Silk fibroin film and adhesion preventive material containing the same

Abstract

Provided is a silk fibroin film suitable for use as an anti-adhesion material. The silk fibroin film may have at least the film surface of silk fibroin dissolved in water within 24 hours after floating on water. Further, in the measurement of the contact angle of water on the film surface, the silk fibroin film may have a contact angle change of -5° / sec or less within 5 seconds after dropping a 5 μL water droplet of ultrapure water onto the film surface. Further, the silk fibroin film may have a weight average molecular weight of silk fibroin of 150 kDa or less.

Description

Related Applications

[0001] This application claims the priority of Japanese Patent Application No. 2023-139250 filed on August 29, 2023, and the entire disclosure of which is incorporated herein by reference in its entirety and made a part of this application.

Technical Field

[0002] The present invention relates to a silk fibroin film used for an adhesion prevention material and an adhesion prevention material containing the same.

Background Art

[0003] In clinical fields such as gastrointestinal surgery, cardiac surgery, orthopedic surgery, and obstetrics and gynecology, adhesions may occur after surgery, where tissues and organs that should originally be separated become joined and do not separate. The occurrence of adhesions may require reoperation for adhesion detachment, which requires a great deal of time and effort, and may also cause new damage during detachment. In addition, complications such as intestinal obstruction and infertility may also occur.

[0004] Various methods have been studied to prevent adhesions between tissues and organs, and adhesion prevention materials have been developed that physically prevent contact between tissues and organs by applying them to the surfaces of tissues and organs. Adhesion prevention materials have been developed in the form of film-like or spray-like materials, etc., and in the fields of gastrointestinal surgery and obstetrics and gynecology, film-like adhesion prevention materials have been clinically applied. For example, as film-like adhesion prevention materials, Seprafilm (registered trademark) containing hyaluronic acid and carboxymethyl cellulose, TenariF (registered trademark) containing gelatin, etc. can be mentioned. However, since these adhesion prevention materials cannot be used for patients with a history of hypersensitivity to the components contained therein, it is necessary to expand the options available for clinical use.

[0005] On the one hand, in recent years, silk fibroin has attracted attention as a biomaterial because of its excellent biocompatibility. For example, Patent Document 1 (Japanese Patent Application Laid-Open No. 2016-517443) discloses a low-molecular-weight silk fibroin composition containing a population of silk fibroin fragments having a certain range of molecular weights, wherein 15% or less of the total number of the silk fibroin fragments in the population has a molecular weight exceeding 200 kDa, at least 50% of the total number of the silk fibroin fragments in the population has a molecular weight within a specified range, and the specified range is between a lower limit of about 3.5 kDa and an upper limit of about 120 kDa. It is described that it can be used in film form and for medical purposes.

[0006] Also, Patent Document 2 (U.S. Patent No. 9,427,499) discloses a silk fibroin matrix selectively modified with poly(ethylene glycol) (PEG) at a density of about 75 to about 750 μg PEG / cm 2 of the silk fibroin matrix, and it is described that it can be used as an anti-adhesion material.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, although Patent Document 1 describes that the low-molecular-weight silk fibroin can have high water solubility, it does not describe the use of the low-molecular-weight silk fibroin composition as an anti-adhesion material.

[0009] In addition, Patent Document 2 describes that by modifying the surface of a silk fibroin matrix with PEG, the adhesiveness and growth promotion properties are suppressed for mesenchymal stem cells and fibroblasts, respectively. However, it is the surface PEG that contributes to these suppressions, and no effect due to the structure of silk fibroin itself has been found. Also, the anti-adhesion effect in vivo has not been evaluated.

[0010] Therefore, an object of the present invention is to provide a silk fibroin film suitable for use as an anti-adhesion material.

Means for Solving the Problems

[0011] As a result of intensive studies to achieve the above object, the inventors of the present invention have found that a silk fibroin film in which the film surface dissolves in water in a short time has an excellent anti-adhesion effect, and thus completed the present invention.

[0012] That is, the present invention can be configured in the following aspects. 〔Aspect 1〕 A silk fibroin film for an anti-adhesion material, wherein at least the film surface of silk fibroin dissolves in water within 24 hours (preferably within 12 hours, more preferably within 1 hour, still more preferably within 10 minutes) after floating on water. 〔Aspect 2〕 The silk fibroin film according to Aspect 1, wherein in the measurement of the contact angle of water on the film surface, the change in the contact angle within 5 seconds after dropping a 5 μL droplet of ultrapure water on the film surface is -5° / sec or less (preferably -20 to -5° / sec, more preferably -15 to -5° / sec, particularly -10 to -5° / sec). 〔Aspect 3〕 In the measurement of the contact angle of water on the film surface, the change in the contact angle from when a 5 μL droplet of ultrapure water is dropped onto the film surface until 5 seconds later is -5° / sec or less (preferably -20 to -5° / sec, more preferably -15 to -5° / sec, particularly -10 to -5° / sec), a silk fibroin film. [Aspect 4] A silk fibroin film according to any one of Aspects 1 to 3, wherein the water dissolution rate of the film after being immersed in water and incubated at 25°C for 24 hours is 50% or more (preferably 70% or more, more preferably 80% or more, still more preferably 90% or more), a silk fibroin film. [Aspect 5] A silk fibroin film according to any one of Aspects 1 to 4, wherein the water dissolution rate of the film after being immersed in water and incubated at 25°C for 1 hour is 100% or less (preferably 99% or less, more preferably 98% or less), a silk fibroin film. [Aspect 6] A silk fibroin film according to any one of Aspects 1 to 5, wherein the weight average molecular weight of the silk fibroin is 150 kDa or less (preferably 120 kDa or less, more preferably 100 kDa or less, still more preferably 90 kDa or less), a silk fibroin film. [Aspect 7] A silk fibroin film according to any one of Aspects 1 to 6, wherein the silk fibroin present on the film surface is silk fibroin not modified with polyethylene glycol, a silk fibroin film. [Aspect 8] A silk fibroin film according to any one of Aspects 1 to 7, wherein the water content is 30% or less (preferably 1 to 30%, more preferably 3 to 20%, still more preferably 5 to 15%), a silk fibroin film. [Aspect 9] A silk fibroin film containing silk fibroin having a weight average molecular weight of 40 to 150 kDa (preferably 50 to 120 kDa, more preferably 55 to 100 kDa) and a molecular weight distribution (PDI) of 1 to 10 (preferably 1.2 to 9, more preferably 1.5 to 8). [Aspect 10] An adhesion prevention material containing the silk fibroin film according to any one of Aspects 1 to 7. [Aspect 11] A method for producing a silk fibroin film for an adhesion prevention material, comprising an alkali treatment step of dissolving refined silk fibroin in a solvent and reacting it with an alkali treatment agent, and a film forming step of forming a film from the alkali-treated silk fibroin. [Aspect 12] The method for producing a silk fibroin film according to Aspect 11, wherein the weight average molecular weight of the alkali-treated silk fibroin is 150 kDa or less (preferably 120 kDa or less, more preferably 100 kDa or less, still more preferably 80 kDa or less). [Aspect 13] An adhesion prevention method using the adhesion prevention material according to Aspect 10, comprising a step of applying the adhesion prevention material to at least one of the cut surface of an organ after surgery and the surface of the surrounding tissue of the organ after surgery.

