CN117624675A - Regenerated silk fibroin membrane and preparation method and application thereof - Google Patents
Regenerated silk fibroin membrane and preparation method and application thereof Download PDFInfo
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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
Technical Field
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.
Background
The silk fibroin is a high molecular protein mainly containing 18 amino acids, has excellent mechanical property, biocompatibility and biodegradability, and has good application prospect in the fields of biological materials and tissue engineering.
Silk fibroin is currently used in a variety of forms (Lin Nan, zuo Baoji. Preparation and research progress of medical regenerated silk fibroin materials [ J ]. Modern silk science and technology, 2020,35 (3): 9.) including:
(1) The regenerated silk fibroin solution is prepared, and specifically comprises a degumming step and a dissolving step. Degumming is a key step in using silk fibroin as a biological material, since the synergistic effect of silk fibroin and dead-spot proteins produces a certain immune response. The silk can be dissolved after degumming, and the dissolved silk fibroin can be prepared into various dosage forms.
(2) The preparation of silk fibroin materials comprises the preparation of hydrogel, porous scaffolds, films, microspheres and other materials. The preparation method of the hydrogel comprises rotational flow shearing, ultrasonic oscillation, acid treatment, natural gel and the like; the porous bracket is prepared by the steps of freeze drying and particle pore-forming lamp; the film is prepared by casting method and other methods; the microsphere is prepared by spray drying, freeze drying and other methods.
Silk fibroin has excellent film-forming ability, and can be made into a silk fibroin film, which is also known in the art as a silk fibroin film or silk fibroin film (refer to patent CN 103200971B). Bian Ruiqi (Bian Ruiqi. Preparation of regenerated silk fibroin/L-polylactic acid-polycaprolactone copolymer composite nanofiber membrane scaffold and performance research [ D ]. Zhejiang university [2023-10-13 ]) explore a preparation method of a film compounded by regenerated silk fibroin/L-polylactic acid, and research results show that as the genipin content is increased, the diameter of nanofibers is gradually increased, the morphology becomes uneven, the crosslinking degree is gradually increased, the thermal performance of the material is gradually improved, and the mechanical performance at normal temperature is also gradually enhanced. In vitro cell culture experiments show that the addition of genipin has no toxic or side effect on L929 cells. In order to further improve the mechanical property of the electrostatic spinning plain protein nanofiber membrane, a high polymer of the L-polylactic acid-polycaprolactone copolymer [ P (LLA-CL) ] and silk fibroin are selected to be blended according to different mixing ratios, and the electrostatic spinning method is utilized to prepare the L-polylactic acid-polycaprolactone copolymer [ P (LLA-CL) ]/silk fibroin blended nanofiber membrane. In order to improve the mechanical property of the material and keep better biocompatibility, the composite nanofiber membrane with a silk fibroin/P (LLA-CL) skin-core structure is prepared by a coaxial electrostatic spinning method, and in order to further improve the mechanical property of the composite nanofiber membrane, a multi-wall carbon nano tube (MWCNTs) is mixed in a core layer of the material, and the mixing of the MWCNTs is beneficial to improving the biocompatibility of the nanofiber membrane.
By analyzing the prior art, it can be found that although the silk fibroin has good biological material preparation application prospect, the film prepared directly from the silk fibroin has poor mechanical properties and needs improvement. Bian Ruiqi et al have improved the mechanical properties of regenerated silk fibroin films, but have been technology in 2012 and have been in the past years, and the mechanical properties of regenerated silk fibroin films have yet to be improved.
Disclosure of Invention
In order to solve the technical problems, the invention provides a regenerated silk fibroin film, and a preparation method and application thereof.
An object of the present invention is to provide 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; the genipin can be subjected to polymerization reaction under certain conditions, can react with free amino groups on silk fibroin, and forms intramolecular and intermolecular covalent bonds, which are key to film formation. Genipin can also be crosslinked with carboxymethyl chitosan. Glycerin is dispersed in the film; wherein the modifier is carboxymethyl chitosan or glycerol. The modifier can simultaneously improve the mechanical property and the biocompatibility of the regenerated silk fibroin film; preferably, PEG300, PEG400 and PEG600 are used as solvents; the L-sodium glutamate has certain stability to light and heat, and the L-sodium glutamate is added into mulberry silk for treatment, so that the stability of silk fibroin can be improved, and degumming can be accelerated;
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.
