium carbonate solution for 30 minutes, washed with deionized water, and the above procedure was repeated once. And (2) putting the spider silk obtained in the step (a) into 200mL of 75wt% acetic acid solution, heating and reacting for 8 hours at 55 ℃, centrifuging the mixed solution at 10000 r/min for 12 minutes after reaction, collecting supernatant to obtain spider silk protein mixed solution, and dialyzing the solution in deionized water for 35 hours to obtain a purer spider silk protein solution.
20g of silk is taken, washed and then placed in 1.0L of 0.05wt% sodium carbonate solution to be boiled for 30 minutes, and after being washed by deionized water, the steps are repeated once. Dissolving the obtained silk in 1.5L 7M lithium bromide solution to obtain silk fibroin mixed solution, and dialyzing the solution in deionized water for 70 hours to obtain relatively pure silk fibroin solution.
And (3) uniformly mixing the obtained spider silk protein solution and the silk protein solution according to a ratio of 1. Dissolving the obtained silk-spider silk composite silk fibroin fiber in deionized water to enable the concentration of the silk-spider silk composite silk fibroin fiber to be 10wt%, placing the dissolved silk-spider silk composite silk fibroin fiber in a phosphate buffer solution with the pH =7.9 according to the proportion of 1.
After 3g/L of a graphene oxide solution, a 7% by mass chitosan solution, 96% by mass of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 98% by mass of N-hydroxysuccinimide were mixed in a ratio of 3. Obtaining the graphene oxide hydrogel modified by chitosan. After low-temperature storage, the graphene oxide particles modified by chitosan are obtained after freeze drying for 36 hours.
Dispersing the obtained silk-spider silk composite silk fibroin microspheres in a small amount of deionized water to form a suspension, adding 10g/L of graphene oxide solution modified by chitosan according to a proportion of 1.
The silk fibroin and spider silk fibroin used in the method have moderate proportion, and the obtained composite nano-microsphere has appropriate mechanical property, tensile strength of 1.9MPa and elongation of 125.82%. The dosage of the chitosan modified graphene oxide is low, the release in vitro through the release test of the loaded antibacterial agent tetracycline is respectively 16.3%, 39.4%, 68.9% and 83.7% in cumulative release at days 1, 3, 7 and 12, and the cumulative release content at day 24 is about 90.0%. The composite nanometer particle is prepared into dressing to cover the surface of the burned rat epidermis wound, the wound closure rate reaches 85.9 percent after 14 days, and no obvious wound exists in the 4 th week.
Example 3
3g of washed spider silk was taken, placed in 500 mL of 2.5wt% sodium carbonate solution, boiled for 30 minutes, and washed with deionized water, and the above procedure was repeated once. And (2) placing the spider silk obtained in the above step into 100 mL of 85wt% acetic acid solution, heating and reacting for 10 hours at 55 ℃, centrifuging the mixed solution for 15 minutes at 12000 r/min after reaction, collecting supernatant to obtain a spider silk protein mixed solution, and dialyzing the solution in deionized water for 36 hours to obtain a relatively pure spider silk protein solution.
20g of silk is taken, washed and then placed in 1.5L of 0.1wt% sodium carbonate solution to be boiled for 45 minutes, and after being washed by deionized water, the steps are repeated once. Dissolving the obtained silk in 1.0L of 9M lithium bromide solution to obtain silk fibroin mixed solution, and dialyzing the solution in deionized water for 65 hours to obtain relatively pure silk fibroin solution.
And (3) uniformly mixing the obtained spidroin solution and fibroin solution according to the proportion of 1. Dissolving the obtained silk-spider silk composite silk fibroin fiber in deionized water to ensure that the concentration of the silk-spider silk composite silk fibroin fiber is 12wt%, placing the dissolved silk-spider silk composite silk fibroin fiber in a phosphate buffer solution with the pH =7.9 according to the proportion of 1.
After mixing 5g/L of a graphene oxide solution, a 7% by mass chitosan solution, 95% by mass 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 95% by mass N-hydroxysuccinimide in a ratio of 5. Obtaining the graphene oxide hydrogel modified by chitosan. After low-temperature storage, the graphene oxide particles modified by chitosan are obtained by freeze drying for 24 hours.
Dispersing the obtained silk-spider silk composite silk fibroin microspheres in a small amount of deionized water to form a suspension, then adding 12g/L of chitosan modified graphene oxide solution according to the proportion of 1.
