CN117624675B - Regenerated silk fibroin membrane and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of silk fibroin, and particularly relates to a regenerated silk fibroin film, and a preparation method and application thereof. The method comprises the following steps: degumming and drying mulberry silk to obtain dry silk fibroin; preparing a silk fibroin solution by using PEG300-PEG600 as a solvent; adding genipin and modifier into the silk fibroin solution to obtain a mixed solution; placing the mixed solution at 40-45 ℃ and stirring for 10-20h to obtain a pre-reaction solution; and preparing the regenerated silk fibroin film by the pre-reaction liquid. The regenerated silk fibroin film prepared by the invention has good mechanical property, good biocompatibility and high application value.

Description

Regenerated silk fibroin film and preparation method and application thereof

Technical Field

The invention belongs to the technical field of silk fibroin, and in particular relates to a regenerated silk fibroin film and a preparation method and application thereof.

Background technique

Silk fibroin is a high molecular weight protein that mainly contains 18 amino acids. Silk fibroin has excellent mechanical properties, biocompatibility and biodegradability, and has good application prospects in the fields of biomaterials and tissue engineering.

At present, silk fibroin is used in various forms (Lin Nan, Zuo Baoqi. Preparation and research progress of medical regenerated silk fibroin materials [J]. Modern Silk Science and Technology, 2020, 35(3):9.), including:

(1) preparing a regenerated silk fibroin solution, specifically including a degumming step and a dissolving step. Since the synergistic effect of silk fibroin and dead keratin will produce a certain immune response, degumming is a key step in using silk fibroin as a biomaterial. After degumming, silk can be dissolved, and the dissolved silk fibroin can be made into a variety of dosage forms.

(2) Preparation of silk fibroin materials, including preparation of hydrogels, porous scaffolds, films, microspheres and other materials. The preparation methods of hydrogels include cyclonic shearing, ultrasonic oscillation, acid treatment, natural gelation, etc.; porous scaffolds are prepared by freeze drying and particle porogens; films are prepared by casting and other methods; microspheres are prepared by spray drying, freeze drying and other methods.

Silk fibroin has excellent film-forming ability and can be made into silk fibroin membrane, which is also called silk membrane or silk protein membrane in the prior art (reference patent CN103200971B). Bian Ruiqi (Bian Ruiqi. Preparation and performance study of regenerated silk fibroin/L-polylactic acid-polycaprolactone copolymer composite nanofiber membrane scaffold [D]. Zhejiang University of Technology [2023-10-13]) explored the preparation method of the film composited with regenerated silk fibroin/L-polylactic acid. The results showed that with the increase of the content of genipin, the diameter of the nanofibers gradually increased, the morphology became uneven, and the degree of cross-linking gradually increased. The thermal properties of the material gradually improved, and the mechanical properties at room temperature also gradually increased. In vitro cell culture experiments showed that the addition of genipin had no toxic side effects on L929 cells. In order to further improve the mechanical properties of electrospun silk fibroin nanofiber membranes, the polymer poly(lactic acid-polycaprolactone copolymer) [P(LLA-CL)] was selected and blended with silk fibroin at different mixing ratios, and the poly(lactic acid-polycaprolactone copolymer) [P(LLA-CL)]/silk fibroin blended nanofiber membranes were prepared by electrospinning. In order to improve the mechanical properties of the material while maintaining good biocompatibility, the silk fibroin/P(LLA-CL) skin-core structure composite nanofiber membrane was prepared by coaxial electrospinning, and multi-walled carbon nanotubes (MWCNTs) were mixed into the core layer of the material in order to further improve the mechanical properties of the composite fiber membrane. The mixing of MwCNTs is beneficial to improving the biocompatibility of the nanomembrane.

By analyzing the above existing technologies, we can find that although silk fibroin has a good application prospect in the preparation of biomaterials, the mechanical properties of the membrane directly prepared by silk fibroin are poor and need to be improved. Although the method of Bian Ruiqi et al. improved the mechanical properties of the regenerated silk fibroin membrane, it is a technology from 2012. It is too old and the mechanical properties of the regenerated silk fibroin membrane need to be improved.

