Silk fibroin film and Anti-adhesion material containing same

Abstract

Provided is a silk fibroin film suitable for use as an anti-adhesion material. When the silk fibroin film is floated in water, at least the film surface of the silk fibroin may dissolve in water within 24 hours of floating. Further, when the contact angle of water on the film surface of the silk fibroin film is measured, the contact angle change from when a 5 μL drop of ultrapure water is dropped onto the film surface until 5 seconds later may be -5° / sec or less. Additionally, in the silk fibroin film, the weight average molecular weight of the silk fibroin may be150 kDa or less.

Description

Silk fibroin film and anti-adhesion material containing the same Related Applications

[0001] This application claims priority from Japanese Patent Application No. 2023-139250, filed on August 29, 2023, the entire contents of which are incorporated herein by reference.

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

[0003] In clinical fields such as gastrointestinal surgery, cardiac surgery, orthopedics, and obstetrics and gynecology, adhesions can form after surgery, forming bonds between tissues or organs that should be separate. Adhesion formation can sometimes require additional surgery to remove the adhesions, which requires a great deal of time and effort, and can even cause additional damage during the removal process. It can also lead to complications such as intestinal obstruction and infertility.

[0004] Various methods have been investigated to prevent adhesion between tissues and organs, and anti-adhesion materials have been developed that physically prevent contact between tissues or organs by applying them to the surface of tissues or organs. Anti-adhesion materials have been developed in the form of films, sprays, etc., and film-type anti-adhesion materials are used clinically in the fields of gastrointestinal surgery and obstetrics and gynecology. Examples of film-type anti-adhesion materials include Seprafilm (registered trademark), which contains hyaluronic acid and carboxymethylcellulose, and Tenaleaf (registered trademark), which contains gelatin. However, these anti-adhesion materials cannot be used in patients with a history of hypersensitivity to the components contained in them, so there is a need to expand the options available in clinical settings.

[0005] On the other hand, in recent years, silk fibroin has attracted attention as a biomaterial due to its excellent biocompatibility. For example, Patent Document 1 (JP 2016-517443 A) 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 have a molecular weight of more than 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 having a lower limit of about 3.5 kDa or more and an upper limit of about 120 kDa or less. The low-molecular-weight silk fibroin composition is described as being usable in the form of a film and for medical purposes.

[0006] Furthermore, Patent Document 2 (U.S. Pat. No. 9,427,499) describes a method for preparing a PEG-coated membrane having a first surface and a second surface, the first surface being coated with PEG at a concentration of about 75 to about 750 μg PEG / cm ２ A silk fibroin matrix selectively modified with poly(ethylene glycol) (PEG) at the density of the silk fibroin matrix has been disclosed, and it has been stated that it can be used as an adhesion barrier.

[0007] Patent Publication No. 2016-517443 U.S. Patent No. 9,427,499

[0008] However, although Patent Document 1 describes that low molecular weight silk fibroin can be made more water soluble, it does not describe the use of low molecular weight silk fibroin compositions as adhesion prevention materials.

[0009] Furthermore, Patent Document 2 describes that modifying the surface of a silk fibroin matrix with PEG suppresses the adhesiveness and proliferation-promoting properties of mesenchymal stem cells and fibroblasts, respectively, but it is the surface PEG that contributes to these suppressions, and does not reveal any effects due to the structure of the silk fibroin itself.Furthermore, the anti-adhesion effect in vivo was not evaluated.

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

[0011] As a result of extensive research to achieve the above-mentioned objective, the inventors of the present invention discovered that a silk fibroin film whose surface dissolves in water in a short period of time has excellent anti-adhesion effects, leading to the completion of the present invention.

