Silk fibroin-containing composition and method for producing composition

Abstract

A composition comprising a mixture of silk fibroin and a bioabsorbable polymer, wherein the weight average molecular weight of the silk fibroin as measured by gel permeation chromatography is 9.0 × 104 to 5.0 × 105, and the mixing ratio of the silk fibroin to the bioabsorbable polymer is 95 / 5 to 25 / 75 by mass ratio.

Description

Composition containing silk fibroin and method for producing the composition

[0001] The present invention relates to a composition comprising silk fibroin and a bioabsorbable polymer, and a method for producing the composition.

[0002] In the field of cardiovascular diseases, including heart and blood vessel diseases, implantable medical devices such as artificial blood vessels, vascular patches, and cardiac patches are used. While materials such as polyester and stretched PTFE are used, these are foreign bodies to the body and do not regenerate tissue.

[0003] From the above perspective, research and development are being conducted in the field of "tissue engineering," and the use of cell scaffold materials such as nonwoven fabrics made from materials containing highly biocompatible silk fibroin as medical devices is being considered (for example, Patent Document 1).

[0004] Japan Special Table No. 2006-519664

[0005] However, cell scaffold materials like those described in Patent Document 1 have the problem of slow degradation in vivo, severely limiting the tissues and diseases to which they can be applied. Furthermore, from the perspective of tissue regeneration, it is important that cell scaffold materials made of nonwoven fabric maintain their porosity after implantation in the body.

[0006] The present invention has been made in view of the above, and one of its objectives is to provide a composition that has an appropriate biodegradation rate, can maintain a porous structure after implantation in a living organism when a nonwoven fabric is formed, and has excellent suture strength when a nonwoven fabric is formed, as well as a method for producing the same.

[0007] Through diligent research, the inventors have discovered that the above problems can be solved by setting the weight-average molecular weight of silk fibroin and the mixing ratio of silk fibroin to bioabsorbable polymer within an appropriate range in a composition containing a mixture of silk fibroin and a bioabsorbable polymer.

[0008] The present invention includes the following embodiments: [1] A composition comprising silk fibroin and a bioabsorbable polymer, wherein the weight-average molecular weight of the silk fibroin determined by gel permeation chromatography is 9.0 × 10４ ~5.0 x 10 ５ The composition wherein the mixing ratio of the silk fibroin and the bioabsorbable polymer is 95 / 5 to 25 / 75 by mass. [2] The weight-average molecular weight of the silk fibroin is 9.0 × 10 ４ ~2.5 x 10 ５ The composition according to [1]. [3] The composition according to [1] or [2], wherein the bioabsorbable polymer is at least one selected from polycarbonate, polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, polyurethane, and polyoxyalkylene glycol. [4] The weight-average molecular weight of the bioabsorbable polymer is 1.0 × 10 ４ ~1.0 x 10 ６The composition according to any one of [1] to [3]. [5] The composition according to any one of [1] to [4] wherein the bioabsorbable polymer comprises at least one of the following repeating units: repeating unit derived from alkylene carbonate, repeating unit derived from lactic acid, repeating unit derived from caprolactone, repeating unit derived from glycolic acid, repeating unit derived from dioxanone. [6] The composition according to any one of [1] to [5] wherein the bioabsorbable polymer is a homopolymer or copolymer of trimethylene carbonate. [7] The composition according to [6] wherein the bioabsorbable polymer is a copolymer of trimethylene carbonate and lactic acid. [8] The composition according to [7] wherein the ratio of trimethylene carbonate to lactic acid is 90 / 10 to 10 / 90 in molar ratio. [9] The composition according to any one of [1] to [8] which is a fiber, film, or nonwoven fabric.

[10] The composition according to any one of [1] to [9] which is a nonwoven fabric with a suture strength of 0.6 N or higher in a suture strength test.

[11] The composition according to any one of [1] to

[10] , wherein the nonwoven fabric has a fluorine-containing alcohol content of 0.1 to 1000 ppm by mass.

[12] A method for producing a composition, comprising the steps of: dissolving silk fibroin and a bioabsorbable polymer in a fluorine-containing alcohol to obtain a solution; and drying the solution to produce a composition, wherein the weight-average molecular weight of the silk fibroin determined by gel permeation chromatography is 9.0 × 10 ４ ~5.0 x 10 ５Controlled to a mass ratio of 95 / 5 to 25 / 75 of the silk fibroin to the biodegradable polymer, a method for producing a composition.

[13] The method for producing a composition according to

[12] , wherein the fluorinated alcohol is 1,1,1,3,3,3-hexafluoro-2-propanol.

[14] The method for producing a composition according to

[12] or

[13] , wherein the silk fibroin is obtained in a step of refining and solubilizing cocoon balls.

[15] The step of drying the solution to form a composition is a step of electrospinning the solution to obtain fibers, a step of insolubilizing the fibers, and a step of removing the fluorinated alcohol from the insolubilized fibers, and then forming a nonwoven fabric. The method for producing a composition according to any one of

[12] to

[14] .

[16] The step of drying the solution to form a composition is a step of forming a film via a step of drying the solution on a substrate. The method for producing a composition according to any one of

[12] to

[14] .

[0009] According to the present invention, it is possible to provide a composition having an appropriate biodegradation rate in a living body, capable of maintaining a porous structure after implantation into a living body when forming a nonwoven fabric, and having excellent suture strength when forming a nonwoven fabric, and a method for producing the same.

[0010] 〔Composition〕The composition according to an embodiment of the present invention is a composition in which silk fibroin and a biodegradable polymer are mixed. The weight average molecular weight of the silk fibroin by gel permeation chromatography is 9.0×10 ４ to 5.0×10 ５ and the mixing ratio of the silk fibroin and the biodegradable polymer is 95 / 5 to 25 / 75 by mass ratio.

[0011] The composition according to an embodiment of the present invention is a composition in which silk fibroin and a biodegradable polymer are mixed. As long as the weight average molecular weight of the silk fibroin and the mixing ratio of the silk fibroin and the biodegradable polymer are within the above ranges, the shape thereof is not limited. That is, it may be in a solid state or even in a liquid state containing a solvent.

[0012] <Silk fibroin> The composition according to the embodiment of the present invention contains silk fibroin. Because silk fibroin has high mechanical strength and high cell affinity, it can be suitably used as a component material for medical devices that are implanted in living organisms.

[0013] The weight-average molecular weight (Mw) of silk fibroin, determined by gel permeation chromatography (GPC), is 9.0 × 10⁻⁶. ４ ~5.0 x 10 ５ Therefore, Mw is 9.0 x 10 ４ If the Mw is less than 5.0 × 10, then in the case of a composition according to the embodiment of the present invention, if it is a nonwoven fabric, fiber breakage will occur in the biodegradability test, making it difficult to maintain the porous structure. In other words, it is difficult to maintain the fiber shape when implanted in a living body. On the other hand, if Mw is 5.0 × 10 ５ If it exceeds this value, the viscosity of the solution containing the composition according to the embodiment of the present invention (which may be the composition according to the embodiment of the present invention itself) increases, and its solubility deteriorates significantly. As a result, it becomes difficult to mold the composition according to the embodiment of the present invention into a fibrous or nonwoven fabric form. From the viewpoint of suppressing a decrease in production efficiency due to a decrease in the solubility of silk fibroin when preparing the above solution, preferably Mw is 4.0 × 10 ５ The following applies:

[0014] Mw is 9.0 x 10 ４ That is all, 1.2 × 10 ５ Preferably, it is 1.6 × 10 ５ It is more preferable that the above is true. Also, Mw is 5.0 × 10 ５ The following is true: 4.0 × 10 ５ Preferably, it is 2.5 × 10 ５ It is even more preferable that Mw is 9.0 × 10 ４ ~5.0 x 10 ５ Therefore, 9.0 × 10 ４ ~4.0 x 10 ５ Preferably, 9.0 × 10 ４ ~2.5 x 10 ５ It is more preferable that it be 1.2 × 10 ５ ~2.5 x 10５ It is even more preferable that Mw is 1.2 × 10 ５ ~5.0 x 10 ５ , 1.2 × 10 ５ ~4.0 x 10 ５ , or 1.2 × 10 ５ ~2.5 x 10 ５ This may also be the case. In a preferred embodiment, Mw is 1.6 × 10 ５ ~5.0 x 10 ５ , 1.6 × 10 ５ ~4.0 x 10 ５ , or 1.6 × 10 ５ ~2.5 x 10 ５ That's fine.

