CN117624675A - 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 membrane and its preparation method and application

Technical field

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

Background technique

Silk fibroin is a polymer 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.

Currently, 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.), include:

(1) Prepare a regenerated silk fibroin solution, specifically including a degumming step and a dissolving step. Since the synergistic effect of silk fibroin and dead keratin produces a certain immune response, degumming is a key step in using silk fibroin as a biomaterial. Silk can be dissolved after degumming, and the dissolved silk fibroin can be made into various dosage forms.

(2) Preparing silk fibroin materials, including preparing hydrogels, porous scaffolds, films, microspheres and other materials. The preparation methods of hydrogel include cyclonic shearing, ultrasonic oscillation, acid treatment, natural gel, etc.; the porous scaffold is prepared by freeze-drying and particle porogen lamp steps; the film is prepared by casting method and other methods; the microspheres are spray-dried. , freeze-drying and other methods.

Silk fibroin has excellent film-forming ability and can be made into silk fibroin film. The existing technology is also called silk fibroin film or silk protein film (refer to patent CN103200971B). Research by Bian Ruiqi (Bian Ruiqi. Preparation and performance study of regenerated silk fibroin/L-polylactic acid-polycaprolactone copolymer composite nanofiber membrane scaffold [D]. Zhejiang Sci-Tech University [2023-10-13]) A method for preparing a film composed of regenerated silk fibroin/L-polylactic acid composite was developed. The research results show that as the genipin content increases, the diameter of the nanofibers gradually becomes larger, the morphology becomes uneven, and the degree of cross-linking Gradually increasing, the thermal properties of the material gradually improve, and the mechanical properties at room temperature also gradually increase. 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 the electrospun silk fibroin nanofiber membrane, the polymer L-polylactic acid-polycaprolactone copolymer [P(LLA-CL)] was selected to blend with silk fibroin in different mixing ratios. L-polylactic acid-polycaprolactone copolymer [P(LLA-CL)]/silk fibroin blend nanofiber membrane was prepared by electrospinning method. In order to improve the mechanical properties of the material while maintaining good biocompatibility, silk fibroin/P (LLA-CL) skin-core structure composite nanofiber membranes were prepared through coaxial electrospinning. In order to further improve the composite To improve the mechanical properties of the fiber membrane, multi-walled carbon nanotubes (MWCNTs) are mixed into the core layer of the material, and the mixing of MwCNTs is beneficial to improving the biocompatibility of the nanomembrane.

By analyzing the above-mentioned existing technologies, we can find that although silk fibroin has good application prospects in biomaterial preparation, the mechanical properties of membranes directly prepared from silk fibroin are poor and need to be improved. Although the method of Bian Ruiqi and others has improved the mechanical properties of the regenerated silk fibroin membrane, it is already a 2012 technology. It is old and the mechanical properties of the regenerated silk fibroin membrane need to be improved.

Contents of the invention

In order to solve the above technical problems, the present invention provides a regenerated silk fibroin film and its preparation method and application. Compared with the existing technology, the regenerated silk fibroin film prepared by the present invention has better mechanical properties and high application value.

An object of the present invention is to provide a preparation method for regenerated silk fibroin membrane, including:

Silk is degummed and dried to obtain dry silk fibroin;

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

Place the mixed solution at 40-45°C and stir for 10-20 hours to obtain a pre-reaction solution;

The pre-reaction solution is used to prepare a regenerated silk fibroin membrane.

Preferably, the above-mentioned preparation method of the regenerated silk fibroin film is to configure the dry silk fibroin into a silk fibroin aqueous solution with a mass fraction of 8-10%; add genipin and a modifier to the silk fibroin aqueous solution to obtain a mixed solution, and the genipin in the mixed solution The mass concentration of the improver is 2-5%, and the mass concentration of the improver is 0.2-0.4%.

Preferably, the above-mentioned preparation method of regenerated silk fibroin film uses PEG400 as a solvent to configure dry silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and improver to the silk fibroin solution to obtain a mixed solution. The mixed solution The mass concentration of genipin is 2%, and the mass concentration of the modifier is 0.3%.

Preferably, in the above method for preparing a regenerated silk fibroin membrane, the pre-reaction solution adopts a flow-through method to prepare a regenerated silk fibroin membrane.

Preferably, the above-mentioned method for preparing a regenerated silk fibroin film also includes a microwave treatment step: the pre-reaction solution is subjected to microwave treatment, and then a regenerated silk fibroin film is prepared.

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

Preferably, in the above preparation method of the regenerated silk fibroin film, the microwave treatment conditions are 700W, 20s.

