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WO2025204578A1 - Fibroïne de soie solidifiée, solution aqueuse de fibroïne de soie et son procédé de production et matériau de fixation à l'état de gel - Google Patents

Fibroïne de soie solidifiée, solution aqueuse de fibroïne de soie et son procédé de production et matériau de fixation à l'état de gel

Info

Publication number
WO2025204578A1
WO2025204578A1 PCT/JP2025/007726 JP2025007726W WO2025204578A1 WO 2025204578 A1 WO2025204578 A1 WO 2025204578A1 JP 2025007726 W JP2025007726 W JP 2025007726W WO 2025204578 A1 WO2025204578 A1 WO 2025204578A1
Authority
WO
WIPO (PCT)
Prior art keywords
silk fibroin
molecular weight
weight
solidified
gel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/007726
Other languages
English (en)
Japanese (ja)
Inventor
裕介 神戸
誠 佐々木
博貴 渕上
恒徳 亀田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Atsumaru Holdings Inc
Charlie Lab Inc
National Agriculture and Food Research Organization
Original Assignee
Atsumaru Holdings Inc
Charlie Lab Inc
National Agriculture and Food Research Organization
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Atsumaru Holdings Inc, Charlie Lab Inc, National Agriculture and Food Research Organization filed Critical Atsumaru Holdings Inc
Publication of WO2025204578A1 publication Critical patent/WO2025204578A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology

Definitions

  • Silk materials generally have low solubility, and while this is not a problem when added to solid materials such as soap, when dissolving silk materials in water, solubility must be taken into consideration. To make them soluble in water, silk materials are decomposed to reduce their molecular weight.
  • Patent Document 1 Japanese Patent No. 6019506 discloses a method for producing an aqueous solution of high molecular weight silk fibroin, in which the weight-average molecular weight of the silk fibroin contained in the aqueous solution is in the range of 10,000 to 50,000 as measured by gel permeation chromatography (GFC).
  • GFC gel permeation chromatography
  • Patent Document 2 JP 2016-517443 A discloses a low-molecular-weight silk fibroin composition containing a population of silk fibroin fragments having molecular weights within a certain range, wherein 15% or less of the total number of the silk fibroin fragments in the population have a molecular weight exceeding 200 kDa, and at least 50% of the total number of the silk fibroin fragments in the population have a molecular weight within a specified range, the specified range being between a lower limit of approximately 3.5 kDa or more and an upper limit of approximately 120 kDa or less.
  • Patent Document 1 proposes a method for producing a high-molecular-weight silk fibroin aqueous solution with excellent storage stability, but the weight-average molecular weight of the silk fibroin used is at most 50,000 (i.e., 50 kDa), which is insufficient for the degree of molecular weight increase, and therefore the effect of improving the physical properties of the liquid by utilizing the molecular structure of silk fibroin is insufficient.
  • Patent Document 2 although a treatment to reduce the molecular weight of silk material is carried out, the method is characterized by requiring a long scouring time, which limits the degree to which the molecular weight can be reduced.
  • Another object of the present invention is to provide an aqueous silk fibroin solution that contains medium-molecular-weight silk fibroin and has excellent storage stability.
  • Another object of the present invention is to provide a gel-like fixative containing medium-molecular-weight silk fibroin, which is useful for fixing materials.
  • the inventors of the present invention discovered that when silk fibroin has a medium molecular weight, not only can it be stored for a long period of time as a solidified silk fibroin, but the solidified product also has good solubility in water, allowing for improved physical properties in liquids, leading to the completion of the present invention.
  • Aspect 3 3.
  • Aspect 5 5.
  • Aspect 6 A solidified silk fibroin material according to any one of Aspects 1 to 5, having a density of 20 ⁇ 10 ⁇ 3 g/cm 3 or more.
  • Aspect 7 7.
  • Aspect 10 A method for producing a solidified silk fibroin according to aspect 8 or 9, wherein the solidifying step comprises obtaining a freeze-dried silk fibroin product from the aqueous solution containing the silk fibroin.
  • Aspect 11 A method for producing a solidified silk fibroin product according to Aspect 10, wherein the solidifying step further comprises a step of pulverizing the freeze-dried silk fibroin product.
  • Aspect 12 A silk fibroin aqueous solution containing silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel filtration chromatography, and containing at least water as a solvent.
