阅读说明：本技术 一种具有抗炎活性的仿生复合纤维薄膜的制备方法 (Preparation method of bionic composite fiber film with anti-inflammatory activity ) 是由 王秉 邓明 金小康 彭志勤 万军民 于 2021-04-25 设计创作，主要内容包括：本发明涉及生物医用材料领域,本发明公开了一种具有抗炎活性的仿生复合纤维薄膜的制备方法,本发明以多酶级联途径制备出角蛋白,从而提高角蛋白的分子量；又以高分子角蛋白和丝素蛋白为核壳原材料,通过共轴静电纺丝制备出的复合纤维薄膜具有高度生物相容性和优异的力学性能；然后通过交联作用引入亚精胺,赋予了材料医用药理性能。使得这种仿生复合纤维薄膜具有更优异的机械性能和更好的生物医用性能。(The invention relates to the field of biomedical materials, and discloses a preparation method of a bionic composite fiber film with anti-inflammatory activity, which prepares keratin by a multienzyme cascade approach so as to improve the molecular weight of the keratin; the composite fiber film prepared by using high-molecular keratin and silk fibroin as core-shell raw materials through coaxial electrostatic spinning has high biocompatibility and excellent mechanical property; then spermidine is introduced through the crosslinking action, and the medical pharmacological performance of the material is endowed. So that the bionic composite fiber film has more excellent mechanical property and better biomedical property.)

1. A preparation method of a bionic composite fiber film with anti-inflammatory activity is characterized by comprising the following steps:

step 1: preparation of high-molecular wool keratin: soaking wool in acetone to extract to remove surface lipid, washing with water, and drying; shearing broken wool, immersing in water containing keratinase, stirring vigorously at 45-55 deg.C for 20-30 hr, and dissolving to obtain hydrolyzed keratin solution; adding transglutaminase into hydrolyzed keratin solution, culturing at 45-55 deg.C for 40-50h, centrifuging, and dialyzing; finally, freezing and drying the keratin aqueous solution to obtain high-molecular wool keratin powder;

step 2: preparation of coaxial spinning solution: adding polyvinyl alcohol into water, heating in a water bath to dissolve the polyvinyl alcohol to obtain a polyvinyl alcohol solution, and cooling to room temperature; dissolving silk fibroin and high-molecular wool keratin powder in polyvinyl alcohol solution respectively, adding acetic acid, heating and stirring in water bath until all the silk fibroin and the high-molecular wool keratin powder are dissolved, and ultrasonically standing to remove bubbles; respectively obtaining polyvinyl alcohol/silk fibroin spinning solution and polyvinyl alcohol/keratin spinning solution;

and step 3: preparation of coaxial spinning solution: adding titanium dioxide into an inner core spinning solution which takes a polyvinyl alcohol/silk fibroin spinning solution as a coaxial electrostatic spinning solution to obtain a coaxial spinning solution;

and 4, step 4: coaxial spinning: setting the front stop point of the push injection A by using an empty injector, and stopping the slide block at the joint of the slide block and the tail end of the injector; in the same way, the same empty syringe is used for setting the front stop point of the bolus injection B; after the front dead center is set, respectively sucking the spinning solution by using two injectors, wherein the polyvinyl alcohol/keratin spinning solution is used as a core layer, the spinning solution obtained in the step (3) is used as a shell layer, the injector used as the core layer is placed into an injection A track, and the injector used as the shell layer is placed into an injection B track; mounting a coaxial needle head assembly, and mounting a copper wire to supply coaxial spinning voltage; adhering a piece of smooth and wrinkle-free aluminum foil paper to a receiver roller for receiving the electrostatic spinning fibers; the sliding block of the fast forward injection B enables liquid drops to drip out of the needle head, and then the sliding block of the fast forward injection A enables the liquid drops to drip out of the needle head; after confirming the front dead point, clamping the positive high-pressure clamp, and adjusting the receiving distance to ensure that the distance between the metal needle and the receiving roller is 13-17 cm; setting the temperature to be 25-35 ℃ and the relative humidity to be 25-35% until the temperature and the humidity are stable; clicking a linkage button, entering a linkage system window, setting the flow of the push injection B, starting the button, and overflowing the solution at the needle head under the pushing action of the injection pump; starting a voltage button, and adjusting the negative voltage to keep the voltage value at 2.8-3.2 kV; then, adjusting the positive voltage until stable spinning solution jet flow is sprayed out, and then setting and starting a push injection button A in the linkage system to control the flow; under the action of high voltage, the spinning solution is sprayed out from a metal needle of the injector and is deposited on the receiving roller; after the spinning is finished, slowly reducing the positive high pressure parameter, closing the positive high pressure and the negative high pressure, and stopping the injection of the injection pump to finish the spinning;

and 5: and (3) crosslinking: blending terephthalaldehyde and spermidine, dissolving in ethanol to obtain a spermidine/terephthalaldehyde crosslinking agent, soaking a cover glass with a composite fiber film in the crosslinking agent, taking out the crosslinked composite fiber film cover glass, and drying to obtain the bionic composite fiber film with anti-inflammatory activity.