Preferably, in the preparation method of the above-mentioned regenerated silk fibroin membrane, the degumming method includes:

(1) Boil the silk with an alkali solution for 30 minutes, filter and collect the precipitate; wherein the alkali solution is a sodium carbonate aqueous solution with a mass fraction of 0.05% and an L-sodium glutamate aqueous solution with a mass fraction of 0.03% mixed in a volume ratio of 20:1 made;

(2) Continue to boil the mulberry silk with the aqueous alkali solution for 30 minutes to detect whether the degumming is complete;

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

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

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

The invention also provides an application of a 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 alkali solution formula to improve the degumming efficiency of mulberry silk, shorten the degumming time, and is energy-saving and environmentally friendly.

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

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

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 invention is more conducive to cell penetration, growth and proliferation.

Description of drawings

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

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

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

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

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

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

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

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

Figure 9 is a mechanical stretching 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 will be further described below with reference to 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 common techniques in this field.

1. Main experimental reagents and equipment

Mulberry silk: commercially available in Zhejiang, is a natural fiber. It is to cut the mulberry cocoon open, remove the pupa and molt inside, and obtain a white cocoon shell.

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

Carboxymethyl chitosan: CAS number 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. Embodiment

Example 1

A method for preparing a regenerated silk fibroin membrane, including the following steps:

1Degumming

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

Wherein, the alkali solution is a mixture of a sodium carbonate aqueous solution with a mass fraction of 0.05% and an L-sodium glutamate aqueous solution with a mass fraction of 0.03% in a volume ratio of 20:1.

1.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

1.2 Processing

1.2.1 Filter the silk fibroin 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. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and sequentially use 75% ethanol and deionized water to perform suction filtration and elution. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

1.2.2 Using PEG400 as the solvent, prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 2%. The mass concentration of the improver is 0.3%.

The improving agent is carboxymethyl chitosan.

1.3 Film making

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

1.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

1.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 62.8%, and the breaking strength is 34.3MPa. See Figure 1 for the SEM image of the film, and Figure 2 for the mechanical tensile curve.

Example 2

A method for preparing a regenerated silk fibroin membrane, including the following steps:

2.1 Degumming

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

2.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

2.2 Processing

2.2.1 Filter the silk fibroin 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 out with a saturated sulfate solution for 2 hours. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and sequentially use 75% ethanol and deionized water to perform suction filtration and elution. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

2.2.2 Using PEG400 as the solvent, prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 3%. The mass concentration of the improver is 0.3%.

The improving agent is carboxymethyl chitosan.

2.3 Film making

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

2.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

2.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 61.3%, and the breaking strength is 34.9MPa. See Figure 3 for the mechanical tensile curve.

Example 3

A method for preparing a regenerated silk fibroin membrane, including the following steps:

3.1 Degumming

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

3.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

3.2 Processing

3.2.1 Filter the silk fibroin 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 out with a saturated sulfate solution for 2 hours. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and sequentially use 75% ethanol and deionized water to perform suction filtration and elution. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

3.2.2 Use PEG400 as the solvent to prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 4%. The mass concentration of the improver is 0.3%.

The improving agent is carboxymethyl chitosan.

3.3 Film making

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

3.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

3.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 60.1%, and the breaking strength is 36.4MPa. See Figure 4 for the mechanical tensile curve.

Example 4

A method for preparing a regenerated silk fibroin membrane, including the following steps:

4.1 Degumming

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

4.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

4.2 Processing

4.2.1 Filter the silk fibroin 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 out with a saturated sulfate solution for 2 hours. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and perform suction filtration and elution with 75% ethanol and deionized water in sequence. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

4.2.2 Using PEG400 as the solvent, prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 5%. The mass concentration of the improver is 0.3%.

The improving agent is carboxymethyl chitosan.

4.3 Film making

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

4.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

4.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 60.0%, and the breaking strength is 36.5MPa. See Figure 5 for the mechanical tensile curve.

Example 5

A method for preparing a regenerated silk fibroin membrane, including the following steps:

5.1 Degumming

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

5.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

5.2 Processing

5.2.1 Filter the silk fibroin 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 out with a saturated sulfate solution for 2 hours. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and sequentially use 75% ethanol and deionized water to perform suction filtration and elution. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

5.2.2 Using PEG400 as the solvent, prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 2%. The mass concentration of the improver is 0.2%.

The improving agent is carboxymethyl chitosan.

5.3 Film making

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

5.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

5.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 66.8%, and the breaking strength is 32.4MPa. See Figure 6 for the mechanical tensile curve.

Example 6

A method for preparing a regenerated silk fibroin membrane, including the following steps:

6.1 Degumming

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

6.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

6.2 Processing

6.2.1 Filter the silk fibroin 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 out with a saturated sulfate solution for 2 hours. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and sequentially use 75% ethanol and deionized water to perform suction filtration and elution. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

6.2.2 Using PEG400 as the solvent, prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 8%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 2%. The mass concentration of the improver is 0.4%.