Preferably, the preparation method of the regenerated silk fibroin film comprises the steps of preparing dry silk into silk aqueous solution with the 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%.
Preferably, in the preparation method of the regenerated silk fibroin film, PEG400 is used as a solvent, and dry silk is prepared into a silk solution with the 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%.
Preferably, in the preparation method of the regenerated silk fibroin film, the pre-reaction solution adopts a tape casting method to prepare the regenerated silk fibroin film.
Preferably, the preparation method of the regenerated silk fibroin film further comprises the following steps of: the pre-reaction liquid is treated by microwave, and then the regenerated silk fibroin film is prepared.
Preferably, the preparation method of the regenerated silk fibroin film has the microwave treatment condition of 600-800W and 20s.
Preferably, the above preparation method of regenerated silk fibroin film has microwave treatment condition of 700W,20s.
Preferably, the preparation method of the regenerated silk fibroin film comprises the following steps:
(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) Boiling the precipitation with the aqueous alkali solution for 30min, and detecting whether degumming is complete;
(3) Repeating the operation (2) until the complete degumming of the mulberry silk is detected, wherein the degummed material is called silk fibroin solution, and concentrating, drying and obtaining the dry silk fibroin.
The invention also provides a regenerated silk fibroin film prepared by the method.
Preferably, the regenerated silk fibroin film is 60-80 μm in thickness.
The invention also provides the use of a regenerated silk fibroin film, comprising, using the regenerated silk fibroin film to promote cell penetration, growth and proliferation.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts an improved alkali solution formula, improves the degumming efficiency of mulberry silk, shortens the degumming time, saves energy and protects environment.
According to the invention, the modifier is added in the preparation of the mixed solution, and the modifier is carboxymethyl chitosan or glycerol, so that the mechanical property of the silk fibroin film can be improved by adding the modifier.
The invention also adopts microwave treatment, increases the crosslinking effect and improves the mechanical property of the membrane.
In conclusion, the regenerated silk fibroin film prepared by the method has better mechanical property, good biocompatibility and high application value. The regenerated silk fibroin film prepared by the invention is more beneficial to the permeation, growth and proliferation of cells.
Drawings
FIG. 1 is a surface SEM image of a regenerated silk fibroin film prepared in example 1, scale 5 μm.
FIG. 2 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 1.
FIG. 3 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 2.
FIG. 4 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 3.
FIG. 5 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 4.
FIG. 6 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 5.
FIG. 7 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 6.
FIG. 8 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 7.
FIG. 9 is a graph showing the mechanical stretching of regenerated silk fibroin film prepared in example 8.
Detailed Description
In order that those skilled in the art will better understand the technical scheme of the present invention, the present invention will be further described with reference to specific embodiments and drawings.
In the description of the present invention, unless otherwise specified, all reagents are commercially available and methods are conventional in the art.
1. Main experiment reagent and equipment
Mulberry silk: commercial Zhejiang is a natural fiber. The silkworm cocoons are cut off, and pupa and molt in the cocoons are removed, so that white and clean cocoon shells are obtained.
Genipin: CAS number 6902-77-8, WUHCKEMIKE biomedical technology Co., ltd, content > 98%.
Carboxymethyl chitosan: CAS number 83512-85-0, guangdong Weng Jiang chemical agent Co., ltd., BR grade. The biocompatibility and the biosafety are good.
The microwave conditions are carried out in a microwave oven.
In the following examples and experiments, the mixed solution was placed on a magnetic stirrer for heating and stirring during the pre-reaction operation step to satisfy the crosslinking requirement.
2. Examples
Example 1
A method for preparing a regenerated silk fibroin film, comprising the following steps:
1 degumming
1.1.1 boiling mulberry 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.
1.1.2 the precipitate was continuously boiled with 1.1.1 aqueous alkali solution for 30min, the solution was collected and checked for complete degumming.
