Patent Application
20220289652
The present invention relates to the technical field of materials, and more particularly to use of an aromatic alcohol as a structural inducer for silk fibroin and a method for treating silk fibroin materials.
Silk consists of outer sericin (20-30%) and inner silk fibroin (70-80%), from which regenerated silk fibroin can be obtained through dissolution, desalination and the like. Regenerated silk fibroin can be used to prepare materials in the form of particles, fibers, films, gels, tubes, scaffolds, and others. These materials are widely used many research fields such as the textile industry, light industry chemistry, drug control release carriers, and tissue engineering scaffolds.
Water-insoluble silk fibroin materials are usually obtained by physical methods such as pH, temperature, ultrasound, or vortex, chemical methods such as induction with a chemical reagent such as methanol, ethanol, and polyethylene glycol, etc., and chemically cross-linking with Genipin or HRP to realize the intramolecular and intermolecular cross-linking of free silk fibroin molecules.
Currently, a commonly used structural inducer for silk fibroin, such as steam, methanol, and ethanol, can be used to change the secondary structure of silk fibroin molecules in a dry silk fibroin material, to realize the intermolecular and intramolecular cross-linking through a physical force, thus making the material insoluble in water, and have strong mechanical performance and chemical and biological stability.
These methods still need improvement. For example, on the one hand, the mechanical performance (rigidity and toughness) of the prepared silk fibroin material is weak, and fails to meet the requirements in industry. Particularly silk fibroin materials are difficult to be formed into a stable shape at a lower concentration. On the other hand, two or more formed dry silk fibroin materials, cannot be bonded to each other or cannot be bonded to other substrate materials after treatment with the above inducers.
To solve the above technical problems, an object of the present invention is to provide use of an aromatic alcohol as a structural inducer for silk fibroin and a method for treating silk fibroin materials. The present invention discloses use of an aromatic alcohol as a structural inducer for silk fibroin, which provides a new direction for the development of reagents that induce changes in the secondary structure of silk fibroin.
A first object of the present invention is to provide use of an aromatic alcohol as a structural inducer for silk fibroin.
Preferably, the aromatic alcohol is selected from the group consisting of the compounds having a structure below:
wherein m=0-5; X is methylene or oxygen; and R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen (—H), phenyl, hydroxyl (—OH), carboxyl (—COOH), aldehyde group (—CHO), keto (—CO—), ester group (—COO—), ether group (—O—), amino (—NH2), nitro (—NO2), cyano (—CN), amido (—CO—NH2), azo (—N═N—), amidino (HN═C(NH2)—), oximido, a hydrazone group, halo (—X), an acid halide (—CO—X), a sulfonic acid group (—SO3H), a disulfide bond (—S—S—), sulfydryl (—SH), phosphino (—PH2), and a phosphate group (—PO3H2).
Preferably, the aromatic alcohol has a structure of
in which a=1-5, and b=1-5.
Preferably, the aromatic alcohol is in a liquid or vapor state.
Preferably, the aromatic alcohol is selected from the group consisting of benzyl alcohol, phenethyl alcohol, phenylpropanol, phenylbutanol, phenylpentanol, 2-phenoxyethanol and any combination thereof.
A second object of the present invention is to provide a treatment method for obtaining a water-insoluble silk fibroin, which comprises the following steps:
bringing silk fibroin into contact with an aromatic alcohol-containing vapor or liquid to induce the structural transformation of silk fibroin, so as to obtain the water-insoluble silk fibroin.
Preferably, the aromatic alcohol is selected from the group consisting of the compounds having a structure below:
in which m=0-5; X is methylene or oxygen; and R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen (—H), phenyl, hydroxyl (—OH), carboxyl (—COOH), an aldehyde group (—CHO), keto (—CO—), an ester group (—COO—), an ether group (—O—), amino (—NH2), nitro (—NO2), cyano (—CN), amido (—CO—NH2), azo (—N═N—), amidino (HN═C(NH2)—), oximido, a hydrazone group, halo (—X), an acid halide (—CO—X), an sulfonic acid group (—SO3H), a disulfide bond (—S—S—), sulfydryl (—SH), phosphino (—PH2), and a phosphate group (—PO3H2).