[0015] In this specification, the weight-average molecular weight (Mw) of silk fibroin was measured under the following conditions. • Apparatus: Tosoh Corporation HLC-8320GPC • Guard column: TSKgel SuperH-H • Analytical column: TSKgel SuperHM-H • Sample: Silk fibroin dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (approx. 0.02% by mass) • Eluent: 5 mM sodium trifluoroacetate HFIP solution • Molecular weight standard: Polymethyl methacrylate (Resonac Corporation, 7-point calibration, Mp range 3310-1230000) • Column flow rate: 0.300 mL / min • Reference flow rate: 1:1 • Sample pressure upper limit: 25.0 MPa • Sample pressure lower limit: 0 MPa • Reference pressure upper limit: 25.0 MPa • Reference pressure lower limit: 0 MPa • Column oven: 40.0 °C Pump temperature: 40.0°C

[0016] In the compositions according to embodiments of the present invention, the silk fibroin used as a raw material can be obtained by removing impurities from silkworm cocoons or raw silk. The cocoon threads spun by silkworm larvae consist of fibroin filaments and the colloidal sericin coating them, and cocoons are created by bonding fibroin filaments with sericin. The fibroin content in cocoons is 70-80% by mass, the sericin content is 20-30% by mass, and other impurities such as small amounts of fat are also included. Raw silk is thread extracted from cocoons after soaking them in hot water and unraveling them, and it contains impurities.

[0017] The silkworm species that produce silk fibroin used in the composition according to the embodiment of the present invention are not particularly limited. For example, the Saturniidae, Bombycidae, Lasiocampidae, Lymantriidae, Arctiidae, Noctuidae, Noctuidae, Psychidae, Tineidae, Leafminers, Tineidae, Dracosporidae, Zygaenidae, Zygaenidae, Limacodidae, Pyralidae, Papilionidae, Notodontidae, Papilionidae, or genetically modified varieties thereof can be used. In Japan, Saturniidae, Bombycidae, or genetically modified varieties thereof are preferred because they are readily available, and Bombycidae and their genetically modified varieties are particularly preferred.

[0018] In the compositions according to the embodiments of the present invention, for example, silk fibroin obtained through scouring can be used. Scouring is a process of removing the above-mentioned impurities from cocoons, and for example, the above-mentioned impurities can be removed by contacting cocoons or raw silk with an alkaline solution such as soap solution, lye, or soda solution. Alternatively, commercially available silk fibroin used as silk thread, silk fabric, or health food may also be used.

[0019] In the compositions according to embodiments of the present invention, the silk fibroin used as a raw material may be silk fibroin that has undergone solubilization treatment in addition to the above-mentioned scouring. For example, the solubilization treatment can be performed by scouring the cocoons to remove impurities, and then dissolving the silk fibroin by contacting it with an aqueous lithium bromide solution, an aqueous calcium chloride solution, or a calcium chloride / water / ethanol solution. Since salt remains in the resulting aqueous solution containing silk fibroin, it can be removed by dialysis treatment. Dialysis treatment can be performed by general methods, such as a method using a dialysis membrane.

[0020] <Bioabsorbable Polymer> The composition according to the embodiment of the present invention contains a bioabsorbable polymer. In this specification, a bioabsorbable polymer means a polymer compound that is degraded by biodegrading enzymes or metabolic systems in the body, or a polymer compound that is nonspecifically hydrolyzed in the body. Bioabsorbable polymers have superior biodegradability compared to silk fibroin. Therefore, by using a predetermined amount of bioabsorbable polymer in addition to silk fibroin, the biodegradability of the composition can be appropriately adjusted.

[0021] The bioabsorbable polymer may be a homopolymer obtained by polymerizing a single monomer, or a copolymer obtained by polymerizing two or more monomers.

[0022] Examples of bioabsorbable polymers include polycarbonate, polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, polyurethane, polyoxyalkylene glycol, and polyalkylene succinate. The bioabsorbable polymer is preferably at least one selected from polycarbonate, polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, polyurethane, and polyoxyalkylene glycol, and more preferably at least one selected from polycarbonate and polylactic acid.

[0023] In this specification, polycarbonate means a polymer having repeating units with a carbonate structure (-O-C(=O)-O-). Polyurethane means a polymer having repeating units with urethane bonds (-NH-C(=O)-O-). Polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, and polyoxyalkylene glycol mean polymers having repeating units derived from lactic acid, glycolic acid, caprolactone, dioxasanone, and alkylene oxide, respectively.

[0024] Polycarbonates, polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, polyurethanes, and polyoxyalkylene glycols may have only a single repeating unit or may have two or more repeating units. They may also be homopolymers obtained by polymerizing a single monomer or copolymers obtained by polymerizing two or more monomers. In the case of copolymers, they may be random copolymers or block copolymers. The choice depends on the intended use of the composition. Adjusting the ratio of repeating structures of the copolymer to the homopolymer alters the crystallinity and physical properties of the polymer, affecting biodegradability and suture strength. In other words, using copolymers offers the advantage of easily designing compositions with a balanced relationship between biodegradability and suture strength.

[0025] In the various polymers described above, the content ratio of each repeating unit to the total repeating units is 50 mol% or more. For example, polycarbonate is defined as a polymer in which the content ratio of repeating units having a carbonate structure to the total repeating units constituting the polymer is 50 mol% or more. Here, for example, a polymer containing repeating units having a carbonate structure and repeating units derived from lactic acid in a 50 / 50 molar ratio is considered both polycarbonate and polylactic acid.

[0026] The bioabsorbable polymer preferably contains at least one of the following repeating units: repeating units derived from alkylene carbonate, repeating units derived from lactic acid, repeating units derived from caprolactone, repeating units derived from glycolic acid, and repeating units derived from dioxanone.

[0027] The repeating units derived from alkylene carbonates are -[O-R] １ -OC(=O)]-(R １ R is a repeating unit represented by (where R represents an alkylene group). １ The monomer is preferably an alkylene group having 1 to 6 carbon atoms. Examples of alkylene carbonates as raw material monomers include ethylene carbonate, propylene carbonate, butylene carbonate, trimethylene carbonate, tetramethylene carbonate, hexamethylene carbonate, heptamethylene carbonate, etc., with propylene carbonate, trimethylene carbonate, tetramethylene carbonate, and hexamethylene carbonate being preferred, and trimethylene carbonate being more preferred.

[0028] The repeating unit derived from lactic acid is -[CH(CH ３ It is a repeating unit represented by )-C(=O)-O]-. Lactic acid as a raw material monomer has optical isomers, and it may be the D-form, the L-form, or the DL-form (racemic mixture). Among these, it is preferable to use the L-form or DL-form of lactic acid.

[0029] The repeating unit derived from caprolactone is -[O-(CH) ２ ) ５ It is a repeating unit represented by -C(=O)]-. The repeating unit derived from glycolic acid is -[O-CH ２ This is a repeating unit represented by -C(=O)-]-. The repeating unit derived from dioxanone is -[O-C(=O)-CH ２ -O-(CH ２ ) ２ This is a repeating unit represented by ]-.

[0030] The bioabsorbable polymer preferably contains at least one repeating unit selected from alkylene carbonate, lactic acid, caprolactone, glycolic acid, and dioxanone in an amount of 50 mol% or more, more preferably 75 mol% or more, even more preferably 90 mol% or more, particularly preferably 95 mol% or more, and most preferably 100 mol%, i.e., all repeating units being the above repeating units, relative to the total repeating units constituting the bioabsorbable polymer. The end groups of the polymer can be appropriately selected from various ester groups, hydroxyl groups, etc. In one preferred embodiment, the amount of the above repeating units may be 50 mol% to 100 mol%, 75 mol% to 100 mol%, 90 mol% to 100 mol%, 95 mol% to 100 mol%, or 100 mol%, relative to the total repeating units constituting the bioabsorbable polymer.

[0031] In one preferred embodiment, from the viewpoint of making the degradation rate in the body more appropriate and improving the suture strength when a nonwoven fabric is formed, the bioabsorbable polymer preferably has at least two selected from repeating units derived from alkylene carbonate, repeating units derived from lactic acid, repeating units derived from caprolactone, repeating units derived from glycolic acid, and repeating units derived from dioxanone, and more preferably has at least two selected from repeating units derived from alkylene carbonate, repeating units derived from lactic acid, and repeating units derived from caprolactone.

[0032] In one preferred embodiment, the bioabsorbable polymer is preferably a homopolymer or copolymer of trimethylene carbonate. Homopolymers or copolymers of trimethylene carbonate are known to be highly flexible, which helps to reduce the risk of the composition being damaged in vivo. They are also known to decompose gradually when absorbed in vivo, and the degradation products have low toxicity. When the bioabsorbable polymer is a copolymer of trimethylene carbonate, the raw material monomer copolymerized with trimethylene carbonate is preferably at least one selected from lactic acid, caprolactone, glycolic acid, and dioxanone, and more preferably lactic acid. In other words, the bioabsorbable polymer is particularly preferably a copolymer of trimethylene carbonate and lactic acid. When the bioabsorbable polymer is a copolymer of trimethylene carbonate and lactic acid, the ratio of trimethylene carbonate to lactic acid (trimethylene carbonate / lactic acid) is preferably 90 / 10 to 10 / 90 in molar ratio, but may also be 80 / 20 to 20 / 80, 70 / 30 to 30 / 70, or 60 / 40 to 40 / 60. In a preferred embodiment, the above ratio may be 90 / 10 to 20 / 80, 90 / 10 to 30 / 70, or 90 / 10 to 40 / 60 in molar ratio. In a preferred embodiment, the above ratio may be 80 / 20 to 10 / 90, 80 / 20 to 20 / 80, 80 / 20 to 30 / 70, or 80 / 20 to 40 / 60 in molar ratio. In a preferred embodiment, the above ratio may be 70 / 30 to 10 / 90, 70 / 30 to 20 / 80, 70 / 30 to 30 / 70, or 70 / 30 to 40 / 60 in molar ratio. In a preferred embodiment, the above ratio may be 60 / 40 to 10 / 90, 60 / 40 to 20 / 80, 60 / 40 to 30 / 70, or 60 / 40 to 40 / 60 in molar ratio.