  • Aspect 12 is a silk fibroin aqueous solution according to aspect 12, wherein, in the silk fibroin, the proportion of fragments having a molecular weight exceeding 100 kDa in a molecular weight distribution measured by polyacrylamide gel electrophoresis is 15% by weight or less (preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less), and/or the proportion of fragments having a molecular weight of 25 to 100 kDa is 15 to 40% by weight (preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight).
  • Aspect 14 14.
  • Aspect 15 A method for producing an aqueous silk fibroin solution, comprising dissolving the solidified silk fibroin according to any one of Aspects 1 to 7 in a solvent containing at least water.
  • Aspect 16 A gel-like fixative for immobilizing an object to be fixed, the gel-like fixative containing silk fibroin having a weight-average molecular weight of 55 to 150 kDa (preferably 60 to 140 kDa, more preferably 70 to 130 kDa) as measured by gel filtration chromatography, and containing a silk fibroin gel prepared from an aqueous solution containing at least water as a solvent.
  • a gel-like fixative according to Aspect 16 wherein the proportion of fragments having a molecular weight exceeding 100 kDa in the molecular weight distribution of the silk fibroin measured by polyacrylamide gel electrophoresis is 15% by weight or less (preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less), and/or the proportion of fragments having a molecular weight of 25 to 100 kDa is 15 to 40% by weight (preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight).
  • Aspect 18 18.
  • the gel-like fixative according to aspect 16 or 17, wherein the object to be fixed is a biological sample or a microorganism.
  • a “solidified material” refers to a substance that is not easily deformed by an external force, and is distinguished from a semi-solidified material or gel that is easily deformed by an external force.
  • a gel-like fixing material means a semi-solid substance that does not have fluidity even when external force or heat is applied, from the perspective of fixing a material to be fixed therein.
  • aqueous solution refers to a solution that contains at least water as a solvent, and in which no visible precipitates are observed in an aqueous solution below the limit solubility of silk fibroin.
  • the proportion of water in the solvent may be, for example, 50 wt % or more, preferably 70 wt % or more, more preferably 90 wt % or more, and even more preferably 95 wt % or more.
  • the solvent may contain only water (100 wt %).
  • a solution containing only water as a solvent is called an aqueous solution.
  • the silk fibroin constituting the solidified silk fibroin of the present invention has a medium molecular weight, which not only allows for long-term storage in the solidified form (e.g., powder, freeze-dried product), but also has good solubility in water and the effect of improving the physical properties of liquids (e.g., thickening). Furthermore, the silk fibroin aqueous solution of the present invention, which contains silk fibroin with a medium molecular weight, not only improves the physical properties of aqueous solutions but also prevents gelation in the aqueous solution state, allowing for long-term storage (e.g., approximately one to one and a half years).
  • the silk fibroin aqueous solution of the present invention can also be gelled by actively carrying out a specified gelation treatment, and therefore, in its gelled state, it can also be used as a fixative for desired objects to be fixed (e.g., biological samples).
  • the peak top molecular weight (Mp) of silk fibroin measured by GFC is smaller than the weight average molecular weight, and the difference between the peak top molecular weight and the weight average molecular weight may be, for example, 1 to 70 kDa, preferably 10 to 60 kDa, and more preferably 20 to 50 kDa.
  • the peak top molecular weight refers to the molecular weight corresponding to the peak top position of the differential molecular weight distribution curve detected by GFC, and is specifically a value measured by the method described in the Examples below.
  • the medium-molecular-weight silk fibroin of the present invention has a specific weight-average molecular weight, but the molecular weight distribution contains many low-molecular-weight fragments.
  • the proportion of fragments with a molecular weight exceeding 100 kDa in the molecular weight distribution measured by polyacrylamide gel electrophoresis (SDS-PAGE) may be, for example, 15% by weight or less, preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 3% by weight or less.
  • the lower limit is not particularly limited, and may be 0% by weight.
  • the proportion of fragments having a molecular weight of 25 to 100 kDa as measured by polyacrylamide gel electrophoresis may be, for example, 15 to 40% by weight, preferably 18 to 35% by weight, more preferably 20 to 30% by weight, and particularly preferably 20 to 28% by weight.
  • the proportion of fragments refers to the proportion of the weight of fragments present in a specific range of molecular weight relative to the weight of all fragments. More specifically, the molecular weight distribution is a value measured by SDS-PAGE according to the method described in the Examples below.
  • the number average molecular weight (Mn) of the silk fibroin measured by GFC may be 80 kDa or less (e.g., 1 to 80 kDa), preferably 50 kDa or less, more preferably 40 kDa or less, and even more preferably 30 kDa or less. There is no particular lower limit for the number average molecular weight, but it may be, for example, 1 kDa or more. In particular, the number average molecular weight is a value measured by GFC using the method described in the Examples below.