Summary of the invention

In order to solve the above technical problems, the present invention provides a regenerated silk fibroin membrane and a preparation method and application thereof. Compared with the prior art, the regenerated silk fibroin membrane prepared by the present invention has better mechanical properties and high application value.

An object of the present invention is to provide a method for preparing a regenerated silk fibroin film, comprising:

Degumming and drying the silk to obtain dry silk fibroin;

Using PEG300-PEG600 as solvent, dry silk fibroin is prepared into a silk solution; Genipin and a modifier are added to the silk solution to obtain a mixed solution; wherein, Genipin can undergo polymerization reaction under certain conditions, and can react with the free amino groups on the silk fibroin to form intramolecular and intermolecular covalent bonds, which is the key to film formation. Genipin can also be cross-linked with carboxymethyl chitosan. Glycerol is dispersed in the film; wherein, the modifier is carboxymethyl chitosan or glycerol. The modifier can simultaneously improve the mechanical properties and biocompatibility of the regenerated silk fibroin film; preferably, PEG300, PEG400, and PEG600 are used as solvents; Sodium L-glutamate has a certain stability to light and heat, and adding it to mulberry silk for treatment can not only improve the stability of silk fibroin, but also accelerate degumming;

The mixed solution was stirred at 40-45° C. for 10-20 h to obtain a pre-reaction solution;

The regenerated silk fibroin membrane was prepared from the pre-reaction solution.

Preferably, in the method for preparing the above-mentioned regenerated silk fibroin film, dry silk fibroin is configured into a silk fibroin aqueous solution with a mass fraction of 8-10%; genipin and a modifier are added to the silk fibroin aqueous solution to obtain a mixed solution, wherein the mass concentration of genipin in the mixed solution is 2-5%, and the mass concentration of the modifier is 0.2-0.4%.

Preferably, in the preparation method of the above-mentioned regenerated silk fibroin film, PEG400 is used as solvent, and dry silk fibroin is configured into a silk fibroin solution with a mass fraction of 8%; genipin and a modifier are added to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution is 2%, and the mass concentration of the modifier is 0.3%.

Preferably, in the method for preparing the above-mentioned regenerated silk fibroin membrane, the pre-reaction liquid is prepared by a cast-through method.

Preferably, the method for preparing the above-mentioned regenerated silk fibroin membrane further comprises a microwave treatment step: the pre-reaction liquid is subjected to microwave treatment, and then the regenerated silk fibroin membrane is prepared.

Preferably, in the method for preparing the above-mentioned regenerated silk fibroin film, the microwave treatment conditions are 600-800W for 20s.

Preferably, in the method for preparing the above-mentioned regenerated silk fibroin film, the microwave treatment conditions are 700W for 20s.

Preferably, in the method for preparing the above-mentioned regenerated silk fibroin film, the degumming method comprises:

(1) boiling silk with an alkaline solution for 30 minutes, filtering, and collecting precipitates; wherein the alkaline solution is a mixture of a 0.05% by mass sodium carbonate aqueous solution and a 0.03% by mass L-glutamate sodium aqueous solution in a volume ratio of 20:1;

(2) boiling the precipitate with the alkaline solution for 30 minutes to detect whether the degumming is complete;

(3) Repeat the operation of (2) until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution, which is concentrated and dried to obtain dry silk fibroin.

The present invention also provides a regenerated silk fibroin film prepared by the method.

Preferably, the regenerated silk fibroin film has a thickness of 60-80 μm.

The present invention also provides an application of the regenerated silk fibroin membrane, including using the regenerated silk fibroin membrane to promote cell penetration, growth and proliferation.

Compared with the prior art, the present invention has the following beneficial effects:

The invention adopts an improved alkaline solution formula, improves the degumming efficiency of mulberry silk, shortens the degumming time, and is energy-saving and environmentally friendly.

In the present invention, a modifier is added when preparing the mixed solution. The modifier is carboxymethyl chitosan or glycerol. The addition of the modifier can improve the mechanical properties of the silk fibroin film.

The present invention also adopts microwave treatment to increase the cross-linking effect and improve the mechanical properties of the film.