1.1.3 repeating step 1.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
1.2 treatment
1.2.1 the silk fibroin solution of 1.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is separated out and centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% volume fraction ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
1.2.2 preparing dry silk fibroin into silk fibroin solution with the mass fraction of 8% by taking PEG400 as a solvent; 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%.
The modifier is carboxymethyl chitosan.
1.3 film formation
1.3.1 pre-reaction: the mixed solution prepared in 1.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
1.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
1.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 62.8%, the breaking strength was 34.3MPa, the SEM image of the film was shown in FIG. 1, and the mechanical stretch profile was shown in FIG. 2.
Example 2
A method for preparing a regenerated silk fibroin film, comprising the following steps:
2.1 degumming
2.1.1 boiling mulberry 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.
2.1.2 the precipitate was further boiled with 2.1.1 aqueous alkali solution for 30min, the solution was collected and checked for complete degumming.
2.1.3 repeating step 2.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
2.2 treatment
2.2.1 the silk fibroin solution of 2.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% volume fraction ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
2.2.2 preparing the dry silk fibroin into a silk fibroin solution with the mass fraction of 8% by taking PEG400 as a solvent; 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 3%, and the mass concentration of the modifier is 0.3%.
The modifier is carboxymethyl chitosan.
2.3 film formation
2.3.1 pre-reaction: the mixed solution prepared in 2.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
2.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
2.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 61.3% and the breaking strength was 34.9MPa, the mechanical tensile graph being shown in FIG. 3.
Example 3
A method for preparing a regenerated silk fibroin film, comprising the following steps:
3.1 degumming
3.1.1 boiling mulberry 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.
3.1.2 the precipitate is further boiled with 3.1.1 aqueous alkali solution for 30min, and the solution is collected and checked whether degumming is complete.
3.1.3 repeating step 3.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
3.2 treatment
3.2.1 the silk fibroin solution of 3.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% volume fraction ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
3.2.2 preparing the dry silk fibroin into a silk fibroin solution with the mass fraction of 8% by taking PEG400 as a solvent; 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 4%, and the mass concentration of the modifier is 0.3%.
The modifier is carboxymethyl chitosan.
3.3 film formation
3.3.1 pre-reaction: the mixed solution prepared in 3.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
3.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
3.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 60.1% and the breaking strength was 36.4MPa, the mechanical tensile graph being shown in FIG. 4.
Example 4
A method for preparing a regenerated silk fibroin film, comprising the following steps:
4.1 degumming
4.1.1 boiling mulberry 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.
4.1.2 the precipitate was continued to be boiled with aqueous alkali solution of 4.1.1 for 30min with mulberry silk and the solution was collected.
4.1.3 repeating step 4.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
4.2 treatment
4.2.1 the silk fibroin solution of 4.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is separated out and centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
4.2.2 preparing the dry silk fibroin into a silk fibroin solution with the mass fraction of 8% by taking PEG400 as a solvent; 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 5%, and the mass concentration of the modifier is 0.3%.
The modifier is carboxymethyl chitosan.
4.3 film formation
4.3.1 pre-reaction: the mixed solution prepared in 4.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
4.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
4.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 60.0%, the breaking strength was 36.5MPa, and the mechanical tensile diagram was shown in FIG. 5.
Example 5
A method for preparing a regenerated silk fibroin film, comprising the following steps:
5.1 degumming
5.1.1 boiling mulberry 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.
5.1.2 the precipitate is further boiled with 5.1.1 aqueous alkali solution for 30min, and the solution is collected and checked whether degumming is complete.
5.1.3 repeating step 5.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
5.2 treatment
5.2.1 the silk fibroin solution of 5.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% volume fraction ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
5.2.2 preparing the dry silk fibroin into a silk fibroin solution with the mass fraction of 8% by taking PEG400 as a solvent; 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 0.2%.
The modifier is carboxymethyl chitosan.
5.3 film formation
5.3.1 pre-reaction: the mixed solution prepared in 5.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
5.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
5.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 66.8% and the breaking strength was 32.4MPa, and the mechanical tensile diagram is shown in FIG. 6.