Preferably, the aromatic alcohol has a structure of
in which a=1-5, and b=1-5.
Preferably, the aromatic alcohol is selected from the group consisting of benzyl alcohol, phenethyl alcohol, phenylpropanol, phenylbutanol, phenylpentanol and 2-phenoxyethanol.
Preferably, the silk fibroin is in a dry state and has a moisture content of not higher than 30%. If the treatment method of the present invention is applied to a formed dry silk fibroin material, the treated silk fibroin has good viscosity, thereby improving its adhesion to other silk fibroin materials or substrates.
Preferably, the silk fibroin is soaked in an aromatic alcohol-containing liquid or the silk fibroin is fumigated with an aromatic alcohol-containing vapor.
Preferably, when an aromatic alcohol-containing liquid is used for treatment, the treatment time is not less than 5 seconds; and the treatment temperature is 20-200° C., and more preferably, 20-120° C.
Preferably, when an aromatic alcohol-containing vapor is used for treatment, the treatment time is not less than 5 min, and the treatment temperature is 25-200° C., and more preferably, 20-120° C.
Preferably, the aromatic alcohol-containing vapor or liquid also comprises water and a conventional alcohol inducer such as methanol and ethanol.
In the present invention, when an aromatic alcohol-containing vapor or liquid is used for inducing the silk fibroin, not only a neat silk fibroin material, but also a composite materials consisting of silk fibroin and an additional material can be obtained. The additional material includes: inorganic metal materials such as ceramics, glass and cement; polymer materials such as rubber, chemical fibers and plastics; and structural and functional composite materials. The resulting products are applicable to the fields of medicine, environment, textile, light industry and medical instruments.
In the present invention, “inducer” means a chemical reagent that can transform a water-soluble silk protein material into a water-insoluble silk protein material. The secondary structure of silk fibroin molecules in the former material is dominated by disordered and random coils. In the latter material, the secondary structure of silk fibroin is dominated by ordered α-helices or β-sheets.
By means of the above technical solutions, the present invention has the following advantages.
The present invention provides new use of an aromatic alcohol as a structural inducer for silk fibroin, which provides a new direction for the development of new structural inducers for silk fibroin.
The present invention also provides a treatment method for obtaining a water-insoluble silk fibroin, wherein an aromatic alcohol-containing vapor or liquid is used for treatment. The method has simple process, the treated silk fibroin is insoluble in water, and has a higher mechanical strength than a product obtained by a traditional treatment method. After treatment with the inducer of the present invention, the mechanical performance of the silk fibroin material and the bonding strength between the silk fibroin material and a substrate material are over 50% higher than those of a silk fibroin material obtained by a traditional treatment method (induction with methanol, ethanol, and steam).
The above description is only a summary of the technical solutions of the present invention. To make the technical means of the present invention clearer and implementable in accordance with the disclosure of the specification, the preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The specific embodiments of the present invention will be described in further detail with reference to embodiments. The following embodiments are intended to illustrate the present invention, instead of limiting the scope of the present invention.
(1) 250 g of raw silkworm silk was weighed, and cut into segments having a length of about 15 cm each. 100 liters of pure water was heated by an electric heating tank, and 212 g of anhydrous sodium carbonate was slowly added when the water was nearly boiling, stirred until uniform, and continuously heated to boiling. Then raw silk was added, and timing was started when the raw silk was all submerged in the water. The raw silk was stirred every 7 min, and taken out after 30 min. The degummed silk was fed to a washing machine, and rinsed twice with pure water, following a set rinsing procedure. The dehydrated degummed silk was fed to an air dry oven, and dried for 12 to 18 hrs.
