Manufacturing method of a silk fibroin molding body and a molding body

Abstract

The present disclosure relates to a fibroin molding method and a molded body provided therefrom, that enables easy release from a mold even in the case of a complicated mold shape. In the provided method, fibroin film is heated and pressurized inside a mold and is then heat-treated outside the mold.

Description

MANUFACTURING METHOD OF A SILK FIBROIN MOLDING BODY AND AMOLDING BODY

[0001] FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to a manufacturing method of a silk fibroin molded body.

[0003] BACKGROUND

[0004] Japanese unexamined patent application No. 2021 / 80304 provides a method for manufacturing a molded article by laminating thin films comprising a protein containing a polypeptide derived from spider silk protein, which is a kind of silk fibroin, and by heating and pressurizing the laminated films inside a mold. In this method, while sufficient strength is exerted in the direction of laminating a thin film, strength may be insufficient in the direction parallel to the thin film, and as a result, there was a possibility that anisotropy might occur in the strength of the molded body.

[0005] SUMMARY OF THE DISCLOSURE

[0006] The present disclosure provides a method for producing a molded body in which anisotropy of the strength is suppressed even when the molded body is manufactured by laminating thin films and heating and pressurizing them inside a mold.

[0007] In one aspect, in the molding method for laminating a fibroin film and heating and pressurizing the film, the silk fibroin molding method heats and pressurizes while depressurizing the atmosphere.

[0008] In a second aspect, the silk fibroin molding method uses a fibroin film having a P-sheet content of less than 10%.

[0009] In a third aspect, the silk fibroin molding method uses a fibroin film wherein the moisture content of the fibroin film is 2% or more and less than 7%.

[0010] In a fourth aspect, a silk fibroin molded body is formed by laminating fibroin film, and heating and pressurizing it while depressurizing the atmosphere.

[0011] According to the present disclosure, even in case of a molded body manufactured by laminating a thin film, and heating and pressurizing it inside a mold, a molded body in which anisotropy of strength is suppressed is formed.

[0012] These and other embodiments, objects, features, and advantages of the presentdisclosure will become apparent upon reading the following detailed description of exemplary embodiments of the present disclosure, when taken in conjunction with the appended drawings, and provided claims.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate various embodiments, objects, features, and advantages of the present disclosure.

[0015] FIG. 1 is a schematic diagram of a mold for silk fibroin molding.

[0016] FIG. 2 is a schematic diagram of a mold for silk fibroin molding used in an embodiment.

[0017] FIG. 3 is a schematic diagram of a silk fibroin molded body molded in an embodiment and a measurement portion of the flexural modulus.

[0018] Throughout the figures, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components or portions of the illustrated embodiments. Moreover, while the subject disclosure will now be described in detail with reference to the figures, it is done so in connection with the illustrative exemplary embodiments. It is intended that changes and modifications can be made to the described exemplary embodiments without departing from the true scope and spirit of the subject disclosure as defined by the appended claims.

[0019] DETAILED DESCRIPTION

[0020] The present disclosure has several embodiments and relies on patents, patent applications and other references for details known to those of the art. Therefore, when a patent, patent application, or other reference is cited or repeated herein, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited.

[0021] The method for producing a silk fibroin molded body of the present disclosure comprises a process of laminating a silk fibroin film inside a mold and a process of heating and pressurizing the laminated silk fibroin film under a reduced pressure environment. When silk fibroin films are laminated and heated and pressurized, air may remain between the films, and the molded part may become white and cloudy, or themechanical strength may become anisotropic even if it is transparent in appearance. It is presumed that this is because hydrogen bonds are formed and integrated between the polypeptide chains constituting the silk fibroin when it is heated and pressurized, but because air voids remains, the hydrogen bonds are not formed between the thin films as much as between the polypeptides within the thin films. In addition, when the molded body becomes white and cloudy, the air voids between the thin films are large enough to cause light scattering, and when the thin film is transparent, it is presumed that the air voids are small enough to not cause light scattering.

[0022] Therefore, when the film is heated and pressurized under a reduced pressure environment, anisotropy is suppressed due to hydrogen bonds equivalent to those between the polypeptide chains in the thin films also forming between the polypeptide chains in the thin films, due to the air escaping from between the thin films.

[0023] Below, silk fibroin, thin film formation, molding, and measurement method will be described separately.

[0024] SILK FIBROIN

[0025] Silk fibroin is a fibrous protein that can be extracted from cocoons and / or nests.

[0026] The extraction of silk fibroin can be carried out, for example, by the method described in WO 2006 / 101223.