  • Silk fibroin is generally known as a type of fibrous protein, with molecules arranged in a regular pattern and composed of crystalline portions containing mainly glycine, alanine, and serine, and amorphous portions containing tyrosine and other elements.
  • silk fibroin can be obtained from the silk raw materials described below.
  • silk fibroin does not have to be chemically modified, but may be chemically modified to the extent that the effects of the present invention are not impaired.
  • this definition also includes silk fibroin that has been chemically modified to the extent that the effects of the present invention are not impaired.
  • the solidified silk fibroin is not particularly limited as long as it has excellent solubility in water and can improve the physical properties of an aqueous solution. From the viewpoint of easily adjusting the concentration of silk fibroin in the aqueous silk fibroin solution, powdered solidified products and three-dimensional solidified products (e.g., approximately rectangular, cubic, cylindrical, spherical, etc.) that are easy to measure are preferred. From the viewpoint of measurement, the solidified silk fibroin may not preferably include a silk fibroin film.
  • the solidified material may be, for example, a freeze-dried product, a crushed freeze-dried product, a spray-dried product, etc., since it is derived from an aqueous silk fibroin solution.
  • the moisture absorption of the solidified silk fibroin may be 50% or less, preferably 40% or less, after being left for 3 hours under high temperature and humidity conditions, for example, 37°C and 100% RH.
  • the lower limit of moisture absorption is not particularly limited, but may be, for example, 5% or more.
  • the moisture absorption of the solidified silk fibroin may be 25% or less.
  • the moisture absorption of the solidified silk fibroin is specifically the value measured in the examples described below.
  • the density may be, for example, 7 ⁇ 10 ⁇ 3 g/cm 3 or more, preferably 15 ⁇ 10 ⁇ 3 g/cm 3 or more. There is no particular upper limit to the density, but it may be, for example, 130 g/cm 3 or less. Furthermore, from the viewpoint of maintaining solubility in water even when absorbing moisture under high temperature and humidity conditions, the density of the solidified silk fibroin may be 25 ⁇ 10 ⁇ 3 g/cm 3 or more.
  • the density of the solidified silk fibroin is specifically a value measured in the examples described later.
  • the solubility of the solidified silk fibroin in water at 20 ⁇ 5°C according to JIS K 8001 (2017) may be, for example, 1 mL or more but less than 10 mL, as the amount of water required to dissolve 1 g of solidified silk fibroin.
  • the amount of silk fibroin that dissolves within 30 minutes is preferably as high as possible, and may be, for example, 50 mg/mL or more, preferably 110 mg/mL or more, and more preferably 120 mg/mL or more. There is no particular upper limit, but it may be 150 mg/mL or less.
  • the solidified silk fibroin may be stored at low temperatures, or in a specified environment (temperature 15-30°C), and may be used in foods, cosmetics, or experimental materials.
  • Experimental materials may be materials used in connection with experiments conducted on a lab scale, such as various experimental culture media.
  • the method for producing solidified silk fibroin includes an alkali treatment step in which refined silk fibroin is dissolved in a solvent and reacted with an alkali treatment agent, and a step in which the aqueous solution containing the alkali-treated silk fibroin is solidified.
  • the raw silk material for silk fibroin can be cocoons or raw silk produced by insects (Lepidoptera such as domestic silkworms, wild silkworms, and wild silkworms, and silkworms that produce silk threads, such as hornets and honeybees, and other Hymenoptera insects) or spiders, and is not particularly limited as long as it contains fibroin and sericin.
  • Silk fibroin from which sericin has been removed can be obtained by scouring the silk material.
  • Silk fibroin can also be obtained from silk glands.
  • scouring can be carried out by known methods, including methods that swell and remove sericin using alkaline scouring agents such as sodium carbonate, sodium silicate, and sodium phosphate, methods that decompose and remove sericin using sericin-degrading enzymes, and methods that rot and remove sericin. From the perspective of ease of setting conditions, scouring using an alkaline scouring agent is preferred.
  • the main purpose of degumming is to remove sericin, and from the perspective of suppressing the decomposition of silk fibroin, the degumming time using an alkaline degumming agent varies depending on the type of silk raw material and alkaline degumming agent, but may be 5 to 60 minutes, preferably 10 to 50 minutes, and more preferably 15 to 45 minutes.