(2) A 9.3 M lithium bromide solution was prepared, and according to a bath ratio of the degummed silk to the lithium bromide solution of 1/4, the degummed silk was added. The degummed silk was dissolved in a dry oven at 60° C. for 4 hrs, taken out, and cooled to room temperature. The dissolved silk fibroin solution was added to a dialysis bag with a molecular weight cut-off of 3500, and dialyzed against pure water for 48 hrs, during which the water was changed 6 times. The dialyzed silk fibroin solution was filtered with 5 layers of gauze to remove impurities, and stored in a refrigerator at 2-8° C. for later use.
(1) The silk fibroin solution obtained in Example 1 was diluted to a content of 4% by weight, and 2 mL of the solution was added to a plastic petri dish with a diameter of 3.5 cm. The solution was stood for more than 3 hrs, until the internal air bubbles were completely removed. Then, the solution was placed in a fume hood at room temperature, and air dried overnight to form a film. The moisture content (W %) of the silk fibroin film was determined by weight loss method:
W %=(W1−W2)/W1*100% (1)
where W1 represents the initial dry weight of the newly prepared silk fibroin film, and W2 represents the weight of the silk fibroin film after thorough drying (at 120° C. for 12 hrs). The experiment was repeated 5 times to improve the reliability of the experiment.
Experimental results show that the internal moisture content is about 3.64%, after the silk fibroin film is naturally air-dried for 12 hrs at room temperature.
(1) The silk fibroin films with a moisture content of about 3.64% prepared in Example 2 were respectively soaked in water, methanol, benzyl alcohol, ethanol, phenethyl alcohol, 2-phenoxyethanol, 3-Phenyl-1-propanol, 4-phenylbutanol and 1,3-butanediol, for 5 min at room temperature. Then the silk fibroin film soaked in each reagent was taken out, the remaining reagent on the surface was absorbed with filter paper, and the resulting sample was weighed. The weight change of the sample after treatment is shown in
(2) Then, the silk fibroin films of the present invention soaked in various alcohols were washed with ethanol to remove the non-specifically bound aromatic alcohol, and then washed with deionized water to remove residual ethanol. The sample was placed in a fume hood and dried overnight at room temperature.
The dried silk fibroin films of the above groups were dried, the dry weight W3 of each silk fibroin film was determined, and the weight change rate (Wm %) of each group of sample was calculated from the weights of the sample before and after treatment.
W
m %=(W1−W3)/W1*100% (2)
where W1 represents the initial dry weight of the silk fibroin film in Example 2. The experiment was repeated 5 times to improve the reliability of the experiment.
(3) The silk fibroin films with a moisture content of about 3.64% prepared in Example 2 were respectively placed on an upper layer of a specialized evaporating dish, and the lower layer was a certain amount of water, methanol, benzyl alcohol, ethanol, phenethyl alcohol, 2-phenoxyethanol, 3-Phenyl-1-propanol, 4-phenylbutanol and 1,3-butanediol respectively. The temperature of each group of experiments was set to 60° C., and the time was 3 hrs. Under the above conditions, the various reagents became a vapor, and were brought into contact with the silk fibroin films in the form of vapors.
(4) The silk fibroin film treated by the above method was taken out, the remaining reagent on the surface was removed by blow drying with an air blower, and the silk fibroin film was weighed immediately. The dry weight W4 of each silk fibroin film was determined, and The moisture content (W %) of the silk fibroin film was determined by weight loss method:
W
n %=(W1−W4)/W1*100% (3)
where W1 represents the initial dry weight of the silk fibroin film in Example 2. The experiment was repeated 5 times to improve the reliability of the experiment.
Further, the performance of the silk fibroin film after the above treatment was characterized as follows.
A. According to the test standard, the silk fibroin film treated with a liquid or vapor of different reagents in Step (1) and Step (3) was cut into a silk fibroin film of a certain area, and scanned for the wave number by an infrared spectrometer in a range of 400-4000 cm−1.