[0013] As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms including "at least one" unless the context clearly indicates otherwise. As used herein, the terms "and / or", "at least 1", and "1 or more" include any and all combinations of the related listed items.

[0014] Also, when referred to as "about", a range of ±10%, preferably ±5% with respect to the numerical value may be included.

[0015] In addition, any combination of at least two components disclosed in the claims and / or the specification and / or the drawings is included in the present invention. In particular, any combination of two or more of the claims recited in the claims is included in the present invention.

Advantages of the Invention

[0016] The silk fibroin film of the present invention can prevent adhesions between tissues and organs after surgery, and since it contains components different from those of conventional film-like adhesion prevention materials, it can be used as an option for selecting an adhesion prevention material according to the allergies of patients.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] (Silk fibroin film) The silk fibroin film has at least the film surface of silk fibroin dissolved in water within 24 hours after floating on water. In this specification, the dissolution of the film surface in water is observed by the method described in the following examples, and can be confirmed by the fact that all six straight lines (line width 1 mm) of the square and its diagonal lines drawn with an oil-based pen (black) on the film surface of a 1 cm-sided square are all broken. That is, when a line of an oil-based pen is drawn on the first surface of the film, and the film is floated on water with the first surface on top and the second surface on the bottom, as the film surface (the first surface) dissolves in water, each of the six straight lines of the oil-based pen drawn on the first surface peels off from the film, separates from each other, and each straight line is divided by cracks. Therefore, after floating the film with a straight line of an oil-based pen drawn on the film surface on water, by confirming that the straight line of the oil-based pen peels off together with the film surface and finally all six straight lines are divided, the water solubility of the film surface can be evaluated. By confirming that the upper surface of the film dissolves when the film is floated on water, the film surface of the lower surface (the second surface) of the film can also be regarded as dissolved. Note that it is only necessary that at least the film surface dissolves in water within 24 hours, and the portion other than the film surface may or may not dissolve. Therefore, the entire film may dissolve in water within 24 hours. In the present invention, due to the high water solubility of the film surface of the silk fibroin film, it is possible to prevent the film surface from being in contact with cells, fibrin, etc. for a long time, and it is considered that the adhesion of cells, fibrin, etc. is inhibited, or because the cells, fibrin, etc. that try to adhere and adsorb peel off together with the film surface, an anti-adhesion effect can be exhibited. The time for the film surface to dissolve in water is preferably within 12 hours after floating on water, more preferably within 1 hour, and even more preferably within 10 minutes. Also, the lower limit of the time for the film surface to dissolve in water is not particularly limited, but for example, it may be 1 second or more, and from the viewpoint of facilitating reattachment, it is preferably 30 seconds or more, more preferably 50 seconds or more, and particularly preferably 100 seconds or more.

[0019] From the perspective of the effect of decomposition and absorption as an adhesion-preventing material, the silk fibroin film may have a water dissolution rate of the film after being immersed in water and incubated at 25°C for 24 hours (hereinafter, may be simply referred to as the water dissolution rate after 24 hours) of 50% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. Note that the upper limit value of the water dissolution rate after 24 hours of immersion in water is not particularly limited and may be 100% or less. The water dissolution rate after 24 hours is a value measured by the method described in the examples below.

[0020] Also, from the perspective of maintaining the physical barrier function as an adhesion-preventing material, the silk fibroin film may have a water dissolution rate of the film after being immersed in water and incubated at 25°C for 1 hour (hereinafter, may be simply referred to as the water dissolution rate after 1 hour) of 100% or less, preferably 99% or less, and more preferably 98% or less. Note that the lower limit value of the water dissolution rate after 1 hour of immersion in water is not particularly limited. For example, from the perspective of the effect of decomposition and absorption as an adhesion-preventing material, it may be 45% or more, preferably 55% or more, more preferably 65% or more, and even more preferably 75% or more.

[0021] In the measurement of the contact angle of water on the surface of the silk fibroin film, the change in the contact angle may be -5° / sec or less within 5 seconds after dropping 5 μL of ultrapure water droplets on the film surface. Since the surface of the silk fibroin film is water-soluble, when water droplets are dropped on the film surface, the contact angle changes as the film surface dissolves in the water droplets. However, it is preferable that the contact angle of water changes more towards the negative side (larger in absolute value) in a short time. The change in the contact angle is preferably -20 to -5° / sec, more preferably -15 to -5° / sec, and particularly -10 to -5° / sec. The change in the contact angle of water of the silk fibroin film is a value measured by the method described in the examples below. Figure 3 shows the plot data of the time change of the contact angle of water of the silk fibroin film of Example 4 described below. In the initial stage after dropping water droplets on the film surface, the contact angle of water becomes smaller, and then the contact angle of water becomes substantially constant. Taking the time when water droplets are dropped on the film surface as 0 seconds, the plot data at 0 to 5 seconds, which is the initial stage, is linearly approximated by the least squares method, and the slope of the obtained approximate straight line (the straight line in Figure 3) is taken as the change in the contact angle of water.