[0027] Silk fibroin is generally characterized by high ratios of glycine, alanine, serine, and tyrosine. Silk fibroin can exemplify a silk fibroin derived from an organism classified into the orders of Lepidoptera, Hymenoptera, or Araneae. In addition, it may also be silk fibroin obtained via gene recombination technology.

[0028] Furthermore, additives may be added to silk fibroin to the extent that it does not impair its properties.

[0029] THIN FILM PREPARATION

[0030] Although there are no particular restrictions on the methods for preparing a thin film, a method for preparing a silk fibroin film by casting silk fibroin dissolved in a solvent onto a substrate and then performing a desolventizing treatment can generally be exemplified. As a silk fibroin dissolved in a solvent, for example, an aqueous solution of silk fibroin obtained by dissolving silk fibroin, obtained by refining silkworm cocoons in anaqueous solution of LiBr and then dialyzing it, can be exemplified. In addition, dissolving a powdery silk fibroin in a dimethyl sulfoxide (DMSO) solvent, etc. can be exemplified.

[0031] The substrate used for cast molding can be a flat glass plate or the like.

[0032] Cast molding can be carried out by applying a silk fibroin solution on a substrate by a bar coater, a dispenser, etc., and then removing the solvent by a method of vacuum drying, hot air drying, and air drying.

[0033] The silk fibroin film is obtained by peeling the formed silk fibroin film from the substrate. The thin film for molding can be obtained by punching this silk fibroin film according to the shape of the mold.

[0034] The silk fibroin film can have a 0-sheet ratio of less than 30%. This is because, when the P-sheet ratio is high, the moldability in the molding later on becomes poor. Specifically, the anisotropy of the flexural modulus of the film becomes hard to eliminate, and the layers become easy to peel off. In addition, the -sheet ratio can be 10% or higher.

[0035] In addition, the moisture content of the silk fibroin film can be from about 0.5% or higher to 25% or lower. If the moisture content is too low, the moldability during subsequent molding may deteriorate. If the moisture content is too high, the flexural modulus becomes too low. The moisture content of the silk fibroin film can be from 0.7% or higher to 17% or lower, or from 0.9% or higher to 14% or lower.

[0036] MOLDING

[0037] A silk fibroin molded body can be obtained by laminating thin silk fibroin film inside a mold while depressurizing the atmosphere or by heating and pressurizing it after reducing the pressure. That is, it is sufficient if at least a part of the heating and pressurizing process is performed by reducing the pressure. FIG. 1 is a schematic diagram of an example of a mold that can be used for the molding of silk fibroin. The mold 3 comprises a through- hole and an upper piston 1 and a lower piston 2, either or both of which can be temperature controlled, and a silk fibroin molded body can be obtained by loading silk fibroin into mold 3, decompressing the atmosphere, and moving (the pistons) 1 and 2 up and down to compress the silk fibroin.

[0038] For the atmospheric pressure, 500 Torr can be used, and the lower the pressure, the more air is released. For the timing of depressurizing the atmosphere, it may be reduced from the normal pressure before heating and pressurizing or during heating and pressurizing. For the temperature of the mold in the heating and pressurizing process, it can be performed from 50°C or higher to 180°C or lower, or from 70°C or higher to 150°C orlower. Below 50°C, it takes time for the protein to sufficiently integrate. In addition, the higher the temperature, the shorter the time it takes for integration. On the other hand, at temperatures higher than 180°C, there is a concern that decomposition of the protein may begin and its strength may decrease. For the pressure at the time of pressurization, it can be conducted at 10 MPa or higher. Below 10 MPa, the protein does not sufficiently integrate, so the molded body does not become sufficiently strong. In one embodiment, it is performed at 20 kPa or higher. In addition, the time for maintaining the pressure after the predetermined pressure is reached can be from 0 minutes to 60 minutes. Furthermore, after taking the molded part out of the mold, a heat treatment may be performed from 70°C to 150°C. By maintaining the heating and pressurizing state inside the mold or performing heat treatment after molding, crystallization progresses, and the strength of the silk fibroin molded body improves.

[0039] MEASURING METHOD

[0040] Hereinafter, a measurement method necessary for the present disclosure will be described.

[0041] The water content was measured using thermogravimetric analysis (TGA 851, Mettler Toledo). The silk fibroin thin film prepared by the same operation as the thin silk fibroin film for molding used for the molding was heated up from 25 °C to 130°C at 5°C / min under a nitrogen atmosphere, maintained at 130°C for 30 minutes, and the weight change during the heating was measured. With the weight at the start of measurement represented by Gi and the weight at the end of measurement represented by G2, the calculation was made using the Equation 1 below.