In summary, the regenerated silk fibroin membrane prepared by the present invention has better mechanical properties, good biocompatibility and high application value. The regenerated silk fibroin membrane prepared by the present invention is more conducive to cell penetration, growth and proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a surface SEM image of the regenerated silk fibroin film prepared in Example 1, with a scale of 5 μm.

FIG. 2 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 1.

FIG. 3 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 2.

FIG. 4 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 3.

FIG. 5 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 4.

FIG. 6 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 5.

FIG. 7 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 6.

FIG. 8 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 7.

FIG. 9 is a mechanical tensile curve of the regenerated silk fibroin film prepared in Example 8.

Detailed ways

In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

1. Main experimental reagents and equipment

Mulberry silk: Available in Zhejiang, it is a natural fiber. It is made by cutting open the silkworm cocoons, removing the pupae and molts inside, and obtaining the white cocoon shell.

Genipin: CAS No. 6902-77-8, Wuhan Kemik Biopharmaceutical Technology Co., Ltd., content > 98%.

Carboxymethyl chitosan: CAS No. 83512-85-0, Guangdong Wengjiang Chemical Reagent Co., Ltd., BR grade. Good biocompatibility and biosafety.

Microwave conditions were performed in a microwave oven.

In the following examples and tests, during the pre-reaction operation step, the mixed solution was placed on a magnetic stirrer for heating and stirring to meet the cross-linking requirements.

2. Example

Example 1

A method for preparing a regenerated silk fibroin film comprises the following steps:

1. Degumming

1.1.1 Boil the silk in alkaline solution for 30 minutes, filter and collect the precipitate.

The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1.

1.1.2 Continue to boil the precipitate with the alkaline solution in 1.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

1.1.3 Repeat step 1.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

1.2 Processing

1.2.1 Filter the silk protein solution in 1.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse with 75% ethanol and deionized water in turn. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% volume fraction of polyethylene glycol 20000 aqueous solution. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

1.2.2 Using PEG400 as solvent, dry silk fibroin was prepared into a silk fibroin solution with a mass fraction of 8%; genipin and a modifier were added to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 2%, and the mass concentration of the modifier was 0.3%.

The improver is carboxymethyl chitosan.

1.3 Film making

1.3.1 Pre-reaction: The mixed solution obtained in 1.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

1.3.2 Microwave treatment: The pre-reaction solution was treated with microwaves at 700 W for 20 s.

1.3.3 Preparation of film by cast-cast method: The pre-reaction solution after microwave treatment was placed on a polyethylene plate, and the solution was evenly spread with a glass rod and air-dried at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 62.8%, and the breaking strength is 34.3 MPa. The SEM image of the membrane is shown in FIG1 , and the mechanical tensile curve is shown in FIG2 .

Example 2

A method for preparing a regenerated silk fibroin film comprises the following steps:

2.1 Degumming

2.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

2.1.2 Continue to boil the precipitate with the alkaline solution in 2.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

2.1.3 Repeat step 2.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

2.2 Processing

2.2.1 Filter the silk protein solution in 2.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt it out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse it with 75% ethanol and deionized water by volume. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% aqueous solution of polyethylene glycol 20000 by volume. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

2.2.2 Using PEG400 as solvent, dry silk was prepared into a silk solution with a mass fraction of 8%. Genipin and a modifier were added to the silk solution to obtain a mixed solution, in which the mass concentration of genipin was 3% and the mass concentration of the modifier was 0.3%.

The improver is carboxymethyl chitosan.

2.3 Film making

2.3.1 Pre-reaction: The mixed solution obtained in 2.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

2.3.2 Microwave treatment: The pre-reaction solution was treated with microwaves at 700 W for 20 s.

2.3.3 Preparation of thin film by cast-cast method: The pre-reaction solution after microwave treatment was placed on a polyethylene plate, and the solution was evenly spread with a glass rod and air-dried at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 61.3%, and the breaking strength is 34.9 MPa. The mechanical tensile curve is shown in FIG3 .