[0012] That is, the present invention can be configured in the following aspects. [Aspect 1] A silk fibroin film for use as an anti-adhesion material, wherein at least the silk fibroin film surface dissolves in water within 24 hours (preferably within 12 hours, more preferably within 1 hour, and even more preferably within 10 minutes) after being floated on water. [Aspect 2] The silk fibroin film according to Aspect 1, wherein, in measurement of the water contact angle on the film surface, the change in contact angle from a 5 μL droplet of ultrapure water dropped on the film surface until 5 seconds later is −5° / sec or less (preferably −20 to −5° / sec, more preferably −15 to −5° / sec, and particularly −10 to −5° / sec). [Aspect 3] A silk fibroin film, in which, in measurement of the water contact angle on the film surface, the change in contact angle 5 seconds after a 5 μL droplet of ultrapure water is dropped on the film surface is -5° / sec or less (preferably -20 to -5° / sec, more preferably -15 to -5° / sec, and particularly -10 to -5° / sec). [Aspect 4] A silk fibroin film according to any one of Aspects 1 to 3, wherein the water solubility of the film after immersion in water and incubation at 25°C for 24 hours is 50% or more (preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more). [Aspect 5] A silk fibroin film according to any one of Aspects 1 to 4, wherein the water solubility of the film after immersion in water and incubation at 25°C for 1 hour is 100% or less (preferably 99% or less, more preferably 98% or less). [Aspect 6] The 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, and even more preferably 90 kDa or less). [Aspect 7] The silk fibroin film according to any one of Aspects 1 to 6, wherein the silk fibroin present on the film surface is silk fibroin that is not modified with polyethylene glycol.[Aspect 8] The silk fibroin film according to any one of Aspects 1 to 7, having a water content of 30% or less (preferably 1 to 30%, more preferably 3 to 20%, and even more preferably 5 to 15%). [Aspect 9] A silk fibroin film comprising silk fibroin having a weight-average molecular weight of 40 to 150 kDa (preferably 50 to 120 kDa, and more preferably 55 to 100 kDa) and a polydispersity index (PDI) of 1 to 10 (preferably 1.2 to 9, and more preferably 1.5 to 8). [Aspect 10] An adhesion preventing material comprising 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 preventing material, comprising: an alkali treatment step of dissolving refined silk fibroin in a solvent and reacting the resulting silk fibroin with an alkali treating agent; and a 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, and even more preferably 80 kDa or less). [Aspect 13] A method for preventing adhesions using the adhesion preventing material according to Aspect 10, comprising a step of applying the adhesion preventing material to at least one of the excised surface of a postoperative organ and the surface of tissue surrounding the postoperative organ.

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

[0014] Furthermore, when the term "about" is used, the range of ±10% of the numerical value may be included, and preferably ±5%.

[0015] It should be noted that any combination of at least two elements disclosed in the claims and / or the specification and / or the drawings is included in the present invention, and in particular any combination of two or more of the claims set forth in the claims is included in the present invention.

[0016] The silk fibroin film of the present invention can prevent adhesion between tissues or organs after surgery, and because it contains components different from conventional film-type adhesion barriers, it can be used as an adhesion barrier of choice depending on the patient's hypersensitivity.

[0017] Photographs showing the state of a sample piece of the silk fibroin film of Example 4 immediately after floating in water and about 3 minutes later. Photographs showing the state of a sample piece of the silk fibroin film of Comparative Example 4 immediately after floating in water and about 8 minutes later. Graphs showing plot data of the change in water contact angle with time for the silk fibroin film of Example 4 and an approximation line for the data from 0 to 5 seconds. Box plots showing adhesion score results for the application of saline, the silk fibroin film of Comparative Example 4 (HMW), the silk fibroin film of Example 4 (LMW), Seprafilm, and Tenaleaf. Photographs showing the cecum and abdominal wall two weeks after implantation of the silk fibroin film of Example 4 (adhesion score: 0). Photographs showing the cecum and abdominal wall two weeks after implantation of the silk fibroin film of Comparative Example 4 (adhesion score: 5). Photographs showing adhesions in the cecum and abdominal wall two weeks after implantation of the silk fibroin film of Comparative Example 4. Stained photographs showing the condition of the abdominal wall and cecum two weeks after application of saline to the laceration wound of Comparative Example 1. The scale bar indicates 1 mm. Stained photographs showing the condition of the abdominal wall and cecum two weeks after application of the silk fibroin film (HMW) of Comparative Example 4 to the laceration wound. The scale bar indicates 1 mm. Stained photographs showing the condition of the abdominal wall and cecum two weeks after application of the silk fibroin film (LMW) of Example 4 to the laceration wound. The scale bar indicates 1 mm. Stained photographs showing the condition of the abdominal wall and cecum two weeks after application of Seprafilm to the laceration wound. The scale bar indicates 1 mm. Stained photographs showing the condition of the abdominal wall and cecum two weeks after application of Tenaleaf to the laceration wound. The scale bar indicates 1 mm.

[0018] (Silk Fibroin Film) A silk fibroin film dissolves in water within 24 hours after being floated on water, at least at the silk fibroin film surface. In this specification, the dissolution of a film surface in water can be confirmed by observing, using the method described in the Examples below, whether a square with a side length of 1 cm drawn with a black oil paint pen on the surface of the film and all six diagonal lines (1 mm line width) are broken. That is, when lines are drawn with an oil paint pen on the first side of a film and the film is floated on water with the first side facing up and the second side facing down, the film surface (first side) dissolves in water, causing each of the six lines drawn with an oil paint pen on the first side to peel off from the film and separate from each other, resulting in the lines being broken by cracks. Therefore, the water solubility of a film surface can be evaluated by floating a film with lines drawn with an oil paint pen on its surface on water and then confirming that the lines peel off along with the film surface, ultimately breaking all six lines. By confirming that the upper surface of the film dissolves when the film is floated on water, it can be assumed that the lower surface (second surface) of the film also dissolves. It is sufficient that at least the film surface dissolves in water within 24 hours; portions 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, the high water solubility of the silk fibroin film surface prevents prolonged contact between the film surface and cells, fibrin, etc., thereby inhibiting adhesion of cells, fibrin, etc., or by causing cells, fibrin, etc. that are intended to adhere or adsorb to peel off along with the film surface, thereby achieving an anti-adhesion effect. The time period 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. The lower limit of the time period for the film surface to dissolve in water is not particularly limited, but may be, for example, 1 second or more. From the viewpoint of ease of reapplication, it may be preferably 30 seconds or more, more preferably 50 seconds or more, and particularly preferably 100 seconds or more.

