Silk fibroin molding base and manufacturing method

Abstract

The present disclosure provides a fibroin molding method and a molded product that can be easily released from a mold even when the mold shape is complex, and the fibroin is heated and pressurized inside the mold and then heat-treated outside the mold.

Description

[Technical Field]

[0001] Technical Field

[0002] The present disclosure relates to a silk fibroin molding element and a method for producing the same.

[0003] Background technology

[0004] International Patent Application Publication No. 2017 / 047503 provides a molded body obtained by filling a mold with a powder of a protein containing a natural spider silk protein, which is silk fibroin, or a polypeptide derived from a natural spider silk protein, and then heating and pressurizing it.

[0005] Disclosure Overview

[0006] In conventional methods, when the mold shape becomes complex, the fibroin is so strong that it cannot be removed from the mold, or the molded body may be damaged when removed from the mold.

[0007] The present disclosure provides a fibroin molding method and molded product that enable easy demolding even in the case of a complex mold shape.

[0008] Specifically, the present disclosure proposes a method for producing a fibroin molded body, the method including the steps of pouring fibroin into a mold, heating and pressurizing the fibroin, removing the pressurized fibroin, and heat-treating the removed fibroin molded body.

[0009] Therefore, even if the mold has a complex shape, the fibroin molded body can be easily released from the mold.

[0010] These and other embodiments, objects, features, and advantages of the present disclosure will become apparent from the following detailed description of exemplary embodiments of the present disclosure, read in conjunction with the accompanying drawings and the appended claims. [Brief explanation of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate various embodiments, objects, features, and advantages of the present disclosure.

[0013] FIG. 1 is a schematic diagram of a mold for molding silk fibroin.

[0014] FIG. 2 is a schematic diagram of a silk fibroin mold used in at least one embodiment.

[0015] FIG. 3 is a schematic diagram of a silk fibroin molding upper piston used in at least one embodiment.

[0016] FIG. 4 is a schematic diagram of a silk fibroin mold used in at least one embodiment.

[0017] FIG. 5 is a schematic diagram of a freeze-drying block used in at least one embodiment.

[0018] FIG. 6 is a schematic diagram of a freeze-drying block used in at least one embodiment.

[0019] FIG. 7 is a schematic diagram of a freeze-drying block used in at least one embodiment.

[0020] FIG. 8 is a schematic diagram of a freeze-drying block used in at least one embodiment.

[0021] FIG. 9 is a schematic diagram of a freeze-drying block used in at least one embodiment.

[0022] FIG. 10 is a schematic diagram of a silk fibroin mold used in at least one embodiment.

[0023] FIG. 11 is a schematic diagram of a silk fibroin mold used in at least one embodiment.

[0024] FIG. 12 is a schematic diagram of a silk fibroin mold used in at least one embodiment.

[0025] FIG. 13 is a schematic diagram of a silk fibroin mold used in at least one embodiment.

[0026] Throughout the drawings, unless otherwise stated, the same reference numerals and characters are used to denote like features, elements, components, or portions of the illustrated embodiments. Moreover, while the present disclosure will now be described in detail with reference to the drawings, it is done so in connection with the exemplary embodiments for purposes of illustration. It is intended that changes and modifications can be made to the exemplary embodiments described without departing from the true scope and spirit of the present disclosure, as defined by the appended claims. Moreover, each described embodiment can be manufactured or used in combination with any other described embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Embodiments

[0028] This disclosure describes several embodiments and relies on patents, patent applications, and other references for details known to those skilled in the art. Accordingly, when a patent, patent application, or other reference is cited or repeated herein, it should be understood that it is incorporated herein by reference in its entirety for all purposes, not just as provided herein.

[0029] The method for producing a fibroin molded body in the present disclosure includes the steps of pouring fibroin into a mold, heating and pressurizing the fibroin, removing the pressurized fibroin, and heat-treating the removed fibroin molded body.

[0030] When pressure is applied to fibrous silk fibroin at a temperature above its glass transition temperature, it deforms to conform to the shape of the mold, while simultaneously expelling the air between the silk fibroin and integrating it into a transparent molded object. At this stage, the silk fibroin is integrated, but has a low flexural modulus. However, it has a high elongation at the yield point and large plastic deformation, allowing it to be easily removed from complex molds. When the silk fibroin molded object removed from the mold is placed in an environment above its glass transition temperature, β-sheets form between the silk fibroin, and the β-sheets then assemble into crystals, resulting in a molded object with a high flexural modulus.

[0031] Below, we will explain silk fibroin, mold molding, heat treatment, and measurement methods individually.