[0033] The weight-average molecular weight of the bioabsorbable polymer is 1.0 × 10⁻⁶. ４ ~1.0 x 10 ６ Preferably, 2.0 × 10 ４ ~5.0 x 10 ５ More preferably, 5.0 × 10 ４ ~3.5 x 10 ５A weight-average molecular weight of 1.0 × 10⁻⁶ is even more preferable. ４ If the molecular weight is smaller, there is a concern that it will degrade too quickly in the body. Also, a weight-average molecular weight of 1.0 × 10⁻⁶ is a concern. ６ If it is larger, the processability of the composition may deteriorate. In one preferred embodiment, the weight-average molecular weight of the bioabsorbable polymer is 1.0 × 10⁻⁶. ４ ~5.0 x 10 ５ , or 1.0 × 10 ４ ~3.5 x 10 ５ This may also be the case. In a preferred embodiment, the weight-average molecular weight of the bioabsorbable polymer is 2.0 × 10 ４ ~1.0 x 10 ６ , 2.0 x 10 ４ ~5.0 x 10 ５ , or 2.0 × 10 ４ ~3.5 x 10 ５ This may also be the case. In a preferred embodiment, the weight-average molecular weight of the bioabsorbable polymer is 5.0 × 10 ４ ~1.0 x 10 ６ , 5.0 x 10 ４ ~5.0 x 10 ５ , or 5.0 x 10 ４ ~3.5 x 10 ５ That's fine.

[0034] In this specification, the weight-average molecular weight (Mw) of a bioabsorbable polymer can be measured by GPC measurement, for example, in the same manner as the weight-average molecular weight (Mw) of silk fibroin described above.

[0035] In the compositions according to the embodiments of the present invention, only one bioabsorbable polymer may be used, or two or more may be used.

[0036] In the compositions according to the embodiments of the present invention, the content of the bioabsorbable polymer relative to the total amount of silk fibroin and bioabsorbable polymer is 5% by mass or more. If it is less than 5% by mass, the effect of adding the bioabsorbable polymer is difficult to obtain, and the biodegradability of the composition may be poor (the rate of degradation in the body is slow). In addition, the suture strength when formed into a nonwoven fabric may be poor. The content of the bioabsorbable polymer is preferably 10% by mass or more, and more preferably 20% by mass or more. On the other hand, the content of the bioabsorbable polymer relative to the total amount of silk fibroin and bioabsorbable polymer is 75% by mass or less. If it exceeds 75% by mass, it may be difficult to maintain a porous structure after implantation in the body when a nonwoven fabric is formed. The content of the bioabsorbable polymer is preferably 70% by mass or less, more preferably 60% by mass or less, and even more preferably 50% by mass or less. The mixing ratio of silk fibroin to bioabsorbable polymer (silk fibroin / bioabsorbable polymer) is 95 / 5 to 25 / 75 by mass ratio, preferably 90 / 10 to 30 / 70, more preferably 80 / 20 to 40 / 60, and even more preferably 80 / 20 to 50 / 50. In a preferred embodiment, the above mixing ratio may be 95 / 5 to 30 / 70, 95 / 5 to 40 / 60, or 95 / 5 to 50 / 50 by mass ratio. In a preferred embodiment, the above mixing ratio may be 90 / 10 to 25 / 75, 90 / 10 to 30 / 70, 90 / 10 to 40 / 60, or 90 / 10 to 50 / 50 by mass ratio. In a preferred embodiment, the above mixing ratio may be 80 / 20 to 25 / 75, 80 / 20 to 30 / 70, 80 / 20 to 40 / 60, or 80 / 20 to 50 / 50 by mass.

[0037] As described above, the composition according to the embodiment of the present invention may be in solid or liquid form, but is preferably in solid form, more preferably in the form of fibers, films, or nonwoven fabrics, and even more preferably in the form of nonwoven fabrics.

[0038] The composition according to the embodiment of the present invention is preferably a nonwoven fabric with a suture strength of 0.6 N or higher in a suture strength test. More preferably, the suture strength is 0.7 N or higher, and even more preferably, 0.8 N or higher. The upper limit of the suture strength is, for example, 10.0 N or less, 3.0 N or less, 2.5 N or less, 2.0 N or less, and 1.5 N or less. In a preferred embodiment, the suture strength may be 0.6 N to 10.0 N, 0.6 N to 3.0 N, 0.6 N to 2.5 N, 0.6 N to 2.0 N, or 0.6 N to 1.5 N. In a preferred embodiment, the suture strength may be 0.7 N to 10.0 N, 0.7 N to 3.0 N, 0.7 N to 2.5 N, 0.7 N to 2.0 N, or 0.7 N to 1.5 N. In a preferred embodiment, the suture strength may be 0.8N to 10.0N, 0.8N to 3.0N, 0.8N to 2.5N, 0.8N to 2.0N, or 0.8N to 1.5N.

[0039] The suture strength test can be performed in accordance with ISO 7198:2016 under the following conditions. The test will be performed with n=3, and the load at the time the sample breaks will be recorded as the suture strength, along with the displacement. • Testing machine: AG-IS (manufactured by Shimadzu Corporation) • Sample shape: Approximately 7 x 14 mm, threaded 2 mm from the edge of the short side • Suture: PP suture (thread: U.S.P. 6-0 (diameter: 0.07-0.1 mm), manufactured by Matsukaze Co., Ltd.) • Test speed: 50 mm / min • Grip length: 10 mm • Test environment: Air (23°C)

[0040] The compositions according to the embodiments of the present invention may contain a fluorine-containing alcohol, and it is preferable that the nonwoven fabric contains 0.1 to 1000 ppm by mass of fluorine-containing alcohol. More preferably, the content of fluorine-containing alcohol is 0.1 to 500 ppm by mass, and even more preferably 0.1 to 300 ppm by mass.

[0041] Combustion ion chromatography is used to quantify the fluorine-containing alcohol content in nonwoven fabrics. Anion mixed standard solution IV (Kanto Chemical Co., Ltd.) is used as the calibration curve, and the amount of fluorine-containing alcohol in the nonwoven fabric is calculated from the obtained fluoride ion amount. If the fluorine-containing alcohol is HFIP, it can be calculated using formula I. Combustion ion chromatography measurements are performed under the following conditions. HFIP (ppm) = (fluoride ion concentration) × (168 / (19 × 6)) ... Calculation formula I • Combustion device: AQF-2100H, manufactured by Mitsubishi Chemical Analytech Co., Ltd. • Ion chromatograph: DIONEX ICS5000+, manufactured by Thermo Fisher Scientific • Column: AG11-HC (4 × 50 mm) / AS11-HC (4 × 250 mm), manufactured by Thermo Fisher Scientific • Eluent: KOH aqueous solution

[0042] When the composition is a nonwoven fabric, the thickness of the nonwoven fabric is preferably 1 μm or more and less than 1000 μm. The thickness of the nonwoven fabric can be controlled to a desired value by, for example, appropriately adjusting the amount and concentration of the fluorine-containing alcohol solution of silk fibroin and bioabsorbable polymer in the composition manufacturing method described later.