[0019] From the viewpoint of the effect of decomposition and absorption as an anti-adhesion material, the water solubility of the silk fibroin film after immersion in water and incubation at 25°C for 24 hours (hereinafter sometimes simply referred to as the water solubility after 24 hours) may be 50% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. The upper limit of the water solubility after 24 hours after immersion in water is not particularly limited, and may be 100% or less. The water solubility after 24 hours is a value measured by the method described in the Examples below.

[0020] Furthermore, from the viewpoint of maintaining the physical barrier function as an anti-adhesion material, the water solubility of the silk fibroin film after immersion in water and incubation at 25°C for 1 hour (hereinafter sometimes simply referred to as the water solubility after 1 hour) may be 100% or less, preferably 99% or less, and more preferably 98% or less. Note that the lower limit of the water solubility after 1 hour after immersion in water is not particularly limited, but from the viewpoint of the effect of decomposition and absorption as an anti-adhesion material, for example, it may be 45% or more, preferably 55% or more, more preferably 65% ​​or more, and even more preferably 75% or more.

[0021] In measuring the water contact angle of the silk fibroin film, the change in the contact angle may be -5° / sec or less within 5 seconds after a 5 μL droplet of ultrapure water is dropped onto the film surface. Because the silk fibroin film has a water-soluble film surface, when a water droplet is dropped onto the film surface, the film surface dissolves in the water droplet, causing a change in the contact angle. Preferably, the water contact angle changes more negatively (larger in absolute value) in a short period of time. The change in the contact angle may be preferably -20 to -5° / sec, more preferably -15 to -5° / sec, and particularly -10 to -5° / sec. The change in the water contact angle of the silk fibroin film is a value measured by the method described in the Examples below. Figure 3 shows plot data of the change in the water contact angle over time for the silk fibroin film of Example 4 below. Initially, after a water droplet is dropped onto the film surface, the water contact angle decreases, and thereafter, the water contact angle remains approximately constant. The time when a water droplet is dropped onto the film surface is set to 0 seconds, and the plot data from 0 to 5 seconds, which is the initial stage, is linearly approximated using the least squares method, and the slope of the obtained approximate line (the line in Figure 3) is taken as the change in water contact angle.

[0022] Although the surface of a silk fibroin film is water-soluble, it does not necessarily have to exhibit high wettability immediately after contact with water. For example, even if the water contact angle is large immediately after a water droplet is dropped on the film surface, the above-mentioned contact angle change may occur due to water solubility. For example, in measuring the water contact angle of a silk fibroin film, the contact angle at the time when a water droplet is dropped on the film surface (0 seconds) may be 50 to 90°, preferably 55 to 85°, and more preferably 60 to 80°. The water contact angle of a silk fibroin film at 0 seconds is a value measured by the method described in the Examples below. The time when a water droplet is dropped on the film surface is set to 0 seconds, and the measured data for the contact angles from 0 to 5 seconds are linearly approximated by the least-squares method, and the value represents the intercept of the line obtained.

[0023] The silk fibroin film contains silk fibroin. Silk fibroin mainly contains glycine, alanine, serine, and tyrosine, and is composed of a crystalline portion in which the molecules are regularly arranged and an amorphous portion in which the molecules are randomly arranged. It is generally known as a type of fibrous protein. As long as silk fibroin has this structure, there are no particular limitations on the raw material from which it is obtained. For example, silk fibroin can be obtained from the silk raw material described below. Furthermore, silk fibroin may be chemically modified to the extent that the effects of the present invention are not impaired. In this specification, when the term "silk fibroin" is simply used, 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] The silk fibroin film preferably contains unmodified silk fibroin, from the viewpoint of utilizing the properties of silk fibroin as an anti-adhesion material. In this specification, unmodified silk fibroin means silk fibroin that has not been subjected to a reaction to introduce functional groups or crosslinking, or a grafting reaction, and in which the side chains of the amino acid residues that constitute the silk fibroin have not been chemically modified.

