KR20150078594A - Silk fibroin nano fiber/regenerated silk composite film and its preparation method - Google Patents
Silk fibroin nano fiber/regenerated silk composite film and its preparation method Download PDFInfo
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Abstract
The present invention relates to a method for producing a silk fibroin nanofiber / regenerated silk composite film. More particularly, the present invention relates to a silk fibroin nanofiber / regenerated silk composite film including a silk fibroin nanofiber and a regenerated silk and having greatly improved crystallinity and mechanical properties, and a method for producing the same.
Description
The present invention relates to a silk fibroin nanofiber / regenerated silk composite film excellent in mechanical properties and a method for producing the same.
Silk has been widely used as an excellent textile material for mankind based on excellent strength, gloss and touch. However, recently silk has excellent biocompatibility and excellent cell activity, And application technology is being actively studied.
Recently, a silk artificial eardrum has been commercialized using a regenerated silk film. In the case of recycled silk films, the development of technologies to be applied to biotechnology such as medical devices such as artificial meninges, burn dressings, and wound dressings, cosmetic materials such as functional packs, and biosensors through enzyme immobilization Efforts are continuously being made.
However, in the case of the conventional regenerated silk film, the mechanical properties are significantly lower than that of the natural silk fiber due to the decrease of the molecular weight and the crystallinity in the process of being regenerated through the dissolution process, and it is disadvantageous in that it is brittle , Excellent biocompatibility and cell activity. However, the application of the regenerated silk film has been limited due to its low mechanical properties.
To improve this, there have been studies on the production of recycled silk films by adding glycerol and other additives to regenerated silk solutions, or the production of synthetic and natural polymers and blends with excellent mechanical properties. However, when such other components other than silk are used, there may be a problem that the excellent characteristics of the silk may be somewhat changed, and examples of commercialization using the regenerated silk film produced by this method are few It is true.
Therefore, due to the limitations of existing regenerated silk films, it is necessary to develop a silk film excellent in mechanical properties and excellent in biocompatibility because it is composed only of silk.
Disclosed is a silk fibroin nanofiber / regenerated silk composite film excellent in crystallinity and mechanical properties as well as excellent biocompatibility and a method for producing the same.
The present invention provides a regenerated silk composite film comprising silk fibroin nanofiber and regenerated silk, wherein the silk fibroin nanofiber is contained in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of regenerated silk.
The present invention also relates to a method of producing a recycled silk fibroin nanofiber comprising mixing silk fibroin nanofibers with a regenerated silk solution and drying the resulting mixture in the form of a film, wherein the silk fibroin nanofiber is used in an amount of 0.1 to 30 parts by weight per 100 parts by weight of recycled silk solid, A method for producing a composite film is provided.
Hereinafter, a silk fibroin nano fiber / regenerated silk composite film according to a specific embodiment of the present invention and a method for producing the same will be described in detail. It is to be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
In addition, throughout this specification, "comprising" or "containing ", unless specifically stated, refers to including any and all components (or components) Can not be interpreted as excluding.
The inventors of the present invention have found that when a composite film is produced by adding silk fibroin nanofibers to a regenerated silk in the course of repeated studies to improve the physical properties of the regenerated silk film, unlike a conventional regenerated silk film, it has high crystallinity and mechanical strength To complete the present invention.
According to one embodiment of the invention, a silk fibroin nanofiber / regenerated silk composite film excellent in crystallinity and mechanical properties and excellent in biocompatibility is provided. The silk fibroin nanofiber / regenerated silk composite film includes silk fibroin nanofiber and regenerated silk, and the silk fibroin nanofiber may be dispersed in the regenerated silk.
Silk yarn obtained from silkworms, silk yarn and silk textile produced by using silkworm silk silk retains the crystallinity and molecular weight of silkworm cocoons produced by silkworms and is called "natural silk". In the present invention, the term "regenerated silk" refers to a state in which a natural silk is prepared as a silk solution by using various solvents, and both the hydrogen bond and the hydrophobic bond between the silk main chains are cleaved and each silk main chain is loosened in the solvent, Is referred to as "regenerated silk ". In this regenerated silk, all of the loosened silk main chains are rearranged again to form new hydrogen bonds and hydrophobic bonds, and the crystallinity is also lower than that of the natural silk, resulting in deterioration of mechanical properties. When the natural silk is dissolved in a solvent, , So that the recycled silk has a lower molecular weight than natural silk. In particular, such regenerated silk may serve as a polymer matrix in which the silk fibroin nanofibers are dispersed as the base material of the composite film.
The regenerated silk may be composed of fibroin, sericin, or a mixture thereof extracted from silk. In particular, the regenerated silk contains fibroin as an essential ingredient in terms of securing excellent mechanical properties in the final film product, and may optionally contain sericin.
In the silk fibroin / regenerated silk composite film of the present invention, the silk fibroin nanofiber may be added in an amount of 0.1 to 30 parts by weight, preferably 0.5 to 25 parts by weight, relative to 100 parts by weight of the regenerated silk, More preferably 1 part by weight to 20 parts by weight. When the content of the silk fibroin nanofibers is less than 0.1 part by weight, the amount of the nanofibers is too small to improve mechanical properties. In addition, when the silk fibroin nanofibers are contained in an amount exceeding 30 parts by weight, the nanofibers are not uniformly dispersed in the regenerated silk, resulting in a problem of a decrease in mechanical properties.