[0043] If the composition is a nonwoven fabric, the average fiber diameter of the fibers constituting the nonwoven fabric is, for example, 10 nm or more, 20 nm or more, 30 nm or more, 50 nm or more, 100 nm or more, or 500 nm or more, and 1 mm or less, 500 μm or less, 250 μm or less, 10 μm or less, 5 μm or less, 3 μm or less, 2 μm or less, 1.5 μm or less, or 1 μm or less. The average fiber diameter can be measured by the method described in the examples. In a preferred embodiment, the average fiber diameter may be 10 nm or more and 1 mm or less, 10 nm or more and 500 μm or less, 10 nm or more and 250 μm or less, 10 nm or more and 10 μm or less, 10 nm or more and 5 μm or less, 10 nm or more and 3 μm or less, 10 nm or more and 2 μm or less, 10 nm or more and 1.5 μm or less, or 10 nm or more and 1 μm or less. In a preferred embodiment, the average fiber diameter may be 20 nm to 1 mm, 20 nm to 500 μm, 20 nm to 250 μm, 20 nm to 10 μm, 20 nm to 5 μm, 20 nm to 3 μm, 20 nm to 2 μm, 20 nm to 1.5 μm, or 20 nm to 1 μm. In a preferred embodiment, the average fiber diameter may be 30 nm to 1 mm, 30 nm to 500 μm, 30 nm to 250 μm, 30 nm to 10 μm, 30 nm to 5 μm, 30 nm to 3 μm, 30 nm to 2 μm, 30 nm to 1.5 μm, or 30 nm to 1 μm. In a preferred embodiment, the average fiber diameter may be 50 nm to 1 mm, 50 nm to 500 μm, 50 nm to 250 μm, 50 nm to 10 μm, 50 nm to 5 μm, 50 nm to 3 μm, 50 nm to 2 μm, 50 nm to 1.5 μm, or 50 nm to 1 μm. In a preferred embodiment, the average fiber diameter may be 100 nm to 1 mm, 100 nm to 500 μm, 100 nm to 250 μm, 100 nm to 10 μm, 100 nm to 5 μm, 100 nm to 3 μm, 100 nm to 2 μm, 100 nm to 1.5 μm, or 100 nm to 1 μm.In a preferred embodiment, the average fiber diameter may be 500 nm or more and 1 mm or less, 500 nm or more and 500 μm or less, 500 nm or more and 250 μm or less, 500 nm or more and 10 μm or less, 500 nm or more and 5 μm or less, 500 nm or more and 3 μm or less, 500 nm or more and 2 μm or less, 500 nm or more and 1.5 μm or less, or 500 nm or more and 1 μm or less. The average fiber diameter of the fibers constituting the nonwoven fabric can be controlled to a desired value, for example, by appropriately adjusting the concentration of the fluorine-containing alcohol solution of silk fibroin and bioabsorbable polymer and the spinning rate in the method for producing the composition described later.

[0044] When the composition is a nonwoven fabric, the average inter-fiber distance between the fibers constituting the nonwoven fabric is, for example, 0.1 μm or more, 0.2 μm or more, 0.3 μm or more, 0.4 μm or more, or 0.5 μm or more, and 100 μm or less, 50 μm or less, 10 μm or less, 5 μm or less, 3 μm or less, 2 μm or less, or 1.5 μm or less. The average inter-fiber distance can be measured by the method described in the examples. In a preferred embodiment, the average inter-fiber distance may be 0.1 μm or more and 100 μm or less, 0.1 μm or more and 50 μm or less, 0.1 μm or more and 10 μm or less, 0.1 μm or more and 5 μm or less, 0.1 μm or more and 3 μm or less, 0.1 μm or more and 2 μm or less, or 0.1 μm or more and 1.5 μm or less. In a preferred embodiment, the average interfiber distance may be 0.2 μm to 100 μm, 0.2 μm to 50 μm, 0.2 μm to 10 μm, 0.2 μm to 5 μm, 0.2 μm to 3 μm, 0.2 μm to 2 μm, or 0.2 μm to 1.5 μm. In a preferred embodiment, the average interfiber distance may be 0.3 μm to 100 μm, 0.3 μm to 50 μm, 0.3 μm to 10 μm, 0.3 μm to 5 μm, 0.3 μm to 3 μm, 0.3 μm to 2 μm, or 0.3 μm to 1.5 μm. In a preferred embodiment, the average inter-fiber distance may be 0.4 μm to 100 μm, 0.4 μm to 50 μm, 0.4 μm to 10 μm, 0.4 μm to 5 μm, 0.4 μm to 3 μm, 0.4 μm to 2 μm, or 0.4 μm to 1.5 μm. In a preferred embodiment, the average inter-fiber distance may be 0.5 μm to 100 μm, 0.5 μm to 50 μm, 0.5 μm to 10 μm, 0.5 μm to 5 μm, 0.5 μm to 3 μm, 0.5 μm to 2 μm, or 0.5 μm to 1.5 μm. The average inter-fiber distance between the fibers constituting the nonwoven fabric can be controlled to a desired value by appropriately adjusting, for example, the applied voltage and discharge speed in the composition manufacturing method described later.

[0045] If the composition is a nonwoven fabric, the basis weight of the nonwoven fabric is, for example, 1 g / m². ２ Above, 5g / m ２ Above, 10g / m ２20 g / m or more ２ 30 g / m or more ２ or 40 g / m or more ２ and 1000 g / m or less ２ 300 g / m or less ２ 100 g / m or less ２ or 80 g / m or less ２ That is, it is as follows. In a preferred embodiment, the basis weight of the non-woven fabric is 1 g / m ２ or more and 1000 g / m ２ or more and 1 g / m ２ or more and 300 g / m ２ or more and 1 g / m ２ or more and 100 g / m ２ or 1 g / m ２ or more and 80 g / m ２ or less. In a preferred embodiment, the basis weight of the non-woven fabric is 5 g / m ２ or more and 1000 g / m ２ or more and 5 g / m ２ or more and 300 g / m ２ or more and 5 g / m ２ or more and 100 g / m ２ or 5 g / m ２ or more and 80 g / m ２ or less. In a preferred embodiment, the basis weight of the non-woven fabric is 10 g / m ２ or more and yet 1000 g / m ２ or more and 10 g / m ２ or more and 300 g / m ２ or more and 10 g / m ２ or more and 100 g / m ２ or 10 g / m ２ or more and 80 g / m ２ or less. In a preferred embodiment, the basis weight of the non-woven fabric is 20 g / m ２ or more and 1000 g / m ２ or more and 20 g / m ２ or more and 300 g / m ２ or more and 20 g / m ２ or more and 100 g / m ２ or 20 g / m ２ or more and 80 g / m ２ or less. In a preferred embodiment, the basis weight of the non-woven fabric is 30 g / m ２ or more and 1000 g / m２ Below, 30g / m ２ More than 300g / m ２ Below, 30g / m ２ More than 100g / m ２ The following, or 30 g / m² ２ 80g / m or more ２ The following may also apply. In one preferred embodiment, the basis weight of the nonwoven fabric is 40 g / m². ２ More than 1000g / m ２ Below, 40g / m ２ More than 300g / m ２ Below, 40g / m ２ More than 100g / m ２ The following, or 40 g / m² ２ 80g / m or more ２ The following may also apply. The basis weight of the nonwoven fabric can be controlled to a desired value by appropriately adjusting the solution concentration, spinning time, etc., in the composition manufacturing method described later.

[0046] [Method for producing the composition] A method for producing the composition according to an embodiment of the present invention comprises the steps of: dissolving silk fibroin and a bioabsorbable polymer in a fluorine-containing alcohol to obtain a solution; and drying the solution to produce a composition, wherein the weight-average molecular weight of the silk fibroin determined by gel permeation chromatography is 9.0 × 10 ４ ~5.0 x 10 ５ The method for producing a composition is to control the mixing ratio of the silk fibroin and the bioabsorbable polymer to such a ratio that is 95 / 5 to 25 / 75 by mass.

[0047] <Step to obtain a fluorine-containing alcohol solution of silk fibroin and bioabsorbable polymer> The method for producing the composition according to the embodiment of the present invention includes the step of dissolving silk fibroin and a bioabsorbable polymer in a fluorine-containing alcohol to obtain a solution.

[0048] As for silk fibroin, the description of silk fibroin in the composition of the present invention described above can be directly applied. That is, silk fibroin obtained in the process of scouring and solubilizing cocoons can be used. The weight-average molecular weight of silk fibroin determined by gel permeation chromatography is 9.0 × 10⁻⁶. ４ ~5.0 x 10 ５ It is controlled to the weight-average molecular weight. The weight-average molecular weight can be controlled, for example, by adjusting processing conditions such as the processing time in scouring. In this case, while removing sericin, which is the purpose of scouring, the weight-average molecular weight of silk fibroin is reduced at the same time, so there is a risk that sericin will remain in fibroin with a weight-average molecular weight that is larger than necessary. For this reason, the weight-average molecular weight of silk fibroin is 9.0 × 10⁻⁶. ４ ~4.0 x 10 ５ Preferably, 9.0 × 10 ４ ~2.5 x 10 ５ This is preferable.

[0049] As for the bioabsorbable polymer, the description of the bioabsorbable polymer in the composition of the present invention described above can be directly applied.

[0050] The mixing ratio of silk fibroin to bioabsorbable polymer (silk fibroin / bioabsorbable polymer) is 95 / 5 to 25 / 75 by mass ratio, preferably 90 / 10 to 30 / 70, more preferably 80 / 20 to 40 / 60, and even more preferably 80 / 20 to 50 / 50. In a preferred embodiment, the above mixing ratio may be 95 / 5 to 30 / 70, 95 / 5 to 40 / 60, or 95 / 5 to 50 / 50 by mass ratio. In a preferred embodiment, the above mixing ratio may be 90 / 10 to 25 / 75, 90 / 10 to 30 / 70, 90 / 10 to 40 / 60, or 90 / 10 to 50 / 50 by mass ratio. In a preferred embodiment, the above mixing ratio may be 80 / 20 to 25 / 75, 80 / 20 to 30 / 70, 80 / 20 to 40 / 60, or 80 / 20 to 50 / 50 by mass.

