CN107298766B - Regenerated tussah silk fibroin solution and preparation method thereof - Google Patents
Regenerated tussah silk fibroin solution and preparation method thereof Download PDFInfo
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- silk fibroin
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000000502 dialysis Methods 0.000 claims abstract description 33
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- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims description 60
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 38
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- 238000009210 therapy by ultrasound Methods 0.000 claims description 22
- 239000007864 aqueous solution Substances 0.000 claims description 17
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- 235000019253 formic acid Nutrition 0.000 claims description 16
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- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 1
- ZJZXSOKJEJFHCP-UHFFFAOYSA-M lithium;thiocyanate Chemical compound [Li+].[S-]C#N ZJZXSOKJEJFHCP-UHFFFAOYSA-M 0.000 description 1
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- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/09—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in organic liquids
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Abstract
The invention relates to a regenerated tussah silk fibroin solution and a preparation method thereof, the solution is composed of pure tussah silk fibroin, the silk fibroin is mainly of a nanofiber structure, the diameter of the silk fibroin is less than 50nm, and the length of the silk fibroin is between 100 nm and 500 nm. The preparation method of the solution directly takes natural tussah silk as a raw material, and the natural tussah silk is obtained after the dissolution of an organic solvent and the dialysis treatment of deionized water. The preparation method disclosed by the invention has the advantages that the organic solvent is directly used for dissolving, the problem of strong degradation caused by high-temperature dissolution of high-concentration neutral salt is avoided, the process is simple, the obtained solution has no reagent residue, and meanwhile, the nano bionic structure is contained, so that the biological safety is excellent.
Description
Technical Field
The invention relates to a natural polymer solution and a preparation method thereof, in particular to a regenerated tussah silk fibroin solution and a preparation method thereof.
Background
Biomedical materials are one of the leading research hotspots in common life science and material science at present. At present, many degradable high molecular materials applied to the biomedical field such as tissue engineering are developed, but most of the original research works of the materials belong to foreign countries and apply for a plurality of patents in China. Therefore, appropriate raw materials are actively searched, the biological materials with the independent intellectual property rights in China are developed, and the method has important significance for reducing the dependence of the biological materials such as tissue engineering scaffolds and the like in China on foreign countries, cultivating new high and new technology industries and realizing the sustainable development of national economy. Tussah silk (Antheraea pernyi) is natural silk fiber which is reeled by taking Tussah cocoons as raw materials and is a valuable resource in China. The annual production of 3-4 ten thousand tons of tussah cocoons in China accounts for more than 90% of the total output of the tussah cocoons in the world, which is a precious resource peculiar to China in the world, but the tussah silk is mainly used as a common textile raw material at present, and the embodied economic value is very limited. In addition to low-value textile materials, tussah silk is increasingly in demand as biomedical materials, such as enzyme immobilization carriers, cell culture substrates, wound dressings, artificial skin, tissue engineering scaffolds, and the like.
From sixty years to date, researchers at home and abroad have conducted more researches on amino acid composition, molecular structure, physical properties and the like of tussah silk fibroin. Compared with the silk of the family, the tussah silk has stronger hygroscopicity, heat resistance and acid and alkali resistance, and the amino acid composition is also different: the main amino acid content sequence of the tussah silk fibroin is alanine (Ala) > glycine (Gly) > serine (Ser) > tyrosine (Tyr), and the tussah silk fibroin is rich in aspartic acid (Asp) and arginine (Arg), so that the tussah silk fibroin contains an arginine-glycine-aspartic acid (RGD) tripeptide sequence. The RGD sequence is used as a recognition site for the combination of a cell membrane integrin receptor and an extracellular ligand, mediates the interaction between cells and extracellular matrix and between cells, and can promote the adhesion of the cells to the bracket. The excellent characteristics endow the tussah silk fibroin with unique advantages and wide application prospect in the field of biological materials such as tissue engineering scaffolds and the like.