Further, the regenerated silk composite film of the present invention contains only silk fibroin nanofiber and regenerated silk, and is composed of only silk and has excellent biocompatibility. The recycled silk composite film may contain 0.1 to 23 wt% and 77 to 99.9 wt%, preferably 0.5 to 20 wt% and 80 to 99.5 wt% of silk fibroin nanofiber and regenerated silk, respectively, and more preferably 1 to 17 wt% % And 83 to 99% by weight.
The silk fibroin nanofiber may have an average fiber diameter of 30 to 900 nm, preferably 50 to 800 nm, more preferably 70 to 700 nm, further preferably 100 to 550 nm. The silk fibroin nanofiber may be present in the natural silk fibroin fiber at a certain diameter and may be obtained by physicochemical treatment and separated from the natural silk fibroin fiber in nanoscale fiber form. Since the average diameter of the fibroin nanofibers present in the fibroin fiber is at least 30 nm, the average diameter of the fibroin nanofibers to be extracted may be 30 nm or more, and the fibroin nanofibers are extracted and extracted according to the extraction conditions The average diameter of the fibroin nanofibers may increase. However, in the case where the average fiber diameter of the silk fibroin nanofibers exceeds 900 nm, fibroin nanofibers of 900 nm or less can be used for the production of the composite material because the advantages as the nanofiber are somewhat lost.
In addition, the silk fibroin nanofiber may have an IR crystallinity of 60% or more, or 60% to 65%, and preferably 61% to 64%, according to infrared analysis. The IR crystallinity index of the silk fibroin nanofiber can be 60% or more because the IR crystallinity index of the natural silk fibroin microfibers is 60% or more.
The silk fibroin nanofiber / regenerated silk composite film of the present invention can be used not only as a biomaterial such as an artificial skin, an anti-adherence agent, an artificial eardrum, a dermal epithelium, a biosensor and a cosmetic pack, but also a biodegradable reinforcing material ) Can also be used for the purpose.
The IR crystallization index of the silk fibroin nanofiber / regenerated silk composite film may be 51% to 70%, preferably 51.5% to 65% by IR analysis. As the mechanical properties of the composite film, the breaking strength may be 65 MPa or more or 65 to 1,000 MPa, preferably 80 MPa or 80 to 500 MPa. The cut-off elongation of the composite film may be 2.5% or more, or 2.5% to 10%, preferably 3% or 3% to 8%. Particularly, the crystallinity index, the breaking strength, and the cutting elongation of the silk fibroin nanofiber / regenerated silk composite film were measured by using 30 mL of a 1% (w / w) regenerated silk formic acid solution in a petridish of 85 mm in diameter It can be measured on the basis of a film produced by ventilation drying in a hood.
Further, the transparency of the silk fibroin nanofiber / regenerated silk composite film may be 60% or more, or 60% to 95%, preferably 65% or more, more preferably 70% or more. The transparency of the composite film may be measured by transmitting light of 700 nm using an ultraviolet visible ray spectrometer on the basis of a 50 탆 thick film. In particular, the silk fibroin nanofiber / regenerated silk composite film exhibits an excellent transparency of 60% or more, and thus it is possible to obtain an advantage that a wound healing state can be grasped when used as a medical material such as an artificial eardrum, dermal epithelial replication, and an image treatment agent.
On the other hand, according to another embodiment of the present invention, a method for producing a silk fibroin nanofiber / regenerated silk composite film as described above is provided. The method for producing the silk fibroin nanofibers / regenerated silk composite film includes a step of mixing the silk fibroin nanofibers into a regenerated silk solution and then drying the resulting mixture into a film.
The method for producing a silk fibroin / recycled silk composite film of the present invention is characterized in that, when a silk fibroin nanofiber is mixed with a regenerated silk solution and dried in a film to produce a composite film, unlike a conventional regenerated silk film, Mechanical strength, excellent biocompatibility and cell activity, and has excellent properties as a medical material.
Particularly, in the present invention, the silk fibroin nanofiber may be added in an amount of 0.1 to 30 parts by weight, preferably 0.5 to 25 parts by weight, more preferably 1 to 5 parts by weight, per 100 parts by weight of the recycled silk, To 20 parts by weight. When the silk fibroin nanofiber is used in an amount of less than 0.1 part by weight, the content of the nanofiber is too small to improve mechanical properties. In addition, when the silk fibroin nanofibers are used in an excess amount exceeding 30 parts by weight, the nanofibers are not uniformly dispersed in the regenerated silk, resulting in a problem that the mechanical properties are rather reduced.
The method for producing a silk fibroin / recycled silk composite film of the present invention may further comprise the step of producing silk fibroin nanofibers on the fibroin surface through a refining process of silk.