[0032] Silk fibroin

[0033] Silk fibroin is a fibrous protein that can be extracted from the cocoon and / or nest.

[0034] Silk fibroin can be extracted, for example, by the method described in WO2006 / 101223.

[0035] Silk fibroin is generally characterized by a high proportion of glycine, alanine, serine, and tyrosine. Examples of silk fibroin include silk fibroin derived from organisms classified as Lepidoptera, Hymenoptera, or Araneae. Silk fibroin may also be silk fibroin obtained by genetic engineering.

[0036] In addition, additives may be added to silk fibroin as long as the properties of the silk fibroin are not impaired.

[0037] There are no particular limitations on the form of silk fibroin to be placed in the mold. Examples include a silk fibroin aqueous solution obtained by dissolving refined silk fibroin in a freeze-dried LiBr aqueous solution and then dialysis, a pulverized freeze-dried silk fibroin, and silk fibroin compressed into a cylindrical, polygonal, spherical, or hemispherical shape. The bulk density of these silk fibroins is approximately 0.50 g / cm. 3 More than ~ approx. 1.20g / cm 3 If the bulk density is low, the tableting strength is low, and the tablet tends to break down when put into a mold and scatter around. Therefore, the bulk density is set to about 0.70 g / cm. 3 More than about 0.90g / cm 3 or more, or about 1.00 g / cm 3 However, the upper limit of the bulk density is 1.20 g / cm because the surface transferability decreases during molding. 3 Less than or equal to 1.10 g / cm 3 It can be less than.

[0038] Furthermore, since the bulk density of silk fibroin, which tends to scatter when handled in air due to its low bulk density, can be adjusted, it may be beneficial to instead weigh out a silk fibroin aqueous solution containing 1 / n (n is an integer) of the amount of silk required for fibroin molding, place the weighed solution in a freeze-drying block with empty through-holes, freeze-dry the weighed solution in the freeze-drying block, compress the silk fibroin in the freeze-drying block, and adjust the bulk density. The freeze-drying block may also be a molding die. The number of through-holes provided in the freeze-drying block may be single or multiple.

[0039] Exemplary Configurations for Molding

[0040] As described herein, various configurations for molding can be used. In certain embodiments, an aqueous fibroin solution is first converted into a molding element by heat and pressure or freeze-drying. The molding element can then be compressed into a final shape using a mold. Specific exemplary configurations for such molding processes are described with the accompanying drawings. It should be noted that the description of one configuration is equally applicable to other configurations having the same functionality and use. For example, if a component is described as being able to adjust its temperature in one configuration, the same is possible in other configurations utilizing the same component.

[0041] Figure 1 is a schematic diagram of an example of a mold that can be used to mold silk fibroin. The mold consists of a temperature-controllable part (mold) 3 with a through-hole, an upper piston 1, and a lower piston 2. Silk fibroin is introduced into part 3 and compressed by moving pistons 1 and 2 up and down to obtain a silk fibroin molded product. Alternative configurations of molds with this or similar activation method can be used, as shown in Figure 2. In Figure 2, part 13 with a through-hole is provided along with upper and lower pistons 11 and 12. As can be seen, upper piston 11 has specific protrusions along its lower edge that contact the silk fibroin, thereby changing the shape of the silk fibroin molded product when compressed. Figure 3 provides a bottom view of upper piston 11, further illustrating the protrusions as cylindrical. As shown in Figure 3, the upper piston 11 used had a 50 mm long piston with a cylindrical protrusion measuring 2 mm in diameter and 1 mm in height, positioned 5 mm from the end. The lower piston 12 was used without protrusions. Protrusions of any shape or form can be utilized from either or both the upper and lower pistons based on the desired compact.

[0042] In one embodiment, as shown in Fig. 4, a molding element 24 is placed in a part 13 having an upper piston 11 and a lower piston 12. The element 24 is then pressurized and heated to form a compact. As shown in Fig. 3, the upper piston 11 in Fig. 4 has a cylindrical protrusion. A mold (part) 13 having a rectangular pillar-shaped through-hole 50 mm long and 15 mm wide is used.

[0043] FIG. 5 shows freeze-drying blocks 31, 32 that can be used to freeze-dry a single or multiple doses of an aqueous silk fibroin solution. The doses are injected into through-holes 33. As shown in FIG. 6, the freeze-drying blocks 31, 32 have fibroin solution holders 34 at the distal ends of the through-holes 33, which hold the aqueous fibroin solution 21 within the through-holes 33. As shown in FIG. 7, the entire blocks 31, 32 can be freeze-dried, resulting in freeze-dried silk fibroin 22 within the through-holes 33, which is held within the through-holes 33 of the blocks 31, 32 by the fibroin solution holders 34. The freeze-dried silk fibroin 22 can then be compressed by pistons 35 to produce a molding blank 24, as shown in FIG. 8. A piston 35 can be applied through the upper opening of the through-hole 33 in the blocks 31, 32, which then compresses the freeze-dried silk fibroin 22 between the piston 35 and the fibroin solution holder 34, resulting in a molding element 24.