The improving agent is carboxymethyl chitosan.

6.3 Film making

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

6.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

6.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 61.5%, and the breaking strength is 38.9MPa. See Figure 7 for the mechanical tensile curve.

Example 7

A method for preparing a regenerated silk fibroin membrane, including the following steps:

7.1 Degumming

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

7.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

7.2 Processing

7.2.1 Filter the silk fibroin 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. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and sequentially use 75% ethanol and deionized water to perform suction filtration and elution. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

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

The improving agent is carboxymethyl chitosan.

7.3 Film making

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

7.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

7.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 61.3%, and the breaking strength is 37.5MPa. See Figure 8 for the mechanical tensile curve.

Example 8

A method for preparing a regenerated silk fibroin membrane, including the following steps:

8.1 Degumming

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

8.1.2 Continue to boil the mulberry silk with the alkali 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. Among them, picric acid carmine solution is used to check whether the degumming is complete.

8.2 Processing

8.2.1 Filter the silk fibroin 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 out with a saturated sulfate solution for 2 hours. The precipitate is precipitated and centrifuged to obtain a protein precipitate. Transfer the protein precipitate to a sand core suction filtration funnel equipped with a microporous filter membrane with a pore size of 0.2 μm, and perform suction filtration and elution with 75% ethanol and deionized water in sequence. The rinsed filter cake was ultrasonically dispersed in deionized water and moved into a dialysis bag with a molecular weight cutoff of 3500 Da. The solution in the dialysis bag was concentrated with an aqueous solution of polyethylene glycol 20000 with a volume fraction of 30%. Put the concentrated liquid into a freeze dryer, pre-freeze it at -30°C, and dry it under vacuum for 10 hours in the frozen state to obtain dry silk fibroin.

8.2.2 Use PEG400 as the solvent to prepare the dried silk fibroin into a silk fibroin solution with a mass fraction of 10%; add genipin and modifier to the silk fibroin solution to obtain a mixed solution. The mass concentration of genipin in the mixed solution is 2%. The mass concentration of the improver is 0.3%.

The improving agent is carboxymethyl chitosan.

8.3 Film making

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

8.3.2 Microwave treatment: Microwave the pre-reaction solution. The microwave treatment conditions are 700W, 20s.

8.3.3 Preparation of films by the casting method: Place the microwave-treated pre-reaction solution 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. The thickness of the regenerated silk fibroin film is 60μm.

The tensile elongation at break prepared in this example is 60.1%, and the breaking strength is 39.0 MPa. See Figure 9 for the mechanical tensile curve.

3. Explanation of key innovation points of the present invention

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

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

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

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

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

Table 1 Comparative 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% Trial 1-3 240min -

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

The results in Table 1 show that the degumming time of mulberry silk is the shortest when boiled using a mixture of sodium carbonate aqueous solution and L-sodium glutamate aqueous solution. Therefore, we also explored the degumming time of different 0.05% sodium carbonate aqueous solution and 0.03% L-sodium glutamate aqueous solution in order to obtain the best ratio. See Table 2 for results. The results in Table 2 show that as the amount of sodium carbonate gradually decreases, the degumming time first decreases and then increases. Among them, when the sodium carbonate aqueous solution and L-sodium glutamate aqueous solution are in the range of 10-30:1, the degumming time The time is shorter, which helps save time and cost.

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

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

(2) Innovation point 2: In the present invention, an improving agent is added when preparing the mixed solution. The improving agent is carboxymethyl chitosan or glycerol. The addition of the improving agent can improve the mechanical properties of the silk fibroin membrane. The experimental settings are as follows.

Test 2-1: Use the method of Example 1 to prepare completely degummed dried silk fibroin. Using PEG400 as the solvent, the dried 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. The mass concentration of genipin in the mixed solution was 2%, and the modifier was 2%. The mass concentration is 0.3%, and the improving agent is carboxymethyl chitosan. The prepared mixed solution was stirred at 40°C for 10 hours to obtain a pre-reaction solution. In the pre-reaction step, the mixed solution is heated and stirred on a magnetic stirrer to meet the cross-linking requirements. Place the pre-reaction solution 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.

Test 2-2: Use the method of Example 1 to prepare completely degummed dried silk fibroin. Using PEG400 as the solvent, the dried 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. The mass concentration of genipin in the mixed solution was 2%, and the modifier was 2%. The mass concentration is 0.3%, and the improving agent is glycerin. The prepared mixed solution was stirred at 40°C for 10 hours to obtain a pre-reaction solution. In the pre-reaction step, the mixed solution is heated and stirred on a magnetic stirrer to meet the cross-linking requirements. Place the pre-reaction solution 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.