The method uses less silk fibroin and more spider silk fibroin, and the obtained composite nano-microsphere has strong mechanical property, tensile strength of 2.1MPa and elongation of 126.31%. The chitosan-modified graphene oxide was used in an appropriate amount, which cumulatively released about 14.3%, 38.9%, 61.6% and 79.1% on days 1, 3, 7 and 12, respectively, and accumulated at about 89.3% on day 24 in an in vitro release test with the loaded antimicrobial tetracycline. The composite nanometer particle is prepared into dressing to cover the surface of the burned rat epidermis wound, the wound closure rate reaches 82.7 percent after 14 days, and no obvious wound exists in the 4 th week.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (9)
1. A preparation method of a silk-spider silk composite silk fibroin nano microsphere containing chitosan modified graphene oxide is characterized by comprising the following steps:
step 1): boiling cleaned spider silk in sodium carbonate solution to remove gum, and cleaning with water to obtain degummed spider silk;
step 2): placing degummed spider silk in acetic acid solution for heating reaction, centrifuging the obtained mixed solution after reaction, collecting supernatant to obtain spider silk protein mixed solution, and dialyzing in water to obtain relatively pure spider silk protein solution;
step 3): taking silk, cleaning, placing in a sodium carbonate solution, boiling for degumming, and cleaning with water to obtain degummed silk;
step 4): dissolving degummed silk in a lithium bromide solution to obtain a silk fibroin mixed solution, and dialyzing in water to obtain a relatively pure silk fibroin solution;
step 5): uniformly mixing the spidroin solution obtained in the step 2) and the silk protein solution obtained in the step 4), storing at low temperature, and freeze-drying to obtain silk-spidroin composite silk fibroin;
step 6): putting the silk-spider silk composite silk fibroin obtained in the step 5) into water, dissolving the silk-spider silk composite silk fibroin, putting the dissolved silk-spider silk composite silk fibroin into a phosphate buffer solution, then adding a protease solution, heating the obtained mixed solution in water bath, heating to inactivate the protease, cooling to room temperature, standing, and freeze-drying to obtain silk-spider silk composite silk fibroin microspheres;
step 7): mixing a graphene oxide solution, a chitosan solution, a cross-linking agent and a catalyst, and then stirring for reaction to covalently bond graphene oxide and chitosan to obtain a graphene oxide hydrogel modified by chitosan; after low-temperature storage, freeze drying to obtain chitosan modified graphene oxide particles;
step 8): dispersing the chitosan modified graphene oxide particles and the silk-spider silk composite fibroin microspheres in water to form a suspension, performing ultrasonic treatment to uniformly mix the suspension, centrifuging the obtained mixed solution, taking down the precipitate of the lower layer, and performing freeze drying to obtain the silk-spider silk composite fibroin nanospheres containing the chitosan modified graphene oxide.
2. The method of claim 1, wherein: in the step 1), the concentration of sodium carbonate is 0.3-3.0wt%, and the dosage ratio of the spider silk to the sodium carbonate solution is 1g: (100-300) mL, and the boiling degumming time is 20-50 minutes.
3. The method of claim 1, wherein: in the step 2), the concentration of the acetic acid solution is 70-90wt%, and the dosage ratio of the spider silk to the acetic acid solution is 1g: (30-70) mL, the heating reaction temperature is 35-72 ℃, the reaction time is 4-12 hours, the centrifugal speed is 8500-14000 r/min, the centrifugal time is 10-20 minutes, and the dialysis time is 30-50 hours.
4. The method of claim 1, wherein: in the step 3), the concentration of sodium carbonate is 0.04-0.1wt%, and the dosage ratio of silk to sodium carbonate solution is 1g: (200-800) mL, and the boiling degumming time is 20-50 minutes.
5. The method of claim 1, wherein: in the step 4), the concentration of lithium bromide is 7-10M, and the dosage ratio of silk to lithium bromide solution is 1g: (20-150) mL, and the dialysis time is 45-85 hours.
6. The method of claim 1, wherein: in the step 5), the mass ratio of the spider silk protein solution to the fibroin solution is 1: (5-20), the low-temperature storage temperature is (-60) - (20 ℃), the storage time is 8-20 hours, and the freeze-drying time is 16-30 hours.
7. The method of claim 1, wherein: in the step 6), the concentration of the silk-spider silk composite silk fibroin solution is 10-15wt%, the pH of the phosphate buffer solution is =7.5-8.0, and the mixing volume ratio of the phosphate buffer solution to the silk-spider silk composite silk fibroin solution is 1: (2-5), the mixing volume ratio of the protease solution to the silk-spider silk composite silk fibroin solution is 1: (30-80), heating in water bath at 40-70 deg.C for 2-5 hours, heating to 105 deg.C for 5-15 minutes, cooling to room temperature, standing for 9-20 hours, and freeze drying for 16-30 hours.
8. The method of claim 1, wherein: in the step 7), the concentration of the graphene oxide solution is 2-9g/L, the mass concentration of the chitosan solution is 1-10%, the cross-linking agent is a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the mass concentration of 90-98%, the catalyst is an N-hydroxysuccinimide solution with the mass concentration of 95-100%, and the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and chitosan in the mixed solution is 1: (3-8); stirring the mixed solution for reaction for 12-24 hours; the freeze-drying time is 24-48 hours.
9. The method of claim 1, wherein: in the step 8), the concentration of the chitosan modified graphene oxide suspension is 1-20g/L, and the mixing volume ratio of the chitosan modified graphene oxide suspension to the silk-spider silk composite silk fibroin suspension is 1: (10-80), the ultrasonic time is 20-50 minutes, the centrifugal speed is 8000-15000 r/min, the centrifugal time is 20-40 minutes, and the freeze-drying time is 24-48 hours.
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