Example 3

A method for preparing a regenerated silk fibroin film comprises the following steps:

3.1 Degumming

3.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

3.1.2 Continue to boil the precipitate with the alkaline solution in 3.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

3.1.3 Repeat step 3.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

3.2 Processing

3.2.1 Filter the silk protein solution in 3.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt it out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse it with 75% ethanol and deionized water by volume. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% aqueous solution of polyethylene glycol 20000 by volume. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

3.2.2 Using PEG400 as solvent, dry silk fibroin was prepared into a silk fibroin solution with a mass fraction of 8%; genipin and a modifier were added to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 4% and the mass concentration of the modifier was 0.3%.

The improver is carboxymethyl chitosan.

3.3 Film making

3.3.1 Pre-reaction: The mixed solution obtained in 3.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

3.3.2 Microwave treatment: The pre-reaction solution was treated with microwaves at 700 W for 20 s.

3.3.3 Preparation of thin film by cast-cast method: The pre-reaction solution after microwave treatment was placed on a polyethylene plate, and the solution was evenly spread with a glass rod and air-dried at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 60.1%, and the breaking strength is 36.4 MPa. The mechanical tensile curve is shown in FIG4 .

Example 4

A method for preparing a regenerated silk fibroin film comprises the following steps:

4.1 Degumming

4.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

4.1.2 Continue to boil the precipitate with the alkaline solution in 4.1.1 for 30 minutes and collect the solution.

4.1.3 Repeat step 4.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

4.2 Processing

4.2.1 Filter the silk protein solution in 4.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt it out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse it with 75% ethanol and deionized water in turn. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% volume fraction of polyethylene glycol 20000 aqueous solution. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

4.2.2 Using PEG400 as solvent, dry silk fibroin was prepared into a silk fibroin solution with a mass fraction of 8%; genipin and a modifier were added to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 5% and the mass concentration of the modifier was 0.3%.

The improver is carboxymethyl chitosan.

4.3 Film preparation

4.3.1 Pre-reaction: The mixed solution obtained in 4.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

4.3.2 Microwave treatment: The pre-reaction solution was subjected to microwave treatment at 700 W for 20 s.

4.3.3 Preparation of thin film by cast-cast method: The pre-reaction solution after microwave treatment was placed on a polyethylene plate, and the solution was evenly spread with a glass rod and air-dried at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 60.0%, and the breaking strength is 36.5 MPa. The mechanical tensile curve is shown in FIG5 .

Example 5

A method for preparing a regenerated silk fibroin film comprises the following steps:

5.1 Degumming

5.1.1 Boil the silk for 30 minutes with an alkaline solution, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

5.1.2 Continue to boil the precipitate with the alkaline solution in 5.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

5.1.3 Repeat step 5.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

5.2 Processing

5.2.1 Filter the silk protein solution in 5.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt it out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse it with 75% ethanol and deionized water in turn. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% aqueous solution of polyethylene glycol 20000. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

5.2.2 Using PEG400 as solvent, dry silk fibroin was prepared into a silk fibroin solution with a mass fraction of 8%. Genipin and a modifier were added to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 2% and the mass concentration of the modifier was 0.2%.

The improver is carboxymethyl chitosan.

5.3 Film making

5.3.1 Pre-reaction: The mixed solution obtained in 5.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

5.3.2 Microwave treatment: The pre-reaction solution was subjected to microwave treatment at 700 W for 20 s.

5.3.3 Preparation of thin film by cast-cast method: The pre-reaction solution after microwave treatment was placed on a polyethylene plate, and the solution was evenly spread with a glass rod and air-dried at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 66.8%, and the breaking strength is 32.4 MPa. The mechanical tensile curve is shown in FIG6 .

Example 6

A method for preparing a regenerated silk fibroin film comprises the following steps:

6.1 Degumming

6.1.1 Boil the silk for 30 minutes with an alkaline solution, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

6.1.2 Continue to boil the precipitate with the alkaline solution in 6.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

6.1.3 Repeat step 6.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, the picric acid carmine solution is used to detect whether the degumming is complete.

6.2 Processing

6.2.1 Filter the silk protein solution in 6.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt it out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse it with 75% ethanol and deionized water in turn. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% aqueous solution of polyethylene glycol 20000. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

6.2.2 Using PEG400 as solvent, prepare a silk solution with a mass fraction of 8% by weight from dry silk fibroin; add genipin and a modifier to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution is 2% and the mass concentration of the modifier is 0.4%.

