CN113622044B

CN113622044B - Method for preparing silk protein-based fiber by dry spinning, silk protein-based fiber prepared by method and application of silk protein-based fiber

Info

Publication number: CN113622044B
Application number: CN202111056708.5A
Authority: CN
Inventors: 郭成辰; 江瑞; 孙子扬
Original assignee: Westlake University
Current assignee: Westlake University
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2023-04-28
Anticipated expiration: 2041-09-09
Also published as: CN113622044A

Abstract

The invention provides a method for preparing silk protein-based fibers by dry spinning, silk protein-based fibers prepared by the method and application of the silk protein-based fibers. The method for preparing silk protein based fiber by dry spinning comprises the following steps: (1) Concentrating the aqueous silk fibroin-based solution without the need to add a foreign substance; (2) And preparing the silk fibroin-based aqueous solution prepared by adopting extrusion equipment as a spinning solution into fibers. The method has the advantages of simplicity in operation, environment friendliness, high efficiency, rapidness and the like, and the silk fibroin-based fiber prepared by the method is uniform in size and controllable in structure, and simultaneously shows improved breaking strength and breaking elongation.

Description

Method for preparing silk protein-based fiber by dry spinning, silk protein-based fiber prepared by method and application of silk protein-based fiber

Technical Field

The invention belongs to the field of protein-based high polymer materials, and particularly relates to a method for preparing high-performance silk protein-based fibers by a dry method, the silk protein-based fibers prepared by the method and medical instruments prepared by the silk protein-based fibers.

Background

The protein-based fiber is a fiber material which is formed by taking protein-based materials such as silk protein, collagen, keratin and the like as raw materials and adopting different processing modes, and the basic structural unit of the fiber material is amino acid. The protein-based fibers commonly found in nature mainly include silk, spider silk, animal hair, collagen microfibers, and the like. These natural protein-based fibers generally have excellent mechanical properties, biocompatibility and bioresorbability, and thus have been widely paid attention in recent years and applied to biomedical fields such as surgical sutures and tissue engineering scaffolds. Besides directly utilizing the native natural protein-based fiber, the method for preparing the regenerated protein-based fiber with specific functions by dissolving the native protein through a reverse engineering technology is an important development direction, and can improve the limitation of the native natural protein-based fiber in aspects of biological functions, degradation performance regulation and the like to a great extent.

At present, four methods for preparing regenerated protein-based fibers are respectively electrostatic spinning, wet spinning, dry spinning and dry-jet wet spinning. Among them, the electrostatic spinning and wet spinning have been studied more, while the dry spinning has been studied relatively less. The dry spinning method is based on the principle that under the action of drafting force, spinning solution is directly pumped out from a spinning hole and solidified into fiber after solvent is volatilized quickly. The spinning mode is a spinning mode of silkworms or spiders in nature, and the obtained fiber has excellent performance. Therefore, the development of an artificial dry spinning method for preparing the high-performance regenerated protein-based fiber by simulating the natural spinning mode of silkworms and spiders has important significance.

At present, the method for preparing the regenerated protein-based dry spinning solution mainly comprises two methods: (1) Dissolving solid protein by using an organic solvent (such as hexafluoroisopropanol) to prepare high-concentration spinning solution; (2) Concentrating the protein water solution to obtain high protein concentration spinning solution, wherein inorganic salt ions and TiO are required to be added in the method ₂ And metal oxides or buffer solution for regulating pH value to make it meet the spinning requirement.

CN101724920B, CN102134757B, CN102220661B discloses that regenerated silk protein fibers are produced by adding metal ions to an aqueous silk protein solution and adjusting pH. CN108396425a discloses the preparation of silk fibroin/carbon nanotube filament yarns by dissolving silk fibroin using an organic acid/salt ion system. CN103572395B discloses that the composite fiber is prepared by mixing and concentrating a silk fibroin aqueous solution and graphene oxide, and adding calcium ions for adjustment.