B. Considering the different boiling points and saturated vapor pressures of different reagents, the influence of fumigation temperature on the induction of silk fibroin films was further investigated. In the present invention, the silk fibroin films prepared in Example 2 were respectively fumigated with water, methanol, benzyl alcohol, ethanol, phenethyl alcohol, 2-phenoxyethanol, 3-Phenyl-1-propanol, 4-phenylbutanol, and 1,3-butanediol and other reagents. The fumigation temperature was 25° C., 60° C. and 90° C., and the fumigation time was 2 hrs.
According to the test standard, the silk fibroin film treated following the above method was prepared into samples, and the breaking strength of the silk fibroin film was tested by Instron-3365 testing machine.
C. The silk fibroin films treated with vapors of different reagents at 25° C., 60° C. and 90° C. were respectively adhered to a round platform of an electron microscope, and coated with a metal (10 mA, 90 s). At a voltage of 3 kV and a current of 10 μA, the surface topography was observed by cold-field-emission scanning electron microscopy, to determine the influence of different reagents on the surface morphology of silk fibroin.
(1) 2 silk fibroin films with a moisture content of about 3.64% prepared in Example 2 were laminated, placed horizontally, and pressed by a counterweight of 500 g, to laminate them together. Multiple groups of parallel experiments were performed, each group having at least two silk fibroin films. The silk fibroin film in each group was respectively soaked in water, methanol, benzyl alcohol, ethanol, phenethyl alcohol, 2-phenoxyethanol, 3-Phenyl-1-propanol, 4-phenylbutanol and 1,3-butanediol for 3 hrs, at room temperature.
(2) 2 silk fibroin films with a moisture content of about 3.64% prepared in Example 2 were laminated, placed horizontally, and pressed by a counterweight of 500 g, to laminate them together. Multiple groups of parallel experiments were performed, each group having at least two silk fibroin films. The silk fibroin film in each group was respectively fumigated with water, methanol, benzyl alcohol, ethanol, phenethyl alcohol, 2-phenoxyethanol, 3-Phenyl-1-propanol, 4-phenylbutanol and 1,3-butanediol at 60° C. for 3 hrs.
(3) The same group of silk fibroin films treated in the Steps (1) and (2) were laminated to each other, and a sample was prepared according to the test standard. The maximum bonding strength upon separation from each other was tested by the Instron-3365 testing machine to determine the specificity of different reagents in the treatment of silk fibroin, and distinguish the effect of reagents with or without a phenyl group on the bonding strength of silk fibroin.
(1) The silk fibroin films with a moisture content of about 3.64% prepared in Example 2 were mechanically clamped, and forced to be laminated together by an external force. The silk fibroin films were fumigated with ethanol (control) or phenethyl alcohol at 60° C. for 1, 3, 6 and 12 hrs. Each group had at least two silk fibroin films.
(2) The same group of silk fibroin films treated in the above step were laminated to each other, and a sample was prepared according to the test standard. The maximum bonding strength upon separation from each other was tested by the Instron-3365 testing machine to determine the specificity of different alcohol reagents in the treatment of silk fibroin, and distinguish the effect of reagents with or without a phenyl group on the bonding strength of silk fibroin.
(1) The silk fibroin solution obtained in Example 1 was added with deionized water stepwise according to the calculated mass fraction to dilute the mass fraction of the solution to 4%.
(2) 2 mL of the solution was added to a plastic petri dish with a diameter of 3.5 cm. The solution was stood for more than 3 hrs, until the internal air bubbles were completely removed. Then, the solution was placed in a fume hood at room temperature, and air dried overnight to form a film.
(3) The silk fibroin film prepared above was respectively clamped with smooth, and frosted glass, and forced to be laminated together by an external force. The samples were fumigated with ethanol (control) or phenethyl alcohol at 60° C. for 3 hrs.
(4)
(1) The silk fibroin solution obtained in Example 1 was added with deionized water stepwise according to the calculated mass fraction to dilute the mass fraction of the solution to 0.1-5%.