The improver is carboxymethyl chitosan.

6.3 Film Forming

6.3.1 Pre-reaction: The mixed solution obtained in 6.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

6.3.2 Microwave treatment: The pre-reaction solution was subjected to microwave treatment at 700 W for 20 s.

6.3.3 Preparation of thin film by cast-cast method: The pre-reaction solution after microwave treatment was placed on a polyethylene plate, and the solution was evenly spread with a glass rod and air-dried at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 61.5%, and the breaking strength is 38.9 MPa. The mechanical tensile curve is shown in FIG7 .

Example 7

A method for preparing a regenerated silk fibroin film comprises the following steps:

7.1 Degumming

7.1.1 Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

7.1.2 Continue to boil the precipitate with the alkaline solution in 7.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

7.1.3 Repeat step 7.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, use picric acid carmine solution to detect whether the degumming is complete.

7.2 Processing

7.2.1 Filter the silk protein solution in 7.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse with 75% ethanol and deionized water in turn. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% aqueous solution of polyethylene glycol 20000. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

7.2.2 Using PEG400 as solvent, prepare the dry silk into a silk solution with a mass fraction of 9%; add genipin and a modifier to the silk solution to obtain a mixed solution, in which the mass concentration of genipin is 2% and the mass concentration of the modifier is 0.3%.

The improver is carboxymethyl chitosan.

7.3 Film Preparation

7.3.1 Pre-reaction: The mixed solution obtained in 7.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

7.3.2 Microwave treatment: The pre-reaction solution was treated with microwaves at 700 W for 20 s.

7.3.3 Preparation of thin film by cast-cast method: Place the pre-reaction solution after microwave treatment on a polyethylene plate, spread the solution evenly with a glass rod, and air-dry at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 61.3%, and the breaking strength is 37.5 MPa. The mechanical tensile curve is shown in FIG8 .

Example 8

A method for preparing a regenerated silk fibroin film comprises the following steps:

8.1 Degumming

8.1.1 Boil the silk for 30 minutes with an alkaline solution, filter and collect the precipitate. The alkaline solution is a mixture of a 0.05% sodium carbonate aqueous solution and a 0.03% sodium L-glutamate aqueous solution in a volume ratio of 20:1.

8.1.2 Continue to boil the precipitate with the alkaline solution in 8.1.1 for 30 minutes, collect the solution and check whether the degumming is complete.

8.1.3 Repeat step 8.1.2 until the mulberry silk is completely degummed. The completely degummed material is called silk fibroin solution. Here, use picric acid carmine solution to detect whether the degumming is complete.

8.2 Processing

8.2.1 Filter the silk protein solution in 8.1.3 with a sand core funnel, adjust the pH value of the filtrate to 4 with acetic acid solution, and then salt it out with a saturated sulfate solution for 2 hours. Centrifuge the precipitate to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel with a microporous filter membrane with a pore size of 0.2 μm, and filter and rinse it with 75% ethanol and deionized water in turn. Ultrasonic disperse the filter cake after elution in deionized water, transfer it into a dialysis bag with a molecular weight cutoff of 3500Da, and concentrate the solution in the dialysis bag with a 30% volume fraction of polyethylene glycol 20000 aqueous solution. Place the concentrated solution in a freeze dryer, pre-freeze it at -30°C, and vacuum dry it for 10 hours in a frozen state to obtain dry silk.

8.2.2 Using PEG400 as solvent, prepare a 10% silk solution with dry silk fibroin; add genipin and a modifier to the silk fibroin solution to obtain a mixed solution, in which the mass concentration of genipin is 2% and the mass concentration of the modifier is 0.3%.

The improver is carboxymethyl chitosan.

8.3 Film Forming

8.3.1 Pre-reaction: The mixed solution obtained in 8.2.2 is placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution is placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements.

8.3.2 Microwave treatment: The pre-reaction solution was treated with microwaves at 700 W for 20 s.

8.3.3 Preparation of thin film by cast-cast method: Place the pre-reaction solution after microwave treatment on a polyethylene plate, spread the solution evenly with a glass rod, and air-dry at room temperature to obtain a regenerated silk fibroin film with a thickness of 60 μm.