Natural tussah silk has limited application due to its single morphology (fibrous) and high crystallinity and is difficult to degrade. A number of salt solutions are known to dissolve natural mulberry silk, such as calcium chloride (CaCl)2) Magnesium chloride (MgCl)2) And lithium bromide (LiBr), etc., so that a mulberry silk fibroin (B silk fibroin) solution can be easily prepared. However, tussah silk is more difficult to dissolve than mulberry silk due to the strong intermolecular interaction, and the tussah silk is difficult to dissolve in a system for dissolving mulberry silk. Strong acid and strong base can cause the tussah silk fibroin to be severely hydrolyzed, the molecular weight is greatly reduced, for example, the molecular weight after being dissolved by hydrochloric acid, phosphoric acid and sulfuric acid is about 200-5000D, so that the silk peptide and the silk fibroin powder are usually prepared by only using the strong acid and the strong base, and the application of the silk peptide and the silk fibroin powder is greatly limited.
The molecular weight of the tussah silk fibroin prepared by the microwave hydrolysis method in recent years is between 2000-3000D, and the tussah silk fibroin with the molecular weight still can not meet the requirements of medical materials, so that other dissolving methods need to be explored to obtain the tussah silk fibroin with larger molecular weight, and the requirements of reshaping processing of a tussah silk fibroin solution and the physical and mechanical properties of a finished product can be met. The current methods for obtaining the tussah silk fibroin solution mainly comprise two methods: the first method is to directly extract the liquid tussah silk fibroin from the silk gland behind the mature tussah, the method has simple operation steps, but is only suitable for preparing tussah silk fibroin solution in a small amount and carrying out related research, and can not be industrialized; in addition, the method is to obtain a regenerated tussah silk fibroin solution by using a neutral salt dissolving method, the neutral salt capable of dissolving tussah silk at present mainly comprises calcium nitrate and lithium thiocyanate (LiSCN), although the method can improve the acquisition amount of the tussah silk fibroin solution, the dissolving efficiency is low, the process is complex, and more importantly, the original multi-stage structure of the tussah silk can be seriously damaged, so that the performance of the regenerated tussah silk fibroin material is poor, and the application requirement is difficult to meet.
Disclosure of Invention
Aiming at the defects of the prior art, the invention solves the technical problem of providing a regenerated tussah silk fibroin solution and a preparation method thereof, and the tussah silk fibroin obtained by the method keeps the original multi-stage fibril structure.
In order to solve the technical problem, the technical scheme of the invention is realized as follows:
a preparation method of a regenerated tussah silk fibroin solution comprises the following steps:
(1) removing impurities from tussah silk, degumming and performing ultrasonic treatment to obtain pretreated tussah silk;
(2) placing the pretreated tussah silk into trifluoroacetic acid aqueous solution containing formic acid to prepare mixed solution, and stirring and dissolving at normal temperature to obtain regenerated tussah silk fibroin organic solution;
(3) and (3) putting the regenerated tussah silk fibroin organic solution into deionized water for dialysis to obtain the regenerated tussah silk fibroin solution.
In the technical scheme, the power of the ultrasonic treatment is 1-10 kW, and the time is 30-50 minutes; preferably, the power of the ultrasonic treatment is 3-6 kW, and the time is 35-40 minutes.
In the technical scheme, the mass concentration of the trifluoroacetic acid aqueous solution is 80-90%; preferably, the mass concentration of the trifluoroacetic acid aqueous solution is 85-88%.
In the technical scheme, in the mixed solution, the mass of the formic acid is 3-5% of that of the trifluoroacetic acid.
In the technical scheme, in the mixed solution, the concentration of the pretreated tussah silk (silk dissolution concentration) is 5-10 wt%; the preferable silk dissolution concentration is 7-8 wt%.
In the technical scheme, the stirring and dissolving time is 5-20 minutes.
In the technical scheme, the dialysis temperature is 4-10 ℃, and the dialysis time is 3-5 days.