[0042] Water content (%) =— — - X 100 Equation 1G

[0043] The 0-sheet ratio was measured using an FTIR (Frontier MIR NIR / Spotlight 400, Perkin Elmer). The absorbance was measured in the range from 1500 to 1800 cm1, the peak wavelength N at 1600 ~ 1650 cm1was read out, and it was calculated with the calculation formula of Equation 2.-1 41 — z

[0044] p — sheet ratio (%) = - x 100 Equation 2

[0045] It was calculated with P-sheet ratio (%) = (1641 - N) / 20 x 100. This is because the peak wavelength is 1641 cm1in the amorphous state and becomes 1621 cm1in the crystallization state.

[0046] The flexural modulus was measured using an Instron universal testing machine (Model 5582, Instron). The distance between the fulcrums of three-point bending was fixed at 27 mm, and the measurement speed was set at 1 mm / min. The flexural modulus was determined from the displacement (strain) of 0.05 up to 0.25%. In addition, the flexural modulus in the direction parallel to the thin film was defined as the transverse flexural modulus, the flexural modulus in the direction perpendicular to the thin film was defined as the longitudinal flexural modulus, and the longitudinal flexural modulus / trans verse flexural modulus was calculated to form a table of anisotropy. The closer the value is to 1, the lower the anisotropy, and the lower the value, the lower the longitudinal flexural modulus and the higher the anisotropy compared with the transverse flexural modulus.

[0047] EXAMPLES

[0048] EXAMPLE 1

[0049] Silkworm cocoons were washed with water and then boiled in a 0.02 mol / L sodium carbonate aqueous solution for 30 minutes to conduct degumming. The degummed silk fibroin was put into a 9.3 mol / L LiBr aqueous solution and dissolved by agitating it at 60°C for 4 hours. Cellulose tubes 30 / 32 (molecular weight cut off of 12000 ~ 14000) manufactured by Sekisui Chemical Co., Ltd. were used for desalination. The concentration of the fibroin aqueous solution after desalination was diluted with pure water so it became 5%.

[0050] A silk fibroin solution was applied onto a glass substrate using a bar coater (#22, manufactured by Mitsui Electric Seiki), and then dried at room temperature for 1 hour, and then at 80°C for 5 hours. The dried film was peeled off from the substrate and when the film thickness was measured, it was 3.2 pm. Upon measuring the water content, it was 12%. When the -sheet ratio was measured, it was 8%.

[0051] Subsequently, it was molded by heating and pressurizing the base material for molding under a reduced pressure atmosphere. For the mold, a mold 13 having a square columnar through- hole of a length of 80 mm and a width of 15 mm was used, as depicted in FIG. 2. For the lower piston 12, as shown in FIG. 2, a piston having a groove of a width of 2 mm and a depth of 35 mm carved in the center was used. The upper piston 11 was did not contain such a groove.

[0052] Two types of thin silk fibroin films, 15 mm in length and 2 mm in width, and 80 mm in length and 15 mm in width, were prepared, and the former was set into the groove portion of the lower piston and the latter was set between the upper and lower pistons. At this time, the total weight of the film set was 4.49 g. The mold was set in a vacuum press apparatus, and after the pressure was reduced to 100 Torr at 125°C, it a pressure of 600 MPa was applied. After that, the mold was taken out, cooled to room temperature, and then the silk fibroin molded body was obtained. When the portions 21 and 22 in FIG. 3 were cut out of the silk fibroin molded body 25 and the longitudinal flexural modulus and the transverse flexural modulus were measured, they were 7.6 GPa and 7.6 GPa, respectively. The longitudinal modulus / transverse modulus was 1.00, and no anisotropy was confirmed.

[0053] COMPARATIVE EXAMPLE 1

[0054] When the molded body was heated and pressurized without depressurizing the atmosphere after it was obtained with the same operation as in Embodiment 1, the longitudinal flexural modulus was 4.6 GPa, the transverse flexural modulus was 7.6 GPa, and the longitudinal flexural modulus / transverse flexural modulus was 0.61, and a large anisotropy was confirmed.

[0055] EXAMPLE 2

[0056] The molded body was prepared with the same operation as in Embodiment 1 , except that the atmospheric pressure at the time of heating and pressurization was set to 200 Torr. The longitudinal flexural modulus was 7.2 GPa, the transverse flexural modulus was7.5 GPa, the longitudinal flexural modulus / transverse flexural modulus was 0.96, and the anisotropy was slight.

