WO2025047047A1 - Silk fibroin film and anti-adhesion material containing same - Google Patents

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 same Related Applications

This application claims priority to Patent Application No. 2023-139250, filed in Japan on August 29, 2023, the entire contents of which are incorporated herein by reference as a part of this application.

The present invention relates to a silk fibroin film for use in an anti-adhesion material and to an anti-adhesion material containing the same.

In clinical fields such as gastrointestinal surgery, cardiac surgery, orthopedics, and obstetrics and gynecology, adhesions can form after surgery, bonding tissues and organs that should normally be separate and becoming inseparable. The formation of adhesions can necessitate a second surgery to remove the adhesions, which can take a lot of time and effort, and can even cause new damage during the removal process. It can also lead to complications such as intestinal obstruction and infertility.

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 surfaces of tissues or organs. Anti-adhesion materials have been developed in the form of films or sprays, 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 on 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.

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 Patent Publication 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 have a molecular weight exceeding 200 kDa, and at least 50% of the total number of the silk fibroin fragments in the population have a molecular weight within a specified range, the specified range being between a lower limit of about 3.5 kDa or more and an upper limit of about 120 kDa or less, and the composition is described as being usable in the form of a film and for medical purposes.

Furthermore, Patent Document 2 (U.S. Pat. No. 9,427,499) discloses a silk fibroin matrix in which at least a portion of a first surface is selectively modified with poly(ethylene glycol) (PEG) at a density of about 75 to about 750 μg PEG/ ^cm2 silk fibroin matrix, and describes that the silk fibroin matrix can be used as an adhesion barrier.

Special Publication No. 2016-517443 U.S. Pat. No. 9,427,499

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.

Patent Document 2 also 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 PEG on the surface that contributes to these inhibitions, and the paper does not find any effect due to the structure of the silk fibroin itself. Furthermore, the paper did not evaluate the adhesion prevention effect in vivo.

The object of the present invention is therefore to provide a silk fibroin film suitable for use as an adhesion prevention material.

As a result of extensive research into achieving the above objective, the inventors of the present invention discovered that a silk fibroin film, the surface of which dissolves in water in a short period of time, has excellent adhesion prevention effects, leading to the completion of the present invention.

That is, the present invention can be configured in the following manner.
[Aspect 1]
The silk fibroin film is an anti-adhesion material, in which at least the surface of the silk fibroin film 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]
A silk fibroin film according to embodiment 1, wherein, in a measurement of 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 on the film surface until 5 seconds later is −5°/sec or less (preferably −20 to −5°/sec, more preferably −15 to −5°/sec, particularly preferably −10 to −5°/sec).
[Aspect 3]
A silk fibroin film, in which, in measurement of 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 on the film surface until 5 seconds later is −5°/sec or less (preferably −20 to −5°/sec, more preferably −15 to −5°/sec, particularly preferably −10 to −5°/sec).
[Aspect 4]
The 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]
The 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]
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, and even more preferably 90 kDa or less).
[Aspect 7]
7. The silk fibroin film according to any one of claims 1 to 6, wherein the silk fibroin present on the film surface is silk fibroin that is not modified with polyethylene glycol.
[Aspect 8]
A silk fibroin film according to any one of aspects 1 to 7, wherein the silk fibroin film has a moisture 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, 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 preventing material comprising the silk fibroin film according to any one of aspects 1 to 7.
[Aspect 11]
The present invention relates to a method for producing a silk fibroin film for use as an anti-adhesion material, the method comprising the steps of: dissolving refined silk fibroin in a solvent and reacting the resulting solution with an alkali treating agent; and forming a film from the alkali-treated silk fibroin.
[Aspect 12]
A 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 the step of applying the adhesion preventing material to at least one of the detached surface of a postoperative organ and the surface of tissue surrounding the postoperative organ.

As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms "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.

Furthermore, when referring to "about," the range may include ±10% of the numerical value, and preferably ±5%.

It should be noted that 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 described in the claims is included in the present invention.