The tensile elongation at break prepared in this embodiment is 60.1%, and the breaking strength is 39.0 MPa. The mechanical tensile curve is shown in FIG9 .

3. Description of the key innovative features of the present invention

(1) Innovation 1: The present invention adopts an improved alkaline solution formula to improve degumming efficiency and shorten degumming time. Different methods are designed as follows:

Test 1-1: Boil the silk with an alkaline solution for 30 minutes, filter and collect the precipitate. The alkaline solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and a sodium L-glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1. Boil the precipitate with the alkaline solution for another 30 minutes to check whether the degumming is complete. Repeat the step of "boil the silk with the alkaline solution for 30 minutes to check whether the degumming is complete" until the silk is completely degummed.

Test 1-2: Boil the silk with 0.05% sodium carbonate aqueous solution for 30 minutes, filter and collect the precipitate. Boil the precipitate with 0.05% sodium carbonate aqueous solution for 30 minutes to check whether the degumming is complete. Repeat the step of "boil the silk with 0.05% sodium carbonate aqueous solution for 30 minutes to check whether the degumming is complete" until the silk is completely degummed.

Test 1-3: Boil the silk with tap water for 30 minutes, filter and collect the precipitate. Boil the precipitate with tap water for another 30 minutes to check whether the silk is completely degummed. Repeat the step of "boil the silk with tap water for 30 minutes and check whether the silk is completely degummed" until the silk is completely degummed.

In this test, picric acid carmine solution was used to detect whether the degumming was complete. The comparison results of degumming time of different methods are shown in Table 1. Among them, the prior art 0.05% sodium carbonate aqueous solution and tap water treatment were used as control groups.

Table 1 Comparison results of degumming time of different methods

experiment method Degumming time (min) Efficiency improvement rate (%) Test 1-1 120min 50% Test 1-2 180min 25% Tests 1-3 240min -

Note: In Table 1, the efficiency improvement rate is calculated based on the time of Tests 1-3, so the meaning of “-” is that the efficiency improvement rate of Tests 1-3 is not calculated.

The results in Table 1 show that the degumming time of mulberry silk is the shortest when the mixture of sodium carbonate aqueous solution and sodium L-glutamate aqueous solution is boiled. Therefore, we also explored the degumming time of different 0.05% sodium carbonate aqueous solution and 0.03% sodium L-glutamate aqueous solution in order to obtain the best ratio. The results are shown in Table 2. The results in Table 2 show that as the amount of sodium carbonate is gradually reduced, the degumming time shows a trend of first decreasing and then increasing. Among them, when the ratio of sodium carbonate aqueous solution to sodium L-glutamate aqueous solution is in the range of 10-30:1, the degumming time is shorter, which is conducive to saving time cost.

Table 2 Degumming time of different sodium carbonate aqueous solutions and sodium L-glutamate aqueous solutions

Proportion Degumming time (min) 40:1 240min 30:1 150min 20:1 120min 10:1 150min 5:1 240min

(2) Innovation 2: In the present invention, a modifier is added when preparing the mixed solution, and the modifier is carboxymethyl chitosan or glycerol. The addition of the modifier can improve the mechanical properties of the silk fibroin film. The experimental setting is as follows.

Experiment 2-1: The method of Example 1 was used to prepare completely degummed dry silk fibroin. Using PEG400 as solvent, the dry silk fibroin was prepared into a silk fibroin aqueous solution with a mass fraction of 8%; genipin and a modifier were added to the silk fibroin aqueous solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 2%, and the mass concentration of the modifier was 0.3%, and the modifier was carboxymethyl chitosan. The prepared mixed solution was placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution was placed on a magnetic stirrer for heating and stirring to meet the cross-linking requirements. The pre-reaction solution was placed on a polyethylene plate, and the solution was evenly spread with a glass rod, and air-dried at room temperature to obtain a regenerated silk fibroin protein film with a thickness of 60 μm.