[0022] The silk fibroin film has a water-soluble surface, but it does not necessarily have to show high wettability immediately after contacting water. For example, even if the contact angle of water is large immediately after dropping water droplets on the film surface, it may have the above-mentioned contact angle change due to its water solubility. For example, in the measurement of the contact angle of water on the surface of the silk fibroin film, the contact angle at the time (0 seconds) when water droplets are dropped on the film surface may be 50 to 90°, preferably 55 to 85°, and more preferably 60 to 80°. The contact angle of water of the silk fibroin film at 0 seconds is a value measured by the method described in the examples below. Taking the time when water droplets are dropped on the film surface as 0 seconds, the measurement data of the contact angle at 0 to 5 seconds is linearly approximated by the least squares method, and the intercept of the obtained straight line is shown.

[0023] The silk fibroin film contains silk fibroin. Silk fibroin mainly contains glycine, alanine, serine, and tyrosine, and is composed of a crystalline part where the molecules are regularly arranged and an amorphous part where the molecules are randomly arranged, and is usually known as a kind of fibrous protein. As long as silk fibroin has such a structure, its raw material is not particularly limited. For example, silk fibroin can be obtained from the silk raw materials described below. In addition, silk fibroin may be chemically modified as long as the effects of the present invention are not inhibited. In this specification, when simply described as "silk fibroin", the definition also includes chemically modified silk fibroin. However, the silk fibroin present on the film surface may be silk fibroin that has not been modified with polyethylene glycol (PEG).

[0024] From the viewpoint of exerting the characteristics of silk fibroin as an anti-adhesion material, the silk fibroin film preferably contains unmodified silk fibroin. In this specification, unmodified silk fibroin means silk fibroin in which a reaction for introducing a functional group, a cross-linking bond, etc. or a grafting reaction has not been performed, and the side chains of the amino acid residues constituting the silk fibroin have not been chemically modified.

[0025] From the viewpoint of improving the water solubility on the film surface, the weight average molecular weight (Mw) of silk fibroin may be 150 kDa or less (for example, 10 to 150 kDa), preferably 120 kDa or less, more preferably 100 kDa or less, and even more preferably 90 kDa or less. The lower limit value of the weight average molecular weight is not particularly limited, but may be, for example, 10 kDa or more, preferably 40 kDa or more, and more preferably 50 kDa or more. The weight average molecular weight of silk fibroin is a value measured by the method described in the examples below.

[0026] In addition, the silk fibroin may have a number average molecular weight (Mn) of 80 kDa or less (for example, 1 to 70 kDa), preferably 60 kDa or less, more preferably 50 kDa or less, still more preferably 40 kDa or less, and even more preferably 30 kDa or less. The lower limit of the number average molecular weight is not particularly limited, and for example, it may be 1 kDa or more, preferably 5 kDa or more. The number average molecular weight of silk fibroin is a value measured by the method described in the examples below.

[0027] The molecular weight distribution of silk fibroin may be 1 to 10 (for example, 1 to 4), preferably 1.2 to 9 (for example, 1.2 to 3.8), more preferably 1.5 to 8 (for example, 1.5 to 3.5), still more preferably 2 to 6, and particularly preferably 3 to 5. The molecular weight distribution indicates the polydispersity (PDI) obtained by dividing the value of the weight average molecular weight (Mw) by the value of the number average molecular weight (Mn).

[0028] The silk fibroin may have a peak top molecular weight (Mp) of 250 kDa or less (for example, 10 to 250 kDa), preferably 150 kDa or less, more preferably 100 kDa or less, even more preferably 80 kDa or less, particularly preferably 60 kDa or less, and still more preferably 50 kDa or less. The lower limit of the peak top molecular weight is not particularly limited, and for example, it may be 5 kDa or more, preferably 10 kDa or more. The peak top molecular weight refers to the molecular weight corresponding to the position of the peak top detected in the chromatogram, and is a value measured by the method described in the examples below.

[0029] Preferred silk fibroin films include, for example, silk fibroin having a weight average molecular weight of 40 to 150 kDa, preferably 50 to 120 kDa, more preferably 55 to 100 kDa, and a molecular weight distribution (PDI) of 1 to 10 (for example, 1 to 4), preferably 1.2 to 9 (for example, 1.2 to 3.8), more preferably 1.5 to 8 (for example, 1.5 to 3.5). Also included are silk fibroin films. The silk fibroin film may have the various characteristics described above.

[0030] The water content of the silk fibroin film may be 30% or less, preferably 1 to 30%, more preferably 3 to 20%, and even more preferably 5 to 15%. The water content of the silk fibroin film is the weight ratio of water contained in the silk fibroin film (the ratio of the weight of water contained to the weight of the silk fibroin film) and is a value measured by the method described in the examples below.

[0031] The silk fibroin film may contain components other than silk fibroin and water (such as additives like colorants), but the content of silk fibroin may be 90% or more based on the solid content weight, preferably 95% or more, more preferably 98% or more, and even more preferably 99.9% or more.

[0032] For example, when a coloring agent is included in the silk fibroin film, it may be advantageous as it makes it easier for medical staff to visually confirm the area where the adhesion prevention material has been applied on the surface of organs, etc.

[0033] The thickness of the silk fibroin film can be appropriately selected according to the clinical field of use, the location of use, etc. of the adhesion prevention material. For example, it can be selected from a wide range of 1 μm to 5 mm. However, considering its use in digestive surgery and obstetrics and gynecology fields, the thickness of the silk fibroin film may be, for example, 1 μm to 1 mm, preferably 5 to 200 μm (for example, 7 to 180 μm), and more preferably 10 to 150 μm.