Example 6
A method for preparing a regenerated silk fibroin film, comprising the following steps:
6.1 degumming
6.1.1 boiling mulberry 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.
6.1.2 the precipitate is further boiled with 6.1.1 aqueous alkali solution for 30min, and the solution is collected and checked whether degumming is complete.
6.1.3 repeating step 6.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
6.2 treatment
6.2.1 the silk fibroin solution of 6.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is separated out and centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% volume fraction ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
6.2.2 preparing the dry silk fibroin into a silk fibroin solution with the mass fraction of 8% by taking PEG400 as a solvent; 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 0.4%.
The modifier is carboxymethyl chitosan.
6.3 film formation
6.3.1 pre-reaction: the mixed solution prepared in 6.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
6.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
6.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 61.5%, the breaking strength was 38.9MPa, and the mechanical tensile diagram was shown in FIG. 7.
Example 7
A method for preparing a regenerated silk fibroin film, comprising the following steps:
7.1 degumming
7.1.1 boiling mulberry 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.
7.1.2 the precipitate was further boiled with 7.1.1 aqueous alkali for 30min, the solution was collected and checked for complete degumming.
7.1.3 repeating step 7.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
7.2 treatment
7.2.1 the silk fibroin solution of 7.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is separated out and centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% volume fraction ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
7.2.2 preparing a silk fibroin solution with a mass fraction of 9% by taking PEG400 as a solvent; 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%.
The modifier is carboxymethyl chitosan.
7.3 film formation
7.3.1 pre-reaction: the mixed solution prepared in 7.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
7.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
7.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 61.3% and the breaking strength was 37.5MPa, and the mechanical tensile diagram is shown in FIG. 8.
Example 8
A method for preparing a regenerated silk fibroin film, comprising the following steps:
8.1 degumming
8.1.1 boiling mulberry 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.
8.1.2 the precipitate is further boiled with 8.1.1 aqueous alkali solution for 30min, and the solution is collected and checked whether degumming is complete.
8.1.3 repeating step 8.1.2 until the silk is degummed to completion, the degummed complete material being called silk fibroin solution. Wherein, picric acid carmine solution is used for detecting whether degumming is complete.
8.2 treatment
8.2.1 the silk fibroin solution of 8.1.3 is filtered by a sand core funnel, the pH value of the filtrate is adjusted to 4 by acetic acid solution, and then the filtrate is salted out by sulfate saturated solution for 2 hours, and the precipitated precipitate is centrifugally separated to obtain protein precipitate. The protein precipitate was transferred to a sand core suction filter funnel with a microporous filter membrane of 0.2 μm pore size and rinsed by suction filtration with 75% ethanol and deionized water in sequence. The filter cake after leaching is dispersed in deionized water by ultrasonic, and is moved into a dialysis bag with the molecular weight cutoff of 3500Da, and the solution in the dialysis bag is concentrated by using 30 percent of polyethylene glycol 20000 water solution by volume fraction. And (3) placing the concentrated solution into a freeze dryer, pre-freezing at the temperature of minus 30 ℃, and vacuum drying for 10 hours in a frozen state to obtain the dry silk fibroin.
8.2.2 preparing a silk fibroin solution with a mass fraction of 10% by taking PEG400 as a solvent; 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%.
The modifier is carboxymethyl chitosan.
8.3 film formation
8.3.1 pre-reaction: the mixed solution prepared in 8.2.2 is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement.
8.3.2 microwave treatment: and carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s.
8.3.3 preparation of film by casting method: and placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The tensile elongation at break prepared in this example was 60.1% and the breaking strength was 39.0MPa, and the mechanical tensile diagram is shown in FIG. 9.
3. Description of the key innovation points of the present invention
(1) Innovation point 1: the invention adopts an improved alkali solution formula, improves the degumming efficiency and shortens the degumming time. The different methods are designed as follows:
test 1-1: boiling mulberry 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. And (3) continuously boiling the mulberry silk for 30min by using the aqueous alkali solution to detect whether degumming is complete. Repeating the steps of boiling the mulberry silk for 30min by using the aqueous alkali solution, and detecting whether degumming is complete or not until the degumming of the mulberry silk is complete.