  • the silk fibroin may be dissolved in a mixed solution containing a neutral salt and an alkali treatment agent, and the alkali treatment may be carried out at the same time.
  • the silk fibroin may be dissolved in a neutral salt solution, and then the alkali treatment may be carried out by adding the alkali treatment agent after the silk fibroin has been dissolved.
  • the neutral salts and alkali treatment agents may be removed by known methods such as dialysis or ultrafiltration. Furthermore, if necessary, it is preferable to purify or sterilize the silk fibroin before performing the freeze-drying process in order to remove impurities other than the neutral salts and alkali treatment agents. Regarding sterilization, known methods such as autoclave sterilization and filter sterilization can be used, but autoclave sterilization is preferred.
  • a step of solidifying the silk fibroin aqueous solution (preferably the silk fibroin aqueous solution after purification and/or sterilization) is carried out.
  • the obtained silk fibroin aqueous solution can be solidified by freeze-drying, spray-drying, or the like.
  • freeze-drying is a drying method in which an aqueous silk fibroin solution is frozen at a temperature below its eutectic point and water is removed by sublimation.
  • Freeze-drying can be performed using a known freeze-drying device, and may be performed in multiple stages, for example.
  • the solution may be pre-freezed before using the freeze-drying device.
  • the pre-freezing may involve freezing the aqueous silk fibroin solution at a temperature of, for example, about ⁇ 80 to ⁇ 30° C.
  • a freeze-dried product may be obtained by freeze-drying in a freeze-drying apparatus at a trap cooling temperature of, for example, ⁇ 100° C. to ⁇ 60° C., preferably ⁇ 90° C. to ⁇ 70° C.
  • the freeze-dried product may be powdered to a desired size, or may be used as is without being powdered.
  • powdering can be carried out using a grinder, mill mixer, ball mill, hammer mill, roller mill, etc.
  • the powdered silk fibroin powder may be classified as needed and may be further packaged in a sterilized pack or the like.
  • silk fibroin aqueous solution contains silk fibroin having a weight-average molecular weight of 55 to 150 kDa as measured by gel permeation chromatography (GFC), and also contains at least water as a solvent. Furthermore, silk fibroin may have, either alone or in combination, various properties described in the above-described solidified silk fibroin product, such as the weight-average molecular weight (Mw), peak-top molecular weight (Mp), number-average molecular weight (Mn), polydispersity (Mw/Mn) measured by GFC, and molecular weight distribution measured by SDS-PAGE. Unless subjected to an active gelation treatment described below, the aqueous silk fibroin solution will not gel and can have excellent storage stability.
  • Mw weight-average molecular weight
  • Mp peak-top molecular weight
  • Mn number-average molecular weight
  • Mw/Mn polydispersity
  • the silk fibroin aqueous solution may be used as an oral aqueous solution or as a transdermal aqueous solution.
  • Oral aqueous solutions can be used as drinks, etc., and in addition to such silk fibroin aqueous solutions, they may contain various active ingredients (various beauty ingredients, hair growth ingredients, blood circulation promoting ingredients, etc.) and additives, as described below, depending on the desired use, such as nutritional tonic or cosmetic use.
  • the transdermal aqueous solution may be used as a cosmetic.
  • the silk fibroin aqueous solution may be used as an aqueous cosmetic, or may be used as an oil-based cosmetic by combining the silk fibroin aqueous solution with an oily base.
  • the aqueous cosmetic may be a cosmetic containing 50 wt% or more, preferably 60 wt% or more, of water.
  • the oil-based cosmetic may be a cosmetic containing 50 wt% or more, preferably 60 wt% or more, of an oily base.
  • the cosmetic may contain various active ingredients (various beauty ingredients, hair growth ingredients, blood circulation promoting ingredients, etc.), additives as described below, etc., depending on the intended use.
  • the oily base can be any known or conventional oil, such as natural oils such as coconut oil, palm oil, squalane, olive oil, castor oil, almond oil, jojoba oil, orange oil, camellia oil, argan oil, bergamot oil, lavender oil, rosemary oil, rosehip oil, tea tree oil, shea oil, macadamia nut oil, hazelnut oil, kukui nut oil, meadowfoam oil, persic oil, peppermint oil, and lanolin; mineral oils such as mineral oil, petrolatum, paraffin, and liquid paraffin; and synthetic oils such as silicone oil.