[0025] From the viewpoint of improving the water solubility of the film surface, the silk fibroin may have a weight-average molecular weight (Mw) of 150 kDa or less (e.g., 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 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] Furthermore, the number average molecular weight (Mn) of silk fibroin may be 80 kDa or less (e.g., 1 to 70 kDa), preferably 60 kDa or less, more preferably 50 kDa or less, even 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, but may be, for example, 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 (e.g., 1 to 4), preferably 1.2 to 9 (e.g., 1.2 to 3.8), more preferably 1.5 to 8 (e.g., 1.5 to 3.5), even more preferably 2 to 6, and particularly preferably 3 to 5. The molecular weight distribution indicates the polydispersity index (PDI) calculated by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn).

[0028] The peak top molecular weight (Mp) of silk fibroin may be 250 kDa or less (e.g., 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 even more preferably 50 kDa or less. The lower limit of the peak top molecular weight is not particularly limited, but may be, for example, 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 a chromatogram, and is a value measured by the method described in the Examples below.

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

[0030] The silk fibroin film may have a moisture content of 30% or less, preferably 1 to 30%, more preferably 3 to 20%, and even more preferably 5 to 15%. The moisture 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 (e.g., additives such as colorants), but the silk fibroin content may be 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99.9% or more, based on the solid weight.

[0032] For example, if the silk fibroin film contains a colorant, it may be advantageous because it makes it easier for medical professionals to visually confirm the area on the surface of an organ or the like where the adhesion barrier has been applied.

[0033] The thickness of the silk fibroin film can be selected appropriately depending on the clinical field and location of use of the adhesion barrier, and can be selected from a wide range, for example, from 1 μm to 5 mm. However, when considering use in the fields of gastrointestinal surgery and obstetrics and gynecology, the thickness of the silk fibroin film may be, for example, 1 μm to 1 mm, preferably 5 to 200 μm (e.g., 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 (e.g., 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, the film is easy to handle as an anti-adhesion material. There is no particular upper limit to the puncture strength of the silk fibroin film, but it 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] (Anti-adhesion material) The silk fibroin film is used as an anti-adhesion material. In this specification, the term "anti-adhesion material" refers to a biomaterial that is applied to sites of organ or tissue damage due to trauma, the dissected surface of an organ after surgery, the surface of tissues surrounding an organ after surgery, and other areas where adhesions may occur, for the purpose of suppressing "adhesion," a phenomenon in which tissue surfaces or organ surfaces that should normally be separated become bonded to other tissue surfaces or organ surfaces and become inseparable due to inflammation after surgery or the like.

[0036] As used herein, the term "trauma" refers to damage to an organ or tissue caused by an external force (mechanical, physical, or chemical).

[0037] As used herein, "organ" refers to an internal organ, which is an anatomical unit with a unique structure and each having a specific function, such as the brain, heart, esophagus, stomach, bladder, small intestine, large intestine, liver, kidneys, pancreas, spleen, uterus, etc.

[0038] As used herein, "tissue" refers to a unit that performs a certain function by combining related cells and substances produced by cells, and examples include skin, muscles, tendons, bones, joints, ligaments, blood vessels, pancreatic islets, and cornea.

[0039] As used herein, the term "dissected surface" refers to the surface that is exposed to the inside of the body when a part of a tissue or organ is excised or peeled off by surgery or the like.

[0040] Anti-adhesion materials can be used to prevent adhesions in any tissue or organ where adhesions may occur in surgeries in clinical fields such as gastrointestinal surgery, cardiac surgery, orthopedic surgery, and obstetrics and gynecology, and are particularly suitable for use in surgeries in gastrointestinal surgery and obstetrics and gynecology. For example, they can be applied to damaged areas of the intestine or peritoneum, or damaged areas of the uterus or appendages. They can also be used in surgeries on animals (non-humans) such as pets, as well as in surgeries on humans. Examples of non-human animals include non-human mammals, such as apes, other primates, mice, rats, hamsters, guinea pigs, horses, cows, pigs, sheep, goats, dogs, cats, and rabbits. In this specification, adhesion prevention refers not only to completely preventing the occurrence of adhesions, but also to reducing the degree, extent, and frequency of adhesions.

[0041] The anti-adhesion material may consist essentially of the silk fibroin film, or may be a laminate containing a support necessary for maintaining the shape of the film.

[0042] The anti-adhesion material may preferably be applied to the excised surface of an organ or tissue after surgery, the surface of tissue surrounding the organ after surgery, etc., during the finishing step of a surgical operation such as an abdominal surgery.

[0043] (Method for producing silk fibroin film) The method for producing 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 step of forming a film from the alkali-treated silk fibroin.