2. The method of claim 1, wherein: in the step 1, the extraction temperature is 70-75 ℃; the drying condition is 60-70 deg.C, 20-30 h.

3. The method of claim 1, wherein: in the step 1, the consumption of the broken wool is 5-8 g; the amount of keratinase is 90-110 kU; the amount of water containing keratinase is 80-120mL, and the amount of transglutaminase is 20-40U/g keratin.

4. The method of claim 1, wherein: in the step 1, the centrifugation time is 10-20 min; the dialysis frequency is 6000-10000r/min, the dialysis time is 2-4d, and the extracorporeal water is replaced 3-5 times per day.

5. The method of claim 1, wherein: in the step 2, the dosage of the polyvinyl alcohol is 8-12g, and the dosage of the water is 88-92 g; the first water bath heating temperature is 55-65 ℃, the rotating speed is 600-; the dosages of the silk fibroin and the keratin are respectively 0.6-1.0g and 0.1-0.3g, the volume fraction of the added acetic acid is 1.5-2.5%, the heating temperature of the second water bath is 55-65 ℃, the rotating speed is 600-; the ultrasonic time is 10-15 h.

6. The method of claim 1, wherein: in the step 3, the dosage of the titanium dioxide is 0.08-0.12g/10 mL.

7. The method of claim 1, wherein: in step 4, the flow rate of the bolus injection B is set to be 0.7-0.9mm/min, the flow rate of the bolus injection A is set to be 0.5-0.7mm/min, and the positive voltage is set to be 23-27 kV.

8. The method of claim 1, wherein: in step 4, the specification of the needle head is 25-18G, the inner diameter is 0.2-0.3mm, and the outer diameter is 0.90-0.92 mm.

9. The method of claim 1, wherein: in the step 5, the dosage of the terephthalaldehyde is 0.100-0.105g, 95-105 mu L of spermidine and 18-022mL of ethanol; soaking the cover glass for 5-15s, and drying at 55-65 deg.C.

Technical Field

The invention relates to the field of biomedical materials, in particular to a preparation method of a bionic composite fiber film with anti-inflammatory activity.

Background

In recent years, silkworm Silk Fibroin (SF) has been widely used in the biomedical field due to its excellent mechanical properties, biodegradability, hemostatic properties, non-cytotoxicity, low antigenicity and non-inflammatory properties. Furthermore, SF also exhibits good compatibility with a wide variety of cells and tissues, and is considered to be a biomaterial with potential for the manufacture of wound dressings of various formulations, as SF promotes the adhesion and proliferation of a variety of cells, including keratinocytes and fibroblasts.

The biological characteristics of keratin are similar to those of silk fibroin, and the keratin has the properties of biocompatibility, biodegradability and the like, is widely applied to biomaterials, but the keratin prepared by a general physical or chemical method has relatively low molecular weight and poor mechanical properties.

Spermidine (SPD) of formula H₂N(CH₂)₃NH(CH₂)₄NH₂Is a natural polyamine small molecular compound. Spermidine is widely distributed in the organism and is biosynthesized from putrescine (butanediamine) and adenosylmethionine. Spermidine has effects of resisting oxidation, inhibiting inflammation, and inhibiting cell necrosis. Naturally occurring polyamines, such as spermidine and putrescine thereof, are believed to play important controlling roles in cells, from basic DNA synthesis to regulation of cell proliferation and differentiation. Chemically, polyamines are positively charged cationic molecules that can electrostatically interact with polyanionic macromolecules in living cells.

Disclosure of Invention

The invention provides a preparation method of a bionic composite fiber film with anti-inflammatory activity, which prepares high molecular weight keratin by a multienzyme cascade approach, takes the high molecular weight keratin and silk fibroin as core-shell raw materials, and the composite fiber film prepared by coaxial electrostatic spinning has high biocompatibility and excellent mechanical property; then spermidine is introduced through the crosslinking action, and the medical pharmacological property of the material is endowed, so that the bionic composite fiber film has more excellent mechanical property and better biomedical property.