Test 1-2: boiling mulberry silk with 0.05% sodium carbonate aqueous solution for 30min, filtering, and collecting precipitate. And (3) continuously boiling the mulberry silk for 30min by using 0.05% sodium carbonate aqueous solution to detect whether degumming is complete. Repeating the steps of boiling the mulberry silk with 0.05% sodium carbonate aqueous solution for 30min, and detecting whether degumming is complete or not until the mulberry silk is completely degummed.
Test 1-3: boiling mulberry silk with tap water for 30min, filtering, and collecting precipitate. And (5) continuously boiling the mulberry silk for 30min by tap water to detect whether degumming is complete. Repeating the step of boiling the mulberry silk for 30min by tap water, and detecting whether degumming is complete or not until the mulberry silk is completely degummed.
In this test, a picric acid carmine solution was used to determine if degumming was complete. The comparative results of the degumming times of the different processes are shown in table 1. Wherein, sodium carbonate aqueous solution with mass fraction of 0.05% and tap water treatment in the prior art are used as a control group.
Table 1 comparison of degumming time results for different methods
| Test method | Degumming time (min) | Efficiency improvement rate (%) |
| Run 1-1 | 120min | 50％ |
| Run 1-2 | 180min | 25％ |
| Runs 1-3 | 240min | - |
Note that: in Table 1, the efficiency improvement ratio was calculated by taking the time of test 1-3 as a control, so the meaning of "-" is not to calculate the efficiency improvement ratio of test 1-3.
The results in Table 1 show that boiling mulberry silk using a mixture of aqueous sodium carbonate and aqueous sodium L-glutamate has the shortest degumming time. We have therefore also explored different degumming times for aqueous 0.05% sodium carbonate and 0.03% sodium l-glutamate solutions in order to obtain the optimal ratio. The results are shown in Table 2. The results in Table 2 show that the degumming time shows a trend of decreasing before increasing with the gradual decrease of the sodium carbonate dosage, wherein, when the sodium carbonate aqueous solution and the L-sodium glutamate aqueous solution are in the range of 10-30:1, the degumming time is shorter, which is beneficial to saving the time cost.
TABLE 2 degumming time of different aqueous sodium carbonate solutions and aqueous sodium L-glutamate solutions
| Proportion of | Degumming time (min) |
| 40:1 | 240min |
| 30:1 | 150min |
| 20:1 | 120min |
| 10:1 | 150min |
| 5:1 | 240min |
(2) Innovation point 2: according to the invention, the modifier is added in the preparation of the mixed solution, and the modifier is carboxymethyl chitosan or glycerol, so that the mechanical property of the silk fibroin film can be improved by adding the modifier. The experimental setup is as follows.
Test 2-1: the degummed, fully dried silk fibroin was prepared using the procedure of example 1. Preparing a dry silk fibroin into 8% silk fibroin aqueous solution by taking PEG400 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%, the mass concentration of the modifier is 0.3%, and the modifier is carboxymethyl chitosan. The prepared mixed solution is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement. And placing the pre-reaction solution on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain the regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
Test 2-2: the degummed, fully dried silk fibroin was prepared using the procedure of example 1. Preparing a dry silk fibroin into 8% silk fibroin aqueous solution by taking PEG400 as a solvent; adding genipin and modifier into silk fibroin aqueous solution to obtain mixed solution, wherein the mass concentration of genipin in the mixed solution is 2%, the mass concentration of the modifier is 0.3%, and the modifier is glycerin. The prepared mixed solution is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement. And placing the pre-reaction solution on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain the regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
Test 2-3: the degummed, fully dried silk fibroin was prepared using the procedure of example 1. Preparing a dry silk fibroin into 8% silk fibroin aqueous solution by taking PEG400 as a solvent; adding genipin into the silk fibroin aqueous solution to obtain a mixed solution, wherein the mass concentration of the genipin in the mixed solution is 2%. The prepared mixed solution is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement. And placing the pre-reaction solution on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain the regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The comparative results of the mechanical properties of regenerated silk fibroin films prepared with different modifiers are shown in table 3. Among them, the method without modifier (test 2-3) was used as a control group. The results in Table 3 show that the film of test 2-2 has good mechanical properties, indicating that glycerol and carboxymethyl chitosan can improve the mechanical properties of the film.