[0051] The fluorine-containing alcohol can be any alcohol capable of dissolving silk fibroin and bioabsorbable polymers, and examples include 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). HFIP is preferred. The fluorine-containing alcohol may be used alone or in combination with other solvents. Furthermore, the fluorine-containing alcohol may be used in combination with one or more other solvents. The other solvents should be those that do not hinder dissolution without causing deterioration or a decrease in molecular weight of the silk fibroin and bioabsorbable polymers. Examples of such solvents include water, N-methylmorpholine N-oxide, 2,2,2-trifluoroethanol, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, 2-butanone, 2-hexanone, 4-methyl-2-pentanone, 1-methoxy-2-propanol, 1-ethoxy-2-propanol, tetrahydrofuran, N,N-dimethylacetamide, N,N-dimethylformamide, toluene, or dichloromethane. These solvents can be used in obtaining a solution of silk fibroin and a bioabsorbable polymer (hereinafter also simply referred to as the solution) in a relative ratio of 30 or less (preferably 10 or less) with the mass of the fluorine-containing alcohol set to 100. In a preferred embodiment, the fluorine-containing alcohol is a solvent containing 70% or more by mass of 1,1,1,3,3,3-hexafluoro-2-propanol.

[0052] The solution is obtained by dissolving the raw materials, silk fibroin and a bioabsorbable polymer, in a fluorine-containing alcohol. The container used when dissolving the silk fibroin and bioabsorbable polymer in the fluorine-containing alcohol is not particularly limited as long as it is airtight and pressure-resistant. For example, a sealed pressure-resistant container made of glass, stainless steel, or these materials lined with resin can be used. In one preferred embodiment, the solution is obtained by dissolving silk fibroin and a bioabsorbable polymer in a solvent containing 70% by mass or more of 1,1,1,3,3,3-hexafluoro-2-propanol. In another preferred embodiment, the solution is obtained by first preparing solution I, in which a predetermined amount of silk fibroin is dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, and solution II, in which a predetermined amount of a bioabsorbable polymer is dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, and then mixing solution I and solution II to a desired composition ratio. The dissolution temperature when dissolving silk fibroin and / or bioabsorbable polymers in a solvent containing 70% by mass or more of 1,1,1,3,3,3-hexafluoro-2-propanol is not particularly limited.

[0053] In a preferred embodiment, the dissolution time of silk fibroin and / or a bioabsorbable polymer in a solvent containing 70% by mass or more of 1,1,1,3,3,3-hexafluoro-2-propanol is 0.5 hours or more and 24 hours or less. A dissolution time of 0.5 hours or more is preferred because it ensures sufficient dissolution of silk fibroin and does not increase the amount of insoluble matter recovered. A dissolution time of 24 hours or less is preferred because it is productive and practical. Preferably, it is 12 hours or less, and particularly preferably 6 hours or less. In this case, stirring or heating may be performed to shorten the dissolution time. In a preferred embodiment, the dissolution time may be 0.5 hours or more and 12 hours or 0.5 hours or more and 6 hours or less.

[0054] The total concentration of silk fibroin and bioabsorbable polymer in the above solution is preferably 0.01% by mass or more and 50% by mass or less relative to the entire solution. More preferably, it is 0.1% by mass or more and 40% by mass or less, even more preferably 1% by mass or more and 30% by mass or less, and particularly preferably 1% by mass or more and 20% by mass or less. If the concentration is 0.01% by mass or more, it is easier to ensure the productivity of molded articles such as nonwoven fabrics containing the obtained silk fibroin (silk fiber) and bioabsorbable polymer, and if it is 50% by mass or less, the viscosity of the solution does not become too high, making it easy to mold. In a preferred embodiment, the above total concentration may be 0.01% by mass or more and 40% by mass or less, 0.01% by mass or more and 30% by mass or less, or 0.01% by mass or more and 20% by mass or less relative to the entire solution. In a preferred embodiment, the above total concentration may be 0.1% by mass or more and 50% by mass or less, 0.1% by mass or more and 40% by mass or less, 0.1% by mass or more and 30% by mass or less, or 0.1% by mass or more and 20% by mass, relative to the entire solution. In a preferred embodiment, the above total concentration may be 1% by mass or more and 50% by mass or less, 1% by mass or more and 40% by mass or less, 1% by mass or more and 30% by mass or less, or 1% by mass or more and 20% by mass, relative to the entire solution.

[0055] The method for producing the above solution according to one embodiment of the present invention can be carried out in either an inert gas atmosphere such as nitrogen or helium, or in an air atmosphere.

[0056] After preparing a dilute solution, a high-concentration solution can be obtained by concentrating it by distilling off a portion of the fluorine-containing alcohol in the solution. For example, after preparing a 1% by mass HFIP solution of silk fibroin and bioabsorbable polymer, an evaporator can be used to obtain an HFIP solution of 10% by mass or more of silk fibroin and bioabsorbable polymer. Although the amount of HFIP used increases, this method has the effect of shortening the dissolution time.

[0057] <Step of drying the solution to produce the composition> The method for producing the composition according to the embodiment of the present invention includes a step of drying the solution to produce the composition. "Drying" refers to removing liquid from a target by applying heat or the like to make it dry, or being in a dry state, but in this specification, "drying" refers to removing the fluorine-containing alcohol and making it dry. From the viewpoint of composition quality, the fluorine-containing alcohol content of the composition obtained after the drying treatment is preferably 1000 ppm by mass or less, 500 ppm by mass or less, or 300 ppm by mass or less. Also, from the viewpoint of manufacturing cost, it is, for example, 0.1 ppm by mass or more, 1 ppm by mass or more, or 5 ppm by mass or more. In a preferred embodiment, the content of fluorine-containing alcohol may be 0.1 ppm by mass or more and 1000 ppm by mass or less, 1 ppm by mass or more and 1000 ppm by mass or less, 5 ppm by mass or more and 1000 ppm by mass or less, 0.1 ppm by mass or more and 500 ppm by mass or less, 1 ppm by mass or more and 500 ppm by mass or less, 5 ppm by mass or more and 500 ppm by mass or less, 0.1 ppm by mass or more and 300 ppm by mass or less, 1 ppm by mass or more and 300 ppm by mass or less, or 5 ppm by mass or more and 300 ppm by mass or less. The composition obtained after drying treatment may be fibers, films, or nonwoven fabrics.

[0058] In one preferred embodiment, the step of drying the solution to produce a composition is to produce a nonwoven fabric by following the steps of electrospinning the solution to obtain fibers, insolubilizing the fibers, and removing the fluorine-containing alcohol from the insolubilized fibers.

[0059] Electrospinning is a method of spinning a polymer solution by applying a voltage to the solution, causing it to be ejected and spun in the direction of the collector. The voltage applied to the polymer solution is often a positive voltage via a nozzle (needle), disk, or wire, while the voltage applied to the collector is often a negative voltage or ground. The voltage can be either alternating current (AC) or direct current (DC).

[0060] Specifically, in the electrospinning method using a nozzle, a polymer solution is placed in a syringe, and while applying a voltage of approximately 1 to 100 kV between the needle at the tip of the syringe and the collector, the polymer solution is ejected from the needle towards the collector. When the voltage exceeds a threshold, the repulsive force of the charges overcomes the surface tension of the polymer solution droplets, generating a charged jet. As the jet extends within the electric field, the solvent evaporates, forming thin fibers, and these fibers accumulate on the collector to obtain a nonwoven fabric. The Japanese Industrial Standards (JIS) define a nonwoven fabric as "a fabric in which fibers are oriented in one direction or randomly, and the fibers are bonded together by entanglement, fusion, or adhesion" (JIS L 0222:2001).

[0061] The preferred applied voltage when performing electrospinning is 1 kV or more and 100 kV or less. More preferably, it is 5 kV or more and 80 kV or less. Particularly preferred is 10 kV or more and 50 kV or less. If the applied voltage is 1 kV or more, when a fluorine-containing alcohol solution of silk fibroin and bioabsorbable polymer is sprayed from the needle, an charged jet is easily generated, improving productivity. If the applied voltage is 100 kV or less, the speed and shape of the jet are easier to control. In one preferred embodiment, the applied voltage may be 1 kV or more and 80 kV or less, or 1 kV or more and 50 kV or less. In one preferred embodiment, the applied voltage may be 5 kV or more and 100 kV or less, 5 kV or more and 80 kV or less, or 5 kV or more and 50 kV or less. In one preferred embodiment, the applied voltage may be 10 kV or more and 100 kV or less, 10 kV or more and 80 kV or less, or 10 kV or more and 50 kV or less.

[0062] The preferred distance between the needle and collector when performing electrospinning (also called the spinning distance) is 5 cm or more and 50 cm or less. More preferably, it is 7.5 cm or more and 40 cm or less. Particularly preferred is 10 cm or more and 30 cm or less. If the distance between the needle and collector is 5 cm or more, the solvent evaporates sufficiently easily and dripping is less likely to occur. If the distance between the needle and collector is 50 cm or less, a jet is easily generated. In a preferred embodiment, the spinning distance may be 5 cm or more and 40 cm or 5 cm or more and 30 cm or less. In a preferred embodiment, the spinning distance may be 7.5 cm or more and 50 cm or less, 7.5 cm or more and 40 cm or less, or 7.5 cm or more and 30 cm or less. In a preferred embodiment, the spinning distance may be 10 cm or more and 50 cm or less, 10 cm or more and 40 cm or less, or 10 cm or more and 30 cm or less.