In the process of preparing the spinning solution, inorganic salt is added or an organic solvent is utilized, and the prepared silk fibroin aqueous solution is not pure silk fibroin aqueous solution, which has a certain difference from a natural spinning mode and influences the performance of the prepared regenerated silk fibroin fiber.

Disclosure of Invention

Based on the problems, the continuous preparation of the high-performance silk fibroin-based fiber filaments is successfully realized by preparing a high-concentration pure silk fibroin aqueous solution and designing and optimizing a dry spinning method. The prepared fiber filament has excellent mechanical property, biocompatibility and biological absorbability. In addition, by adding active biomolecules such as enzymes, drugs, antibiotics, etc. to the spinning solution, silk protein-based fibers having both bioactivity, biocompatibility and bioresorbability can be prepared and applied to the relevant biomedical field.

It is a technical object of the present invention to provide a method for preparing silk fibroin-based fibers by dry process.

It is another technical object of the present invention to provide a silk fibroin-based fiber prepared by the above method.

Another technical object of the present invention is to provide a medical device made of the above silk fibroin-based fibers.

In one aspect, the present invention provides a method of dry spinning silk fibroin-based fibers, the method comprising the steps of:

(1) Concentrating the aqueous silk protein-based solution comprising the steps of:

1': dialyzing the initial silk fibroin aqueous solution in a dialysis bag, centrifuging the dialyzed solution at a first centrifugal speed under the ambient pressure, and discarding the precipitate to obtain a silk fibroin-based aqueous solution with the concentration of 4.0-8.0w/v%, preferably 4.5-6.5 w/v%;

2': placing the silk fibroin-based aqueous solution obtained in the step 1' in a vacuum environment for centrifugal concentration at a second centrifugal rotating speed, thereby obtaining silk fibroin-based aqueous solution with concentration of more than 10 w/v%;

(2) Preparing fibers: and preparing the silk fibroin-based aqueous solution prepared by adopting extrusion equipment as a spinning solution into fibers.

In particular embodiments, the starting aqueous silk fibroin solution in step 1' can be an aqueous natural silk fibroin solution; an aqueous solution of the regenerated silk protein derivative obtained through chemical modification; the regenerated silk protein/additive (such as medicine, growth factor, antibiotic, etc.) composite silk protein base solution is obtained through physical mixing; recombinant silk protein aqueous solutions, and the like.

In particular embodiments, the starting aqueous natural silk protein solution is prepared by the following method: placing the cut cocoons into 0.02M Na ₂ CO ₃ Boiling in water solution at 60-100deg.C for 30-180min to degumm silk cocoon, and drying the obtained glue solution to obtain degummed silk; the degummed silk is then dissolved in 9-10M, preferably 9.3M LiBr in water and placed in an oven at 60-150℃for 2-8h to allow complete dissolution.

In a specific embodiment, in step 1', the aqueous starting silk protein solution is dialyzed under the following dialysis conditions: the dialysis time is 2-5 days, the dialysate is deionized water, and the cut-off molecular weight of the dialysis bag is 3500 daltons (Da).

In a specific embodiment, in step 1', the first centrifugation speed is in the range of 5000-11000rpm, preferably 9000rpm. The aforementioned centrifugation is mainly used to remove the precipitate generated during the dialysis process.

In particular embodiments, additives for improving properties of the silk protein based material product selected from glycerol, inorganic salts, bioactive molecules (e.g. enzymes, antibiotics, drugs, etc.), inorganic materials, organic molecules may be added to the silk protein aqueous solution prior to step 2' or step (2). The concentration of the additive may be 0.1 to 50wt%. By adding additives, properties of the silk fibroin-based material product, such as physicochemical properties and biological properties, can be improved without affecting the effect of concentration.

In a specific embodiment, in step 2', vacuum centrifugation is performed under the following conditions: the second centrifugation speed is 100-3000rpm, preferably 300-2000rpm, the concentration temperature is 5-60 ℃, the vacuum degree is 0.1-1000mbar, and the concentration time is 3-48h. The main purpose of this step is to concentrate the aqueous silk protein solution.