The invention also discloses a regenerative tussah silk fibroin material and a preparation method thereof, and the regenerative tussah silk fibroin material comprises the following steps:
(1) removing impurities from tussah silk, degumming and performing ultrasonic treatment to obtain pretreated tussah silk;
(2) placing the pretreated tussah silk into trifluoroacetic acid aqueous solution containing formic acid to prepare mixed solution, and stirring and dissolving at normal temperature to obtain regenerated tussah silk fibroin organic solution;
(3) putting the regenerated tussah silk fibroin organic solution into deionized water for dialysis to obtain a regenerated tussah silk fibroin aqueous solution;
(4) and preparing the regenerated tussah silk fibroin aqueous solution to obtain the regenerated tussah silk fibroin material.
In the technical scheme, the regenerative tussah silk fibroin material comprises a regenerative tussah silk fibroin film and regenerative tussah silk fibroin gel.
The invention substantially adopts trifluoroacetic acid as a solvent, adds a small amount of formic acid, directly and quickly dissolves the tussah silk at normal temperature, removes the organic solvent through dialysis to obtain a regenerated tussah silk fibroin aqueous solution, and takes the nano-fiber as a main structure; the regenerated silk fibroin material prepared by the method has good mechanical property.
Compared with the prior art, the invention has the following advantages:
the preparation method disclosed by the invention uses an ultrasonic pretreatment process, loosens the compact structure in the silk, is favorable for solvent permeation to accelerate the dissolution of the silk, and is favorable for regulating and controlling the dissolution degree of silk protein by the solvent.
The regenerated tussah silk fibroin solution and the preparation method thereof disclosed by the invention only adopt the organic solvent, avoid the serious degradation of high-concentration neutral salt to silk under the high-temperature condition, and realize the purpose of regulating and controlling the silk fibroin dissolving degree through the mixing proportion of the organic solvent.
Compared with the traditional tussah silk fibroin solution, the regenerated tussah silk fibroin solution disclosed by the invention is remarkably characterized in that tussah silk fibroin nano fibrils are used as main units, the nano fibrils are natural tussah silk, and are formed by self-assembly in a non-regeneration process and are natural tussah silk fibroin nano fibrils.
The regenerated tussah silk fibroin solution disclosed by the invention can be prepared by taking an organic solvent as a dissolving solution for the first time and stirring at normal temperature.
Drawings
FIG. 1 is a scanning electron micrograph of the form of fibroin in a solution prepared in accordance with one embodiment of the present invention;
FIG. 2 is an infrared spectrum of the regenerated silk fibroin membrane prepared in the first embodiment of the present invention before and after treatment;
FIG. 3 is a scanning electron microscope image of silk fibroin morphology in the solution prepared in the second embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Boiling natural tussah silk with 0.5wt% sodium bicarbonate solution for 60min, washing with deionized water, and air drying to obtain degummed silk. Placing the degummed silk in a beaker, adding deionized water, and then carrying out ultrasonic treatment with the ultrasonic power of 5kW and the ultrasonic treatment time of 40 minutes. Placing degummed silk in 86wt% trifluoroacetic acid water solution containing 3% formic acid (trifluoroacetic acid mass is base number) at room temperature, wherein the mass fraction of the silk is 7%, and magnetically stirring for 10min to obtain regenerated silk fibroin trifluoroacetic acid solution. And then injecting the solution into a dialysis bag, putting the dialysis bag into flowing deionized water for dialysis for 3d, and obtaining the purified regenerated tussah silk fibroin nanofiber aqueous solution at the dialysis temperature of 10 ℃. The silk fibroin in the solution is in a nano-fiber shape, the length of the fiber is 0.1-15 mu m, the diameter is 0.01-1 mu m (shown in figure 1), and the molecular weight distribution range of the silk fibroin is 150-300 kDa. Injecting the regenerated silk fibroin solution into a polyethylene dish, and naturally drying to obtain a regenerated tussah silk fibroin film, wherein the protein structure is an amorphous structure, and is converted into a beta-folding crystal structure (shown in figure 2) after being treated by an ethanol water solution, and the film has excellent mechanical properties, the breaking strength is 98MPa, and the breaking elongation is 6.5%; the regenerated tussah silk fibroin gel with good biocompatibility can be obtained through gelation treatment; the wet compression modulus is 96 MPa; can be applied to the preparation of tissue engineering scaffolds.