[0044] As the silk fibroin raw material, cocoons and raw silk produced by insects (such as Lepidoptera insects as domesticated silkworms, wild silkworms, and wild silkworms, and other silkworms producing insects such as hornets and honeybees, and other Hymenoptera insects such as wasps and honeybees) or spiders can be used, and there are no particular limitations on the silk raw material as long as it contains fibroin and sericin. Silk fibroin from which sericin has been removed can be obtained by scouring the silk raw material. Silk fibroin can also be obtained from silk glands. Scouring can be performed by known methods, including, for example, a method of removing sericin by swelling it using an alkaline scouring agent such as sodium carbonate, sodium silicate, or sodium phosphate; a method of removing sericin by decomposing it using a sericin-degrading enzyme; and a method of removing sericin by decaying it. From the viewpoint 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 viewpoint 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.

[0045] Next, the refined silk fibroin can be subjected to an alkali treatment to hydrolyze the silk fibroin and improve its water solubility. From the viewpoint of molecular weight adjustment, it is preferable to perform the hydrolysis of silk fibroin in an alkali treatment step. The alkali treatment can be performed by dissolving silk fibroin in a solvent and reacting it with an alkali treatment agent (e.g., 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. The alkali treatment can be performed by dissolving silk fibroin in a mixed solution containing a neutral salt and an alkali treatment agent, or by dissolving silk fibroin in a neutral salt solution and then adding an alkali treatment agent to perform the alkali treatment.

[0046] The concentration of the neutral salt in the solution may be, for example, 3 to 15 M, preferably 4 to 14 M, and more preferably 3 to 12 M. The concentration of the alkali metal hydroxide in the solution may be, for example, 0.05 to 2 M, and preferably 0.08 to 1.5 M. The concentration of ammonia in the solution may be, for example, 0.05 to 1.5 M, and 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 may be 40°C or lower (e.g., 5 to 40°C), preferably 10 to 40°C, and more preferably 15 to 35°C. The alkali treatment time may be longer than 3 hours, preferably 4 hours or longer, more preferably 10 hours or longer, and even more preferably 15 hours or longer. The upper limit of the alkali treatment time is not particularly limited, but may be, for example, 48 hours or shorter. In this specification, the alkali treatment time refers to the time during which the silk fibroin is in contact with the alkali treatment agent. For example, when a mixed solution containing a neutral salt and an alkali treatment agent is used, the time refers to the time from the addition of the mixed solution to the silk fibroin (or from the silk fibroin to the mixed solution). When the alkali treatment agent is added after dissolving the silk fibroin in a neutral salt solution, the time refers to the time from the addition of the alkali treatment agent.

[0048] The silk fibroin obtained by 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 silk fibroin film having a weight-average molecular weight (Mw) of 150 kDa or less (preferably 120 kDa or less, more preferably 100 kDa or less, and even more preferably 80 kDa or less).

[0049] After the alkali treatment, the neutral salt and the alkali treatment agent may be removed by a known method such as dialysis or ultrafiltration. Furthermore, from the viewpoint of use as a biomaterial, it is preferable to purify or sterilize the silk fibroin before membrane formation (film formation) in order to remove impurities other than the neutral salt and the alkali treatment agent. Regarding the sterilization, known methods such as autoclave sterilization and filter sterilization can be used, but autoclave sterilization is preferred.

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

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

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited by these examples. 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 adjusted to a concentration of 1% (w / v) was used as a measurement sample. Using this measurement sample, measurements were performed by gel filtration chromatography (GFC) using the following measurement equipment 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 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. Apparatus: GFC apparatus "AKTAgo" manufactured by Cytiva Separation column: "HiLoad16 / 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: Room temperature (24.0°C) or less; when simply referring to room temperature, 24.0°C is used as the representative value.

[0054] [Water Solubility of Film Surface] A 1 cm square and its diagonal were drawn on a silk fibroin film stored overnight or longer in an indoor environment (temperature 22.4-29.1°C, humidity 32-64 RH) using a water-resistant permanent marker (Zebra Corporation, product name "Mackie Extra Fine," black). The line thickness was approximately 1 mm. The film was cut along the square lines to obtain sample pieces measuring 1 cm in length and 1 cm in width. The sample pieces were floated in ultrapure water (24-28°C) in a Petri dish at room temperature with the side drawn with the permanent marker facing up. Starting from the time of floating, the time it took for all six lines drawn with the permanent marker (four sides, two diagonals) to break was measured. Measurements were conducted on three sample pieces, and the average time it took for all six lines drawn with the permanent marker to break was calculated as the time it took for the film surface to dissolve in water. If the six straight lines drawn with the oil-based pen were not broken even after one day or more, it was determined that the film surface was not water-soluble.