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

Photographs showing the state of a sample piece of the silk fibroin film of Example 4 immediately after being floated 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 being floated in water and about 8 minutes later. 1 is a graph showing plot data of the change over time in the contact angle of water of the silk fibroin film of Example 4 and an approximation line of the data from 0 to 5 seconds. FIG. 13 is a box plot showing the adhesion score results when saline, the silk fibroin film of Comparative Example 4 (HMW), the silk fibroin film of Example 4 (LMW), Seprafilm, and Tenaleaf were applied, respectively. 13 is a photograph showing the cecum and abdominal wall (adhesion score: 0) two weeks after implantation of the silk fibroin film of Example 4. 13 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. 13 is a photograph showing adhesions between the cecum and abdominal wall two weeks after implantation of the silk fibroin film of Comparative Example 4. 1 is a stained photograph showing the state of the abdominal wall and cecum two weeks after application of saline to the laceration in Comparative Example 1. The scale bar indicates 1 mm. 1 is a stained photograph showing the state of the abdominal wall and cecum two weeks after application of the silk fibroin film (HMW) of Comparative Example 4 to a laceration wound. The scale bar indicates 1 mm. 1 is a stained photograph showing the state of the abdominal wall and cecum two weeks after application of the silk fibroin film (LMW) of Example 4 to a laceration wound. The scale bar indicates 1 mm. 1 is a stained photograph showing the condition of the abdominal wall and cecum two weeks after application of Seprafilm to the laceration. Scale bar indicates 1 mm. 1 is a stained photograph showing the condition of the abdominal wall and cecum two weeks after application of Tenaleaf to the laceration. Scale bar indicates 1 mm.

(Silk fibroin film)
The silk fibroin film dissolves in water at least at the surface of the silk fibroin within 24 hours after floating on water. In this specification, the dissolution of the film surface in water can be confirmed by observing the method described in the examples below, and by confirming that the square drawn with an oil pen (black) on the surface of a square film with sides of 1 cm and the six straight lines (line width 1 mm) of the square drawn with an oil pen are all broken. That is, when a line is drawn with an oil pen on the first surface of the film and the film is floated on water with the first surface facing up and the second surface facing down, the film surface (first surface) dissolves in water, and each of the six straight lines drawn with an oil pen on the first surface peels off from the film and separates from each other, and each straight line is broken by cracks. Therefore, the water solubility of the film surface can be evaluated by confirming that the straight lines drawn with an oil pen peel off together with the film surface after the film is floated on water, and finally all six straight lines are broken. By confirming that the upper surface of the film dissolves when the film is floated on water, the film surface of the lower surface (second surface) of the film can also be considered to dissolve. It should be noted that at least the film surface needs to dissolve in water within 24 hours, and the 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 film surface of the silk fibroin film can prevent the film surface from being in contact with cells, fibrin, etc. for a long time, and the adhesion of cells, fibrin, etc. is inhibited, or the cells, fibrin, etc. that are intended to be adhered or adsorbed are peeled off together with the film surface, so that the adhesion prevention effect can be exerted. The time for which the film surface dissolves in water is preferably within 12 hours after floating on water, more preferably within 1 hour, and even more preferably within 10 minutes. In addition, the lower limit of the time for which the film surface dissolves in water is not particularly limited, but may be, for example, 1 second or more, and from the viewpoint of easy re-application, it may be preferably 30 seconds or more, more preferably 50 seconds or more, and particularly preferably 100 seconds or more.

From the viewpoint of the effect of decomposition and absorption as an adhesion prevention 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.

Furthermore, from the viewpoint of maintaining the physical barrier function as an adhesion prevention material, the water solubility of the silk fibroin film after immersion in water and incubation at 25°C for 1 hour (hereinafter, may be 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. The lower limit of the water solubility after immersion in water for 1 hour is not particularly limited, but from the viewpoint of the effect of decomposition and absorption as an adhesion prevention 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.

In the measurement of the contact angle of water on the surface of the silk fibroin film, the change in the contact angle from dropping a 5 μL drop of ultrapure water on the film surface until 5 seconds later may be -5°/sec or less. Since the surface of the silk fibroin film is water-soluble, when a drop of water is dropped on the film surface, the film surface dissolves in the drop of water, causing the contact angle to change. It is preferable that the contact angle of water changes more negatively (larger in absolute value) in a short 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 contact angle of water on 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 contact angle of water over time for the silk fibroin film of Example 4 described below. In the early stage after dropping a drop of water on the film surface, the contact angle of water is small, and thereafter, the contact angle of water is 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 resulting approximate line (the line in Figure 3) is the change in the contact angle of water.

The silk fibroin film has a water-soluble film surface, but does not necessarily have to show high wettability immediately after contact with water. For example, the contact angle of water may be large immediately after a water droplet is dropped on the film surface, but the film may have the above-mentioned contact angle change due to its water-solubility. For example, in measuring the contact angle of water on the surface of a silk fibroin film, the contact angle 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 measurement data of the contact angle from 0 to 5 seconds is linearly approximated by the least squares method, and the intercept of the obtained line is shown.