The scouring agent used in the scouring process may be any scouring agent commonly used for removing sericin from silk. For example, it can be carried out using a polishing agent comprising at least one selected from the group consisting of an aqueous solution of soap, an aqueous alkali solution, an aqueous acid solution, an aqueous enzyme solution and an aqueous amine solution. Here, the soap may be selected from the group consisting of Tallow Oliver oil, Coconut oil, Caster oil, Arachis oil, Cotton seed oil, Sodium laurate, sodium laurate, sodium myristate, sodium stearate, sodium arachidate, sodium oleate, sodium ricinoleate, And sodium ricinoleate. It is also possible to refine it in an alkali aqueous solution or an acidic aqueous solution, and also to apply a refining method using an enzyme. The alkali aqueous solution may be sodium hydroxide, sodium carbonate, calcium carbonate, sodium silicate, sodium phosphate, sodium bicarbonate, borax, ammonia or the like. The acidic aqueous solution may be lactic acid, tartaric acid, citric acid, oxalic acid, malonic acid, succinic acid, acetic acid, chloroacetic acid, dichloroacetic acid trichloroacetic acid and the like. Trypsin, Papain and the like may be used as the enzyme. In addition, refining using methylamine, ethylamine or the like is also applicable as an amine compound.
(Bath ratio, w / v, g / mL), i.e., silk weight (g): refining bath volume (mL) to a degumming bath containing the refining agent is 1: 5 or more 1: 5 to 1: 100. If the bath ratio of the silk is less than 1: 5 w / v (g / mL), the cleansing liquid may not be sufficiently immersed in the silk, so sericin removal may not be effectively performed. In addition, the bath ratio of the silk to the refining bath may preferably be 1:10 w / v (g / mL) or more. However, the bath ratio of the silk to the degumming bath is preferably 1: 100 w / v (g / mL) or less, preferably 1:50 w / v (g / mL).
The refining process may be performed at 70 to 150 ° C, preferably 90 to 130 ° C. The refining time may be 0.3 to 5 hours, preferably 0.5 to 4 hours. The refining time can be optimally performed in the temperature range described above in terms of effectively producing the fibroin nanofibers without damaging the silk through the refining process. In particular, when the temperature of the refining step is less than 70 캜, sericin is not sufficiently removed, silk fibroin nanofibers are not sufficiently formed, or the time required for sericin removal and generation of silk fibroin nanofibers is greatly increased, Can be increased. If the temperature of the refining step exceeds 150 ° C, the molecular chains of the silk may be cut off during the refining process, or the silk may be injured.
In addition, the method for producing a silk fibroin nanofiber / regenerated silk composite film may further include a step of performing ultrasonic treatment, physical stirring, or physical blowing to the silk fibroin after the refining step.
The silk fibroin nanofibers used in the present invention can be obtained by, for example, treating excessively a scouring agent to produce nanofibers on the surface of silk fibroin, and then adding the nanofibers to water, treating the ultrasonic wave to freeze-dry the aqueous solution containing the silk fibroin nanofibers Can be manufactured. The ultrasonic treatment may be performed under a power condition of 200 W to 1,200 W, preferably 250 W to 1,000 W, and more preferably 300 W to 800 W. In addition, the ultrasonic treatment may be performed for 0.3 to 15 hours, preferably 0.4 to 12 hours, more preferably 0.5 to 10 hours. For example, in the case of ultrasonic treatment, the ultrasonic treatment is performed for 30 seconds at a power of 600 W, and the treatment is performed for 0.3 to 15 hours in a manner of resting for 30 seconds. At this time, if the ultrasonic treatment time is less than 0.3 hour, the silk fibroin nanofiber produced on the surface of the silk fibroin can not be sufficiently extracted and the silk fibroin nanofiber may not be obtained. Further, when the ultrasonic treatment time exceeds 15 hours, the nanofibers on the surface of the silk fibroin are no longer extracted so that the process cost is increased. In addition, the silk fibroin is crushed in a direction perpendicular to the fiber axis It is difficult to obtain only pure silk fibroin nanofibers, and silk fibroin nanofibers are mixed with micrometer (mu m) sized silk fibroin.
In addition to the above ultrasonic treatment, the refined silk fibroin can be increased and collected by physical stirring or physical blowing. Here, the physical blowing can be performed by manually blowing the silk fibroin having the nanofiber formed on the surface after refining alternately with two rods. In addition, the physical stirring may be carried out under the conditions of 150 to 1,500 rpm, preferably 180 to 1,300 rpm, more preferably 200 to 850 rpm. Further, the physical stirring may be carried out for 0.3 to 15 hours, preferably 0.4 to 12 hours, more preferably 0.5 to 10 hours. For example, in the case of physical stirring, a 2-liter beaker may be filled with 1 liter of water and then treated with a scouring agent-treated silk fibroin at 500 rpm for 0.3 to 15 hours. If the agitation time is less than 0.3 hour, silk fibroin nanofiber may not be sufficiently generated. If the agitation time exceeds 15 hours, the nanofibers are not further generated, and thus the process cost may increase.
In the present invention, the silk fibroin nanofiber may have an average fiber diameter of 30 to 900 nm, preferably 50 to 800 nm, more preferably 70 to 700 nm, further preferably 100 to 550 nm. The average fiber diameter of the silk fibroin nanofibers can be somewhat controlled by variously adjusting the refining conditions, but it may be 30 nm or more considering the limit of the minimum diameter of the silk nanofibers present on the surface of the silk fibroin. In addition, when the diameter of the silk fibroin nanofiber is too large, it is preferable to use a material having a diameter of 900 nm or less from the viewpoint that the difference in physical properties with respect to conventional microfibers is not large. In addition, the silk fibroin nanofiber of the present invention may have an IR crystallization degree of 60% or 60% to 65%, preferably 61% to 64%, according to infrared analysis.