In a specific embodiment, the concentration of the aqueous silk fibroin-based solution obtained in step 2' is 10-50w/v%, preferably 25-40w/v%, preferably the concentration of the aqueous silk fibroin-based solution obtained in step (2) is 6 times the concentration of the aqueous silk fibroin-based solution obtained in step (1).

In a specific embodiment, the method further comprises: after step 2', a third centrifugation operation at a third centrifugation speed is performed to separate the precipitated silk proteins.

In a specific embodiment, the third centrifugation is performed under the following conditions: the centrifugation time is 5-30min, the centrifugation temperature is 4-30deg.C, and the third centrifugation speed is 5000-14000rpm, preferably 9000rpm. The third centrifugation is mainly used to remove the precipitate generated during step 2'.

In a specific embodiment, in step (2), the silk fibroin-based aqueous solution obtained in step (1) is prepared into a fiber as a spinning dope by: transferring the spinning solution into an injector, wherein the port of the injector is connected with a stainless steel needle, the diameter of the needle is 0.2-2.0mm, and the flow rate of the solution is controlled by an injection pump and is set to be 1-2000 mu L/h; the diameter of the collecting device is 5-1000mm, the rotating speed is 1-5000rpm, the horizontal distance between the needle tip and the collecting device is 1-1000mm, the temperature is 5-200 ℃ and the humidity is 5-90% in the injection process.

The method of the present invention may further comprise: (3) post-treatment: the fiber structure is regulated and controlled by placing the fiber prepared in the above way in methanol water solution, ethanol vapor or acetic acid vapor for post treatment or in the air, alcohol bath, ethanol vapor or acetic acid vapor environment for post drawing.

In the invention, the post-treatment or post-drawing treatment in the step (3) can increase the beta-sheet content in the fiber to a certain extent so as to further improve the mechanical properties of the fiber and increase the application range of the fiber.

In a specific embodiment, in step (3), in the case of the post-treatment with an aqueous methanol solution, the methanol concentration is 50 to 95v/v% and the treatment time is 1 to 600min.

In a specific embodiment, in step (3), in the case of the post-treatment with acetic acid vapor, the acetic acid vapor concentration is 0.1 to 17.5M and the treatment time is 1 to 180s, for example 10s,30s.

In a specific embodiment, in step (3), the resulting fiber is first placed in a 3.5M acetic acid vapor environment for a treatment time of 30s, and then the fiber is immersed in a 95v/v% methanol solution for a treatment time of 120min.

In another aspect, the present invention provides a silk fibroin-based fiber prepared by the above method.

In yet another aspect, the present invention provides a medical device made from the silk fibroin-based fibers described above.

In a specific embodiment, the medical device is made from the silk fibroin-based fibers described above by a post-processing process of twisting, sizing, or weaving.

In specific embodiments, the medical device is selected from one of a surgical suture, an artificial ligament, a tissue scaffold, and a wound dressing.

Advantageous effects

The invention has the advantages compared with the prior art that:

(1) The time efficiency of the spinning solution preparation is high, and the property is stable;

(2) Can be continuously spun;

(3) The prepared fiber filament has uniform size, controllable structure and more excellent mechanical property.

(5) The method of the invention has the advantages of simple operation, environmental protection, high efficiency, rapidness and the like.

(6) The fibers obtained by dry spinning in the present application have a breaking strength of 0.05-1.0GPa and an elongation at break of 1% -800% and, since no chemicals, such as inorganic salts or organic solvents, which may adversely affect the properties of the fibers or additives may be added during the fiber preparation process, provide convenience for subsequent processing into biocompatible materials.

(7) The fiber preparation method of the invention can also be applied to the preparation of fibers taking collagen and keratin as raw materials, so as to prepare fibers similar to those in the application, and the prepared fibers are also expected to be applied to medical instruments or other fields.