[0034] The silk fibroin film may have a puncture strength of 0.01 N or more (for example, 0.01 to 5 N), preferably 0.05 N or more, and more preferably 0.1 N or more. When the puncture strength is within the above range, it is easy to handle as an adhesion prevention material. The upper limit of the puncture strength of the silk fibroin film is not particularly limited, and may be, for example, 5 N or less. The puncture strength of the silk fibroin film is a value measured by the method described in the examples below.

[0035] (Adhesion prevention material) The silk fibroin film is used as an adhesion prevention material containing the same. In this specification, the "adhesion prevention material" is for the purpose of suppressing "adhesion", which is a phenomenon in which tissue surfaces or organ surfaces that should originally be separated become bound to other tissue surfaces or organ surfaces and do not separate due to inflammation after surgery, etc. It refers to a biomaterial applied to sites of organ or tissue damage caused by trauma where adhesion may occur, the cut surface of an organ after surgery, and the surrounding tissue surfaces of an organ after surgery.

[0036] In this specification, "trauma" refers to damage to an organ or tissue caused by external forces (mechanical, physical, chemical).

[0037] In this specification, "organ" means an internal organ that has a unique structure and each has a specific function, and examples include the brain, heart, esophagus, stomach, bladder, small intestine, large intestine, liver, kidney, pancreas, spleen, uterus, etc.

[0038] In this specification, "tissue" refers to a unit in which cells and cell-derived substances related to each other combine to form a certain function, and examples include skin, muscle, tendon, bone, joint, ligament, blood vessel, islet, cornea, etc.

[0039] In this specification, the "cut surface" refers to the surface that is exposed inside the body by excising or peeling off a part of a tissue or organ by surgery or the like.

[0040] The adhesion prevention material can be used for preventing adhesion of any tissues and organs where adhesion may occur in surgeries in clinical fields such as digestive surgery, cardiac surgery, orthopedic surgery, and obstetrics and gynecology, and can be preferably used in surgeries in digestive surgery and obstetrics and gynecology in particular. For example, it can be used by attaching it to damaged sites of the intestine or peritoneum, damaged sites of the uterus or adnexa, etc. Further, it can be used not only in human surgeries but also in surgeries of animals (non-humans) such as pets. For example, non-human animals include non-human mammals, and examples of non-human mammals include apes, other primates, mice, rats, hamsters, guinea pigs, horses, cows, pigs, sheep, goats, dogs, cats, rabbits, etc. In addition, in this specification, adhesion prevention includes not only completely preventing the occurrence of adhesion but also reducing the degree, range, and frequency of adhesion.

[0041] The adhesion prevention material may consist substantially only of the above-mentioned silk fibroin film, or the adhesion prevention material may be a laminate including a support necessary for maintaining the shape of the film.

[0042] The adhesion prevention material may preferably be attached to the cut surface of the postoperative organ or tissue and the surface of the surrounding tissue of the postoperative organ, etc. in the finishing process of a surgical operation such as laparotomy.

[0043] (Method for manufacturing silk fibroin film) The method for manufacturing a silk fibroin film may include an alkali treatment step of dissolving refined silk fibroin in a solvent and reacting it with an alkali treatment agent, and a film-forming step of forming a film from the alkali-treated silk fibroin.

[0044] As silk fibroin raw materials, cocoons and raw silk produced by insects (Lepidoptera insects such as silkworms, wild silkworms, and tussah silkworms, and Hymenoptera insects such as wasps and honeybees, i.e., silk-producing insects) or spiders can be used, and there is no particular limitation as long as it is a silk raw material containing fibroin and sericin. By refining the silk raw material, silk fibroin from which sericin has been removed can be obtained. Also, silk fibroin can be obtained from silk glands. Note that refining can be performed by known methods. For example, methods such as swelling and removing sericin using an alkaline refining agent such as sodium carbonate, sodium silicate, or sodium phosphate, decomposing and removing sericin using a sericin-degrading enzyme, or putrefying and removing sericin can be mentioned. From the viewpoint of simplicity in setting conditions, refining using an alkaline refining agent is preferred. In refining, the main purpose is to remove sericin, and from the viewpoint of suppressing the decomposition of silk fibroin, the refining time using an alkaline refining agent varies depending on the type of silk raw material and alkaline refining agent, etc., but it may be 5 to 60 minutes, preferably 10 to 50 minutes, more preferably 15 to 45 minutes.

[0045] Next, by subjecting the refined silk fibroin to an alkali treatment, the silk fibroin can be hydrolyzed to improve its water solubility. From the viewpoint of molecular weight adjustment, it is preferable to perform the hydrolysis of silk fibroin in the alkali treatment step. The alkali treatment can be carried out by dissolving silk fibroin in a solvent and reacting it with an alkali treatment agent (for example, an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, or ammonia). For example, silk fibroin may be dissolved in a neutral salt solution containing a neutral salt such as lithium bromide or calcium chloride. Silk fibroin can be dissolved using a mixed solution containing a neutral salt and an alkali treatment agent and the alkali treatment can be carried out, or the alkali treatment agent can be added after dissolving silk fibroin in a neutral salt solution to carry out the alkali treatment.

[0046] The concentration in the solution of the neutral salt may be, for example, 3 to 15 M, preferably 4 to 14 M, more preferably 3 to 12 M. Also, the concentration in the solution of the alkali metal hydroxide may be, for example, 0.05 to 2 M, preferably 0.08 to 1.5 M. The concentration in the solution of ammonia may be, for example, 0.05 to 1.5 M, preferably 0.1 to 1 M.

[0047] By performing the alkali treatment under relatively mild conditions, the weight-average molecular weight can be effectively reduced. The temperature of the alkali treatment at this time may be 40°C or lower (for example, 5 to 40°C), preferably 10°C to 40°C, more preferably 15°C to 35°C. Also, the time for which the alkali treatment is performed may be a time exceeding 3 hours, preferably 4 hours or more, more preferably 10 hours or more, still more preferably 15 hours or more. The upper limit of the time for which the alkali treatment is performed is not particularly limited, but may be, for example, 48 hours or less. In this specification, the time for which the alkali treatment is performed means the time during which the silk fibroin is in contact with the alkali treatment agent. For example, when using a mixed solution containing a neutral salt and an alkali treatment agent, it indicates the time after adding the mixed solution to the silk fibroin (or adding the silk fibroin to the mixed solution), and when adding the alkali treatment agent after dissolving the silk fibroin in the neutral salt solution, it indicates the time after adding the alkali treatment agent.