  • natural oils such as coconut oil, palm oil, squalane, olive oil, castor oil, almond oil, jojoba oil, orange oil, camellia oil, argan oil, bergamot oil, lavender oil, rosemary oil, rosehip oil, tea tree oil, shea oil, macadamia nut oil, hazelnut oil, kukui nut oil, meadowfoam oil, pers
  • liquid cosmetics e.g., lotion, emulsion, serum, cosmetic liquid (e.g., all-in-one cosmetic liquid), emulsified foundation, oil-based foundation, two-layer foundation, nail enamel, shampoo, rinse, conditioner, treatment, tonic, hair growth agent, perm agent, hair manicure, liquid soap, various oils (e.g., oil cleanser, moisturizing skin oil, hair oil, etc.), foam, mist, etc.), and semi-solid cosmetics (e.g., cream, gel, grease, coating agent, ointment, etc.).
  • Liquid cosmetics may be used as a sheet impregnated with the liquid cosmetic.
  • various oils preferably hair oil
  • cream etc.
  • silk fibroin is preferred because it can moisturize the hair while protecting it from heat damage caused by dryers, hair irons, etc.
  • a liquid cosmetic When a liquid cosmetic contains water and oil, it may be an emulsion cosmetic such as oil-in-water (O/W), water-in-oil (W/O), W/O/W, or O/W/O.
  • O/W oil-in-water
  • W/O water-in-oil
  • W/O/W W/O/W
  • O/W/O O/W/O
  • the additives can be appropriately selected depending on the application, and examples thereof include surfactants, antioxidants, antiseptics, disinfectants, preservatives, pH adjusters, ultraviolet absorbers, chelating agents, refreshing agents, stabilizers, fragrances, colorants, etc. These additives may be used alone or in combination of two or more.
  • the silk fibroin may have, either alone or in combination of two or more of the various properties described in the above-mentioned solidified silk fibroin, such as the weight-average molecular weight (Mw) measured by GFC, the molecular weight distribution measured by SDS-PAGE, the peak-top molecular weight (Mp), the number-average molecular weight (Mn), and the polydispersity (Mw/Mn).
  • Mw weight-average molecular weight
  • Mp peak-top molecular weight
  • Mn number-average molecular weight
  • Mw/Mn polydispersity
  • the desired fixable object e.g., biological sample, microorganism, etc.
  • the fixable object can be fixed according to its physical properties.
  • Biological samples include, for example, cells, tissues, blood (including, for example, whole blood, plasma, umbilical cord blood, platelets, and serum), milk products (e.g., milk), amniotic fluid, sputum, urine, saliva, mucus, semen, cerebrospinal fluid, bronchial aspirate, sweat, nasal secretion, vaginal fluid, liquefied feces, synovial fluid, lymphatic fluid, tears, tracheal aspirate, or fractions thereof (e.g., proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins), or combinations thereof.
  • milk products e.g., milk
  • amniotic fluid e.g., milk
  • sputum e.g., milk
  • urine saliva
  • mucus semen
  • cerebrospinal fluid e.g
  • Biological samples may be derived from either plants or animals.
  • Animals may be human or non-human animals.
  • Non-human animals include, for example, non-human mammals, such as apes, other primates, mice, rats, hamsters, guinea pigs, horses, cows, pigs, sheep, goats, dogs, cats, and rabbits.
  • Microorganisms are a general term for all minute living organisms, and in this specification include viruses, bacteria, fungi (yeast, mold, mushrooms, etc.), microalgae, and protozoa (amoeba, paramecium, etc.).
  • the method for producing a gel-like fixative may include a dispersion step in which a predetermined amount of the desired object to be fixed is dispersed in an aqueous silk fibroin solution to obtain a dispersion of the aqueous silk fibroin solution and the object to be fixed, and a gelation step in which a gelation treatment is performed on the dispersion.
  • the method for producing a gel-like fixative may include a gelation induction step for inducing gelation in an aqueous silk fibroin solution, a mixing step for mixing the desired object to be fixed directly or as a suspension with the aqueous silk fibroin solution that has been subjected to the gelation induction treatment to obtain a dispersion, and a gelation completion step for leaving the dispersion to complete gelation.
  • the object to be fixed is first dispersed in a highly fluid liquid, and then a gelling process is performed to fix it in place with a network of entangled silk fibroin molecules, allowing the object to be fixed in a uniformly dispersed state within the silk fibroin gel.
  • the gelation treatment can be carried out using various known methods.
  • the gelation treatment may also be comprised of a gelation-inducing step and a gelation-completion step.