Drawings

FIG. 1 is a surface scanning electron microscope image of the dry-process regenerated yarn obtained in step (2) of example 1.

FIG. 2 is a cross-sectional scanning electron microscope image of the dry-process regenerated yarn obtained in step (2) of example 2.

FIG. 3 is an infrared spectrum of degummed silk, dry-process regenerated silk, and dry-process regenerated silk after 120min of soaking treatment with methanol solution in example 2.

FIG. 4 is a tensile stress-strain curve of degummed silk and dry-process regenerated silk in example 3.

Fig. 5 is an infrared spectrum of the dry-process regenerated silk of example 4 and its fiber after post-drawing treatment under conditions of ethanol vapor (example 4) and acetic acid vapor 5 (example).

FIG. 6 is a tensile stress-strain curve of degummed silk, dry-process regenerated silk, and dry-process regenerated silk treated with methanol solution for 30min in example 7.

Detailed Description

The term "silk protein" may be used interchangeably with "silk fibroin" herein.

Hereinafter, the technical contents of the present invention will be described in detail through specific embodiments so that those skilled in the art can better understand the present invention, but these embodiments are not intended to limit the contents of the present invention.

The instruments and reagent sources used in the examples and test examples below are shown in Table 1 below.

TABLE 1 Main Experimental reagents and laboratory instruments

Example 1

(1) Silkworm cocoons are treated with 0.02M Na ₂ CO ₃ Boiling in water solution at 60-100deg.C for 30-180min, degumming cocoon, and drying the obtained glue solution to obtain degummed silk.

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, then the completely dissolved solution was placed in a dialysis bag having a molecular weight cutoff of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure, the supernatant was aspirated and the precipitate was separated to obtain an aqueous silk protein solution having an initial concentration of 4.5 wt.%.

The above solution was then subjected to centrifugal concentration under vacuum to prepare a high-concentration silk fibroin aqueous solution. Wherein the concentration temperature is 30 ℃, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, and the concentration time is 13h.

The concentrated solution was centrifuged at 6000rpm for 10min at 4 ℃. Finally, a concentrated silk protein solution with a mass fraction of 25wt.% is obtained.

(2) The 25wt.% silk fibroin concentrated solution was added to a 5mL syringe, the syringe port was connected to a stainless steel needle having a diameter of 0.4mm. The solution flow rate was controlled by a syringe pump at a rate of 40. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 140rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 25℃and the humidity was 45%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 3.6+/-0.3 mu m.

(3) The dry-process regenerated silk prepared above was immersed in a 95% (v/v) methanol solution, treated at room temperature for 30min, and then dried.

Example 2

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, then the completely dissolved solution was placed in a dialysis bag having a molecular weight cutoff of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure, the supernatant was aspirated and the precipitate was separated to obtain an aqueous silk protein solution having an initial concentration of 5.3 wt.%.

The above solution was then subjected to centrifugal concentration under vacuum to prepare a high-concentration silk fibroin aqueous solution. Wherein the concentration temperature is 45 ℃, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, and the concentration time is 14h.

The concentrated solution was centrifuged at 6000rpm for 10min at 4 ℃. Finally, a concentrated silk protein solution with a mass fraction of 27wt.% was obtained.

(2) The concentrated silk fibroin solution with the mass fraction of 27wt.% is added into a 5mL syringe, the port of the syringe is connected with a stainless steel needle, and the diameter of the needle is 0.4mm. The solution flow rate was controlled by a syringe pump at a rate of 40. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 140rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 25℃and the humidity was 35%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 6.5+/-0.7 mu m.

(3) Immersing the dry-process regenerated silk prepared in the above into 95% (v/v) methanol solution, treating at room temperature for 120min, and drying.

Example 3

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, then the completely dissolved solution was placed in a dialysis bag having a molecular weight cutoff of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure, the supernatant was aspirated and the precipitate was separated to obtain an aqueous silk protein solution having an initial concentration of 5.8 wt.%.