Experiment 2-2: The method of Example 1 was used to prepare completely degummed dry silk fibroin. Using PEG400 as solvent, the dry silk fibroin was prepared into a silk fibroin aqueous solution with a mass fraction of 8%; genipin and a modifier were added to the silk fibroin aqueous solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 2%, and the mass concentration of the modifier was 0.3%, and the modifier was glycerol. The prepared mixed solution was placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution was placed on a magnetic stirrer for heating and stirring to meet the cross-linking requirements. The pre-reaction solution was placed on a polyethylene plate, and the solution was evenly spread with a glass rod, and air-dried at room temperature to obtain a regenerated silk fibroin protein film with a thickness of 60 μm.

Experiment 2-3: The method of Example 1 was used to prepare completely degummed dry silk fibroin. Using PEG400 as solvent, the dry silk fibroin was prepared into a silk fibroin aqueous solution with a mass fraction of 8%; Genipin was added to the silk fibroin aqueous solution to obtain a mixed solution, and the mass concentration of Genipin in the mixed solution was 2%. The prepared mixed solution was placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution was placed on a magnetic stirrer and heated and stirred to meet the cross-linking requirements. The pre-reaction solution was placed on a polyethylene plate, and the solution was evenly spread with a glass rod, and air-dried at room temperature to obtain a regenerated silk fibroin protein film with a thickness of 60 μm.

The mechanical properties of the regenerated silk fibroin membranes prepared with different modifiers are compared in Table 3. Among them, the method without modifier (experiment 2-3) is used as the control group. The results in Table 3 show that the membrane of experiment 2-2 has good mechanical properties, indicating that glycerol and carboxymethyl chitosan can improve the mechanical properties of the membrane.

Table 3 Mechanical properties of regenerated silk fibroin films prepared with different modifiers

Taking carboxymethyl chitosan as an example, we also explored the mechanical properties of regenerated silk fibroin membranes prepared with different concentrations of modifiers in the mixed solution, and the results are shown in Table 4. The results showed that the mechanical properties of the regenerated silk fibroin membranes at a concentration of 0.2-0.4% were better.

Table 4 Mechanical properties of regenerated silk fibroin films prepared with different modifiers

(3) Innovation point 3: The microwave treatment of the present invention increases the cross-linking effect and improves the mechanical properties of the membrane.

The different methods are designed as follows:

Experiment 3-1: The method of Example 1 was used to prepare completely degummed dry silk fibroin. PEG400 was used as a solvent to prepare a silk fibroin aqueous solution with a mass fraction of 8%. Genipin and a modifier were added to the silk fibroin aqueous solution to obtain a mixed solution, in which the mass concentration of Genipin in the mixed solution was 2%, and the mass concentration of the modifier was 0.3%, and the modifier was carboxymethyl chitosan. The prepared mixed solution was placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution was placed on a magnetic stirrer for heating and stirring to meet the cross-linking requirements. The pre-reaction solution was microwave-treated, and the microwave treatment conditions were 700W for 20s. The microwave-treated pre-reaction solution was placed on a polyethylene plate, and the solution was evenly spread with a glass rod, and air-dried at room temperature to obtain a regenerated silk fibroin protein film with a thickness of 60μm.

Experiment 3-2: The method of Example 1 was used to prepare completely degummed dry silk fibroin. Using PEG400 as solvent, the dry silk fibroin was prepared into a silk fibroin aqueous solution with a mass fraction of 8%; genipin and a modifier were added to the silk fibroin aqueous solution to obtain a mixed solution, in which the mass concentration of genipin in the mixed solution was 2%, and the mass concentration of the modifier was 0.3%, and the modifier was carboxymethyl chitosan. The prepared mixed solution was placed at 40°C and stirred for 10 hours to obtain a pre-reaction solution. In the pre-reaction operation step, the mixed solution was placed on a magnetic stirrer for heating and stirring to meet the cross-linking requirements. The pre-reaction solution was placed on a polyethylene plate, and the solution was evenly spread with a glass rod, and air-dried at room temperature to obtain a regenerated silk fibroin protein film with a thickness of 60 μm.

The comparison results of mechanical properties of regenerated silk fibroin membranes treated by different methods are shown in Table 5. The results show that microwave treatment helps to enhance the mechanical properties of the membrane.