Comparative example one:
see the process of example one, wherein formic acid is not contained, and the molecular weight distribution range of the silk fibroin is 50-70 kDa; the breaking strength and the breaking elongation of the regenerated tussah silk fibroin film are 46MPa and 3.5 percent respectively; the wet compression modulus of the regenerated tussah silk fibroin gel is 36 MPa.
Example two:
boiling natural tussah silk with 0.05wt% sodium bicarbonate solution for 60min, fully washing with deionized water, and repeatedly drying for three times to obtain degummed silk. Placing the degummed silk in a beaker, adding deionized water, and then carrying out ultrasonic treatment, wherein the ultrasonic power is 3kW, and the ultrasonic treatment time is 35 minutes. Placing degummed silk in 85wt% trifluoroacetic acid water solution containing 5% formic acid (trifluoroacetic acid mass is base number) at room temperature, magnetically stirring the silk with mass fraction of 8% for 5min to obtain regenerated silk fibroin organic solution. And then injecting the solution into a dialysis bag, putting the dialysis bag into flowing deionized water for dialysis for 5d, and obtaining the purified regenerated tussah silk fibroin nanofiber aqueous solution at the dialysis temperature of 4 ℃. The silk fibroin in the solution is in a nano-fiber shape, as shown in figure 3, the molecular weight distribution range of the silk fibroin is 140-200 kDa; naturally drying to obtain the regenerated tussah silk fibroin film with the breaking strength of 95MPa and the breaking elongation of 6.1 percent; the regenerated tussah silk fibroin gel with good biocompatibility can be obtained through gelation treatment; the wet compression modulus is 94 MPa; can be applied to the preparation of tissue engineering scaffolds.
Comparative example two:
see the process of example two, wherein formic acid is not contained, and the molecular weight distribution range of the silk fibroin is 40-65 kDa; the breaking strength and the breaking elongation of the regenerated tussah silk fibroin film are 40MPa and 3.2 percent respectively; the wet compression modulus of the regenerated tussah silk fibroin gel is 38 MPa.
Example three:
boiling natural tussah silk with 0.25wt% sodium bicarbonate solution for 60min, fully washing with deionized water, and repeatedly drying for three times to obtain degummed silk. Placing the degummed silk in a beaker, adding deionized water, and then carrying out ultrasonic treatment with the ultrasonic power of 6kW and the ultrasonic treatment time of 35 minutes. Placing degummed silk in 88wt% trifluoroacetic acid water solution containing 4% formic acid (trifluoroacetic acid mass as base) at room temperature, wherein the mass fraction of the silk is 7%, and magnetically stirring for 20min to obtain regenerated silk fibroin organic solution. Then, injecting the solution into a dialysis bag, placing the dialysis bag in flowing deionized water for dialysis for 3d, and obtaining a purified regenerated tussah silk fibroin nanofiber aqueous solution at the dialysis temperature of 4 ℃, wherein the molecular weight distribution range of the silk fibroin is 110-180 kDa; naturally drying to obtain a regenerated tussah silk fibroin film, wherein the protein structure is an amorphous structure, is converted into a beta-folding crystal structure after being treated by an ethanol water solution, and has the breaking strength of 89MPa and the breaking elongation of 6.1 percent; the regenerated tussah silk fibroin gel with good biocompatibility can be obtained through gelation treatment; the wet compression modulus is 95 MPa; can be applied to the preparation of tissue engineering scaffolds.