[0055] [Film Water Solubility] Silk fibroin film stored overnight or longer at room temperature was cut out to obtain a sample piece measuring 0.5 cm in length and 0.5 cm in width, and the sample piece was weighed (this weight was designated the "film weight before dissolution"). The sample piece was immersed in 1 mL of ultrapure water in a tube and incubated at 25°C for 1 hour or 24 hours. Using a micropipette (Eppendorf, "Reference 2, 4920000.083"), the remaining film was sucked onto the tip of the pipette, and the film was transferred to a 2 mL tube that had been pre-weighed (this weight was designated the "tube weight"). If the film could not be confirmed at this point, the water solubility was designated as 100%. The tube was then placed in a constant temperature dryer (KM-600V, manufactured by AS ONE Corporation) set to 50°C and incubated overnight to evaporate the water retained in the undissolved film, and the combined weight of the tube and dried silk fibroin film was measured (this weight was designated as the "tube + dry film weight"). The water solubility (%) of the silk fibroin film was then calculated according to the following formula: Water solubility (%) = 100 x [film weight before dissolution (mg) - {tube weight + dry film weight (mg) - tube weight (mg)}] / film weight before dissolution (mg).

[0056] [Water Contact Angle] Using a contact angle meter (Excimer Corporation, "SImageAUTO100"), approximately 5 μL of ultrapure water was dropped onto the surface of a silk fibroin film at room temperature, following the sessile drop method of JIS R 3257:1999. The time change in the water contact angle measured by the tangent method was measured (measurement interval: 1 / 15 second). The time when the water droplet was dropped was set to 0 seconds, and measurements were continued up to 30 seconds. The contact angle measurement data from 0 to 5 seconds were linearly approximated using the least squares method, and the slope and intercept of the resulting line were calculated. Measurements were taken at three locations on the silk fibroin film, and the average value of the slope of the approximated line was calculated as the contact angle change (° / sec), and the average value of the intercept was calculated as the contact angle (°) at the time when the water droplet was dropped onto the film surface (0 seconds).

[0057] [Moisture Content] The moisture content (%) of silk fibroin films was measured using a thermogravimetric differential thermal analyzer (TG-DTA; Rigaku Corporation, "Thermoplus TG8120"). Silk fibroin film samples (2.5 to 8.5 mg) stored overnight or longer at room temperature were placed in aluminum pans, and nitrogen was passed through them at a flow rate of 200 ml / min. The weight change was measured as the temperature rose from 30°C at a rate of 10°C / min. The weight of the silk fibroin film at the start of the measurement (30°C) (referred to as "sample weight") and the weight lost during the temperature rise from 30°C to 180°C (referred to as "water weight") were measured. The moisture content of the silk fibroin film was then measured according to the following formula. Three individually prepared samples were similarly measured, and the average of these values ​​was calculated as the moisture content (%) of the silk fibroin film. Moisture content (%) = 100 × water weight (mg) / sample weight (mg)

[0058] [Thickness] The thickness (μm) of the silk fibroin film was measured using a digital micrometer (Nikon Corporation, "MFC-101A"). The zero point was set with the measurement head lowered to the base. Then, a silk fibroin film sample stored overnight or longer in an indoor environment was placed on the base, and the measurement head was lowered to the sample to measure the thickness of the silk fibroin film. In the same manner, measurements were taken for each of three individually prepared samples, and the average of these measurements was calculated as the thickness (μm) of the silk fibroin film.

[0059] [Puncture Strength] Using silk fibroin film samples stored overnight or longer in an indoor environment, the puncture strength (N) was measured using the following measuring device and conditions. Similarly, measurements were taken for each of three individually prepared samples, and the average value was calculated as the puncture strength (N) of the silk fibroin film. - Device: Compact tabletop testing machine (Shimadzu Corporation, "EZTest") - Load cell capacity: 10 N - Puncture jig: Diameter φ3 mm, tip angle 60° - Puncture speed: 50 mm / min - Temperature: Room temperature

[0060] Example 1 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.

[0061] 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.

[0062] The obtained silk fibroin aqueous solution was dialyzed against deionized water using a dialysis membrane (molecular weight cutoff: 6-8 kJ). Each dialysis session was performed at room temperature for 6-12 hours, and this was repeated six times (bath ratio: 160). This removed the lithium bromide and sodium hydroxide contained in the silk fibroin aqueous solution.

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

[0064] The concentrated silk fibroin aqueous solution was subjected to autoclave sterilization using an autoclave device (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.

[0065] The autoclave-sterilized silk fibroin aqueous solution was centrifuged (40,000×g, 20° C., 30 minutes) using a centrifugal separator (Beckman Coulter, "Avanti 20I") to remove precipitated insoluble matter.

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

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

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

[0069] [Example 3] After freeze-drying, a silk fibroin aqueous solution was prepared so that the concentration was 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 in a polystyrene petri dish.

[0070] [Example 4] After freeze-drying, a silk fibroin aqueous solution was prepared so that the concentration was 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 in a polystyrene petri dish.

[0071] [Example 5] After freeze-drying, a silk fibroin aqueous solution was prepared 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 in 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 aqueous solution of lithium bromide (50 mL) that did not contain NaOH.

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

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

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

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

[0077] The results of various measurements carried out on each of the Examples and Comparative Examples are shown in Table 1.