The specific technical scheme of the invention is as follows: a preparation method of a bionic composite fiber film with anti-inflammatory activity comprises the following steps:

step 1: preparation of high-molecular wool keratin: soaking wool in acetone to extract to remove surface lipid, washing with water, and drying; shearing broken wool, immersing in water containing keratinase, stirring vigorously at 45-55 deg.C for 20-30 hr, and dissolving to obtain hydrolyzed keratin solution; adding transglutaminase into hydrolyzed keratin solution, culturing at 45-55 deg.C for 40-50h, centrifuging, and dialyzing; and finally, freezing and drying the obtained keratin aqueous solution to obtain the high-molecular wool keratin powder.

Keratinase is more effective in hydrolyzing insoluble keratin than pepsin, trypsin and other common proteases. Compared with physical or chemical methods, the keratin hydrolysis by the keratinase is an environment-friendly process, can keep the functional characteristics of the keratin under mild treatment conditions, and has the advantages of low energy consumption, little damage to the protein backbone of the product and the like. The enzyme cross-linking method is low in toxicity and high in efficiency, and the transglutaminase is a cross-linked substance capable of introducing covalent cross-linking between the peptide fragment and the protein, so that the stability of the protein is enhanced. In addition, the keratin prepared by the method has higher molecular weight and good mechanical property.

Step 2: preparation of coaxial spinning solution: adding polyvinyl alcohol into water, heating in a water bath to dissolve the polyvinyl alcohol to obtain a polyvinyl alcohol solution, and cooling to room temperature; dissolving silk fibroin and high-molecular wool keratin powder in polyvinyl alcohol solution respectively, adding acetic acid, heating and stirring in water bath until all the silk fibroin and the high-molecular wool keratin powder are dissolved, and ultrasonically standing to remove bubbles; respectively obtaining the polyvinyl alcohol/silk fibroin spinning solution and the polyvinyl alcohol/keratin spinning solution.

The optimal mixing ratio of the spinning solution is adopted to carry out spinning experiments, and the fiber with the optimal performance can be generated.

And step 3: preparation of coaxial spinning solution: adding titanium dioxide into an inner core spinning solution which takes polyvinyl alcohol/silk fibroin spinning solution as coaxial electrostatic spinning solution to obtain coaxial spinning solution.

The nanofiber with the core-shell structure and better performance and structure is prepared by adopting a coaxial spinning method. As the coaxial spinning needs the double-track connection injector to act simultaneously, the concentrations of the spinning solution required by the inner core layer and the outer shell layer, experimental parameters and the like are different, the components and the proportion of the spinning solution of the inner core layer and the outer shell layer are determined at first, and the flow rate of the two tracks, the related voltage parameters and the like are set. In order to be better applied to the research on the aspects of cell compatibility and functionality, silk fibroin is selected as a raw material of an outer shell layer during coaxial spinning, so that the silk fibroin has better biocompatibility; the inner core layer is selected from keratin, so that the inner core layer can better support the spatial structure of the fiber, and the spinning forming of the fiber is facilitated.

And 4, step 4: coaxial spinning: setting the front stop point of the push injection A by using an empty injector, and stopping the slide block at the joint of the slide block and the tail end of the injector; in the same way, the same empty syringe is used for setting the front stop point of the bolus injection B; after the front dead center is set, respectively sucking the spinning solution by using two injectors, wherein the polyvinyl alcohol/keratin spinning solution is used as a core layer, the spinning solution obtained in the step (3) is used as a shell layer, the injector used as the core layer is placed into an injection A track, and the injector used as the shell layer is placed into an injection B track; mounting a coaxial needle head assembly, and mounting a copper wire to supply coaxial spinning voltage; adhering a piece of smooth and wrinkle-free aluminum foil paper to a receiver roller for receiving the electrostatic spinning fibers; the sliding block of the fast forward injection B enables liquid drops to drip out of the needle head, and then the sliding block of the fast forward injection A enables the liquid drops to drip out of the needle head; after confirming the front dead point, clamping the positive high-pressure clamp, and adjusting the receiving distance to ensure that the distance between the metal needle and the receiving roller is 13-17 cm; setting the temperature to be 25-35 ℃ and the relative humidity to be 25-35% until the temperature and the humidity are stable; clicking a linkage button, entering a linkage system window, setting the flow of the push injection B, starting the button, and overflowing the solution at the needle head under the pushing action of the injection pump; starting a voltage button, and adjusting the negative voltage to keep the voltage value at 2.8-3.2 kV; then, adjusting the positive voltage until stable spinning solution jet flow is sprayed out, and then setting and starting a push injection button A in the linkage system to control the flow; under the action of high voltage, the spinning solution is sprayed out from a metal needle of the injector and is deposited on the receiving roller; and after the spinning is finished, slowly reducing the positive high-pressure parameter, closing the positive high pressure and the negative high pressure, and stopping injecting by the injection pump to finish the spinning.