The above solution was then subjected to centrifugal concentration under vacuum to prepare a high-concentration silk fibroin aqueous solution. Wherein the concentration temperature is 45 ℃, the centrifugal speed is 1500rpm, the vacuum degree is 20mbar, and the concentration time is 17h.

The concentrated solution was centrifuged at 9000rpm at 4℃for 15min. Finally, a concentrated silk protein solution with a mass fraction of 40wt.% was obtained.

(2) The 40wt.% silk fibroin concentrate solution was added to a 5mL syringe, the syringe port was connected to a stainless steel needle having a diameter of 0.6mm. The solution flow rate was controlled by a syringe pump at 60. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 80rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 27℃and the humidity was 40%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 20.7+/-1.5 mu m.

(3) The dry-process regenerated silk prepared above was immersed in a 95% (v/v) methanol solution, treated at room temperature for 60min, and then dried.

Example 4

(1) The 25wt.% silk fibroin concentrated solution prepared in step (1) of example 1 was added to a 5mL syringe, the syringe port was connected to a stainless steel needle, and the diameter of the needle was 0.4mm. The solution flow rate was controlled by a syringe pump at a rate of 40. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 165rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 25℃and the humidity was 45%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 3.0+/-0.2 mu m.

(2) And (3) carrying out post-drawing treatment on the dry-method regenerated silk prepared in the above way under the condition of 95% (v/v) ethanol vapor for 30s, and then drying.

Example 5

(1) The dry-process regenerated silk produced in step (1) of example 4 was subjected to post-drawing treatment under 17.5M acetic acid vapor for 30s, and then dried.

Example 6

(1) The 25wt.% silk fibroin concentrated solution prepared in step (1) of example 1 was added to a 5mL syringe, the syringe port was connected to a stainless steel needle, and the diameter of the needle was 0.4mm. The solution flow rate was controlled by a syringe pump at a rate of 40. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 180rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 25℃and the humidity was 45%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 2.7+/-0.3 mu m.

(2) The dry-process regenerated silk prepared above was placed under 3.5M acetic acid vapor environment for 30s, then immersed in 95% (v/v) methanol solution for 120min, and then dried.

Example 7

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, then the completely dissolved solution was placed in a dialysis bag having a molecular weight cutoff of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure, the supernatant was aspirated and the precipitate was separated to obtain an aqueous silk protein solution having an initial concentration of 5.5 wt.%.

The glycerol and calcium chloride solution was then added to a 5.5wt.% aqueous silk fibroin solution, such that the mass ratio of glycerol to calcium chloride to silk fibroin in the solution was 4:1:15. Followed by centrifugation and concentration under vacuum to prepare a high concentration silk protein aqueous solution. Wherein the concentration temperature is 30 ℃, the centrifugal speed is 2000rpm, the vacuum degree is 20mbar, and the concentration time is 15h.

The concentrated solution was centrifuged at 6000rpm for 10min at 4 ℃. Finally, a concentrated solution of composite silk proteins with a mass fraction of 45wt.% was obtained.

(2) The 45wt.% silk fibroin concentrate solution was added to a 5mL syringe, the syringe port was connected to a stainless steel needle having a diameter of 0.4mm. The solution flow rate was controlled by a syringe pump at a rate of 40. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 100rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 23℃and the humidity was 50%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 13+/-1.5 mu m.

Example 8

(1) The 25wt.% silk fibroin concentrated solution prepared in step (1) of example 1 was added to a 5mL syringe, the syringe port was connected to a stainless steel needle, and the diameter of the needle was 0.6mm. The solution flow rate was controlled by a syringe pump at a rate of 40. Mu.L/h. The diameter of the collecting device was 12mm and the rotational speed was 100rpm. The distance between the needle tip and the collection device was 150mm. The temperature during the experiment was 25℃and the humidity was 45%. Finally, the dry-process regenerated silk is produced, and the fiber diameter is 11.6+/-1.1 mu m.