[0044] A further embodiment is shown in Figure 9, in which freeze-drying blocks 31, 32 contain a fibroin solution weighed out 25, which is 1 / n of the amount of the molded article, and which is contained in through-holes 33 and held in through-holes 33 by fibroin solution holders 34. Blocks 31, 32 are then freeze-dried, and the freeze-dried fibroin solution weighed out 26, which is 1 / n of the amount of the molded article, is compressed in through-holes 33 by pistons 35, resulting in a molding body 27 which is 1 / n of the amount of the molded article.

[0045] As shown in Figure 10, n portions (shown as two portions in Figure 10) of molding material 27, which is 1 / n of the molded product, are placed in mold (part) 13, and then compressed by upper piston 11 and lower piston 12 to form molded product 23.

[0046] In a further embodiment, as shown in FIG. 11 , an aqueous fibroin solution 15 can be poured into a mold 13 and held in place by a fibroin solution holder 14, and the entire mold can then be freeze-dried. As shown in FIG. 12 , this results in the fibroin solution 15 being transformed into freeze-dried silk fibroin 16, which resides within the mold 13 to become the final molded body. Such freeze-dried silk fibroin 16 is held within the mold 13 by the fibroin solution holder 14, which can then be removed and replaced with a lower piston 12, as shown in FIG. 13 . The upper piston 11 can be positioned at the top opening of the through-hole 33 so that the freeze-dried silk fibroin is bracketed at both ends of the through-hole 33 by the upper piston 11 and the lower piston 12. The upper piston 11 and the lower piston 12 can apply pressure to the freeze-dried silk fibroin 16, resulting in a molded body 17.

[0047] Mold forming

[0048] Silk fibroin with adjusted bulk density is placed in a mold and heated and pressurized to obtain a silk fibroin molded product. As mentioned above, Figure 1 is a schematic diagram of an example of a mold that can be used to mold silk fibroin. The mold is composed of a temperature-controllable part 3 having a through-hole, an upper piston 1, and a lower piston 2. Silk fibroin is placed in part 3 and compressed by moving pistons 1 and 2 up and down to obtain a silk fibroin molded product.

[0049] The mold temperature during the heating and pressurizing process can be 70°C to 200°C or 100°C to 150°C. Below 70°C, the proteins do not fully integrate. On the other hand, at temperatures above 200°C, the proteins begin to decompose, potentially reducing strength. Pressurization can be performed at 10 MPa or higher. Below 10 MPa, the proteins do not fully integrate, resulting in insufficient strength for the molded product. Optionally, pressurization can be performed at 50 MPa or higher. Additionally, the applied pressure can be approximately 1000 MPa or lower. After reaching a predetermined pressure, the pressure is maintained for 1 to 300 seconds or 1 to 30 seconds. The time from placing the silk fibroin into the heated mold to applying pressure and removing it from the mold can be less than 500 seconds or less than 300 seconds. Longer times can promote crystallization, increasing the flexural modulus (flexural modulus) of the molded product, potentially requiring a large force for release from the mold or resulting in breakage.

[0050] Furthermore, since there is no need to adjust the bulk density and there is no need to move the molding body, the molding process can be reduced and the weight of the molded body can be accurately determined.Therefore, it is possible to weigh out a silk fibroin aqueous solution, pour the weighed amount of silk required for the fibroin molded body into a mold, freeze-dry it while it is still in the weighed state, and then heat-compress the silk fibroin in the freeze-dried mold to obtain a molded body.

[0051] Heat Treatment

[0052] The heat treatment method is not particularly limited. It can be performed by heating with heated air in a thermostatic oven, by heating on a hot plate using heat transfer from the top plate, or by infrared heating using an infrared heater. The heat treatment temperature can be 70°C to 200°C or 100°C to 150°C. Below 70°C, the silk fibroin is not sufficiently integrated, resulting in an insufficient strength of the molded article. On the other hand, above 200°C, the silk fibroin may begin to decompose, resulting in a decrease in strength. The heat treatment time can be 600 seconds or longer. If it is less than 600 seconds, crystallization does not proceed sufficiently, resulting in an insufficient strength of the molded article. Furthermore, the heat treatment time should be sufficiently long, e.g., longer than the heating and pressurizing time in a mold. This shortens the heating and pressurizing time in a mold.