[0063] The collector may be fixed during electrospinning, or it may be rotated during electrospinning. The rotational speed of the collector is not particularly limited, but is preferably 30 rpm or more and 500 rpm or less, and more preferably 50 rpm or more and 150 rpm or less. In one preferred embodiment, the rotational speed of the collector may be 30 rpm or more and 150 rpm or less. In one preferred embodiment, the rotational speed of the collector may be 50 rpm or more and 500 rpm or less, or 50 rpm or more and 150 rpm or less.

[0064] The temperature in the electrospinning method is not particularly limited, but is preferably 0°C or higher and 80°C or lower, and more preferably 15°C or higher and 40°C or lower, which is close to room temperature (20-25°C). In one preferred embodiment, the temperature may be 0°C or higher and 40°C or lower. In one preferred embodiment, the temperature may be 15°C or higher and 80°C or lower, or 15°C or higher and 40°C or lower. The relative humidity in the electrospinning method is not particularly limited, but is preferably 10% or higher and 80% or lower, and more preferably 20% or higher and 70% or lower. In one preferred embodiment, the relative humidity may be 10% or higher and 70% or lower. In one preferred embodiment, the relative humidity may be 20% or higher and 80% or lower, or 20% or higher and 70% or lower. Regarding the pressure during electrospinning, unless it is necessary to pressurize or depressurize the electrospinning apparatus to adjust the evaporation rate of the solvent in the solution, there is no need to pressurize or depressurize it, and it is preferable to perform the process at atmospheric pressure (1013.25 hPa at sea level). Furthermore, there is no need to provide a pressurization / depressurization mechanism in the electrospinning apparatus, making it economical.

[0065] In this specification, the NANON-03 nonwoven fabric electrospinning apparatus manufactured by MEC Corporation is used as the apparatus for electrospinning.

[0066] As described above, the fibers (nonwoven fabric) containing silk fibroin and bioabsorbable polymer, produced by electrospinning, are subjected to insolubilization treatment by contact with water or alcohol (e.g., immersion or vapor exposure), and then dried to remove the fluorine-containing alcohol. The alcohol is not particularly limited, but examples include water-soluble alcohols such as methanol, ethanol, n-propyl alcohol, and isopropanol. Ethanol is preferred because it is readily available and easy to handle. The alcohol may also be an aqueous solution. Known contact methods for contacting the nonwoven fabric with water or alcohol include immersing the nonwoven fabric in alcohol, exposing the nonwoven fabric to alcohol vapor, and spraying the nonwoven fabric with alcohol. From the viewpoint of uniform insolubilization treatment, the method of exposing the nonwoven fabric to alcohol vapor is preferred. The contact time with water or alcohol is not particularly limited, but is usually preferably 10 minutes to 48 hours, more preferably 10 minutes to 24 hours, and even more preferably 10 minutes to 12 hours.

[0067] The contact temperature with water or alcohol is not particularly limited, but is preferably 5°C or higher and 50°C or lower, and more preferably 10°C or higher and 40°C or lower. Furthermore, a contact temperature lower than the glass transition temperature of the bioabsorbable polymer is desirable because it makes it easier to prevent deformation of the fibers. The above contact with alcohol may also be carried out under pressurized conditions. In one preferred embodiment, the above contact temperature may be 5°C or higher and 40°C or lower. In another preferred embodiment, the above contact temperature may be 10°C or higher and 50°C or lower, or 10°C or higher and 40°C or lower. The pressurized conditions are not particularly limited, but are preferably atmospheric pressure (1013.25 hPa at sea level) or higher and twice atmospheric pressure (2026.5 hPa) or lower. The drying is not particularly limited as long as the insolubilized nonwoven fabric is dry, but examples include constant temperature drying, hot air drying, etc. The insolubilization treatment may also serve as drying.

[0068] The nonwoven fabric described above is preferably a nonwoven fabric for blood compatibility materials. The present invention also relates to blood compatibility materials, including a nonwoven fabric for blood compatibility materials containing silk fibroin and bioabsorbable polymer fibers. Blood compatibility materials are materials used in contact with blood. Examples of blood compatibility materials, though not particularly limited, include artificial blood vessels, catheters, blood filters, adhesion prevention materials, stents, cell scaffold materials, artificial organs, artificial dura mater, artificial valves, artificial chordae tendineae, vascular patches, cardiac patches, hemodialysis machines, sutures, blood storage containers, and blood transfusion devices. The nonwoven fabric containing silk fibroin and bioabsorbable polymer fibers according to one embodiment of the present invention can be particularly suitably used in artificial blood vessels, artificial valves, vascular patches, and cardiac patches that are always in contact with blood. These blood compatibility materials contain at least a portion of silk fibroin and bioabsorbable polymer fibers and may also contain other constituent members. For example, these blood-compatible materials may be multilayered bodies in which a nonwoven fabric for blood-compatible materials containing silk fibers is coated onto a substrate (e.g., a polymer member containing synthetic polymers or biopolymers, or a metal member).

[0069] The nonwoven fabric described above is preferably a wound dressing.

[0070] The nonwoven fabric described above is preferably a cell scaffold material. Examples of cells include living tissue cells, mesenchymal stem cells, pluripotent stem cells, differentiated stem cells, and their progenitor cells. As pluripotent stem cells, iPS cells may also be used.

[0071] iPS cells are induced pluripotent stem cells, and are a type of pluripotent stem cell. A "stem cell" is a cell that has the ability to self-renew and differentiate. Among stem cells, those that have the ability to self-renew and can differentiate into all cells of the endoderm, mesoderm, and ectoderm from a single cell are called "pluripotent stem cells."

[0072] The origin of the cells is not particularly limited; for example, if they are mammals, they could be derived from humans, monkeys, chimpanzees, cows, horses, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats, and so on.

[0073] In one preferred embodiment, the step of drying the aforementioned solution to produce a composition may also involve a step of drying the aforementioned solution on a substrate to produce a film.

[0074] The substrate on which the solution is spread can be any flat or curved surface that is not affected by the solution and can spread the solution uniformly. Depending on the desired shape of the film, examples include a flat plate, mold, petri dish, or cylindrical shape, and the substrate can be made of resin, metal, glass, or ceramics.

[0075] Furthermore, "unfolding" refers to the act of pouring a fluid into a mold to obtain the desired shape of a molded object. For example, this refers to pouring the solution into a cylindrical mold, such as a petri dish.

[0076] The temperature at which the solution is spread onto the substrate depends on the boiling point of the solvent used, but is between 10°C and 120°C. Preferably, it is between 15°C and 80°C, and more preferably between 20°C and 40°C. In one preferred embodiment, the above temperature may be between 10°C and 80°C, or between 10°C and 40°C. In one preferred embodiment, the above temperature may be between 15°C and 120°C, between 15°C and 80°C, or between 15°C and 40°C. In one preferred embodiment, the above temperature may be between 20°C and 120°C, between 20°C and 80°C, or between 20°C and 40°C.

[0077] When the solvent is HFIP, since the boiling point of HFIP is 58.6°C, the temperature at which the solution is spread onto the substrate is 58.6°C or lower, preferably 20°C or higher and 40°C or lower. In a preferred embodiment, the above temperature may be 20°C or higher and 58.6°C or 20°C or higher and 40°C or lower.

[0078] Next, the solution is dried on the substrate. By drying, the fluorine-containing alcohol is removed from the solution, thereby enabling the production of a film.

[0079] The ambient temperature when drying the solution on the substrate depends on the boiling point of the solvent used, but is, for example, 10°C or higher and 120°C or lower. Preferably, it is 15°C or higher and 40°C or lower, and more preferably 15°C or higher and 35°C or lower. A temperature of 10°C or higher is preferable because it does not require an excessively long drying time. A temperature of 120°C or lower is preferable because the film precursor is less likely to deform due to heat. In a preferred embodiment, the ambient temperature may be 10°C or higher and 40°C or lower, or 10°C or higher and 35°C or lower. In a preferred embodiment, the ambient temperature may be 15°C or higher and 120°C or lower, 15°C or higher and 40°C or lower, or 15°C or higher and 35°C or lower.

[0080] The relative humidity when drying the solution spread on the substrate is, for example, 5% or more and 95% or less.

[0081] The drying time for the solution spread on the substrate is not particularly limited. It depends on the ambient temperature, relative humidity, and the concentration and amount of the solution used, but is preferably 0.1 seconds to 96 hours, more preferably 1 second to 72 hours. If the drying time is shorter than 1 second, the evaporation of the solvent may be insufficient. If the dissolution time is longer than 72 hours, the productivity will be low and it will not be practical. Drying is preferably completed when it is confirmed that there is no change in weight when the obtained film is weighed (for example, when the film and substrate are placed on a balance and weighed). In one preferred embodiment, the drying time may be 0.1 seconds to 72 hours. In one preferred embodiment, the drying time may be 1 second to 96 hours, or 1 second to 72 hours.