In the aspect of selecting the spinning solution in the injector, the silk fibroin and the keratin with the optimal concentration ratio are coaxially spun, so that the best spinning can be performed. And obtaining the composite fiber film with the best performance by adopting the best spinning conditions.

And 5: and (3) crosslinking: blending terephthalaldehyde and spermidine, dissolving in ethanol to obtain a spermidine/terephthalaldehyde crosslinking agent, soaking a cover glass with a composite fiber film in the crosslinking agent, taking out the crosslinked composite fiber film cover glass, and drying to obtain the bionic composite fiber film with anti-inflammatory activity.

The chemical structure and unique biological activity of the diamino of the spermidine are comprehensively considered, and the spermidine is selected as a raw material to prepare the cross-linking agent. The application of the spermidine crosslinking agent can quickly realize the crosslinking of the film through an alkali reaction to form a stable fiber structure, and can also introduce spermidine into the fiber film to endow the material with anti-inflammatory activity.

In conclusion, the keratin is prepared by a multienzyme cascade way, so that the molecular weight of the keratin is improved; the composite fiber film prepared by using high-molecular keratin and silk fibroin as core-shell raw materials through coaxial electrostatic spinning has high biocompatibility and excellent mechanical property; then spermidine is introduced through the crosslinking action, and the medical pharmacological performance of the material is endowed. So that the bionic fiber film has more excellent mechanical property and better biomedical property.

Preferably, in the step 1, the extraction temperature is 70-75 ℃; the drying condition is 60-70 deg.C, 20-30 h.

Preferably, in the step 1, the amount of the broken wool is 5-8 g; the amount of keratinase is 90-110 kU; the amount of water containing keratinase is 80-120mL, and the amount of transglutaminase is 20-40U/g keratin.

Preferably, in the step 1, the centrifugation time is 10-20 min; the dialysis frequency is 6000-10000r/min, the dialysis time is 2-4d, and the extracorporeal water is replaced 3-5 times per day.

Preferably, in the step 2, the dosage of the polyvinyl alcohol is 8-12g, and the dosage of the water is 88-92 g; the first water bath heating temperature is 55-65 ℃, the rotating speed is 600-; the dosages of the silk fibroin and the keratin are respectively 0.6-1.0g and 0.1-0.3g, the volume fraction of the added acetic acid is 1.5-2.5%, the heating temperature of the second water bath is 55-65 ℃, the rotating speed is 600-; the ultrasonic time is 10-15 h.

Preferably, in step 3, the amount of titanium dioxide used is 0.08 to 0.12g/10 mL.

Preferably, in step 4, the flow rate of bolus B is set to 0.7-0.9mm/min, the flow rate of bolus A is set to 0.5-0.7mm/min, and the positive voltage is set to 23-27 kV.

Preferably, in step 4, the needle has a gauge of 25-18G, an inner diameter of 0.2-0.3mm and an outer diameter of 0.90-0.92 mm.

Preferably, in the step 5, the dosage of the terephthalaldehyde is 0.100-0.105g, 95-105 μ L of spermidine and 18-022mL of ethanol; soaking the cover glass for 5-15s, and drying at 55-65 deg.C.

Compared with the prior art, the invention has the beneficial effects that: the invention uses two natural protein macromolecules common in the nature to prepare the biomedical materials, reduces the expensive cost of medical raw materials, and enables common people to enjoy cheap and high-quality medical materials; meanwhile, a bionic biomaterial for artificial skin is designed by applying a coupling bionic idea, and a bionic active film is prepared by using a coaxial electrostatic spinning technology and simulating the components and the structure of human skin, and has high biocompatibility and excellent mechanical property; and then, the biomolecular spermidine is utilized to prepare the cross-linking agent, so that the bionic composite fiber film which has a stable structure, anti-inflammatory and other pharmacological functions and anti-inflammatory activity is obtained, and the excellent performance of the film can be used as a skin implant material to enable chronic wounds and wounds which are difficult to heal quickly, and can be used as a wound dressing to enable the wound surface to heal quickly and simultaneously have an anti-inflammatory effect, so that the wound is prevented from being infected in the healing process.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

1) Preparation of high molecular wool keratin

The surface lipids of the wool were extracted in acetone for 24h at 72 ℃, then rinsed with high purity water and oven dried for 24h at 65 ℃. Cut 5g of the broken wool was immersed in 100 ml of water containing 90kU of keratinase. Dissolved after 24 hours of vigorous stirring at 50 ℃. Transglutaminase was added to the hydrolyzed keratin solution at a dose of 20U/g keratin and incubated at 50 ℃ for 48 hours. Centrifuging for 10min, dialyzing at 8000 rpm for 3d, and replacing extracorporeal water 4 times per day. The aqueous keratin solution thus obtained is finally dried in a freeze-dryer, collecting approximately 4g of keratin powder. Potassium and appropriate amount of keratin powder were mixed with 5 times of polyacrylamide gel electrophoresis buffer and boiled for 2 minutes. The loaded sample was then subjected to electrophoresis under standard procedures to give a keratin average molecular weight of approximately 115 kDa.