Comparative example three:
referring to the process of the third embodiment, ultrasonic treatment is not adopted, and the molecular weight distribution range of the silk fibroin is 90-120 kDa; the breaking strength and the breaking elongation of the regenerated tussah silk fibroin film are 68MPa and 5.5 percent respectively; the wet compression modulus of the regenerated tussah silk fibroin gel is 82 MPa.
Example four:
boiling natural tussah silk with 0.5wt% sodium bicarbonate solution for 60min, fully washing with deionized water, and repeatedly drying for three times to obtain degummed silk. Placing the degummed silk in a beaker, adding deionized water, and then carrying out ultrasonic treatment with the ultrasonic power of 1kW and the ultrasonic treatment time of 50 minutes. Placing degummed silk in 80wt% trifluoroacetic acid water solution containing 4% formic acid (trifluoroacetic acid mass is base number) at room temperature, wherein the mass fraction of the silk is 5%, and magnetically stirring for 15min to obtain regenerated silk fibroin organic solution. Then, injecting the solution into a dialysis bag, placing the dialysis bag in flowing deionized water for dialysis for 5d, and obtaining a purified regenerated tussah silk fibroin nanofiber aqueous solution at the dialysis temperature of 10 ℃, wherein the molecular weight distribution range of the silk fibroin is 160-320 kDa; naturally drying to obtain a regenerated tussah silk fibroin film, wherein the protein structure is an amorphous structure, is converted into a beta-folding crystal structure after being treated by an ethanol water solution, and has the breaking strength of 78MPa and the breaking elongation of 5.5 percent; the regenerated tussah silk fibroin gel with good biocompatibility can be obtained through gelation treatment; the wet compression modulus is 82 MPa; can be applied to the preparation of tissue engineering scaffolds.
Comparative example four:
referring to the process of the fourth embodiment, the amount of formic acid is 8%, and the molecular weight distribution range of the silk fibroin is 8-10 kDa; the breaking strength and the breaking elongation of the regenerated tussah silk fibroin film are 40MPa and 2.9 percent respectively; the wet compression modulus of the regenerated tussah silk fibroin gel is 31 MPa.
Example five:
boiling natural tussah silk with 0.25wt% of sodium carbonate solution for 60min, fully washing with deionized water, and repeatedly airing twice to obtain the degummed silk. Placing the degummed silk in a beaker, adding deionized water, and then carrying out ultrasonic treatment with the ultrasonic power of 10kW and the ultrasonic treatment time of 30 minutes. Placing degummed silk in 90wt% trifluoroacetic acid aqueous solution containing 4% formic acid (trifluoroacetic acid mass is base number) at room temperature, wherein the silk mass fraction is 5%, and magnetically stirring for 10min to obtain regenerated silk fibroin organic solution. Then, injecting the solution into a dialysis bag, putting the dialysis bag into flowing deionized water for dialysis for 3d, and obtaining a purified and regenerated tussah silk fibroin nanofiber aqueous solution at the dialysis temperature of 4 ℃, wherein the molecular weight distribution range of the silk fibroin is 130-200 kDa; naturally drying to obtain a regenerated tussah silk fibroin film, wherein the protein structure is an amorphous structure, is converted into a beta-folding crystal structure after being treated by an ethanol water solution, and has the breaking strength of 75MPa and the breaking elongation of 5.6 percent; the regenerated tussah silk fibroin gel with good biocompatibility can be obtained through gelation treatment; the wet compression modulus is 83 MPa; can be applied to the preparation of tissue engineering scaffolds.
Comparative example five:
see the process of the fifth embodiment, wherein the ultrasonic power is 20kW, and the molecular weight distribution range of the silk fibroin is 30-80 kDa; the breaking strength and the breaking elongation of the regenerated tussah silk fibroin film are 58MPa and 4.5 percent respectively; the wet compression modulus of the regenerated tussah silk fibroin gel is 49 MPa.