[0082] The environment for drying the solution spread on the substrate is not particularly limited. It may be a normal environment such as a fume hood in a laboratory, or a desiccator equipped with a desiccant, a hot plate, an electric furnace, or an environmental testing machine may be used.

[0083] The above film is preferably an adhesion prevention material or a cell scaffold material.

[0084] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

[0085] [Preparation of Silk Fibroin Sponge (SF7)] <Preparation of Silk Fibroin (Degumming of Cocoon Balls)> 1365 g of an aqueous sodium carbonate solution prepared to a concentration of 1.0% by mass and 55.0 g of cocoon balls (variety, Kinshu x Showa) were placed in a 3.0 L reaction apparatus with a constant temperature bath, and the temperature was raised to 90°C. After heating for 4 hours, the mixture was allowed to stand and cooled to 40°C, after which the contents were washed with purified water. Next, the cocoon balls were returned to the reaction apparatus, 1365 g of purified water was added to the apparatus, and the temperature was raised to 90°C for boiling and washing. After boiling for 10 minutes, the mixture was cooled to 40°C, and the contents were washed with purified water. After repeating this washing operation twice, the cocoon balls were placed in a drying oven and dried at a temperature of 50°C for 20 hours to obtain 32.2 g of silk fibroin (1).

[0086] <Preparation of Silk Fibroin Sponge> 20.8 g of silk fibroin (1) was placed in a 1000 mL glass flask equipped with a Liebig reflux apparatus, and 457 g of calcium chloride / ethanol aqueous solution (calcium chloride:ethanol:water = 1:2:8 (molar ratio)) was added. The bottom of the flask was then immersed in an oil bath at 73°C and heated for 1 hour to dissolve the silk fibroin and obtain an aqueous silk fibroin solution. The aqueous silk fibroin solution was then subjected to pressure filtration, and the filtrate was collected.

[0087] The filtrate was added to a dialysis tube (manufactured by Spectrum Laboratories, USA, regenerated cellulose membrane, product name: Spectra / por3, molecular weight fraction 12-14 kDa), and dialysis was performed using purified water as the dialysis buffer to remove calcium chloride and ethanol. The dialysis buffer was replaced a total of eight times. The aqueous solution in this dialysis tube was freeze-dried to obtain a porous silk fibroin sponge (SF7).

[0088] The yield of silk fibroin sponge was 17.6 g, and the yield was 84.6%. The weight-average molecular weight (Mw) of the obtained silk fibroin was measured by gel permeation chromatography (GPC), and the measured value was 7.0 × 10⁻⁶. ４The GPC measurements were performed under the aforementioned measurement conditions.

[0089] [Preparation of Silk Fibroin Sponge (SF10)] Silk fibroin sponge (SF10) was obtained in the same manner as the preparation of silk fibroin sponge (SF7), except that the heating temperature in the <Preparation of Silk Fibroin> was changed from 90°C to 80°C. The weight-average molecular weight (Mw) of the obtained silk fibroin was measured by GPC, and the measured value was 1.0 × 10⁻⁶. ５ That was the case.

[0090] [Preparation of Silk Fibroin Sponge (SF14)] Silk fibroin sponge (SF14) was obtained in the same manner as the preparation of silk fibroin sponge (SF7), except that the concentration of the sodium carbonate aqueous solution in the <Preparation of Silk Fibroin> was changed from 1.0% by mass to 0.40% by mass. The weight-average molecular weight (Mw) of the obtained silk fibroin was measured by GPC, and the measured value was 1.4 × 10⁻⁶. ５ That was the case.

[0091] [Preparation of Silk Fibroin Sponge (SF22)] Except for changing the heating time in the <Preparation of Silk Fibroin> from 4 hours to 8 hours and changing the concentration of the sodium carbonate aqueous solution from 1.0% by mass to 0.20% by mass, silk fibroin sponge (SF22) was obtained in the same manner as the preparation of silk fibroin sponge (SF7). The weight-average molecular weight (Mw) of the obtained silk fibroin was measured by GPC, and the measured value was 2.2 × 10⁻⁶. ５ That was the case.

[0092] [Preparation of Silk Fibroin Sponge (SF45)] Except for changing the heating time in the <Preparation of Silk Fibroin> from 4 hours to 0.5 hours and changing the concentration of the sodium carbonate aqueous solution from 1.0% by mass to 0.20% by mass, silk fibroin sponge (SF45) was obtained in the same manner as the preparation of silk fibroin sponge (SF7). The weight-average molecular weight (Mw) of the obtained silk fibroin was measured by GPC measurement, and the measured value was 4.5 × 10⁻⁶. ５ That was the case.

[0093] [Preparation of Silk Fibroin Sponge (SF51)] Except for changing the heating temperature in the <Preparation of Silk Fibroin> from 90°C to 80°C, changing the heating time from 4 hours to 0.5 hours, and changing the concentration of the sodium carbonate aqueous solution from 1.0% by mass to 0.20% by mass, silk fibroin sponge (SF51) was obtained in the same manner as the preparation of silk fibroin sponge (SF7).

[0094] [Example 1] <Preparation of HFIP solution of silk fibroin> 3.2 g of silk fibroin (SF22) and 36.8 g of HFIP were weighed into a glass vial equipped with a stirring bar and stirred at room temperature to obtain an HFIP solution with a silk fibroin concentration of 8% by mass.

[0095] <Preparation of HFIP solution of bioabsorbable polymer> 3.2 g of a copolymer of trimethylene carbonate and DL lactic acid (PTDL; trimethylene carbonate / DL lactic acid = 50 / 50 (molar ratio)) and 36.8 g of HFIP were weighed into a glass vial equipped with a stirring bar and stirred at room temperature to obtain an HFIP solution with a bioabsorbable polymer concentration of 8% by mass.

[0096] <Preparation of HFIP mixed solution of silk fibroin and bioabsorbable polymer> The HFIP solution of silk fibroin and the HFIP solution of bioabsorbable polymer prepared by the above method were weighed into a glass vial equipped with a stirring bar in a mass ratio of 50 / 50 and mixed by stirring to obtain an HFIP solution of silk fibroin and bioabsorbable polymer.

[0097] <Spinning by electrospinning> Spinning was performed using a nonwoven electrospinning apparatus NANON-03 manufactured by MEC Corporation from the HFIP solution of silk fibroin and bioabsorbable polymer obtained above. The nozzle inner diameter was set to 0.22 mm, the applied voltage to 16 kV, the spinning speed to 0.7 mL / h, and the spinning distance to 20 cm. The spinning apparatus temperature was maintained at 23-24°C and the relative humidity at 50-60%. Aluminum foil was used as the electrode. The prepared nonwoven fabric was left to stand for 24 hours at 15°C under saturated vapor of 30% ethanol (EtOH) aqueous solution, and then dried to obtain an immobilized nonwoven fabric. The obtained nonwoven fabric had a thickness of 196 μm, an average fiber diameter of 0.9 nm, an average interfiber distance of 0.8 μm, and a basis weight of 58 g / m². ２ That was the case.

[0098] [Examples 2-16, Comparative Examples 1-8] Nonwoven fabrics for Examples 2-16 and Comparative Examples 1-8 were obtained by using the conditions described in Table 2, including the type of silk fibroin, the type of bioabsorbable polymer, the mixing ratio (mass ratio) of silk fibroin and bioabsorbable polymer, the total solid content concentration of the HFIP mixed solution of silk fibroin and bioabsorbable polymer, and the spinning conditions for each. When changing the total solid content concentration, HFIP solutions of silk fibroin and bioabsorbable polymer with the same concentration as the total solid content concentration were prepared and mixed.

[0099] [Comparative Example 9] When SF22 in Example 1's <Preparation of Silk Fibroin HFIP Solution> was replaced with SF51 to prepare a silk fibroin HFIP solution, there was a large amount of undissolved material, and a uniform solution could not be prepared.

[0100] <Measurement of Weight-Average Molecular Weight of Silk Fibroin> The weight-average molecular weight (Mw) of the silk fibroin used was determined by GPC measurement. The GPC measurement was performed under the measurement conditions described above.

[0101] <Average Fiber Diameter of Nonwoven Fabrics> The average fiber diameter of nonwoven fabrics was determined by obtaining secondary electron images using a field emission scanning electron microscope (FE-SEM) and measuring the fiber diameter using software (ImageJ). Measurements were performed for n=10 or more samples, and the average value was taken as the average fiber diameter.

[0102] <Average inter-fiber distance of nonwoven fabric> The average inter-fiber distance of nonwoven fabric was calculated using the following formula (1).