The method for producing a silk fibroin nano fiber / regenerated silk composite film of the present invention may further comprise the step of preparing a regenerated silk solution by dissolving the regenerated silk solid in water or an acidic solvent.
The regenerated silk solid used in the production of the regenerated silk solution for producing the regenerated silk composite film is a regenerated silk aqueous solution obtained by drying an aqueous solution containing fibroin, sericin, or a mixture thereof extracted from silk, It may be obtained by drying a regenerated silk aqueous solution obtained by dissolving fibroin containing sericin. The recycled silk solid can be any solid state, and can be used regardless of the granularity or properties. For example, the regenerated silk solid content of the present invention may be in the form of powder or particles prepared by freeze-drying the regenerated silk aqueous solution. The regenerated silk solid may be a film or a sheet, a particle, or a powder, which is prepared by thermally drying a regenerated silk aqueous solution. The regenerated silk solids may differ in the crystallinity of the regenerated silk which is finally obtained according to the drying method, and the difference in the thickness of the resulting film or the particles of the powder may cause the dissolution rate to vary when the regenerated silk is dissolved Although the solution preparation time may vary, there is little difference in the characteristics of the regenerated silk solution or film to be finally produced. When the regenerated silk powder is prepared by a method such as freeze-drying, the dissolution rate of the regenerated silk solid component is increased and the process speed can be improved. However, since the production cost of the recycled silk powder is increased, it is possible to select a method for producing the recycled silk solid product in terms of productivity. However, both the regenerated silk solid and the regenerated silk powder do not greatly affect the properties of the regenerated silk solution and the film ultimately produced.
In addition, the regenerated silk used in the present invention is largely produced through a step of removing all or part of sericin through refining and a step of dissolving. The refining can be performed using a refining agent usually used for removing sericin, for example, a refining agent comprising at least one selected from the group consisting of an aqueous solution of soap, an aqueous alkali solution, an aqueous acid solution, an aqueous enzyme solution and an aqueous amine solution, The sericin can be completely or partially removed by adjusting the amount of refining time, type of refining agent and amount of refining agent.
The production of the regenerated silk according to the present invention may further include a step of dissolving and refining the refined silk after performing the refining step. Particularly, after the refining process is performed, the refined silk is dissolved in the metal salt aqueous solution, and the silk aqueous solution thus obtained can be dialyzed with water. At this time, the metal salt aqueous solution may be an alcohol-mixed aqueous solution further containing an alcohol component. The metal salt may be any salt compound containing an alkali metal, an alkaline earth metal, a transition metal, or the like, as long as it is a neutral salt compound that does not become acidic or alkaline when dissolved in water. In particular, the metal salt may be at least one of calcium chloride, calcium nitrate, lithium bromide (LiBr), lithium thiocyanate (LiSCN), magnesium nitrate, zinc nitrate, zinc chloride, and mixtures thereof.
The molar ratio of the metal salt: water to the alcohol may be 1: 2: 8 to 16: 2 in the case of using an alcohol, To 4 can be used. In addition, the aqueous metal salt solution may be dissolved at 70-100 ° C for 3 minutes to 3 hours, preferably at 80-90 ° C for 20 minutes to 2 hours. If the melting temperature is less than 70 ° C, the silk may be difficult to dissolve. If the melting temperature is more than 85 ° C, the silk molecular chain may be severed and the physical properties may be deteriorated. If the dissolution time is less than 3 minutes, dissolution of the silk may not occur completely or may be uneven. If the dissolution time exceeds 3 hours, the property may be deteriorated due to silk molecular chain breaking.
In addition, the dialysis step is a step for removing calcium chloride and ethanol used for dissolution. As described above, after refining the refined silk in an aqueous solution of a metal salt aqueous solution, the solution was placed in a cellulose dialysis membrane (molecular weight cutoff 12,000 to 14,000) and dialyzed for three days or more in flowing distilled water to obtain a silk aqueous solution in which calcium chloride and ethanol had been removed Can be obtained. The silk aqueous solution may be used in the production of a silk fibroin nanofiber / regenerated silk composite film. The silk aqueous solution may be dried and then dissolved in formic acid to prepare a silk formic acid solution, which may then be used for producing a nanofiber / regenerated silk composite film.
The silk fibroin / regenerated silk composite film of the present invention is obtained by adding silk fibroin nanofibers to a regenerated silk solution (aqueous solution or formic acid solution) and then uniformly mixing them by stirring or ultrasonic treatment to obtain a silk fibroin nanofiber / And drying it to produce a silk fibroin nanofiber / regenerated silk composite film. At this time, the concentration of the regenerated silk solution in the silk fibroin nanofiber / regenerated silk mixed solution can be preferably used in the range of 0.01% to 25%, more preferably 0.1% to 20%. When the concentration of the regenerated silk solution is less than 0.01%, the amount of the solvent (water or formic acid) to be used is excessively large and the solvent is volatilized, so that it takes a long time to produce the final composite film, Problems may arise. Thus, when the regeneration silk concentration is too low, the film formability is improved, and the film can be easily broken. If the concentration of the regenerated silk solution is more than 25%, it becomes easy to gel in the aqueous silk solution and the viscosity is too high, so that uniform mixing with the fibroin nanofiber is difficult, and the strength and elongation can be reduced. Further, in the silk formic acid solution, it may be difficult to dissolve silicate in formic acid to a concentration of 30%.