[0053] Measurement method

[0054] The measurement method required for this disclosure will be described below.

[0055] The bulk density was measured using the bead displacement method. Specifically, a pre-weighed amount of quartz sand (0.3-0.5 mm) was placed in a volumetric scale, and then silk fibroin was added to the scale. The bulk density was estimated from the weight of the scale and the increase in volume.

[0056] The flexural modulus was measured using an Instron universal testing machine (Model 5582, Instron). The distance between the three-point bending supports was fixed at 27 mm, and the measurement speed was 1 mm / min. The flexural modulus was calculated from the displacement (strain) from 0.05 to 0.25%.

[0057] The demolding force was measured using an Instron universal testing machine (5582 model, Instron Corporation). The mold was placed in the machine, and the upper and lower pistons were pulled apart at a speed of 10 mm / sec to release the mold. The maximum stress at this time was recorded as the demolding force.

[0058] Example

[0059] Example 1

[0060] After washing the silkworm cocoons, they were boiled in a 0.02 mol / L aqueous solution of sodium carbonate for 30 minutes for degumming. The degummed silk fibroin was placed in a 9.3 mol / L aqueous solution of LiBr and dissolved by stirring at 60°C for 4 hours. The silk fibroin was desalted using a Sekisui Chemical cellulose tube 30 / 32 (molecular weight cutoff: 12,000-14,000). The desalted fibroin solution was diluted with pure water to a concentration of 5%.

[0061] The resulting 5% fibroin aqueous solution was divided into 39.0 ml portions and placed in containers. The divided fibroin solutions were freeze-dried using a Tokyo Rikakikai freeze dryer (FD-550P). The freeze-drying conditions were as follows: after freezing at -30°C, the atmosphere was reduced in pressure, the temperature was raised to -6°C, and freeze-drying was continued for 100 hours. Next, one of the divided freeze-dried silk fibroin particles was placed in a mold with a cylindrical through-hole of 5 mm diameter, pressurized at 25°C and 30 MPa, and then removed to obtain a molding element.

[0062] The bulk density of the obtained molding body was measured and found to be 1.02 g / cm 3 It was.

[0063] Next, the molding element was heated and pressurized as shown in Figure 4. A die having a rectangular prism-shaped through-hole 50 mm long and 15 mm wide was used. The upper piston had a length of 50 mm and a cylindrical protrusion 2 mm in diameter and 1 mm high located 5 mm from the end of the piston, as shown in Figure 3. The lower piston had no protrusion.

[0064] One molding element was placed into a mold previously adjusted to a temperature of 120°C, and after applying a pressure of 500 MPa for 10 seconds, it was placed in a mold release force measuring device and released 30 seconds after the start of heating. The mold release force was 0.76 kN, and the mold was easily released. In this example, the mold was pressurized in a preheated state, but preheating is not necessarily required. However, preheating the mold in advance has the advantage of shortening the molding time.

[0065] The obtained molded body was heat-treated for 600 seconds in a temperature-lowering furnace at 120° C. When the flexural modulus after the heat treatment was measured, it was found to have a sufficient strength of 6.2 GPa.

[0066] Comparative example A

[0067] After obtaining a molded product in the same manner as in Example 1, the flexural modulus was measured without heat treatment, and it was found to be 1.8 GPa, which was not a sufficient strength.

[0068] Example 2

[0069] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the pressure during molding was 20 MPa. The mold release force was 0.56 kN, and the mold was easily released. The flexural modulus was 4.2 MPa, and sufficient strength was obtained.

[0070] Example 3

[0071] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the pressure during molding was 10 MPa. The mold release force was 0.41 kN, and the mold was easily released. The flexural modulus was 2.8 MPa, and sufficient strength was obtained.

[0072] Example 4

[0073] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the temperature during molding was 70°C. The mold release force was 0.54 kN, and the mold could be easily released. The flexural modulus was 4.5 MPa, and sufficient strength was obtained.

[0074] Example 5

[0075] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the temperature during molding was 50°C. The mold release force was 0.45 kN, and the mold was easily released. The flexural modulus was 2.3 MPa, and sufficient strength was obtained.

[0076] Example 6

[0077] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the temperature during molding was 150°C. The mold release force was 0.71 kN, and the mold could be easily released. The flexural modulus was 5.9 MPa, and sufficient strength was obtained.