TABLE 3 mechanical Properties of regenerated silk fibroin films prepared with different improvers
Taking carboxymethyl chitosan as an example, we also explore the mechanical properties of regenerated silk fibroin films prepared by modifying agents with different concentrations in the mixed solution, and the results are shown in table 4. The results show that the regenerated silk fibroin film with the concentration of 0.2-0.4% has better mechanical properties.
TABLE 4 mechanical Properties of regenerated silk fibroin films prepared with different improvers
(3) Innovation point 3: the invention improves the crosslinking effect and the mechanical property of the membrane by microwave treatment.
The different methods are designed as follows:
test 3-1: the degummed, fully dried silk fibroin was prepared using the procedure of example 1. Preparing a dry silk fibroin into 8% silk fibroin aqueous solution by taking PEG400 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%, the mass concentration of the modifier is 0.3%, and the modifier is carboxymethyl chitosan. The prepared mixed solution is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement. And carrying out microwave treatment on the pre-reaction liquid, wherein the microwave treatment condition is 700W and 20s. And placing the pre-reaction solution after microwave treatment on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain a regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
Test 3-2: the degummed, fully dried silk fibroin was prepared using the procedure of example 1. Preparing a dry silk fibroin into 8% silk fibroin aqueous solution by taking PEG400 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%, the mass concentration of the modifier is 0.3%, and the modifier is carboxymethyl chitosan. The prepared mixed solution is placed at 40 ℃ 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 for heating and stirring so as to meet the crosslinking requirement. And placing the pre-reaction solution on a polyethylene plate, uniformly spreading the solution by using a glass rod, and air-drying at room temperature to obtain the regenerated silk fibroin film, wherein the thickness of the regenerated silk fibroin film is 60 mu m.
The comparative results of the mechanical properties of regenerated silk fibroin films of different treatments are shown in table 5. The results show that the microwave treatment helps to enhance the mechanical properties of the film.
TABLE 5 mechanical Properties of regenerated silk fibroin films prepared by different treatments
We have also explored the mechanical properties of regenerated silk fibroin films prepared by different microwave power treatments, and the results are shown in table 6. The results show that the film has better mechanical property under the microwave power of 600-800W.
TABLE 6 mechanical Properties of regenerated silk fibroin films prepared by different microwave Power treatments
4. Application of
4.1 test cells: SD rat bone marrow mesenchymal stem cell
4.2 cell seeding
Cutting the regenerated silk fibroin film into small discs with the diameter of 8mm, and performing ultraviolet irradiation for 20min for sterilization. Immersing regenerated silk fibroin film in the culture medium for growth of mesenchymal stem cells of rat (SD rat bone marrow mesenchymal stem cells complete culture medium, siro biosciences Co., ltd.) for 4 hr, transferring into 6-well plate, and removing the same amount of culture medium as the inoculated cell suspension with a pipette to ensure that the regenerated silk fibroin film can fully absorb cell suspension, the inoculation amount is 1×10 5 cell/well. Then placing the regenerated silk fibroin film after inoculating the cells into an incubator,after 3 hours incubation, 5m1 SD rat bone marrow mesenchymal stem cells were added to complete medium for culture.
The cytocompatibility of the material was evaluated by studying the cell proliferation and growth in regenerated silk fibroin films. Bone marrow mesenchymal stem cells were cultured on regenerated silk fibroin membrane, and growth of cells on the membrane was observed on days 1 and 6. The results showed that cells were uniformly distributed on the regenerated silk fibroin film 1 day after cell inoculation, and cell density increased after 6 days of culture. As can be seen from the above-mentioned cell adhesion and proliferation experiments, the regenerated silk fibroin film prepared by the present invention is more favorable for cell permeation, growth and proliferation.
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
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.
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| WO2012154136A1 (en) * | 2011-05-06 | 2012-11-15 | Jesadanont, Sukanya | Method for preparing silk sericin-pva scaffold using genipin as crosslinking agent |
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