[0103]

[0104] In equation (1), D, ρ, t, and W represent the following: • D: Average fiber diameter (μm) • ρ: Resin density of the fiber (g / cm³) ３ ) ・t: Thickness of nonwoven fabric (mm) ・W: Basis weight (g / m ２ )

[0105] <Thickness of Nonwoven Fabric> The thickness of the nonwoven fabric was measured using a constant-pressure thickness measuring instrument (product name: PG-02, manufactured by Teclock Co., Ltd.) with a measuring force of 120 gf and a probe diameter of 5 mmΦ.

[0106] <Balance of Nonwoven Fabric> The basis weight of the nonwoven fabric was calculated by measuring the dimensions and mass of a rectangular piece of nonwoven fabric.

[0107] <Fluorine-containing alcohol content in nonwoven fabric> The amount of fluorine-containing alcohol (HFIP) in the obtained nonwoven fabric was quantified. Combustion ion chromatography was used to quantify the solvent. Anion mixed standard solution IV (Kanto Chemical Co., Ltd.) was used as the calibration curve, and the amount of fluorine-containing alcohol in the nonwoven fabric was calculated from the obtained amount of fluoride ions using the above calculation formula I. Combustion ion chromatography measurements were performed under the above conditions.

[0108] <Biodegradability Test of Nonwoven Fabric> A sample of nonwoven fabric cut to 5 x 30 mm was vacuum-dried at room temperature (23°C), and its mass (W) was measured. ｉ The mass (W) was measured. Next, the above sample was immersed for 120 hours in a 200 U / mL lipase solution (lipase powder (Sigma-Aldrich, 38.6 U / mg) dissolved in 0.1 M PBS (Fujifilm Wako Pure Chemical Industries, pH = 7.4)). The immersion was carried out in a 37°C incubator while shaking with a seesaw shaker. After immersion, the sample was vacuum dried and its mass (W) was measured. ｄ ) was measured.

[0109] (Mass loss rate) The mass loss rate was calculated using the following formula (2). Mass loss rate (%) = {(W ｉ -W ｄ) / W ｉ} × 100 ... (2) The test was conducted with n=3 and the average value was calculated.

[0110] (Mass loss rate per bioabsorbable polymer composition ratio) The mass loss rate per bioabsorbable polymer composition ratio was calculated using the following formula (3): Mass loss rate per bioabsorbable polymer composition ratio (%) = (Mass loss rate (%) / Composition ratio (%)) × 100 ... (3) In formula (3), the composition ratio (%) is the ratio (mass%) of the bioabsorbable polymer to the total of silk fibroin and the bioabsorbable polymer. The obtained mass loss rate per bioabsorbable polymer composition ratio (%) was evaluated according to the following evaluation criteria: A: Over 95% B: Over 80% and 95% or less C: Over 1% and 80% or less D: Less than 1% In this test, B and C are the passing standards.

[0111] (Evaluation of Porosity Preservation) After immersion, the samples were vacuum-dried, and images of the surface morphology were taken using a field emission scanning electron microscope (FE-SEM). The samples were compared with the samples before the immersion test and evaluated according to the following criteria: A: Fiber shape and porous structure were preserved. B: Fiber breakage, reduction of voids, and loss of porous structure were observed.

[0112] <Nonwoven Fabric Suture Strength Test> The suture strength test was conducted in accordance with ISO 7198:2016 under the following conditions. The test was performed with n=3, and the load at the time the sample broke was recorded as the suture strength, along with the displacement. ・Test machine: AG-IS (manufactured by Shimadzu Corporation) ・Sample shape: Approximately 7 x 14 mm, threaded 2 mm from the edge of the short side ・Suture thread: PP suture thread (thread: U.S.P. 6-0 (diameter: 0.07-0.1 mm), manufactured by Matsukaze) ・Test speed: 50 mm / min ・Grip length: 10 mm ・Test environment: Air (23°C) In this test, a suture strength of 0.6 N or higher is considered acceptable.

[0113] Table 1 below shows the results of the biodegradation test and suture strength test for each example and comparative example.

[0114] The abbreviations used in Table 1 are as follows: <Silk fibroin> ・SF7: Silk fibroin sponge obtained by the above preparation method (SF7) ・SF10: Silk fibroin sponge obtained by the above preparation method (SF10) ・SF14: Silk fibroin sponge obtained by the above preparation method (SF14) ・SF22: Silk fibroin sponge obtained by the above preparation method (SF22) ・SF45: Silk fibroin sponge obtained by the above preparation method (SF45) <Bioabsorbable polymer> ・PTMC: Poly(trimethylene carbonate), Mw=50,000, Apollo Scientific (Sincere Chemicals Co., Ltd. PTDL: Copolymer of trimethylene carbonate and DL lactic acid, trimethylene carbonate / DL lactic acid = 50 / 50 (molar ratio), Mw = 250,000, trade name "CARBOMAXX TDL55", Apollo Scientific (Bezwada Biomedical) Co., Ltd. PTLL: Copolymer of trimethylene carbonate and L lactic acid, trimethylene carbonate / L lactic acid = 50 / 50 (molar ratio), Mw = 230,000, trade name "LACTOMAXX LT55", Apollo Scientific (Bezwada PLCL (manufactured by Biomedical): Copolymer of L-lactic acid and caprolactone, L-lactic acid / caprolactone = 75 / 25 (molar ratio), Mw = 350,000. PLGA (manufactured by Taki Chemical Co., Ltd.): Copolymer of DL-lactic acid and glycolic acid, DL-lactic acid / glycolic acid = 50 / 50 (molar ratio), Mw = 200,000.

[0115]

[0116]

[0117] As shown in Table 1, the compositions (nonwoven fabrics) obtained in the examples exhibited an appropriate biodegradation rate and maintained their porous structure even after biodegradation testing. Furthermore, they demonstrated excellent suture strength.

[0118] According to the present invention, it is possible to provide a composition that has an appropriate biodegradation rate, can maintain a porous structure after implantation in a living organism when a nonwoven fabric is formed, and has excellent suture strength when a nonwoven fabric is formed, as well as a method for producing the same.

[0119] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2025-013369 filed on 29 January 2025, the contents of which are incorporated herein by reference.

Claims

1. A composition comprising silk fibroin and a bioabsorbable polymer, wherein the weight-average molecular weight of the silk fibroin determined by gel permeation chromatography is 9.0 × 10⁻⁶. ４ ~5.0 x 10 ５ The composition wherein the mixing ratio of the silk fibroin and the bioabsorbable polymer is 95 / 5 to 25 / 75 by mass.

2. The weight-average molecular weight of the silk fibroin is 9.0 × 10 ４ ~2.5 x 10 ５ The composition according to claim 1.

3. The composition according to claim 1, wherein the bioabsorbable polymer is at least one selected from polycarbonate, polylactic acid, polyglycolic acid, polycaprolactone, polydioxanone, polyurethane, and polyoxyalkylene glycol.

4. The weight-average molecular weight of the bioabsorbable polymer is 1.0 × 10⁻⁶ ４ ~1.0 x 10 ６ The composition according to claim 1.

5. The composition according to claim 1, wherein the bioabsorbable polymer comprises at least one of the following repeating units: repeating unit derived from alkylene carbonate, repeating unit derived from lactic acid, repeating unit derived from caprolactone, repeating unit derived from glycolic acid, repeating unit derived from dioxanone 6. The composition according to claim 1, wherein the bioabsorbable polymer is a homopolymer or copolymer of trimethylene carbonate.

7. The composition according to claim 6, wherein the bioabsorbable polymer is a copolymer of trimethylene carbonate and lactic acid.

8. The composition according to claim 7, wherein the ratio of trimethylene carbonate to lactic acid is 90 / 10 to 10 / 90 in molar ratio.

9. The composition according to claim 1, which is a fiber, film, or nonwoven fabric.

10. The composition according to claim 1, wherein the nonwoven fabric has a suture strength of 0.6 N or higher in a suture strength test.

11. The composition according to claim 1, wherein the nonwoven fabric has a fluorine-containing alcohol content of 0.1 to 1000 ppm by mass.

12. A method for producing a composition, comprising the steps of: dissolving silk fibroin and a bioabsorbable polymer in a fluorine-containing alcohol to obtain a solution; and drying the solution to produce a composition, wherein the weight-average molecular weight of the silk fibroin determined by gel permeation chromatography is 9.0 × 10 ４ ~5.0 x 10 ５ A method for producing a composition, wherein the mixing ratio of the silk fibroin to the bioabsorbable polymer is controlled to such an extent that the mixing ratio is 95 / 5 to 25 / 75 by mass.

13. A method for producing the composition according to claim 12, wherein the fluorine-containing alcohol is 1,1,1,3,3,3-hexafluoro-2-propanol.

14. The method for producing the composition according to claim 12, wherein the silk fibroin is obtained in a process of scouring and solubilizing cocoons.

15. The method for producing a composition according to claim 12, wherein the step of drying the solution to produce a composition is to produce a nonwoven fabric by electrospinning the solution to obtain fibers, insolubilizing the fibers, and removing fluorine-containing alcohol from the insolubilized fibers.

16. The method for producing a composition according to claim 12, wherein the step of drying the solution to produce a composition is a step of producing a film via a step of drying the solution on a substrate.

Images