Test 2-3: Use the method of Example 1 to prepare completely degummed dried silk fibroin. Using PEG400 as the solvent, the dried 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 stirred at 40°C for 10 hours to obtain a pre-reaction solution. In the pre-reaction step, the mixed solution is heated and stirred on a magnetic stirrer to meet the cross-linking requirements. Place the pre-reaction solution 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 comparative results of the mechanical properties of regenerated silk fibroin membranes prepared with different improvers are shown in Table 3. Among them, the method without adding modifier (Test 2-3) was used as the control group. The results in Table 3 show that the membrane in Test 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 membranes prepared with different improvers

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. The results are shown in Table 4. The results show that the mechanical properties of the regenerated silk fibroin membrane at a concentration of 0.2-0.4% are better.

Table 4 Mechanical properties of regenerated silk fibroin membranes prepared with different improvers

(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:

Test 3-1: Use the method of Example 1 to prepare completely degummed dried silk fibroin. Using PEG400 as the solvent, the dried 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. The mass concentration of genipin in the mixed solution was 2%, and the modifier was 2%. The mass concentration is 0.3%, and the improving agent is carboxymethyl chitosan. The prepared mixed solution was stirred at 40°C for 10 hours to obtain a pre-reaction solution. In the pre-reaction step, the mixed solution is heated and stirred on a magnetic stirrer to meet the cross-linking requirements. Microwave the pre-reaction solution, and the microwave treatment conditions are 700W, 20s. Place the microwave-treated pre-reaction solution 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.

Test 3-2: Use the method of Example 1 to prepare completely degummed dried silk fibroin. Using PEG400 as the solvent, the dried 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. The mass concentration of genipin in the mixed solution was 2%, and the modifier was 2%. The mass concentration is 0.3%, and the improving agent is carboxymethyl chitosan. The prepared mixed solution was stirred at 40°C for 10 hours to obtain a pre-reaction solution. In the pre-reaction step, the mixed solution is heated and stirred on a magnetic stirrer to meet the cross-linking requirements. Place the pre-reaction solution 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 comparative results of the mechanical properties of regenerated silk fibroin membranes treated with different treatment methods are shown in Table 5. The results showed that microwave treatment helped to enhance the mechanical properties of the membrane.

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

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

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

4.Application

4.1 Test cells: SD rat bone marrow mesenchymal stem cells

4.2 Cell seeding

The regenerated silk fibroin film was cut into small discs with a diameter of 8 mm and sterilized by ultraviolet irradiation for 20 minutes. The regenerated silk fibroin membrane was soaked in the growth medium of rat mesenchymal stem cells (the medium used SD rat bone marrow mesenchymal stem cell complete medium, Saiye (Suzhou) Biotechnology Co., Ltd.), and then Transfer to a 6-well plate, and use a pipette to remove the same amount of culture medium as the inoculated cell suspension to ensure that the regenerated silk fibroin membrane can fully absorb the cell suspension. The inoculated volume is 1×10 ⁵ cells/well. Then, the regenerated silk fibroin membrane after seeding with cells was placed in an incubator, and after incubation for 3 hours, 5 ml of SD rat bone marrow mesenchymal stem cell complete culture medium was added for culture.

The material's cytocompatibility was evaluated by studying cell proliferation and growth on regenerated silk fibroin membranes. 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 1 day after cell seeding, and the cell density increased after 6 days of culture. It can be seen from the above cell adhesion and proliferation experiments that the regenerated silk fibroin membrane prepared by the present invention is more conducive to cell penetration, growth and proliferation.

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 numerical 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 preferred embodiments. Although the preferred embodiments of the invention have been described, those skilled in the art will be able to make additional changes and modifications to these embodiments once the basic inventive concepts are apparent. Therefore, it is intended that the appended claims be construed to include the preferred embodiments and all changes and modifications that fall within the scope of the 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 invention. In this way, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims (10)

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

degumming and drying 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; 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;

and preparing the regenerated silk fibroin film by the pre-reaction liquid.

2. The method for producing a regenerated silk fibroin film according to claim 1, wherein the dry silk fibroin is prepared into a silk fibroin aqueous solution with a mass fraction of 8-10%; 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%.

3. The method for preparing a regenerated silk fibroin film according to claim 2, 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%.

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

5. The method for preparing a regenerated silk fibroin film according to claim 4, further comprising the step of microwave treatment: the pre-reaction liquid is subjected to microwave treatment, and then the regenerated silk fibroin film is prepared.

6. The method for producing a regenerated silk fibroin film according to claim 5, wherein the microwave treatment condition is 600 to 800w,20s.

7. The method of claim 6, 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.

8. A regenerated silk fibroin film prepared according to the method of any one of claims 1-7.

9. The regenerated silk fibroin film according to claim 8, wherein the regenerated silk fibroin film has a thickness of 60-80 μm.

10. The use of a regenerated silk fibroin film according to claim 8 for promoting cell penetration, growth and proliferation.