(2) Polyester, polyamide, polyurethane and other common chemical fiber fabrics that are 10 cm in length and width were soaked in the silk fibroin solutions of different mass fractions above, for 5-10 min, and taken out after being completely infiltrated.
(3) The infiltrated chemical fiber fabrics were treated by a double-roll padder, and the pressure of padding was set by a rotary spring to remove excess silk fibroin solution. The fabrics were removed and air dried.
(4) Method 1: A self-made fully enclosed evaporating dish was used, and a phenethyl alcohol solution was held at the bottom. The evaporating dish was placed in an oven, with a temperature set to 45-120° C. After the phenethyl alcohol solution at the bottom was in a vapor state, the treated chemical fiber fabrics were suspended on the opening of the evaporating dish in the horizontal direction, as shown in
(1) The silk fibroin solution obtained in Example 1 was added with deionized water stepwise according to the calculated mass fraction to dilute the mass fraction of the solution to 0.1-5%.
(2) Polyester, polyamide, polyurethane and other common chemical fiber fabrics that are 10 cm in length and width were soaked in the silk fibroin solutions of different mass fractions above, for 5-10 min, and taken out after being completely infiltrated.
(3) The infiltrated chemical fiber fabrics were treated by a double-roll padder, and the pressure of padding was set by a rotary spring to remove excess silk fibroin solution. The fabrics were removed and air dried.
(4) Method 2: A phenethyl alcohol solution was held inside the iron. Distilled water and ethanol were used as a control group. The chemical fiber fabrics treated in Step (3) was air dried at room temperature, and ironed.
(1) The silk fibroin solution obtained in Example 1 was added with deionized water stepwise according to the calculated mass fraction to dilute the mass fraction of the solution to 0.1-5%.
(2) Method 3: The silk fibroin solutions of different mass fractions obtained in Step (1) were mixed with a phenethyl alcohol solution at a volume ratio of 50:1 to 1:1, and the chemical fiber fabrics were soaked in the above mixed solutions for 5-10 min, and taken out after being completely infiltrated.
(3) The infiltrated chemical fiber fabrics were treated by a double-roll padder, and the pressure of padding was set by a rotary spring to remove excess silk fibroin solution. The fabrics were removed and air dried. The treated chemical fiber fabrics was directly ironed with an iron (containing water therein).
The chemical fiber fabrics prepared by the above three methods were tested for surface morphology, air permeability, fabric thickness and moisture regain. 1) Surface morphology test: The chemical fiber fabrics prepared above were adhered onto a round platform of an electron microscope, and coated with a metal (10 mA, 90 s). At a voltage of 3 kV and a current of 10 μA, the surface topography was observed by cold-field-emission scanning electron microscopy. 2) Air permeability test: The chemical fiber fabrics prepared above were cut into a round shape of 20 cm2, and tested by YG461E-III automatic air permeability tester, set to have the following parameters: equilibration time 24 hrs, pressure 100 Pa, temperature 20° C., humidity 65% unit: mm/s. 3) Fabric thickness test: The chemical fiber fabrics prepared above were cut into a round shape of 20 cm2, and tested by YG141D-II fabric thickness meter, set to have the following parameters: equilibration time 24 hrs, test standard GB/T 3820-1997, test time 10 s, single cycle, temperature 20° C., humidity 65% unit: mm; 4) Moisture regain test: The chemical fiber fabrics prepared above were cut into a round shape of 20 cm2, and tested by HF-MS moisture regain tester, set to have the following parameters: equilibration time 24 hrs, temperature 104° C., measurements 10, test standard GB/T 9995-1997, test time 10 s, single cycle, temperature 20° C., humidity 65% unit %.
The above results show that the chemical fiber fabrics treated by the above three methods have a silk imitation rate of up to 95-99%.
While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. It should be appreciated that some improvements and variations can be made by those skilled in the art without departing from the technical principles of the present invention which are also contemplated to be within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201911201913.9 | Nov 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/122715 | 12/3/2019 | WO |