[0078]

[0079] As shown in Table 1, in Examples 1 to 5, in which 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. Figure 1 is a photograph showing the state of a sample piece of the silk fibroin film of Example 4 immediately after floating in water and about 3 minutes later. Figure 2 is a photograph showing the state of a sample piece of the silk fibroin film of Comparative Example 4 immediately after floating in water and about 8 minutes later. In Comparative Examples 1 to 5, six straight lines drawn with an oil-based marker were not broken even after 7 days or more.

[0080] Furthermore, 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 thought to be due to the difference in water solubility of the film surface. In Examples 1 to 5, which were subjected to alkali treatment, the molecular weight was reduced compared to Comparative Examples 1 to 5, and it is thought that the reduction in the molecular weight of silk fibroin also contributes to the water solubility of the film surface.

[0081] Evaluation of Anti-Adhesion Effect in a Rat Cecum Abrasion Model: Under 2-2.5% isoflurane inhalation anesthesia, rats (SD, 7-week-old males, Japan SLC) were subjected to abdominal section to expose the rat cecum. The surface of the exposed cecum was rubbed 50 times with dry gauze to create an abrasion wound of approximately 1 cm x 2 cm. The surface of the abdominal wall was then scraped with tweezers to create a laceration wound of approximately 1 cm x 1 cm. The cecum was then returned to the abdominal cavity, and the wounds on the cecum and abdominal wall surfaces were aligned. One film-type anti-adhesion material (2 cm x 2 cm) was then applied to each of the wounds on the cecum and abdominal wall surfaces, followed by closure. The film-type anti-adhesion materials used were the silk fibroin film of Example 4, the silk fibroin film of Comparative Example 4, Seprafilm (manufactured by Baxter), and Tenaleaf (manufactured by Gunze Co., Ltd.). Four rats were implanted with the silk fibroin film of Example 4, the silk fibroin film of Comparative Example 4, and Tenaleaf, and five rats were implanted with Seprafilm. Four rats were also intraperitoneally administered with 3 mL of saline. Each rat was given an intramuscular injection of 0.6 μg of buprenorphine after the application of the adhesion barrier and closure of the wound, and then allowed to awaken. Two weeks after the surgery, the rats were euthanized, and the abdomen was opened again to evaluate adhesions.

[0082] Adhesion was evaluated by visual observation and scored on a 6-point scale from 0 to 5 as follows: 0: no adhesion; 1: thin membrane-like adhesion; 2: multiple thin membrane-like adhesions; 3: thick adhesions with patches; 4: thick adhesions with surface adhesions; 5: very thick adhesions with neovascularization or adhesions with multiple surfaces.

[0083] After scoring, the abraded portion of the cecum and the contusion portion of the abdominal wall were collected and washed with saline. The collected tissues were immersed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.) and fixed at room temperature for at least 3 days. The fixed tissues were embedded in OCT compound (Sakura Finetech Japan Co., Ltd.) and frozen at -20°C. The tissues were then sliced ​​to a thickness of 10-12 μm using a cryostat (Leica, "CM3050S") and attached to APS-coated glass slides (Matsunami Glass Co., Ltd.). After drying overnight at room temperature, the sections were immersed in 95% (v / v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) at 4°C for 10 minutes and then dried at room temperature. The sections were then immersed in running water for 2 minutes, Mayer's hematoxylin solution (Fujifilm Wako Pure Chemical Industries, Ltd.) for 10 minutes, running water for 15 minutes, 1% eosin Y solution (Fujifilm Wako Pure Chemical Industries, Ltd.) for 2 minutes, 70% (v / v) ethanol for 15 seconds, 99.5% ethanol for 15 seconds, xylene (Fujifilm Wako Pure Chemical Industries, Ltd.) for 1 minute, and xylene for 2 minutes at room temperature for hematoxylin-eosin staining. The stained tissue sections were mounted in mounting medium (Merck, "Entelaneu") and bright-field images were observed using a microscope (Keyence, "BZX710").

[0084] Figure 4 is a boxplot showing the adhesion score results for each adhesion barrier. Specifically, for saline, adhesion scores for four rats were 0, 1, 4, and 4, with an average adhesion score of 2.25. For the silk fibroin film (HMW) of Comparative Example 4, adhesion scores for four rats were 2, 3, 5, and 0, with an average adhesion score of 2.50. For the silk fibroin film (LMW) of Example 4, adhesion scores for four rats were 0, 0, 0, and 4, with an average adhesion score of 1.00. For Seprafilm, two of six rats died within three days after implantation, and four rats received adhesion scores of 0, 0, 5, and 0, with an average adhesion score of 1.25. In the case of Tenaleaf, one of the five rats died within three days after transplantation, and the adhesion scores of three rats were 0, 3, 3, and 0, with an average adhesion score of 1.50.