On the other hand, when an acidic solvent is used in the production of the regenerated silk solution, the acidic solvent may have an acidity range, that is, a pKa of 0.1 to 5.0, preferably 0.5 to 4.5, more preferably 1 to 4.2. When the pKa is more than 5.0, since the acidic solvent does not dissolve the regenerated silk solid, it may be difficult to produce a regenerated silk composite film. When the pKa of the acidic solvent is less than 0.1, the degree of decomposition of the regenerated silk molecular chains is too high by the acid, which may lead to a problem of deteriorating the mechanical properties of the produced regenerated silk composite film.
It is also preferred that the acidic solvent has an optimized volatility in terms of reworking the film after dissolving the regenerated silk solids. In this respect, the acidic solvent preferably has a vapor pressure of 0.01 to 1,000 mmHg, preferably 0.1 to 800 mmHg, more preferably 5 to 500 mmHg, more preferably 5 to 500 mmHg, measured at 20 deg. C according to a measurement method using a manometer May be 20 to 300 mmHg. If the vapor pressure of the acidic solvent is less than 0.01 mmHg, the acid solvent may not volatilize and it may be difficult to produce a regenerated silk composite film. Further, when the vapor pressure of the acidic solvent exceeds 1,000 mmHg, it may be difficult to form a film due to the volatilization of too quickly and the regenerated silk is difficult to form a sufficient shape.
In the present invention, the acidic solvent may include at least one acid component selected from the group consisting of formic acid, citric acid, acetic acid, tetrafluoroacetic acid, benzoic acid, oxalic acid, phosphoric acid, propionic acid, chloroacetic acid, chloric acid and nitrous acid. Of these, it is preferable to use formic acid, acetic acid, propionic acid or the like in view of the possibility of decomposition of regenerated silk by an acid and the volatility of a solvent.
The liquor ratio of the regenerated silk / solvent of the solvent for dissolving the regenerated silk to produce the regenerated silk acid solution may be 1: 5 to 1: 10,000, preferably 1: 7 to 1: 5,000, More preferably from 1:10 to 1: 1,000. When the liquid ratio is less than 1: 5, the amount of the solvent is too small to sufficiently dissolve the regenerated silk. If the liquid ratio exceeds 1: 10,000, too much solvent is consumed to dissolve the regenerated silk, and thus the cost of producing the regenerated silk composite film may be excessively increased.
Also, since the pKa value of the regenerated silk acid solution does not have the alkali and acidic characteristics which greatly change the pKa value of the acid solvent used for producing the regenerated silk solution itself, the pKa value of the regenerated silk acid solution is regenerated The same value as the pKa of the acid solvent used to dissolve the silk. That is, the pKa value of the regenerated silk acid solution may be 0.1 to 5.0, preferably 0.5 to 4.5, more preferably 1 to 4.2.
On the other hand, when the regenerated silk solution is prepared, the regenerated silk solid content can be dissolved in water or an acidic solvent at a temperature of 20 ° C to 95 ° C, preferably 20 ° C to 70 ° C, more preferably 20 ° C to 50 ° C . When the regenerated silk is dissolved at a high temperature, there is an advantage that the solubility is increased. However, when the temperature is unnecessarily high, there is a disadvantage that the silk molecular chain can be cleaved. On the other hand, when the regenerated silk is dissolved at too low a temperature, the silk molecular chain breakage becomes smaller or the solubility becomes lower. Furthermore, when the regenerated silk is composed of fibroin only without sericin, it can be dissolved at 20 占 폚, so that it may be preferable to dissolve at 20 占 폚. However, when sericin is additionally included in the regenerated silk together with fibroin, it may not be completely dissolved at 20 DEG C, so that it can be dissolved while increasing the temperature. However, when the dissolution temperature is increased to 95 DEG C, there is a possibility that all of the regenerated silk having excess sericin remains dissolved. Therefore, in order to prevent unnecessary silk molecular chain breakage, the dissolution temperature of the regenerated silk does not exceed 95 DEG C .
In the present invention, the viscosity of the silk fibroin nanofibers / regenerated silk mixed solution is preferably 1 cps to 20,000 cps, more preferably 1 cps to 10,000 cps. When the viscosity of the mixed solution exceeds 20,000 cps, the stirring is not smoothly performed, or the silk fibroin nanofiber and the regenerated silk solution are not uniformly mixed with each other, so that the mechanical properties There is a problem in that it is lowered.
The silk fibroin nanofiber / regenerated silk mixed solution has a turbidity (τ) according to the following formula 1: 0.001 cm -1 to 1 cm -1 , more preferably 0.1 cm -1 To 0.8 cm <" 1 >.
[Equation 1]
Turbidity (τ) = -ln T / C
Where C is the thickness of the cell and T is the transmittance at 700 nm.
The turbidity of the silk fibroin nanofiber / regenerated silk mixed solution is a value that can not be obtained at less than 0.001 cm -1 when the regeneration silk concentration is 1%. Also, when the turbidity of the silk fibroin / regenerated silk mixed solution exceeds 1 cm -1 , the silk fibroin nanofiber is not homogeneously distributed in the regenerated silk formic acid solution, And thus the physical properties may be deteriorated during the production of the film. When the regenerated silk is completely dissolved in the solvent and the regenerated silk is present in the solution, the regenerated silk shows a high turbidity. When the film is produced by using the regenerated silk, the regenerated silk is completely dissolved and the molecular chains are entangled Otherwise, the film may be cracked or the mechanical properties may deteriorate. On the other hand, when the regenerated silk is dissolved well in a solvent, it exhibits low turbidity. In this case, the fibroin or sericin molecular chains of the regenerated silk are well dissolved and entangled with each other, Strength shows excellent characteristics.