  • the gelation-inducing step is carried out on a silk fibroin aqueous solution, and examples include a step of adding an acidic substance such as citric acid to adjust the pH to an acidic condition, for example, pH 3 to 6, preferably pH 3 to 4; a step of adding an organic solvent such as ethanol (for example, the amount of organic solvent added is 1 volume or less per volume of the silk fibroin aqueous solution); vortex treatment; ultrasonic treatment; and spraying with carbon dioxide gas or the like.
  • These gelation-inducing steps may be carried out alone or in combination.
  • vortex treatment or ultrasonic treatment is preferably performed compared to gelation treatment under acidic conditions or gelation treatment involving the addition of an organic solvent.
  • Vortexing conditions can be set appropriately depending on the silk fibroin concentration of the silk fibroin aqueous solution, but for example, the rotation speed may be 1,000 to 5,000 rpm, preferably 2,000 to 4,000 rpm. Furthermore, the vortexing time may be, for example, 1 to 20 minutes, preferably 2 to 11 minutes.
  • the conditions for ultrasonic treatment can be appropriately set depending on the silk fibroin concentration of the silk fibroin aqueous solution, and the amplitude (Ampl), for example, may be 10 to 40%, preferably 15 to 30%.
  • the ultrasonic treatment time can be set appropriately depending on the amplitude, and may be, for example, 10 to 60 seconds, preferably 15 to 50 seconds.
  • the liquid to be treated that has been treated in the gelation induction step may be allowed to stand at a predetermined temperature, for example, 15 to 40°C, preferably 35 to 40°C, for 100 minutes or less (for example, 2 to 90 minutes), preferably 80 minutes or less (for example, 3 to 70 minutes), and even more preferably 60 minutes or less (for example, 4 to 50 minutes), to complete the gelation process.
  • a predetermined temperature for example, 15 to 40°C, preferably 35 to 40°C, for 100 minutes or less (for example, 2 to 90 minutes), preferably 80 minutes or less (for example, 3 to 70 minutes), and even more preferably 60 minutes or less (for example, 4 to 50 minutes).
  • the gel-like fixative is not particularly limited as long as it can uniformly disperse and fix the object to be fixed.
  • the concentration of silk fibroin in the treatment liquid may be, for example, 4 to 15% (w/v), preferably 5 to 9% (w/v).
  • the gel-like fixative can fix the object to be fixed in a desired container.
  • the object to be fixed is a biological sample or a microorganism
  • the object can be fixed with the gel-like fixative in a container such as a multi-well culture plate or culture dish.
  • the therapeutic agent is not particularly limited, but includes, for example, anticancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anticonvulsants, hormones, muscle relaxants, antispasmodics, ophthalmological agents, prostaglandins, antidepressants, antipsychotic substances, nutritional factors, osteoinductive proteins, growth factors, peptide drugs, and vaccines.
  • the molecular weight of silk fibroin was measured using an aqueous silk fibroin solution adjusted to a concentration of 1% (w/v) as the measurement sample. This measurement sample was used for gel filtration chromatography (GFC) measurement using the following measurement equipment and conditions.
  • the weight-average molecular weight (Mw), number-average molecular weight (Mn), polydispersity index (PDI), and peak-top molecular weight (Mp) of silk fibroin were calculated using molecular weight markers included in the low molecular weight (LMW) gel filtration calibration kits and the high molecular weight (HMW) gel filtration calibration kits (both manufactured by Cytiva) as standards. The measured values are the average of three or more measurements.
  • LMW low molecular weight
  • HMW high molecular weight
  • the silk fibroin solution was diluted with a 6M urea aqueous solution to a concentration of 0.5% (w/v) to prepare the measurement sample.
  • a sample buffer (4x Laemmli Sample Buffer, BioRad) and 2-mercaptoethanol were mixed at a volume ratio of 9:1 to prepare a reducing agent-containing sample buffer.
  • the measurement sample and the reducing agent-containing sample buffer were mixed at a volume ratio of 3:1 and heated at 98°C for 5 minutes.
  • the mixture was loaded onto a polyacrylamide gel (EHR-R520L, Atto) at 10 ⁇ L/lane and electrophoresed at a constant current (30 mA) using an electrophoresis apparatus (AE-8155 MyPower II500, Atto).
  • the electrophoresis buffer used was electrophoresis buffer solution (30329-61, manufactured by Nacalai Tesque) diluted 10-fold with deionized water, and Precision Plus Protein Two-Color Standard (manufactured by BioRad) was used as a molecular weight marker.