[0048] The silk fibroin obtained by the alkali treatment may have various molecular weights (Mw, Mn, PDI, and Mp) within the above-mentioned ranges. For example, the method for producing a silk fibroin film may include a step of forming a film from silk fibroin having a weight-average molecular weight (Mw) of 150 kDa or less (preferably 120 kDa or less, more preferably 100 kDa or less, still more preferably 80 kDa or less).

[0049] After the alkali treatment, removal of neutral salts and alkali treatment agents may be performed by known methods such as dialysis and ultrafiltration. Also, from the viewpoint of using as a biological material, it is preferable to purify or sterilize silk fibroin in order to remove impurities other than neutral salts and alkali treatment agents before film formation (forming a film). Regarding the sterilization treatment, known methods such as autoclave sterilization and filter sterilization can be adopted, but autoclave sterilization is preferably performed.

[0050] A silk fibroin film can be obtained by forming a film using an aqueous solution of silk fibroin (preferably an aqueous solution of silk fibroin after purification and / or sterilization treatment). The concentration of the aqueous solution of silk fibroin may be adjusted according to the desired film thickness, physical properties, etc. For example, the concentration of the aqueous solution of silk fibroin may be 0.1 to 10% (w / v), preferably 0.5 to 8% (w / v), more preferably 1 to 6% (w / v).

[0051] Silk fibroin can be formed into a film by known film-forming methods such as the casting method and the coating method. For example, a silk fibroin film can be obtained by casting or coating an aqueous solution of silk fibroin on a substrate and then drying it.

Example

[0052] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto. In the following examples and comparative examples, various physical properties were measured by the following methods.

[0053] [Molecular weight] 500 μL of a silk fibroin aqueous solution with a concentration adjusted to 1% (w / v) was used as the measurement sample. Using this measurement sample, measurements were performed by gel filtration chromatography (GFC) with the following measurement apparatus and conditions. The weight-average molecular weight (Mw), number-average molecular weight (Mn), molecular weight distribution (PDI), and peak-top molecular weight (Mp) of silk fibroin were calculated using molecular weight markers included in Low Molecular Weight (LMW) Gel Filtration Calibration Kits and High Molecular Weight (HMW) Gel Filtration Calibration Kits (both manufactured by Cytiva) as standard substances. · Apparatus: GFC apparatus "AKTAgo" manufactured by Cytiva · Separation column: "HiLoad 16 / 600 Superdex 200pg" manufactured by Cytiva · Mobile phase: 20 mM sodium phosphate buffer containing 500 mM sodium chloride (pH: 7.4) · Flow rate: 1 mL / min · Temperature: Below room temperature (24.0 °C). When simply referred to as room temperature, 24.0 °C is used as the representative value.

[0054] [Water solubility of the film surface] A silk fibroin film stored in an indoor environment (temperature 22.4 - 29.1 °C, humidity 32 - 64 RH) for more than one night was marked with a water-resistant permanent marker (manufactured by Zebra Co., Ltd., product name "Macky 0.25mm", black) to draw a square with a side length of 1 cm and its diagonal. The thickness of the line was approximately 1 mm. The film was cut out along the lines of the square to obtain sample pieces with a length of 1 cm and a width of 1 cm. At room temperature, the sample pieces were floated on ultrapure water (24 - 28 °C) in a petri dish with the surface marked with the permanent marker facing up. Starting from the time of floating, the time when all six straight lines (four sides and two diagonals) marked with the permanent marker were completely broken was measured. The measurement was performed on three sample pieces, and the average time when all six straight lines marked with the permanent marker were completely broken was calculated as the time when the film surface dissolved in water. If the six straight lines marked with the permanent marker were not broken even after more than one day, it was determined that the film surface did not dissolve in water.

[0055] [Water solubility of the film] A silk fibroin film stored in an indoor environment for more than one night was cut out to obtain sample pieces with a length of 0.5 cm and a width of 0.5 cm, and the weight of these sample pieces was measured (this weight is referred to as the "weight of the film before dissolution"). The sample pieces were immersed in 1 mL of ultrapure water in a tube and incubated at 25 °C for 1 hour or 24 hours. Using a micropipette (manufactured by Eppendorf, "Reference 2, 4920000.083"), the remaining undissolved film was adsorbed to the tip of the pipette tip and transferred into a 2 mL tube whose weight had been measured in advance (this weight is referred to as the "tube weight"). If the film could not be confirmed at this point, the water dissolution rate was set to 100%. Then, the tube was placed in a constant temperature dryer (manufactured by AS ONE, "KM-600V") set at 50 °C and incubated overnight to evaporate the moisture retained in the remaining undissolved film, and the combined weight of the tube and the dried silk fibroin film was measured (this weight is referred to as the "tube + dried film weight"). Then, the water dissolution rate (%) of the silk fibroin film was calculated according to the following formula. Water solubility rate (%) = 100 × [film weight before dissolution (mg) - {tube weight + dry film weight (mg) - tube weight (mg)}] / film weight before dissolution (mg)

[0056] [Contact angle of water] Using a contact angle meter (manufactured by Eximer, "SImage AUTO100"), at room temperature, referring to the sessile drop method of JIS R 3257:1999, about 5 μL of ultrapure water droplets were dropped onto the film surface of the silk fibroin film, and the time change of the contact angle of water measured by the tangent method was measured (measurement interval: 1 / 15 second). The time point when the water droplets were dropped was set as 0 second, and the measurement was carried out until 30 seconds. The measurement data of the contact angle from 0 to 5 seconds were linearly approximated by the least squares method, and the slope and intercept of the obtained straight line were calculated. The measurement was carried out at three locations on the silk fibroin film, and the average value of the slope of the approximate straight line was calculated as the contact angle change (° / sec), and the average value of the intercept was calculated as the contact angle (°) when the water droplets were dropped onto the film surface at 0 second.