(2) And (3) placing the prepared dry regenerated silk into an oven at 80 ℃ for 30min, and then cooling to room temperature.

Example 9

The dried degummed silk was then dissolved in 9.3M LiBr aqueous solution, then the completely dissolved solution was placed in a dialysis bag having a molecular weight cutoff of 3500 daltons (Da), wherein the dialysis solution was deionized water, the dialysis time was 3 days, and the dialyzed solution was centrifuged at 9000rpm under normal pressure, the supernatant was aspirated and the precipitate was separated to obtain an aqueous silk fibroin solution having an initial concentration of 5.8 wt.%.

The above solution was then subjected to centrifugal concentration under vacuum to prepare a high-concentration silk fibroin aqueous solution. Wherein the concentration temperature is 45 ℃, the centrifugal rotation speed is 5000rpm, the vacuum degree is 20mbar, and the concentration time is 15.5h.

The concentrated solution was centrifuged at 9000rpm at 4℃for 15min. Finally, a concentrated silk fibroin solution with a mass fraction of 32wt.% was obtained.

(2) Horseradish peroxidase (Horseradish Peroxidase, HRP) was added in a mass fraction of 1 wt%o based on silk protein in a 32wt.% silk protein concentrated solution at room temperature. And (5) fully mixing to obtain the composite spinning solution containing the HRP enzyme for standby.

(3) The procedure of step (1) of example 4 was repeated except that the above-mentioned silk protein concentrated solution having a mass fraction of 32wt.% and the prepared HRP enzyme-containing composite dope were used, respectively, to obtain two kinds of fibers, wherein the fibers to which HRP enzyme was not added in the dope were used as a control group.

(4) The two fibers prepared above were taken in a length of 1cm in an orifice plate, followed by sequentially adding 50. Mu.L of deionized water and 50. Mu.L of 3,3', 5' -Tetramethylbenzidine (TMB) developing solution. After standing for a period of time, the solution with HPR enzyme added to the fiber appeared blue, while the control group was colorless and transparent. Subsequently, 100. Mu.L of 1M HCl solution was added to the well plate to terminate the reaction. After addition of the HCl solution, the solution in the sample with HPR enzyme added changed from blue to bright yellow, while the control solution was always colorless and transparent.

From the above results, it can be confirmed that the spinning process of the present application allows the HRP enzyme to maintain a good biological activity in the fiber. This further reflects the great advantage of the silk fibroin-based fibers of the present application in the subsequent preparation of various biocompatible materials, particularly medical materials or medical devices, as they do not contain additional undesired chemicals, compared to silk fibroin-based fibers of the prior art.

Test example 1

The testing method comprises the following steps: the surface structure and cross-sectional structure of the fiber were observed by Cai Sigao-resolved analytical field emission scanning electron microscopy. Before testing, the fiber sample needs to be subjected to Pt spraying treatment on the surface of the fiber, and the thickness of the fiber sample is 5nm. The test voltage was 3kV and the detector was about 9mm from the sample stage. When calculating the fiber diameter, the statistical fiber diameter was measured by taking 5 SEM images of different parts of each sample using Image pro plus software, and 20 times for each sample, and the average value and standard deviation of the fiber diameter were calculated.

Test results:

FIG. 1 is a surface scanning electron microscope image of the dry-process regenerated yarn obtained in step (2) of example 1. As can be seen from the figure, the surface of the prepared fiber is smoother, and the diameter is uniform. The fiber diameter produced under the conditions of this example was 3.6±0.3 μm, which is close to the diameter of natural spider silk.

FIG. 2 is a cross-sectional scanning electron micrograph of the dry-process regenerated yarn obtained in step (2) of example 2, in which it is seen that the fiber has a cross-section that is nearly circular.