Example six:
boiling natural tussah silk with 0.5wt% of sodium carbonate solution for 60min, fully washing with deionized water, and air drying to obtain degummed silk. Placing the degummed silk in a beaker, adding deionized water, and then carrying out ultrasonic treatment with the ultrasonic power of 6kW and the ultrasonic treatment time of 50 minutes. Placing degummed silk in 85wt% trifluoroacetic acid water solution containing 4% formic acid (trifluoroacetic acid mass is base number) at room temperature, magnetically stirring the silk with the mass fraction of 10% for 10min to obtain regenerated silk fibroin organic solution. Then, injecting the solution into a dialysis bag, putting the dialysis bag into flowing deionized water for dialysis for 3d, and obtaining a purified regenerated tussah silk fibroin nanofiber aqueous solution at the dialysis temperature of 4 ℃, wherein the molecular weight distribution range of the silk fibroin is 160-300 kDa; naturally drying to obtain a regenerated tussah silk fibroin film, wherein the protein structure is an amorphous structure, is converted into a beta-folding crystal structure after being treated by an ethanol water solution, and has the breaking strength of 72MPa and the breaking elongation of 5.5 percent; the regenerated tussah silk fibroin gel with good biocompatibility can be obtained through gelation treatment; the wet compression modulus is 81 MPa; can be applied to the preparation of tissue engineering scaffolds.
Comparative example six:
referring to the process of the sixth embodiment, 90% formic acid is adopted for dissolution, trifluoroacetic acid is not adopted, and the molecular weight distribution range of the silk fibroin is 6-8 kDa; the breaking strength and the breaking elongation of the regenerated tussah silk fibroin film are 28MPa and 2.5 percent respectively; the wet compression modulus of the regenerated tussah silk fibroin gel is 29 MPa.
Claims (5)
1. A preparation method of a regenerated tussah silk fibroin solution is characterized by comprising the following steps:
(1) removing impurities from tussah silk, degumming and performing ultrasonic treatment to obtain pretreated tussah silk, wherein the ultrasonic treatment is to place the degummed silk in a beaker, add deionized water and then perform ultrasonic treatment; the power of the ultrasonic treatment is 1-10 kW, and the time is 30-50 minutes;
(2) placing the pretreated tussah silk into trifluoroacetic acid aqueous solution containing formic acid to prepare mixed solution, and stirring and dissolving at normal temperature to obtain regenerated tussah silk fibroin organic solution; in the mixed solution, the mass of the formic acid is 3-5% of that of the trifluoroacetic acid; the mass concentration of the trifluoroacetic acid aqueous solution is 80-90%; in the mixed solution, the concentration of the pretreated tussah silk is 5-10 wt%;
(3) and (3) putting the regenerated tussah silk fibroin organic solution into deionized water for dialysis to obtain the regenerated tussah silk fibroin solution.
2. The preparation method according to claim 1, wherein the ultrasonic treatment is carried out at a power of 3 to 6kW for a time of 35 to 40 minutes.
3. The preparation method according to claim 1, wherein the mass concentration of the trifluoroacetic acid aqueous solution is 85-88%; in the mixed solution, the concentration of the pretreated tussah silk is 7-8 wt%.
4. The preparation method according to claim 1, wherein the stirring and dissolving time is 5 to 20 minutes; the dialysis temperature is 4-10 ℃, and the dialysis time is 3-5 days.
5. The regenerated tussah silk fibroin solution prepared by the preparation method of claim 1.
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| CN112545910A (en) * | 2020-12-24 | 2021-03-26 | 苏州至明至信生物科技有限公司 | Glabridin water dispersion solution, preparation method and application thereof |
| CN113018203A (en) * | 2020-12-24 | 2021-06-25 | 苏州至明至信生物科技有限公司 | Aqueous retinol dispersion, method for preparing same and use thereof |
| CN113279282A (en) * | 2021-06-25 | 2021-08-20 | 陕西科技大学 | Silk-reinforced aramid nanofiber film and preparation method thereof |
| CN113696562B (en) * | 2021-08-23 | 2022-05-24 | 西南大学 | High-strength transparent silkworm cocoon composite material and preparation method and application thereof |
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