  • the polyacrylamide gel was immersed in gel staining solution (EzStain AQua, manufactured by Atto) in a steel square case (1-4698-12, manufactured by AS ONE Corporation) and shaken at room temperature for 2 hours for staining. The gel staining solution was then removed, and deionized water was added. A washing procedure was then performed in which the gel was shaken overnight at room temperature. The deionized water was exchanged five times during the washing procedure. After washing, the polyacrylamide gel was imaged using a gel imaging device (WSE-5400 Printgraph Classic, Atto) equipped with a white light source (Flatview, Atto) (exposure time: 1 msec, magnification: ⁇ 1.8).
  • a gel imaging device WSE-5400 Printgraph Classic, Atto
  • a white light source Exposure time: 1 msec, magnification: ⁇ 1.8
  • the resulting image data was analyzed using ImageJ software (National Institute of Health). The color tone was set to 8 bits, and background subtraction was performed using the Subtract Background command (Rolling ball radius: 90 pixels). Then, the Gels command was used to obtain the change in brightness relative to the molecular weight in each lane. Based on the brightness peak of the molecular weight marker lane, the amount of silk fibroin distributed between the two molecular weight markers was calculated as an area. The weight ratio of silk fibroin fragments within a certain molecular weight range to the total silk fibroin fragments was calculated by dividing the area by the total area of each lane.
  • the storage stability test for powder is an accelerated test, in which the powder packed in an aluminum bag is tested under conditions of 40°C ⁇ 2°C/75% RH ⁇ 5% at the start, after 2 months (guaranteed for 1 year), and after 3 months (guaranteed for 1.5 years).
  • the color, pH, and moisture content of the powder after storage can be evaluated using the following criteria.
  • Example 3 The silk fibroin powder obtained in Example 1 was redissolved in water at room temperature to obtain a 4% (w/v) silk fibroin aqueous solution. 500 ⁇ L of this solution was placed in a 1.5 mL tube (Eppendorf) and subjected to ultrasonic irradiation (Ampl: 20%, 30 seconds) using an ultrasonic homogenizer (SONICS & MATERIALS "VCX-750"). 300 ⁇ L of this solution was transferred to a 2 mL tube (Eppendorf), and the time until gelation was complete was measured using the tilt method.
  • Silk fibroin powder was prepared in the same manner as in Example 1, except that a 9M lithium bromide aqueous solution was used as the solution for dissolving the refined silk fibroin, as in Patent Document 2.
  • Table 1 shows the physical properties of the silk fibroin powder.
  • Figure 3 shows the state of silk fibroin powder (100 mg) placed in a glass tube (left) and the state after adding deionized water (1 mL) to the tube and shaking vigorously for 30 seconds after 5 minutes (right).
  • the silk fibroin aqueous solution was powdered.
  • the silk fibroin powder was barely soluble in water, and precipitates were visually observed. Note that because precipitates were observed in the silk fibroin powder obtained in Comparative Example 2, experiments using aqueous solutions were not performed.
  • Example 1 As shown in Table 1, the powder obtained in Example 1 had a weight-average molecular weight in the range of 55 to 150 kDa, which indicates that silk fibroin was successfully converted to a medium molecular weight.
  • Example 1 and Comparative Example 1 the solubility in water was good, but in Comparative Example 1, the improvement in physical properties was insufficient.
  • Comparative Example 2 the molecular weight could not be sufficiently reduced, and the solubility in water was poor.
  • the silk fibroin powder obtained in Example 1 had good storage stability, and even when an accelerated storage test was performed, there was little change in color, pH, and moisture content after 6 months (guaranteed period). Furthermore, the silk fibroin aqueous solutions with concentrations of 1% (w/v) and 3% (w/v) obtained in Example 2 also exhibited good storage stability, showing no gelation and little change in color and pH after 6 months of storage.
  • the silk fibroin of Comparative Example 1 did not have a medium molecular weight compared to the silk fibroin of Example 3, and therefore required more than four times the time required for gelation to be completed than in Example 3. Furthermore, in Example 4, gelation was completed in five minutes.
  • Example 5 Silkworm cocoons were cut into small pieces and degummed in a boiling 0.02 M aqueous solution of sodium carbonate for 30 minutes to obtain silk fibroin. 3 g of the refined silk fibroin was immersed in a mixed aqueous solution (50 mL) containing 9 M lithium bromide and 0.1 M sodium hydroxide (NaOH) and left at room temperature for 1 to 6 hours. Next, this mixed aqueous solution was stirred at room temperature for 17 hours to dissolve the silk fibroin in the mixed aqueous solution and to perform an alkali treatment, thereby obtaining a silk fibroin aqueous solution.