2) Preparation of coaxial spinning solutions

Weighing 10g of polyvinyl alcohol, dissolving the polyvinyl alcohol in 90g of purified water, placing a beaker filled with the polyvinyl alcohol solution in a crystallizing dish on a heating stirrer, heating the beaker in a water bath at 60 ℃, rotating at 800r/min, and heating and stirring for 2 hours. The PVA solution was prepared by dissolving the whole of the above components, and was cooled to room temperature. Weighing 0.6g of silk fibroin and 0.1g of keratin, respectively dissolving the silk fibroin and the keratin in a prepared polyvinyl alcohol solution, adding 2% by volume of acetic acid, heating and stirring in a water bath at the temperature of 60 ℃ and at the rotating speed of 800r/min, heating and stirring for 0.3h until the silk fibroin and the keratin are completely dissolved, and preparing 10ml of polyvinyl alcohol/silk fibroin spinning solution and 0.1% polyvinyl alcohol/keratin spinning solution with the mass fractions of about 0.6% respectively. Finally, carrying out ultrasonic treatment for 12h, standing, and removing bubbles in the spinning solution.

3) Preparation of coaxial spinning solution characterized by core-shell structure

After the proportion in the step 2 is obtained, 0.1g of titanium dioxide is added into the core spinning solution (keratin/polyvinyl alcohol spinning solution) of the original coaxial electrostatic spinning solution to prepare the coaxial spinning solution for core-shell structure representation.

4) Preparation of silk fibroin-based nanofiber and high-molecular keratin-based nanofiber co-spun film

Setting a front stop point of the bolus injection A by using an empty 2.5mL injector, and stopping the slide block at a joint of the slide block and the tail end of the injector; similarly, the same empty 2.5mL syringe was used to set the front dead center for bolus B. After the front dead center is set, two injectors of 2.5mL are used for respectively sucking proper spinning solutions, wherein the spinning solution containing polyvinyl alcohol/keratin is used as a core layer, the spinning solution containing polyvinyl alcohol/silk fibroin is used as a shell layer, the injector used as the core layer is placed in a push injection A track, and the injector used as the shell layer is placed in a push injection B track. Mounting a coaxial needle assembly, wherein the specification of the needle is 25G-18G, the inner diameter is about 0.25mm, and the outer diameter is about 0.91 mm; the copper wire is arranged to facilitate the coaxial spinning voltage. A piece of smooth, wrinkle-free aluminum foil paper was glued onto a receiver roller for receiving electrospun fibers. The fast forward injection B sliding block enables liquid drops to drip out of the needle head, and then the fast forward injection A sliding block enables the liquid drops to drip out of the needle head. And clamping the positive high-pressure clamp after confirming the front dead center. The receiving distance is adjusted to make the distance between the metal needle and the receiving roller be 15 cm. The temperature is set to be 30 ℃ and the relative humidity is set to be 30% until the temperature and the humidity are stable. And clicking a linkage button to enter a linkage system window, setting the flow of the bolus injection B to be 0.8mm/min, starting the button, and overflowing the solution at the needle head under the pushing action of the injection pump. Starting a voltage button, and adjusting negative voltage to keep the voltage value at about 3 kV; and then adjusting the positive voltage to 25kV until stable spinning solution jet flow is sprayed out, and then setting and starting a push injection button A in the linkage system to control the flow to be 0.6 mm/min. Under the action of high voltage, the spinning solution is sprayed out from a metal needle of an injector and is deposited on a receiving roller. After the experiment is finished, slowly reducing the parameters of the positive high pressure, closing the positive high pressure and the negative high pressure, stopping injecting by the injection pump, and preparing the coaxial spinning film.

5) Preparation of spermidine-introduced silk fibroin-based nanofiber and high-molecular keratin-based nanofiber bionic film

Weighing 0.103g of terephthalaldehyde on a precise electronic balance, blending the terephthalaldehyde with 100 mu L of spermidine, dissolving the mixture in 20mL of ethanol to prepare a spermidine/terephthalaldehyde crosslinking agent, soaking a cover glass with a composite fiber film in the crosslinking agent for 10s, taking out the crosslinked composite fiber film cover glass, drying the crosslinked composite fiber film cover glass in a 60 ℃ constant-temperature blast drying oven, and preparing the spermidine-introduced silk fibroin-based nanofiber and high-molecular keratin-based nanofiber bionic film.