[0078] Example 7

[0079] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the temperature during molding was 180°C. The mold release force was 0.32 kN, and the mold could be easily released. The flexural modulus was 3.5 MPa, and sufficient strength was obtained.

[0080] Example 8

[0081] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the pressing time during molding was 1 second. The mold release force was 0.74 kN, and the mold was easily released. The flexural modulus was 6.5 MPa, and sufficient strength was obtained.

[0082] Example 9

[0083] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the heating time during molding was 90°C and the time until demolding was 300 seconds. The demolding force was 1.08 kN, and the body was easily demolded. The flexural modulus was 6.6 MPa, and sufficient strength was obtained.

[0084] Example 10

[0085] A molded body was prepared and heat-treated in the same manner as in Example 1, except that the heating time during molding was 85°C and the time until demolding was 350 seconds. The demolding force was 1.36 kN, and the body was easily demolded. The flexural modulus was 6.3 MPa, and sufficient strength was obtained.

[0086] The results of the examples and comparative examples are summarized in Table 1.

[0087] Comparative example B

[0088] A molded body was obtained in the same manner as in Comparative Example A, except that the time until demolding was 500 seconds. The force required for demolding was 1.85 kN, which was very strong. Furthermore, the protrusions were damaged during demolding. When the flexural modulus of the obtained molded body was measured without heat treatment, it was 6.4 GPa, which was insufficient strength.

[0089] Example 11

[0090] A molded body was prepared and heat-treated in the same manner as in Example 1, except that 39.0 ml of fibroin solution was poured into a freeze-drying block having a cylindrical through-hole with a diameter of 5 mm as shown in Figure 5, the block was freeze-dried as shown in Figure 7, and the silk fibroin was compressed within the block as shown in Figure 8 to obtain a molding element. The mold release force was 0.76 kN, and the mold was easily released. The flexural modulus was 6.2 MPa, indicating sufficient strength.

[0091] Example 12

[0092] As shown in Figure 9, 19.5 ml of the fibroin solution, half the original volume of 39 ml, was poured into a freeze-drying block with a cylindrical through-hole having a diameter of 5 mm, and the block was freeze-dried. The silk fibroin was compressed within the block to obtain a molding element. A molding was produced and heat-treated in the same manner as in Example 1, except that two molding elements were placed in a mold as shown in Figure 10. The mold release force was 0.76 kN, and the product was easily demolded. The flexural modulus was 6.2 MPa, indicating sufficient strength.

[0093] Example 13

[0094] As shown in Figure 11, 39.0 ml of fibroin solution was poured into a mold for the final molded body, and the mold was freeze-dried. As shown in Figure 12, the freeze-drying state was changed to a state where freeze-dried silk fibroin was already poured into the mold. As shown in Figure 13, the freeze-dried silk fibroin was pressed between the upper and lower molds without changing the mold to obtain a molded body. A molded body was produced and heat-treated in the same manner as in Example 1, except that the mold release force was 0.76 kN, and the mold was easily released. The flexural modulus was 6.2 MPa, and sufficient strength was obtained. [Table 1A] [Table 1B]

Claims

1. A method for producing a fibroin molded body, pouring the fibroin into a mold; A step of heating and pressurizing the fibroin; removing the pressurized fibroin, wherein the pressurized fibroin forms a fibroin molded body; A method for producing a fibroin molded body, comprising the step of heat-treating the fibroin molded body.

2. 2. The method for producing a fibroin molded product according to claim 1, wherein the heating and pressurizing is carried out at 70° C. to 200° C., 10 MPa or more, for 1 to 300 seconds.

3. 2. The method for producing a fibroin molded body according to claim 1, wherein the molded body is removed within 500 seconds after the start of heating.

4. The method for producing a fibroin molded product according to any one of the preceding claims, characterized in that the heat treatment is carried out at a temperature of 70°C to 200°C.

5. 2. The method for producing a fibroin molded product according to claim 1, wherein the heating time of the heat treatment is 600 seconds or more.

6. 2. The method for producing a fibroin molded body according to claim 1, wherein the heating time for the heat treatment is longer than the heating time for the heating and pressurizing.

7. The fibroin is 0.7 g / cm ３ ~1.2 g / cm ３ 2. The method for producing a fibroin molded product according to claim 1, wherein the fibroin molded product has a bulk density of 1000 or less.

8. 10. The method for producing a fibroin molded product according to claim 9, further comprising the step of drying an aqueous solution containing fibroin in a block to produce the fibroin.

9. 9. The method for producing a fibroin molded product according to claim 8, further comprising a step of pressurizing a dried aqueous solution containing fibroin in a block to produce the fibroin.

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