[0085] The adhesion score evaluation results for the adhesion barrier showed that the silk fibroin film (LMW) of Example 4 had a better average adhesion score than the silk fibroin film (HMW) of Comparative Example 4, demonstrating its adhesion prevention effect. Figure 5 is a photograph showing the cecum and abdominal wall (adhesion score: 0) two weeks after implantation of the silk fibroin film of Example 4. When the silk fibroin film of Example 4 was applied, no film was observed to remain after two weeks, indicating that the film had been degraded and absorbed by the body. Figure 6 is a photograph showing the cecum and abdominal wall (adhesion score: 5) two weeks after implantation of the silk fibroin film of Comparative Example 4, and Figure 7 is a photograph showing the adhesion site. When the silk fibroin film of Comparative Example 4 was applied, a lump was formed at the adhesion site, and the film was observed to remain inside, indicating that it had not been degraded and absorbed by the body (Figure 7).

[0086] Furthermore, the silk fibroin film of Comparative Example 4 had an average adhesion score inferior to that of the reference saline solution, indicating that simply containing silk fibroin is not enough to exert an adhesion prevention effect.

[0087] Furthermore, the silk fibroin film of Example 4 showed a better average adhesion score than commercially available film-type adhesion barriers, Seprafilm and Tenaleaf. Furthermore, although the cause is unknown, some rats receiving Seprafilm and Tenaleaf died within three days of transplantation, whereas no rats receiving the silk fibroin film of Example 4 died, suggesting its superior safety.

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

[0089] On the other hand, Figure 10 is a stained photograph showing the condition of the abdominal wall (AW) and cecum (C) two weeks after application of the silk fibroin film (LMW) of Example 4 to the laceration wound. As shown in Figure 10, the abdominal wall and cecum were separated from each other, and no adhesions were observed.

[0090] 11 and 12 are stained photographs showing the condition of the abdominal wall (AW) and cecum (C) two weeks after application of commercially available Seprafilm and Tenaleaf, respectively, to the laceration. As shown in Fig. 11 and Fig. 12, even when the commercially available adhesion barrier was used, the abdominal wall and cecum remained separated from each other, and no adhesions were observed.

[0091] Therefore, it was histologically confirmed that the silk fibroin film according to the present invention exhibited an anti-adhesion effect equal to or greater than that of commercially available film-type anti-adhesion materials. On the other hand, despite using silk fibroin, the silk fibroin film of Comparative Example 4 failed to exhibit an anti-adhesion effect.

[0092] From the above, the evaluation results of the in vivo test confirmed that the silk fibroin film of the present invention exhibits an adhesion prevention effect equal to or greater than that of commercially available film-type adhesion prevention materials, and it was also confirmed that the silk fibroin film of the present invention is degraded and absorbed by the body within two weeks after implantation, demonstrating its usefulness as an adhesion prevention material.

[0093] The silk fibroin film of the present invention can be used as an anti-adhesion material to prevent adhesion of any tissue or organ where adhesion may occur in various surgeries in the clinical fields of gastrointestinal surgery, cardiac surgery, orthopedic surgery, obstetrics and gynecology, etc. (particularly gastrointestinal surgery and obstetrics and gynecology).

[0094] While the preferred embodiments of the present invention have been described above, those skilled in the art will readily recognize various changes and modifications within the scope of the present invention, which are obvious from the description of the present invention. Therefore, such changes and modifications are to be interpreted as falling within the scope of the invention as defined by the claims.

Claims

1. A silk fibroin film for use as an anti-adhesion material, in which at least the surface of the silk fibroin film dissolves in water within 24 hours after being floated on water.

2. A silk fibroin film in which, in measuring the contact angle of water on the film surface, the change in 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.

3. A silk fibroin film according to claim 1 or 2, wherein the water solubility of the film after immersion in water and incubation at 25°C for 24 hours is 50% or more.

4. A silk fibroin film according to any one of claims 1 to 3, wherein the water solubility of the film after immersion in water and incubation at 25°C for 1 hour is 99% or less.

5. A silk fibroin film according to any one of claims 1 to 4, wherein the weight-average molecular weight of the silk fibroin is 150 kDa or less.

6. A silk fibroin film according to any one of claims 1 to 5, wherein the silk fibroin present on the film surface is silk fibroin that is not modified with polyethylene glycol.

7. A silk fibroin film according to any one of claims 1 to 6, having a moisture content of 30% or less.

8. A silk fibroin film comprising silk fibroin having a weight average molecular weight of 40-150 kDa and a molecular weight distribution (PDI) of 1-10.

9. An adhesion prevention 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 and reacting it with an alkali treatment agent, and a step of forming a film from the alkali-treated silk fibroin.

11. The method for producing a silk fibroin film according to claim 10, wherein the weight-average molecular weight of the alkali-treated silk fibroin is 150 kDa or less.

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