Test example 2

The testing method comprises the following steps: the FT-IR test results were obtained by Fourier transform micro-IR spectroscopy. The test mode adopted is ATR, and the resolution of the instrument is 4cm ^-1 Each spectral line is overlapped by 64 scans, and the spectrum range is 500-4000cm ^-1 . During the test, each sample was tested 3 times. In the spectrum, para-amide I region (1600-1700 cm ^-1 ) And carrying out peak-splitting fitting treatment, and quantitatively analyzing the secondary conformation of the silk protein.

Analysis of results: the beta-sheet content of each of the fibers obtained in examples 1-8 is shown in Table 2 below.

TABLE 2

Examples numbering	Beta-sheet content (%)
		1	21.7
2	25.4
		3	22.4
4	19.6
		5	26.8
6	29.7
		7	23.7
8	24.7

FIG. 3 is an infrared spectrum of degummed silk, dry-process regenerated silk, and dry-process regenerated silk after 120min of soaking treatment with methanol solution in example 2. As shown in FIG. 3, when the dry-process regenerated silk is immersed in the methanol solution for 120min, the infrared spectrogram comparison analysis of the dry-process regenerated silk without the methanol solution shows that the peak of the fiber treated by the methanol solution in the amide I region is 1643cm ^-1 Move to the right to 1622cm ^-1 Here, it is explained that the treatment with methanol solution induces the formation of silk protein structureMore β -sheet structure and β -sheet content increased from untreated 10.6% to 25.4%.

Fig. 5 is an infrared spectrum of the dry-process regenerated yarn of example 4 and after post-drawing treatment under the conditions of acetic acid vapor (example 5) and ethanol vapor (example 4). As can be seen from FIG. 5, after the ethanol vapor and the acetic acid vapor treatment, 1640cm of the infrared spectrum of the dry-process regenerated yarn was compared with that of the dry-process regenerated yarn without any treatment ^-1 The peaks at the positions are all offset to the right, wherein the peaks of the fiber subjected to post-drawing treatment under the condition of acetic acid vapor are offset to 1621cm ^-1 Where it is located. The method shows that the beta-sheet content in the fiber can be improved to a greater extent by carrying out the post-drawing treatment under the condition of acetic acid vapor.

Test example 3

The testing method comprises the following steps: the mechanical properties of the fibers were tested and analyzed using a biomaterials testing instrument from Cell Scale. The corresponding diameter of each test sample was measured by optical microscopy prior to mechanical testing. The test conditions were: the clamping distance is 5mm, the stretching speed is 5mm/min, the temperature is 25+/-3 ℃, and the humidity is 60+/-5%. Each sample was tested 10 times.

Analysis of results:

FIG. 4 is a tensile stress-strain curve of degummed silk and dry-process regenerated silk in example 3. As is clear from the graph, the elongation at break of the dry-process regenerated yarn is greater than that of degummed yarn, up to 58%, and the breaking strength is slightly lower than that of degummed yarn, 103MPa.

Jin et al (Y.Jin, Y.Zhang, Y.Hang, H.Shao, X.Hu, A simple process for dry spinning of regenerated silk fibroin aqueous solution, journal of Materials Research 28 (20) (2013) 2897-2902.) added CaCl to 20wt.% silk fibroin aqueous solution ₂ Aqueous solution to make Ca in spinning solution ²⁺ The ion concentration was 0.3M. And concentrating the spinning solution to 40-60wt.% and performing dry spinning to obtain the silk fibroin-based fiber. The resulting fiber was drawn 4 times in an aqueous 80v/v (%) ethanol solution and soaked under this condition for 3 hours. Compared with the results (elongation at break is 10.6% and breaking strength is 78.9 MPa) obtained in the article of Jin et al, the spinning solution in the application is pure silk protein-based fiber, and other metal ions are not added or regulatedThe pH-adjusted buffer solution gives fibers exhibiting more excellent breaking strength and elongation at break.