[0057] [Water content] Using a thermogravimetric differential thermal analyzer (TG-DTA; manufactured by Rigaku, "Thermoplus TG8120"), the water content (%) of the silk fibroin film was measured. 2.5 - 8.5 mg of the silk fibroin film sample stored in the indoor environment for more than one night was placed in an aluminum pan, nitrogen was flowed at a flow rate of 200 ml / min, and the weight change when the temperature was raised from 30°C at a rate of 10°C / min was measured. The weight of the silk fibroin film at the start of the measurement (30°C) (referred to as "sample weight") and the weight decreased during the temperature increase process from 30 to 180°C (referred to as "water weight") were measured. Then, the water content of the silk fibroin film was measured according to the following formula. In the same way, the measurement was carried out for each of the three individually prepared samples, and the average value of these was calculated as the water content (%) of the silk fibroin film. Water content (%) = 100 × water weight (mg) / sample weight (mg)

[0058] [Thickness] The thickness (μm) of the silk fibroin film was measured using a digital micrometer (manufactured by Nikon Corporation, "MFC-101A"). Zero point setting was performed with the measuring head lowered to the pedestal. Then, a silk fibroin film sample stored in the indoor environment for more than one night was placed on the pedestal, and the measuring head was lowered to the sample to measure the thickness of the silk fibroin film. In the same manner, each of the three individually prepared samples was measured, and the average value of these was calculated as the thickness (μm) of the silk fibroin film.

[0059] [Puncturing strength] Using a silk fibroin film sample stored in the indoor environment for more than one night, the puncturing strength (N) was measured with the following measuring apparatus and conditions. In the same manner, each of the three individually prepared samples was measured, and the average value of these was calculated as the puncturing strength (N) of the silk fibroin film. · Apparatus: Small bench-top testing machine (manufactured by Shimadzu Corporation, "EZTest") · Load cell capacity: 10 N · Puncturing jig: Diameter φ3 mm, tip angle 60° · Puncturing speed: 50 mm / min · Temperature: Room temperature

[0060] [Example 1] Silkworm cocoons were shredded, and these cocoons were scoured in boiling 0.02 M aqueous sodium carbonate solution for 30 minutes to obtain silk fibroin.

[0061] 3 g of the scoured 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 perform an alkali treatment, thereby obtaining a silk fibroin aqueous solution.

[0062] The obtained aqueous silk fibroin solution was dialyzed in deionized water using a dialysis membrane (molecular weight cut-off: 6 - 8 kDa). One dialysis was carried out at room temperature for 6 to 12 hours, and this was repeated 6 times (bath ratio: 160 times). Thereby, lithium bromide and sodium hydroxide contained in the aqueous silk fibroin solution were removed.

[0063] After removing lithium bromide and sodium hydroxide, the aqueous silk fibroin solution was concentrated by air-drying at room temperature. The concentration by air-drying was carried out until the volume of the aqueous silk fibroin solution became 1 / 5 to 1 / 3.

[0064] The concentrated aqueous silk fibroin solution was subjected to autoclave sterilization treatment using an autoclave device (manufactured by Tommy Seiko Co., Ltd., "LBS-245"). Autoclave sterilization treatment at 121 °C for 20 minutes was carried out twice. All subsequent treatments were carried out in a sterilized state.

[0065] The aqueous silk fibroin solution after autoclave sterilization was centrifuged (40000×g, 20 °C, 30 minutes) using a centrifuge (manufactured by Beckman Coulter, "Avanti20I"), and the precipitated insoluble matter was removed.

[0066] The aqueous silk fibroin solution after removing the insoluble matter was freeze-dried and adjusted to a concentration of 1% (w / v).

[0067] 3 mL of the aqueous silk fibroin solution adjusted to a concentration of 1% (w / v) was spread on a polystyrene petri dish (inner diameter 5.4 cm) and left standing at room temperature for 2 days or more to evaporate the moisture, thereby obtaining a silk fibroin film.

[0068] [Example 2] After freeze-drying, a silk fibroin film was obtained in the same manner as in Example 1, except that the aqueous silk fibroin solution was adjusted to a concentration of 5% (w / v) and 3 mL of the aqueous silk fibroin solution was spread on a polystyrene petri dish.

[0069] [Example 3] After freeze-drying, a silk fibroin aqueous solution was adjusted to a concentration of 4% (w / v), and a silk fibroin film was obtained in the same manner as in Example 1 except that 6 mL of the silk fibroin aqueous solution was spread on a polystyrene petri dish.

[0070] [Example 4] After freeze-drying, a silk fibroin aqueous solution was adjusted to a concentration of 5% (w / v), and a silk fibroin film was obtained in the same manner as in Example 1 except that 6 mL of the silk fibroin aqueous solution was spread on a polystyrene petri dish.

[0071] [Example 5] After freeze-drying, a silk fibroin aqueous solution was adjusted to a concentration of 5% (w / v), and a silk fibroin film was obtained in the same manner as in Example 1 except that 10 mL of the silk fibroin aqueous solution was spread on a polystyrene petri dish.

[0072] [Comparative Example 1] A silk fibroin film was obtained in the same manner as in Example 1 except that 3 g of refined silk fibroin was immersed in a 9 M lithium bromide aqueous solution (50 mL) not containing NaOH.

[0073] [Comparative Example 2] A silk fibroin film was obtained in the same manner as in Example 2 except that 3 g of refined silk fibroin was immersed in a 9 M lithium bromide aqueous solution (50 mL) not containing NaOH.

[0074] [Comparative Example 3] A silk fibroin film was obtained in the same manner as in Example 3 except that 3 g of refined silk fibroin was immersed in a 9 M lithium bromide aqueous solution (50 mL) not containing NaOH.