  • a mixed aqueous solution 50 mL
  • LiOH lithium bromide
  • NaOH sodium hydroxide
  • the resulting silk fibroin aqueous solution was dialyzed against deionized water using a dialysis membrane (molecular weight cutoff: 6-8kJ). Each dialysis session took 6-12 hours at room temperature, and this was repeated six times (bath ratio: 160). This removed the lithium bromide and sodium hydroxide contained in the silk fibroin aqueous solution.
  • the silk fibroin aqueous solution was concentrated by air-drying at room temperature. Concentration by air-drying was continued until the volume of the silk fibroin aqueous solution was reduced to 1/5 to 1/3 of its original volume.
  • the concentrated silk fibroin aqueous solution was subjected to autoclave sterilization using an autoclave (Tomy Seiko Co., Ltd., "LBS-245"). Autoclave sterilization was performed twice at 121°C for 20 minutes. All subsequent treatments were performed under sterile conditions.
  • the concentration of the silk fibroin aqueous solution was adjusted as shown in Table 4, and the obtained silk fibroin aqueous solution was left to stand overnight in a deep freezer (VT-78HC, manufactured by Nippon Freezer Co., Ltd.) and pre-frozen at ⁇ 80° C. Then, it was freeze-dried (trap cooling temperature: ⁇ 90° C.) for 3 days using a freeze dryer (FDS-1000, manufactured by Tokyo Rikakikai Co., Ltd.).
  • Table 4 shows the density of the freeze-dried product, the hygroscopicity of the freeze-dried product in a hot and humid environment, and the solubility in water after storage in a hot and humid environment.
  • the freeze-dried products obtained by varying the concentration of the silk aqueous solution had different hygroscopicity and density.
  • the freeze-dried product was able to maintain its solubility in water even after being left under high-temperature, high-humidity conditions for 3 hours.

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Abstract

L'invention concerne : de la fibroïne de soie solidifiée comprenant de la fibroïne de soie ayant une proportion spécifique de poids moléculaires ; une solution aqueuse de fibroïne de soie et son procédé de production ; et un matériau de fixation à l'état de gel. La fibroïne de soie incluse dans la fibroïne de soie solidifiée, la solution aqueuse de fibroïne de soie, et le matériau de fixation à l'état de gel a un poids moléculaire moyen en poids, tel que déterminé par chromatographie par filtration sur gel, de 55 à 150 kDa.
PCT/JP2025/007726 2024-03-28 2025-03-04 Fibroïne de soie solidifiée, solution aqueuse de fibroïne de soie et son procédé de production et matériau de fixation à l'état de gel Pending WO2025204578A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017048302A (ja) * 2015-09-01 2017-03-09 日立化成株式会社 シルクフィブロイン粉末の製造方法
JP2017119647A (ja) * 2015-12-28 2017-07-06 株式会社松田養蚕場 高分子量シルクフィブロイン水溶液の製造方法および高分子量シルクフィブロイン粉末の製造方法
JP2017527612A (ja) * 2014-08-20 2017-09-21 シルク テクノロジーズ， リミテッド フィブロインに由来するタンパク質組成物
JP2021098737A (ja) * 2015-04-29 2021-07-01 エボルブド バイ ネイチャー， インコーポレイテッド 絹ベース湿潤剤組成物、及びその方法
WO2023190130A1 (fr) * 2022-03-31 2023-10-05 キヤノン株式会社 Solution aqueuse de fibroïne, son procédé de production et article comprenant de la fibroïne

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017527612A (ja) * 2014-08-20 2017-09-21 シルク テクノロジーズ， リミテッド フィブロインに由来するタンパク質組成物
JP2021098737A (ja) * 2015-04-29 2021-07-01 エボルブド バイ ネイチャー， インコーポレイテッド 絹ベース湿潤剤組成物、及びその方法
JP2017048302A (ja) * 2015-09-01 2017-03-09 日立化成株式会社 シルクフィブロイン粉末の製造方法
JP2017119647A (ja) * 2015-12-28 2017-07-06 株式会社松田養蚕場 高分子量シルクフィブロイン水溶液の製造方法および高分子量シルクフィブロイン粉末の製造方法
WO2023190130A1 (fr) * 2022-03-31 2023-10-05 キヤノン株式会社 Solution aqueuse de fibroïne, son procédé de production et article comprenant de la fibroïne

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