In addition, the silk fibroin nanofiber / regenerated silk mixed solution prepared by mixing the regenerated silk solution with the silk fibroin nanofibers can be produced as a regenerated silk composite film through a process of drying the film. In particular, the regenerated silk mixed solution obtained by mixing the silk fibroin nanofibers is preferably dried in a ventilated hood to produce a final regenerated silk composite film. In this case, in the case of the silk fibroin nanofiber / regenerated silk mixed solution using the acidic solvent, if the ventilation does not occur, the workers may be exposed to the volatile acidic solvent, thereby posing a problem of threatening the health of the workers.
The drying step may be carried out at a temperature within a range from room temperature to such an extent that the regenerated silk and the like are not decomposed. For example, the drying step may be carried out under a temperature condition of 20 ° C to 95 ° C, preferably 22 ° C to 90 ° C, more preferably 25 ° C to 80 ° C. However, regardless of the drying temperature, it is desirable to provide a ventilation device for removing the solvent, so that the drying temperature can be determined in consideration of the device design cost. If the drying temperature is lower than the room temperature, the volatilization rate of the solvent is lowered, so that not only the time for producing the film is prolonged but also energy for reducing the temperature is required, which may increase the process cost. On the other hand, if the drying temperature exceeds 95 ° C., the silk fibroin nanofiber and the regenerated silk may be decomposed by acid during the drying process, resulting in deterioration of the mechanical properties of the final film. In addition to this, blowing of such a high temperature wind and discharging it for removal of solvent may cause a problem of a significant increase in the process cost.
In the present invention, matters other than those described above can be added or subtracted as needed, and therefore, the present invention is not particularly limited thereto.
According to the present invention, when the silk fibroin nanofiber / regenerated silk composite film is produced, an excellent effect that the mechanical properties and the degree of crystallization are greatly increased as compared with the existing regenerated silk film can be obtained.
1 is a result of infrared spectroscopic analysis of a regenerated silk film produced according to Comparative Example 1 of the present invention.
2 is a result of infrared spectroscopic analysis of a silk fibroin nanofiber / regenerated silk composite film produced according to Example 2 of the present invention.
Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.
[Example]
Production Example 1: Preparation of regenerated silk powder
Silkworm cocoons were treated with 0.3% sodium oleate and 0.2% sodium carbonate aqueous solution for 1 hour to remove sericin, followed by washing with water and drying. After the refining, the silk was treated with a 1: 20 mixture of calcium chloride, water and ethanol (molar ratio 1: 8: 2) at 85 DEG C for 3 minutes to dissolve the silk. After dissolution, the silk was dissolved in a dialysis membrane (molecular weight cut off = 12,000 ~ 14,000) was dialyzed against distilled water flowing for 5 days to remove calcium chloride and ethanol, and a regenerated silk aqueous solution was obtained. This was dried and pulverized to obtain a regenerated silk powder.
Production Example 2: Production of silk fibroin nanofiber
The silkworm cocoons were treated for 1 hour at 100 ° C in an aqueous solution of 0.3% (w / v) sodium carbonate, 0.1% (w / v) sorbitan monostearate for 2 hours, And dried. And then immersed in distilled water at 30 ° C for 10 minutes so that water can be uniformly processed into the inside of the silk fibroin fiber. After immersing, 0.5 g of silk fibroin fiber was placed in a 50-mL beaker, and distilled water was added so that it could be fully immersed. Ultrasonic wave generator (ULH700S, Ulsso Hi-tec, After the treatment, ultrasonic treatment was performed in a state of resting for 5 seconds. After 30 minutes of continuous treatment, ultrasonic waves were treated for 6 hours by cooling the ultrasonic crusher for 30 minutes.
After the ultrasonic treatment as described above, an aqueous solution containing silk was filtered through a nonwoven fabric to obtain a solution in which silk fibroin nanofibers and water were mixed and lyophilized using a freeze dryer to obtain silk fibroin nanofibers.
The thus-produced silk fibroin nanofibers had an average fiber diameter of 230 nm and an IR crystallization degree of 62.5% according to infrared analysis.
Example 1: Composite film production of silk fibroin nanofiber and regenerated silk
1 g of the regenerated silk powder obtained in Production Example 1 was dissolved in 99 g of 98% formic acid to prepare a regenerated silk solution having a concentration of 1% (w / w). The silk fibroin nanofiber obtained in Preparation Example 2 was added to the regenerated silk solution thus prepared in an amount of 5 parts by weight based on 100 parts by weight of the regenerated silk powder and stirred. After that, the nanofiber was uniformly mixed with the regenerated silk solution using the ultrasonic crusher So that a mixed solution of silk fibroin nanofibers / regenerated silk was obtained.
The mixed solution of the obtained silk fibroin nanofibers / regenerated silk was poured into a Petri dish in an amount of 30 mL, and then dried at a temperature of 25 ° C in a hood to remove all the formic acid as a solvent. Then, a silk fibroin nanofiber / regenerated silk composite film .