[0075] [Comparative Example 4] A silk fibroin film was obtained in the same manner as in Example 4, except that 3 g of refined silk fibroin was immersed in a 9 M lithium bromide aqueous solution (50 mL) not containing NaOH.

[0076] [Comparative Example 5] A silk fibroin film was obtained in the same manner as in Example 5, except that 3 g of refined silk fibroin was immersed in a 9 M lithium bromide aqueous solution (50 mL) not containing NaOH.

[0077] Table 1 shows the results of various measurements for each example and comparative example.

[0078]

Table 1

[0079] As shown in Table 1, in Examples 1 to 5 where alkali treatment was performed, the film surface dissolved in water in a short time, whereas in Comparative Examples 1 to 5, the film surface did not dissolve in water. Fig. 1 is a photograph showing the state of a sample piece of the silk fibroin film of Example 4 immediately after floating it on water and after about 3 minutes. Fig. 2 is a photograph showing the state of a sample piece of the silk fibroin film of Comparative Example 4 immediately after floating it on water and after about 8 minutes. In Comparative Examples 1 to 5, the six straight lines drawn with an oil-based pen were not broken even after 7 days or more.

[0080] Also, there is no significant difference in the contact angle at 0 seconds between Examples 1 to 5 and Comparative Examples 1 to 5, but the change in contact angle is smaller in Examples 1 to 5 than in Comparative Examples 1 to 5. This is considered to be due to the difference in water solubility of the film surface. In Examples 1 to 5 where alkali treatment was performed, the molecular weight could be reduced compared to Comparative Examples 1 to 5, and it is considered that the reduction in the molecular weight of silk fibroin also contributes to the water solubility of the film surface.

[0081] [Evaluation of adhesion prevention effect in rat cecal abrasion model] Rats (SD, 7-week-old males, purchased from Japan SLC) were anesthetized by inhalation of 2-2.5% isoflurane, and laparotomy was performed to expose the cecum of the rats. A scratch wound of approximately 1 cm × 2 cm was formed by rubbing the surface of the exposed cecum 50 times with a dry gauze. Subsequently, a laceration wound of approximately 1 cm × 1 cm was formed by pinching the surface of the abdominal wall with forceps. Then, the cecum was returned into the abdominal cavity, and the positions of the wounds on the cecal surface and the abdominal wall surface were aligned. And a film-shaped adhesion prevention material (2 cm × 2 cm) was attached to each of the wounds on the cecal surface and the abdominal wall surface, and the wound was closed. As the film-shaped adhesion prevention materials, the silk fibroin film of Example 4, the silk fibroin film of Comparative Example 4, Seprafilm (manufactured by Baxter), and Tenari F (manufactured by Gunze Co., Ltd.) were used. Four rats were prepared for each of the rats transplanted with the silk fibroin film of Example 4, the silk fibroin film of Comparative Example 4, and Tenari F, and five rats were prepared for the rats transplanted with Seprafilm. Also, four rats were prepared by intraperitoneally administering 3 mL of physiological saline. After intramuscular injection of 0.6 μg of buprenorphine to each rat with the wound closed by applying the adhesion prevention material, the rats were awakened. And two weeks after the above operation, the rats were euthanized, and laparotomy was performed again to evaluate the adhesions.

[0082] The evaluation of adhesions was performed by visual observation, and the score determination was carried out according to the following six-stage evaluation of adhesion scores from 0 to 5. 0: No adhesion 1: Thin membranous adhesion 2: Multiple thin membranous adhesions 3: Thick adhesion with patches 4: Thick adhesion with surface attachment 5: Adhesion with very thick and accompanied by new blood vessels, or adhesion with multiple surface attachments

[0083] After score determination, the portion of the cecum where abrasions were formed and the portion of the abdominal wall where contusions were formed were collected and washed with physiological saline. The collected tissues were immersed in 10% neutral buffered formalin solution (manufactured by Fujifilm Wako Pure Chemical Corporation) and fixed by allowing them to stand at room temperature for 3 days or more. The fixed tissues were embedded in OCT compound (manufactured by Sakura Finetek Japan Co., Ltd.) and frozen at -20°C. Thereafter, using a cryostat (manufactured by Leica, "CM3050S"), the tissues were thinly sliced to a thickness of 10 - 12 μm and adhered to APS-coated slide glasses (manufactured by Matsunami Glass Ind., Ltd.). After drying overnight at room temperature, they were immersed in 95% (v / v) ethanol (manufactured by Fujifilm Wako Pure Chemical Corporation) at 4°C for 10 minutes and then dried at room temperature. Thereafter, at room temperature, they were immersed in running water for 2 minutes, Mayer's hematoxylin solution (manufactured by Fujifilm Wako Pure Chemical Corporation) for 10 minutes, running water for 15 minutes, 1% eosin Y solution (manufactured by Fujifilm Wako Pure Chemical Corporation) for 2 minutes, 70% (v / v) ethanol for 15 seconds, 99.5% ethanol for 15 seconds, xylene (manufactured by Fujifilm Wako Pure Chemical Corporation) for 1 minute, and xylene for 2 minutes in sequence, and hematoxylin-eosin staining was performed. The stained tissue sections were sealed in a mounting medium (manufactured by Merck, "Entellan New"), and bright-field images were observed using a microscope (manufactured by Keyence Corporation, "BZX710").

[0084] Figure 4 is a box plot showing the adhesion score determination results for each anti-adhesion material. Specifically, for Saline, the adhesion scores of 4 rats were 0, 1, 4, and 4, and the average adhesion score was 2.25. For the silk fibroin film (HMW) in Comparative Example 4, the adhesion scores of 4 rats were 2, 3, 5, and 0, and the average adhesion score was 2.50. For the silk fibroin film (LMW) in Example 4, the adhesion scores of 4 rats were 0, 0, 0, and 4, and the average adhesion score was 1.00. For Seprafilm, 2 out of 6 rats died within 3 days after transplantation, and the adhesion scores of the remaining 4 rats were 0, 0, 5, and 0, and the average adhesion score was 1.25. For Tenaleaf, 1 out of 5 rats died within 3 days after transplantation, and the adhesion scores of the remaining 3 rats were 0, 3, 3, and 0, and the average adhesion score was 1.50.