The fiber film prepared by the embodiment randomly selects 80 fibers to carry out diameter measurement according to the conversion of a corresponding scale, so that the diameter length of the fibers is between 200nm and 350nm, and the diameter length of the fibers accounts for about 85 percent of the total fibers; there are of course also fibers of larger and smaller diameter, between 650nm-700nm and 50nm-100nm, respectively, accounting for the remaining 15%; the mechanical properties of the fiber films were measured using an electronic universal tester, and the fiber films were cut into rectangles (2.0 cm. times.4.0 cm) before the tensile test. During the test, the crosshead speed was 2mm/min and the ambient temperature was 25 ℃. The fiber film was tested three times to obtain a Young's modulus of 35.4 MPa and a tensile strength of 2.8 MPa. When the film is applied to a mouse test, the concentration of the interleukin which induces cells to secrete inflammatory factors is lower and is 10 pg/mL.

Example 2

1) Preparation of high molecular wool keratin

The surface lipids of the wool were extracted in acetone for 24h at 72 ℃, then rinsed with high purity water and oven dried for 24h at 65 ℃. Cut 6g of the broken wool was immersed in 100 ml of water containing 100kU of keratinase. Dissolved after 24 hours of vigorous stirring at 50 ℃. Transglutaminase was added to the hydrolyzed keratin solution at a dose of 30U/g keratin and incubated at 50 ℃ for 48 hours. Centrifuging for 15min, dialyzing at 8000 rpm for 3d, and replacing extracorporeal water 4 times per day. The aqueous keratin solution thus obtained is finally dried in a freeze-dryer, collecting approximately 5g of keratin powder. Potassium and appropriate amount of keratin powder were mixed with 5 times of polyacrylamide gel electrophoresis buffer and boiled for 2 minutes. The loaded sample was then subjected to electrophoresis under standard procedures to give a keratin average molecular weight of approximately 120 kDa.

2) Preparation of coaxial spinning solutions

Weighing 10g of polyvinyl alcohol, dissolving the polyvinyl alcohol in 90g of purified water, placing a beaker filled with the polyvinyl alcohol solution in a crystallizing dish on a heating stirrer, heating the beaker in a water bath at 60 ℃, rotating at 800r/min, and heating and stirring for 2 hours. The PVA solution was prepared by dissolving the whole of the above components, and was cooled to room temperature. Weighing 0.8g of silk fibroin and 0.2g of keratin, respectively dissolving the silk fibroin and the keratin in a prepared polyvinyl alcohol solution, adding 2% by volume of acetic acid, heating and stirring in a water bath at the temperature of 60 ℃ and at the rotating speed of 800r/min, heating and stirring for 0.5h until the silk fibroin and the keratin are completely dissolved, and preparing 10ml of polyvinyl alcohol/silk fibroin spinning solution and 0.2% polyvinyl alcohol/keratin spinning solution with the mass fractions of about 0.8%, wherein the volume fractions of the polyvinyl alcohol/silk fibroin spinning solution and the 0.2% polyvinyl alcohol/keratin spinning solution are respectively taken. Finally, carrying out ultrasonic treatment for 12h, standing, and removing bubbles in the spinning solution.

3) Preparation of coaxial spinning solution characterized by core-shell structure

After the proportion in the step 2 is obtained, 0.1g of titanium dioxide is added into the core spinning solution (keratin/polyvinyl alcohol spinning solution) of the original coaxial electrostatic spinning solution to prepare the coaxial spinning solution for core-shell structure representation.

4) Preparation of silk fibroin-based nanofiber and high-molecular keratin-based nanofiber co-spun film