Examples 2 to 8
As shown in the following Table 1, in the same manner as in Example 1 except that the concentration of the regenerated silk solution using the regenerated silk powder obtained in Production Example 1 and the content of the silk fibroin nanofiber obtained in Production Example 2 were different from each other To prepare a silk fibroin nanofiber / regenerated silk composite film.
Comparative Example 1
Except that no recycled silk fibroin nanofibers were added and 1 g of the recycled silk powder obtained in Preparation Example 1 was dissolved in 99 g of 98% formic acid to use only a regenerated silk solution having a concentration of 1% (w / w) 1, a regenerated silk film was produced.
Comparative Examples 2 to 3
As shown in the following Table 1, in the same manner as in Example 1 except that the concentration of the regenerated silk solution using the regenerated silk powder obtained in Production Example 1 and the content of the silk fibroin nanofiber obtained in Production Example 2 were different from each other To prepare a silk fibroin nanofiber / regenerated silk composite film.
Test Example
The properties of the silk fibroin nanofibers / regenerated silk composite films or regenerated silk films prepared according to Examples 1 to 8 and Comparative Examples 1 to 3 were evaluated in the following manner, .
a) Turbidity measurement of mixed solution
To measure the turbidity of the silk fibroin nanofibers / regenerated silk blend solution, transmittance at 700 nm using a 1 cm thick (C) cell using an ultraviolet visible spectrophotometer (Evolution 201, Thermo Fisher Scientific, USA) (T) was measured and the turbidity (τ) was measured according to the following equation (3).
[Equation 1]
Turbidity (τ) = -ln T / C
Wherein,
C is the thickness of the cell,
T is the transmittance at 700 nm.
b) Viscosity measurement of mixed solution
To measure the viscosity of the silk fibroin nanofibers / regenerated silk blends, a 60 mm cone and plate geometry was used to measure the viscosity of the complex (complexes) using a rheometer (MARS III, Thermo Fisher Scientific, Germany) viscosity was measured at 25 ℃ and 0.01% strain. The composite viscosities were measured at frequency of 10 Rad / s and the composite viscosities were measured at the temperature of 25 ℃ and strain of 0.01% Was taken as the viscosity value.
c) Determination of the crystallinity of the film
The crystallinity index of the composite film was measured using an infrared spectrometer (Nicolet 380, Thermo Fisher Scientific, USA) and an attenuated total reflection (ATR) apparatus to obtain an infrared spectral spectrum, The crystallinity index was determined.
[Equation 2]
Crystallinity Index (%) =
In the formula, A 1235cm -1 is the infrared absorption at infrared spectroscopy when 1,235 cm -1, A 1260cm -1 is an infrared absorption at 1,260 cm -1.
d) Strength and elongation measurement of film
Strength and elongation were measured using a universal material testing machine (OTT-003, Oriental TM, Korea) to investigate the mechanical properties of the silk fibroin nano-fiber / regenerated silk film.
First, all samples before measurement were stored in a constant temperature and humidity room at 20 ° C and 40% relative humidity for at least 1 day to reach an equilibrium state, and then the strength and elongation were measured under the same temperature and humidity conditions. At this time, the film samples were prepared in a size of 5 mm in width and 50 mm in length and measured at a tensile speed of 0.2 mm / s using a load cell of 3 kgf under a condition of a gauge length of 3 cm Respectively.
silk
density
(%)
Solid content
Silk fibroin nanofiber
Weight portion
Nano Fiber / Recycled Silk
Mixed solution
Recycled silk composite film
(cm -1 )
(cps)
Hwado
(%)
burglar
(MPa)
Shindo
(%)
The turbidity of the silk fibroin nanofiber / regenerated silk mixed solution tended to increase with increasing the concentration of regenerated silk, which is a natural result because the scattering of light increases with increasing concentration. There was little change in the turbidity until the weight of the silk fibroin nanofiber was increased to 5 (Comparative Example 1, Examples 1 and 6) when the regenerated silk concentration was 1%, but it gradually increased after that, The turbidity was greatly increased to 1.243 (Comparative Example 3). Thus, the turbidity was greatly increased at 30 parts by weight, which is considered to result from the fact that aggregation of the nanofibers became very large and the solution became uneven.
The viscosity of the silk fibroin nano-fiber / regenerated silk mixture solution was such that the viscosity of the silk fibroin / regenerated silk mixture was too low to be measured by the rheological property measuring device used at the regeneration silk concentration of 1% or less and the viscosity value of 85 cps at the regeneration silk concentration of 5% (Example 3). In particular, the crystallinity index of the silk fibroin nanofibers / regenerated silk films tended to increase as the concentration of the regenerated silk films increased and as the weight of the silk fibroin nanofibers increased. That is, as in Comparative Example 1, the crystallinity index of the recycled silk film to which the silk fibroin nanofibers were not added was 50.8%. However, as 5% of the silk fibroin nanofibers were added, the crystallinity index was 57.0% (Example 2). As shown in FIG. 1, in the infrared spectroscopic analysis of the regenerated silk film, infrared rays at 1,260 cm -1 corresponding to the beta sheet crystals were observed in the infrared ray spectroscopic analysis of the regenerated silk and composite film. The absorption peak was weak. However, as shown in FIG. 2, as the 5% silk fibroin nanofiber is added to the regenerated silk, the absorption peak at 1,260 cm -1 becomes more pronounced, which means that the beta sheet crystal content is increased .