[0085] In the adhesion score evaluation results of the anti-adhesion materials, the silk fibroin film (LMW) in Example 4 showed a better average adhesion score than the silk fibroin film (HMW) in Comparative Example 4, indicating an anti-adhesion effect. Figure 5 is a photograph showing the cecum and abdominal wall (adhesion score: 0) 2 weeks after transplantation of the silk fibroin film in Example 4. When the silk fibroin film in Example 4 was applied, no film residue was confirmed 2 weeks later, indicating that it was decomposed and absorbed by the living body. Figure 6 is a photograph showing the cecum and abdominal wall (adhesion score: 5) 2 weeks after transplantation of the silk fibroin film in Comparative Example 4, and Figure 7 is a photograph showing the adhesion part. When the silk fibroin film in Comparative Example 4 was applied, a lump formed at the adhesion part, and film residue was confirmed inside, indicating that it was not decomposed and absorbed by the living body (Figure 7).

[0086] Also, since the silk fibroin film in Comparative Example 4 had a lower average adhesion score than Saline as a reference example, it is shown that simply containing silk fibroin cannot exert an anti-adhesion effect.

[0087] Furthermore, the silk fibroin film of Example 4 showed better average adhesion scores than Seprafilm and Tenaleaf, which are commercially available film-shaped anti-adhesion materials. Also, although the cause is unknown, among the rats to which Seprafilm and Tenaleaf were applied, there were rats that died within 3 days after transplantation, but in the silk fibroin film of Example 4, there were no dead rats, suggesting that it is also excellent in safety.

[0088] Figures 8 and 9 are stained photographs showing the states of the abdominal wall (AW) and cecum (C) 2 weeks after applying the physiological saline (Saline) of Comparative Example 1 and the silk fibroin film (HMW) of Comparative Example 4 to a laceration, respectively. As shown in Figures 8 and 9, the abdominal wall and the cecum were adhered to each other and integrated.

[0089] On the other hand, Figure 10 is a stained photograph showing the states of the abdominal wall (AW) and cecum (C) 2 weeks after applying the silk fibroin film (LMW) of Example 4 to a laceration. As shown in Figure 10, the abdominal wall and the cecum were separated from each other, and no adhered portions were seen.

[0090] Also, Figures 11 and 12 are stained photographs showing the states of the abdominal wall (AW) and cecum (C) 2 weeks after applying commercially available Seprafilm and Tenaleaf to a laceration, respectively. As shown in Figures 11 and 12, even when using commercially available anti-adhesion materials, the abdominal wall and the cecum were separated from each other, and no adhered portions were seen.

[0091] Therefore, it was histologically confirmed that the silk fibroin film according to the present invention exhibits an anti-adhesion effect equal to or better than that of commercially available film-shaped anti-adhesion materials. On the other hand, although silk fibroin was used, the silk fibroin film of Comparative Example 4 could not exhibit an anti-adhesion effect.

[0092] As described above, based on the evaluation results in the in vivo test, it was confirmed that the silk fibroin film according to the present invention exhibits an adhesion prevention effect equal to or better than that of commercially available film-shaped adhesion prevention materials, and it was also confirmed that the silk fibroin film is biodegraded and absorbed by the living body within two weeks after transplantation, indicating that it is useful as an adhesion prevention material.

Industrial Applicability

[0093] The silk fibroin film of the present invention can be used as an adhesion prevention material to prevent adhesion of any tissues and organs that may develop adhesion in various surgeries in clinical fields such as digestive surgery, cardiac surgery, orthopedic surgery, and obstetrics and gynecology (especially digestive surgery and obstetrics and gynecology).

[0094] As described above, the preferred embodiments of the present invention have been described. However, those skilled in the art will easily assume various changes and modifications within the obvious scope upon seeing this specification. Therefore, such changes and modifications are construed as being within the scope of the invention defined by the claims.

Claims

1. The silk fibroin film is an adhesion preventing material, in which at least the surface of the silk fibroin film dissolves in water within 24 hours after floating on water, and the weight-average molecular weight of the silk fibroin is 150 kDa or less.

2. A silk fibroin film as described in claim 1, wherein, in measuring the contact angle of water on the film surface, the change in contact angle from 5 seconds after a 5 μL droplet of ultrapure water is dropped on the film surface is -5° / sec or less, and the weight-average molecular weight of the silk fibroin is 150 kDa or less.

3. 2. The silk fibroin film according to claim 1, wherein the water solubility of the film is 50% or more after being immersed in water and then incubated at 25° C. for 24 hours.

4. 2. The silk fibroin film according to claim 1, wherein the water solubility of the film after immersion in water and incubation at 25° C. for 1 hour is 99% or less.

5. 2. The silk fibroin film according to claim 1, wherein the molecular weight distribution (PDI) of the silk fibroin is 1-10.

6. 2. The silk fibroin film according to claim 1, wherein the silk fibroin present on the film surface is silk fibroin that is not modified with polyethylene glycol.

7. 2. The silk fibroin film according to claim 1, having a moisture content of 30% or less.

8. The weight average molecular weight is 40 to 150 kDa, and the molecular weight distribution (PDI) is 1 to 1 A silk fibroin film for use as an adhesion prevention material, comprising silk fibroin having a viscosity of 0.

9. An adhesion preventing material comprising the silk fibroin film according to any one of claims 1 to 8.

10. A method for producing a silk fibroin film for use as an anti-adhesion material, comprising: an alkali treatment step of dissolving refined silk fibroin in a solvent, reacting the silk fibroin with an alkali treatment agent, and making the weight-average molecular weight of the alkali-treated silk fibroin 150 kDa or less; and a step of forming the alkali-treated silk fibroin into a film.

11. The method for producing a silk fibroin film according to claim 10, wherein the molecular weight distribution (PDI) of the alkali-treated silk fibroin is 1 to 10.

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