Setting a front stop point of the bolus injection A by using an empty 2.5mL injector, and stopping the slide block at a joint of the slide block and the tail end of the injector; similarly, the same empty 2.5mL syringe was used to set the front dead center for bolus B. After the front dead center is set, two injectors of 2.5mL are used for respectively sucking proper spinning solutions, wherein the spinning solution containing polyvinyl alcohol/keratin is used as a core layer, the spinning solution containing polyvinyl alcohol/silk fibroin is used as a shell layer, the injector used as the core layer is placed in a push injection A track, and the injector used as the shell layer is placed in a push injection B track. Mounting a coaxial needle assembly, wherein the specification of the needle is 25G-18G, the inner diameter is about 0.25mm, and the outer diameter is about 0.91 mm; the copper wire is arranged to facilitate the coaxial spinning voltage. A piece of smooth, wrinkle-free aluminum foil paper was glued onto a receiver roller for receiving electrospun fibers. The fast forward injection B sliding block enables liquid drops to drip out of the needle head, and then the fast forward injection A sliding block enables the liquid drops to drip out of the needle head. And clamping the positive high-pressure clamp after confirming the front dead center. The receiving distance is adjusted to make the distance between the metal needle and the receiving roller be 15 cm. The temperature is set to be 30 ℃ and the relative humidity is set to be 30% until the temperature and the humidity are stable. And clicking a linkage button to enter a linkage system window, setting the flow of the bolus injection B to be 0.8mm/min, starting the button, and overflowing the solution at the needle head under the pushing action of the injection pump. Starting a voltage button, and adjusting negative voltage to keep the voltage value at about 3 kV; and then adjusting the positive voltage to 25kV until stable spinning solution jet flow is sprayed out, and then setting and starting a push injection button A in the linkage system to control the flow to be 0.6 mm/min. Under the action of high voltage, the spinning solution is sprayed out from a metal needle of an injector and is deposited on a receiving roller. After the experiment is finished, slowly reducing the parameters of the positive high pressure, closing the positive high pressure and the negative high pressure, stopping injecting by the injection pump, and preparing the coaxial spinning film.

5) Preparation of spermidine-introduced silk fibroin-based nanofiber and high-molecular keratin-based nanofiber bionic film

Weighing 0.103g of terephthalaldehyde on a precise electronic balance, blending the terephthalaldehyde with 100 mu L of spermidine, dissolving the mixture in 20mL of ethanol to prepare a spermidine/terephthalaldehyde crosslinking agent, soaking a cover glass with a composite fiber film in the crosslinking agent for 10s, taking out the crosslinked composite fiber film cover glass, drying the crosslinked composite fiber film cover glass in a 60 ℃ constant-temperature blast drying oven, and preparing the spermidine-introduced silk fibroin-based nanofiber and high-molecular keratin-based nanofiber bionic film.

The fiber film prepared by the embodiment randomly selects 80 fibers to measure the diameter according to the conversion of a corresponding scale, so that the diameter length of the fibers is between 210nm and 320nm, and the diameter length of the fibers accounts for about 81 percent of the total fibers; there are of course also fibers of larger and smaller diameter, between 670nm-710nm and 40nm-100nm, respectively, accounting for the remaining 19%; the mechanical properties of the fiber films were measured using an electronic universal tester, and the fiber films were cut into rectangles (2.0 cm. times.4.0 cm) before the tensile test. During the test, the crosshead speed was 2mm/min and the ambient temperature was 25 ℃. The fiber film was tested three times to obtain a Young's modulus of 42.9 MPa and a tensile strength of 3.2 MPa. When the film is applied to a mouse test, the concentration of the interleukin which induces cells to secrete inflammatory factors is very low and is 7 pg/mL.

Example 3

1) Preparation of high molecular wool keratin

The surface lipids of the wool were extracted in acetone for 24h at 72 ℃, then rinsed with high purity water and oven dried for 24h at 65 ℃. Cut pieces of 8g were immersed in 100 ml of water containing 110kU of keratinase. Dissolved after 24 hours of vigorous stirring at 50 ℃. Transglutaminase was added to the hydrolyzed keratin solution at a dose of 40U/g keratin and incubated at 50 ℃ for 48 hours. Centrifuging for 20min, dialyzing at 8000 rpm for 3d, and replacing extracorporeal water 4 times per day. The aqueous keratin solution thus obtained is finally dried in a freeze-dryer, collecting approximately 6g of keratin powder. Potassium and appropriate amount of keratin powder were mixed with 5 times of polyacrylamide gel electrophoresis buffer and boiled for 2 minutes. The loaded sample was then subjected to electrophoresis under standard procedures to give a keratin average molecular weight of approximately 118 kDa.

2) Preparation of coaxial spinning solutions

Weighing 10g of polyvinyl alcohol, dissolving the polyvinyl alcohol in 90g of purified water, placing a beaker filled with the polyvinyl alcohol solution in a crystallizing dish on a heating stirrer, heating the beaker in a water bath at 60 ℃, rotating at 800r/min, and heating and stirring for 2 hours. The PVA solution was prepared by dissolving the whole of the above components, and was cooled to room temperature. Weighing 1.0g of silk fibroin and 0.3g of keratin, respectively dissolving the silk fibroin and the keratin in a prepared polyvinyl alcohol solution, adding 2% by volume of acetic acid, heating and stirring in a water bath at the temperature of 60 ℃ and at the rotating speed of 800r/min, heating and stirring for 0.6h until the silk fibroin and the keratin are completely dissolved, and preparing 10ml of polyvinyl alcohol/silk fibroin spinning solution and 0.3% polyvinyl alcohol/keratin spinning solution with the mass fractions of about 1.0%, wherein the volume fractions of the polyvinyl alcohol/silk fibroin spinning solution and the 0.3% polyvinyl alcohol/keratin spinning solution are respectively taken. Finally, carrying out ultrasonic treatment for 12h, standing, and removing bubbles in the spinning solution.