As the concentration of the regenerated silk film increases, the degree of crystallization of the film increases. This is because the crystallization degree of the film is increased due to the more dense silk molecular chains, It is believed that the first cause is found in the fibroin nanofiber due to its higher crystallinity than the regenerated silk, and that the addition of nanofibers also plays a role in promoting the crystallization of the regenerated silk.
The mechanical properties of the silk fibroin nanofibers / regenerated silk films were greatly improved as the silk fibroin nanofibers were added to the regenerated silk. That is, the regenerated silk film without nanofibers showed a breaking strength of 51 MPa and an elongation of 1.8% (Comparative Example 1), and there was no significant change in physical properties until the addition of 0.05 part by weight of silk fibroin nanofiber (Comparative Example 2), the addition of 0.5 part by weight of silk fibroin nanofiber significantly increased the breaking strength to 90 MPa and elongation to 3.0% (Example 5). (Examples 6 to 8), the strength and elongation were increased to 23 MPa and 0.9%, respectively, when the amount of the nanofibers was increased to 30 parts by weight, (Comparative Example 3). As can be seen from the turbidity value, it is considered that the nanofiber adversely affects the mechanical properties of the regenerated silk because the solution becomes very heterogeneous when the nanofiber content is increased to 30 parts by weight.
On the other hand, when the regeneration silk concentration was 0.1 to 20%, the strength was 100 MPa or more and the elongation was 3% or more, which showed excellent mechanical properties as compared with the regenerated silk film (Examples 1 to 4). In particular, when silk fibroin nanofibers were added to regenerated silk fibers to produce a silk fibroin / regenerated silk composite film, the crystallinity and maximum breaking strength were 12.5% and 12.5%, respectively, 5.2 times, and the cutting elongation was increased 3.3 times at the maximum, resulting in a significant improvement in crystallinity and mechanical properties. Therefore, it can be said that the silk fibroin nanofiber / regenerated silk composite film can be applied to various biotechnological fields such as high crystallization, wound dressing requiring high strength, artificial eosinophil, functional cosmetic pack, antioxidant, and biosensor.
Claims (18)
Wherein the silk fibroin nanofiber is contained in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of the recycled silk.
Wherein the silk fibroin nanofibers are dispersed in the regenerated silk.
Wherein the silk fibroin nanofiber has an average fiber diameter of 30 to 900 nm.
Wherein the silk fibroin nanofiber has an IR crystallinity of 60% or more according to infrared analysis.
And a crystallinity of 51% to 70%.
A recycled silk composite film having a breaking strength of 65 MPa or more.
A recycled silk composite film having a cutting elongation of 2.5% or more.
A reproduction silk composite film having a transparency of 60% or more.
0.1 to 23% by weight of silk fibroin nanofibers and 77 to 99.9% by weight of regenerated silk.
Wherein the silk fibroin nanofiber is used in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of the recycled silk solid content.
And the regenerated silk solution has a concentration of 0.01% to 25%.
Further comprising the step of dissolving the regenerated silk solid in water or an acidic solvent to prepare a regenerated silk solution.
Wherein the step of dissolving the regenerated silk solid is carried out under a temperature condition of from 20 캜 to 95 캜.
Wherein the silk fibroin nanofiber / regenerated silk mixed solution has a viscosity of 1 cps to 20,000 cps.
Wherein the silk fibroin nano fiber / regenerated silk mixed solution has a turbidity (?) Of 0.001 to 1 cm < -1 > according to the following equation 1:
[Equation 1]
Turbidity (τ) = -ln T / C
Where C is the thickness of the cell and T is the transmittance at 700 nm.
Further comprising the step of producing a silk fibroin nanofiber on the fibroin surface through a refining process of the silk.
Wherein the refining step is carried out at 70 to 150 DEG C for 0.3 to 5 hours.
Further comprising performing ultrasonic treatment, physical stirring, or physical blowing on the silk fibroin after the refining step.
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| JP2018119035A (en) * | 2017-01-24 | 2018-08-02 | 富山県 | Method for producing silk nanofiber, composite material, and silk nanofiber film |
| KR102245960B1 (en) * | 2020-12-23 | 2021-04-30 | 국방과학연구소 | Perylene diimide derivatives, graphene channel and biosensor comprising the same |
| KR20240041599A (en) * | 2022-09-23 | 2024-04-01 | 국립공주대학교 산학협력단 | Manufacturing method for inorganic carbonate fibroin composites with core-shell structure and inorganic carbonate fibroin composites manufactured using the same |
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| JP2008231147A (en) * | 2007-03-16 | 2008-10-02 | National Institute Of Agrobiological Sciences | Compounded silk-molded article and method for producing the same |
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| JP2018119035A (en) * | 2017-01-24 | 2018-08-02 | 富山県 | Method for producing silk nanofiber, composite material, and silk nanofiber film |
| KR102245960B1 (en) * | 2020-12-23 | 2021-04-30 | 국방과학연구소 | Perylene diimide derivatives, graphene channel and biosensor comprising the same |
| KR20240041599A (en) * | 2022-09-23 | 2024-04-01 | 국립공주대학교 산학협력단 | Manufacturing method for inorganic carbonate fibroin composites with core-shell structure and inorganic carbonate fibroin composites manufactured using the same |
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