[0057] EXAMPLE 3

[0058] The molded body was prepared with the same operation as in Embodiment 1, except that the atmospheric pressure at the time of heating and pressurization was set to 500 Torr. The longitudinal flexural modulus was 6.8 GPa, the transverse flexural modulus was7.6 GPa, the longitudinal flexural modulus / transverse flexural modulus was 0.89, and the anisotropy was slight.

[0059] EXAMPLE 4

[0060] A molded body was prepared with the same operation as in Embodiment 1, except that the drying of the thin silk fibroin film was set at room temperature for 4 hours and then at 80°C for 5 hours. The longitudinal flexural modulus was 6.1 GPa, the transverse flexural modulus was 7.2 GPa, the longitudinal flexural modulus / transverse flexural modulus was 0.82, and the anisotropy was slight.

[0061] EXAMPLE 5

[0062] A molded body was prepared with the same operation as in Embodiment 1 , except that the drying of the thin silk fibroin film was set at room temperature for 7 hours and then at 80°C for 5 hours. The longitudinal flexural modulus was 5.8 GPa, the transverse flexural modulus was 7.0 GPa, the longitudinal flexural modulus / transverse flexural modulus was 0.83, and the anisotropy was slight.

[0063] EXAMPLE 6

[0064] A molded body was prepared with the same operation as in Embodiment 1, except that the drying of the thin silk fibroin film was set at room temperature for 1 hour and then at 90°C for 5 hours. The longitudinal flexural modulus was 5.2 GPa, the transverse flexural modulus was 6.8 GPa, the longitudinal flexural modulus / transverse flexural modulus was 0.76, and the anisotropy was slight.

[0065] EXAMPLE 7

[0066] A molded body was prepared with the same operation as in Embodiment 1, except that the drying of the thin silk fibroin film was set at room temperature for 1 hour and then at 80°C for 30 minutes. The longitudinal flexural modulus was 5.5 GPa, the transverse flexural modulus was 7.5 GPa, the longitudinal flexural modulus / transverse flexural modulus was 0.73, and the anisotropy was slight.

[0067] The results of the Embodiments and Comparative Examples are summarized in Table 1.

[0068] Table 1

[0069] Definitions

[0070] In referring to the description, specific details are set forth in order to provide a thorough understanding of the examples disclosed. In other instances, well-known methods, procedures, components and circuits have not been described in detail as not to unnecessarily lengthen the present disclosure.

[0071] It should be understood that if an element or part is referred herein as being "on", "against", "connected to", or "coupled to" another element or part, then it can be directly on, against, connected or coupled to the other element or part, or intervening elements or parts may be present. In contrast, if an element is referred to as being "directly on", "directly connected to", or "directly coupled to" another element or part, then there are no intervening elements or parts present. When used, term "and / or", includes any and all combinations of one or more of the associated listed items, if so provided.

[0072] Spatially relative terms, such as “under” “beneath”, "below", "lower", "above", "upper", “proximal”, “distal”, and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the various figures. It should be understood, however, that the spatially relative terms areintended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, a relative spatial term such as "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein are to be interpreted accordingly. Similarly, the relative spatial terms “proximal” and “distal” may also be interchangeable, where applicable.

[0073] The term “about,” as used herein means, for example, within 10%, within 5%, or less. In some embodiments, the term “about” may mean within measurement error.

[0074] The terms first, second, third, etc. may be used herein to describe various elements, components, regions, parts and / or sections. It should be understood that these elements, components, regions, parts and / or sections should not be limited by these terms. These terms have been used only to distinguish one element, component, region, part, or section from another region, part, or section. Thus, a first element, component, region, part, or section discussed below could be termed a second element, component, region, part, or section without departing from the teachings herein.

[0075] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “includes”, “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Specifically, these terms, when used in the present specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof not explicitly stated. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherw ise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0076] It will be appreciated that the methods and compositions of the instant disclosure can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS:

1. A fibroin film molding method comprising: a process of laminating a fibroin film; and a process of molding by heating and pressurizing the laminated fibroin film, wherein at least a part of the molding process is performed by reducing the pressure of the atmosphere.

2. The method for molding silk fibroin according to Claim 1 , wherein the P-sheet ratio of the fibroin film is less than 30%.

3. The method for molding silk fibroin according to Claim 1, wherein the P-sheet ratio of the fibroin film is 10% or more.

4. The silk fibroin molding method according to Claim 1, wherein the moisture content of the fibroin film is 0.5% or more and 25% or less.

5. A fibroin film molding method comprising the steps of: laminating a fibroin film; and molding the laminated fibroin film by heating and pressurizing the laminated fibroin film, wherein air pressure is reduced during at least part of the molding process to reduce anisotropy in a strength of a molded result.

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