3) Preparation of coaxial spinning solution characterized by core-shell structure

After the proportion in the step 2 is obtained, 0.1g of titanium dioxide is added into the core spinning solution (keratin/polyvinyl alcohol spinning solution) of the original coaxial electrostatic spinning solution to prepare the coaxial spinning solution for core-shell structure representation.

4) Preparation of silk fibroin-based nanofiber and high-molecular keratin-based nanofiber co-spun film

Setting a front stop point of the bolus injection A by using an empty 2.5mL injector, and stopping the slide block at a joint of the slide block and the tail end of the injector; similarly, the same empty 2.5mL syringe was used to set the front dead center for bolus B. After the front dead center is set, two injectors of 2.5mL are used for respectively sucking proper spinning solutions, wherein the spinning solution containing polyvinyl alcohol/keratin is used as a core layer, the spinning solution containing polyvinyl alcohol/silk fibroin is used as a shell layer, the injector used as the core layer is placed in a push injection A track, and the injector used as the shell layer is placed in a push injection B track. Mounting a coaxial needle assembly, wherein the specification of the needle is 25G-18G, the inner diameter is about 0.25mm, and the outer diameter is about 0.91 mm; the copper wire is arranged to facilitate the coaxial spinning voltage. A piece of smooth, wrinkle-free aluminum foil paper was glued onto a receiver roller for receiving electrospun fibers. The fast forward injection B sliding block enables liquid drops to drip out of the needle head, and then the fast forward injection A sliding block enables the liquid drops to drip out of the needle head. And clamping the positive high-pressure clamp after confirming the front dead center. The receiving distance is adjusted to make the distance between the metal needle and the receiving roller be 15 cm. The temperature is set to be 30 ℃ and the relative humidity is set to be 30% until the temperature and the humidity are stable. And clicking a linkage button to enter a linkage system window, setting the flow of the bolus injection B to be 0.8mm/min, starting the button, and overflowing the solution at the needle head under the pushing action of the injection pump. Starting a voltage button, and adjusting negative voltage to keep the voltage value at about 3 kV; and then adjusting the positive voltage to 25kV until stable spinning solution jet flow is sprayed out, and then setting and starting a push injection button A in the linkage system to control the flow to be 0.6 mm/min. Under the action of high voltage, the spinning solution is sprayed out from a metal needle of an injector and is deposited on a receiving roller. After the experiment is finished, slowly reducing the parameters of the positive high pressure, closing the positive high pressure and the negative high pressure, stopping injecting by the injection pump, and preparing the coaxial spinning film.

5) Preparation of spermidine-introduced silk fibroin-based nanofiber and high-molecular keratin-based nanofiber bionic film

Weighing 0.103g of terephthalaldehyde on a precise electronic balance, blending the terephthalaldehyde with 100 mu L of spermidine, dissolving the mixture in 20mL of ethanol to prepare a spermidine/terephthalaldehyde crosslinking agent, soaking a cover glass with a composite fiber film in the crosslinking agent for 10s, taking out the crosslinked composite fiber film cover glass, drying the crosslinked composite fiber film cover glass in a 60 ℃ constant-temperature blast drying oven, and preparing the spermidine-introduced silk fibroin-based nanofiber and high-molecular keratin-based nanofiber bionic film.

The fiber film prepared by the embodiment randomly selects 80 fibers to carry out diameter measurement according to the conversion of a corresponding scale, and the diameter length of the fibers is between 190nm and 330nm and is about 87 percent of the total fibers; there are of course also fibers of larger and smaller diameter, between 690nm-720nm and 35nm-110nm, respectively, accounting for the remaining 13%; the mechanical properties of the fiber films were measured using an electronic universal tester, and the fiber films were cut into rectangles (2.0 cm. times.4.0 cm) before the tensile test. During the test, the crosshead speed was 2mm/min and the ambient temperature was 25 ℃. The fiber film was tested three times to obtain a Young's modulus of 43.2 MPa and a tensile strength of 3.3 MPa. When the film is applied to a mouse test, the concentration of the interleukin which induces cells to secrete inflammatory factors is very low and is 5 pg/mL.

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.