FIG. 6 is a tensile stress-strain curve of degummed silk, dry-process regenerated silk, and dry-process regenerated silk treated with aqueous methanol for 30 minutes in example 7. From the graph, the breaking strength of the regenerated silk produced by the dry method is similar to that of degummed silk and can reach 386MPa. Meanwhile, the dry-method regenerated silk has excellent stretchability, and the elongation at break can reach 700%. After the dry-method regenerated silk is treated by a methanol aqueous solution for 30min, the content of beta-sheet in the fiber is increased according to the FT-IR experimental result, the initial modulus is greatly improved, the elongation at break is reduced, and the breaking strength is not greatly changed.

Claims

1. A method of dry spinning silk protein based fibers, the method comprising the steps of:

1': putting the initial silk fibroin aqueous solution into a dialysis bag for dialysis, centrifuging the dialyzed solution at a first centrifugal speed under the ambient pressure, and discarding the precipitate to obtain a silk fibroin-based aqueous solution with the concentration of 4.0-8.0 w/v%;

(2) Preparing fibers: and (3) preparing the silk fibroin-based aqueous solution prepared in the step (1) into fibers by adopting extrusion equipment as a spinning solution.

2. The method according to claim 1, wherein in step 1', the aqueous silk fibroin-based solution having a concentration of 4.5-6.5w/v% is obtained by a first centrifugation.

3. The method of claim 1, wherein the initial aqueous silk protein solution in step 1' is an aqueous natural silk protein solution; an aqueous solution of the regenerated silk protein derivative obtained through chemical modification; the regenerated silk protein/additive composite silk protein base solution is obtained through physical mixing; or an aqueous solution of recombinant silk proteins.

4. The method according to claim 1, wherein in step 1', the first centrifugal speed is 5000-11000rpm;

in step 2', vacuum centrifugation is performed under the following conditions: the second centrifugal speed is 100-3000rpm, the concentration temperature is 5-60 ℃, the vacuum degree is 0.1-1000mbar, and the concentration time is 3-48h.

5. The method according to claim 4, wherein in step 1', the first centrifugal rotational speed is 9000rpm; the second centrifugal speed is 300-2000rpm.

6. The method according to claim 1, wherein an additive is added to the aqueous silk protein solution before step 2' or (2), the additive being selected from glycerol, inorganic salts, bioactive molecules, the concentration of the additive being 0.1-50wt%.

7. The method according to claim 1, wherein in step (2), the dope is transferred to a syringe, a syringe port is connected to a stainless steel needle having a diameter of 0.2 to 2.0mm, and a solution flow rate is controlled by a syringe pump, the solution flow rate being set to 1 to 2000 μl/h; the diameter of the collecting device is 5-1000mm, the rotating speed is 1-5000rpm, the horizontal distance between the needle tip and the collecting device is 1-1000mm, the temperature is 5-200 ℃ and the humidity is 5-90% in the injection process.

8. The method of claim 1, wherein after step (2), the method further comprises step (3): post-treating the fiber prepared in the step (2) in methanol water solution, ethanol vapor or acetic acid vapor, or post-drafting under air, alcohol bath, ethanol vapor or acetic acid vapor,

optionally, in step (3), in the case of the post-treatment with an aqueous methanol solution, the methanol concentration is 50 to 95v/v% and the treatment time is 1 to 600min;

optionally, in step (3), in the case of post-drawing in acetic acid vapor, the acetic acid vapor concentration is 0.1 to 17.5M and the treatment time is 1 to 180s;

alternatively, in step (3), the resulting fiber is first placed in a 3.5M acetic acid vapor environment for a treatment time of 30s, and then immersed in a 95v/v% methanol solution for a treatment time of 120min.

9. A silk fibroin-based fiber prepared by the method of any one of claims 1-8.

10. A medical device made from the silk fibroin-based fiber of claim 9.

11. The medical device of claim 10, made from the silk fibroin-based fibers by a post-processing process of twisting, sizing, or weaving.

12. The medical device of claim 10, selected from any one of a surgical suture, an artificial ligament, a tissue scaffold, and a wound dressing.