Table 5 Mechanical properties of regenerated silk fibroin films prepared by different treatment methods

We also investigated the mechanical properties of the regenerated silk fibroin membranes prepared by different microwave power treatments, and the results are shown in Table 6. The results show that the mechanical properties of the membranes are better under a microwave power of 600-800 W.

Table 6 Mechanical properties of regenerated silk fibroin films prepared by different microwave power treatments

4. Application

4.1 Test cells: SD rat bone marrow mesenchymal stem cells

4.2 Cell seeding

Cut the regenerated silk fibroin membrane into small discs with a diameter of 8 mm and sterilize them by ultraviolet irradiation for 20 minutes. Soak the regenerated silk fibroin membrane in the medium for the growth of rat mesenchymal stem cells (the medium uses SD rat bone marrow mesenchymal stem cell complete medium, Saiye (Suzhou) Biotechnology Co., Ltd.) for 4 hours, then transfer it to a 6-well plate, and use a pipette to remove the medium equal to the inoculated cell suspension to ensure that the regenerated silk fibroin membrane can fully absorb the cell suspension, and the inoculation amount is 1×10 ⁵ cell/well. Then put the regenerated silk fibroin membrane after inoculation of cells into the incubator, and add 5ml SD rat bone marrow mesenchymal stem cell complete medium for culture after incubation for 3 hours.

The cell compatibility of the material is evaluated by studying the proliferation and growth of cells on the regenerated silk fibroin membrane. Bone marrow mesenchymal stem cells were cultured on the regenerated silk fibroin membrane, and the growth of cells on the membrane was observed on days 1 and 6. The results showed that the cells were evenly distributed on the regenerated silk fibroin membrane after 1 day of cell inoculation, and the cell density increased after 6 days of culture. It can be seen from the above-mentioned cell adhesion and proliferation experiments that the regenerated silk fibroin membrane prepared by the present invention is more conducive to the penetration, growth and proliferation of cells.

It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiment, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims (7)

1. A method for producing a regenerated silk fibroin film, comprising:

Degumming and drying silk to obtain dry silk fibroin;

Preparing a dry silk fibroin into a silk fibroin aqueous solution with the mass fraction of 8-10% by taking PEG300-PEG600 as a solvent; adding genipin and modifier into the silk fibroin aqueous solution to obtain a mixed solution, wherein the mass concentration of the genipin in the mixed solution is 2-5%, and the mass concentration of the modifier is 0.2-0.4%;

Wherein the modifier is carboxymethyl chitosan or glycerol;

Placing the mixed solution at 40-45 ℃ and stirring for 10-20h to obtain a pre-reaction solution;

The pre-reaction liquid is subjected to microwave treatment, and then a regenerated silk fibroin film is prepared;

the microwave treatment condition is 600-800W,20s.

2. The method for preparing a regenerated silk fibroin film according to claim 1, wherein PEG400 is used as a solvent to prepare a silk fibroin solution with a mass fraction of 8%; adding genipin and modifier into the silk fibroin solution to obtain a mixed solution, wherein the mass concentration of the genipin in the mixed solution is 2%, and the mass concentration of the modifier is 0.3%.

3. The method for producing a regenerated silk fibroin film according to claim 2, wherein the pre-reaction solution is prepared into the regenerated silk fibroin film by a casting method.

4. The method of claim 1, wherein the degumming process comprises:

(1) Boiling silk with alkali solution for 30min, filtering, and collecting precipitate; wherein the alkali solution is formed by mixing sodium carbonate aqueous solution with the mass fraction of 0.05% and L-sodium glutamate aqueous solution with the mass fraction of 0.03% according to the volume ratio of 20:1;

(2) The precipitate is further boiled with the aqueous alkali solution until the degumming of the mulberry silk is complete, and the degummed complete material is called silk fibroin solution.

5. A regenerated silk fibroin film prepared according to the method of any one of claims 1-4.

6. The regenerated silk fibroin film according to claim 5, wherein the regenerated silk fibroin film has a thickness of 60-80 μm.

7. The use of regenerated silk fibroin film according to claim 6 for promoting cell penetration, growth and proliferation.