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, and is generally known as a type of fibrous protein. As long as the silk fibroin has such a structure, the raw material is not particularly limited, and silk fibroin can be obtained from, for example, a silk raw material described later. In addition, silk fibroin may be chemically modified within a range that does not impair the effects of the present invention.
In this specification, when the term "silk fibroin" is used, the definition includes chemically modified silk fibroin, although the silk fibroin present on the film surface may be silk fibroin that is not modified with polyethylene glycol (PEG).

The silk fibroin film preferably contains unmodified silk fibroin in order to utilize the properties of silk fibroin as an adhesion prevention 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 the side chains of the amino acid residues that constitute the silk fibroin have not been chemically modified.

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. There is no particular limit to the lower limit of the weight average molecular weight, but it 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.

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. There is no particular limit to the lower limit of the number average molecular weight, but it 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.

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).

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.

Preferred silk fibroin films include silk fibroin films 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 (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). The silk fibroin film may also have the various characteristics described above.

The moisture content of the silk fibroin film may be 30% or less, preferably 1-30%, more preferably 3-20%, and even more preferably 5-15%. 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.

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 by weight of solid matter, preferably 95% or more, more preferably 98% or more, and even more preferably 99.9% or more.

For example, if the silk fibroin film contains a coloring agent, this can be advantageous as it makes it easier for medical professionals to visually confirm the areas on the surface of organs, etc. where the adhesion prevention material has been applied.

The thickness of the silk fibroin film can be appropriately selected depending on the clinical field and location of use of the adhesion prevention material, 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.

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. If the puncture strength is within the above range, it is easy to handle as an adhesion prevention 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.

(Anti-adhesion material)
Silk fibroin films are used as adhesion inhibitors, including the silk fibroin film. In this specification, the term "adhesion inhibitor" refers to a biomaterial that is applied to sites of organ or tissue damage caused by trauma, dissected surfaces of organs after surgery, and tissue surfaces surrounding organs after surgery, where adhesions may occur, for the purpose of suppressing "adhesion," a phenomenon in which tissue surfaces or organ surfaces that should be separated from each other become bonded to other tissue surfaces or organ surfaces and cannot be separated due to inflammation after surgery or the like.

In this specification, "trauma" refers to damage to organs or tissues caused by external forces (mechanical, physical, or chemical).

In this specification, "organ" refers to an internal organ, which is an anatomical unit with its own structure and specific function, such as the brain, heart, esophagus, stomach, bladder, small intestine, large intestine, liver, kidneys, pancreas, spleen, uterus, etc.

In this specification, "tissue" refers to a unit that performs a certain function by combining related cells or substances produced by cells, and examples of such tissue include skin, muscle, tendon, bone, joint, ligament, blood vessel, pancreatic islet, and cornea.

In this specification, the term "dissected surface" refers to the surface that is exposed to the inside of the body after part of a tissue or organ is removed or peeled off during surgery or other procedures.

The adhesion prevention material can be used to prevent adhesion of any tissue or organ where adhesion may occur in surgery in clinical fields such as gastrointestinal surgery, cardiac surgery, orthopedics, and obstetrics and gynecology, and is particularly suitable for use in surgery in gastrointestinal surgery and obstetrics and gynecology. For example, it can be used by applying it to damaged areas of the intestine or peritoneum, or damaged areas of the uterus or appendages. It can also be used not only in human surgery, but also in surgery on animals (non-humans) such as pets. For example, 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 does not only mean completely preventing the occurrence of adhesions, but also includes reducing the degree, range, and frequency of adhesions.

The anti-adhesion material may consist essentially of the silk fibroin film, or it may be a laminate that includes a support necessary for maintaining the shape of the film.

The adhesion prevention material may be preferably applied to the dissected surface of an organ or tissue after surgery and to the surface of tissue surrounding the organ after surgery during the finishing process of a surgical procedure such as an abdominal surgery.

(Method of manufacturing 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.

As the raw material for silk fibroin, cocoons and raw silk produced by insects (Lepidoptera insects such as domestic silkworms, wild silkworms, and wild silkworms, and silkworms producing insects such as hornets and honeybees producing insects such as hymenoptera insects) and spiders can be used, and there is no particular limitation as long as the raw material for silk contains fibroin and sericin. Silk fibroin from which sericin has been removed can be obtained by refining the raw silk material. Silk fibroin can also be obtained from silk glands. Refining can be performed by known methods, and examples of such methods include a method of removing sericin by swelling it using an alkaline refining agent such as sodium carbonate, sodium silicate, or sodium phosphate, a method of decomposing sericin using a sericin-degrading enzyme, and a method of removing sericin by decay. Refining using an alkaline refining agent is preferred from the viewpoint of ease of setting conditions. The main purpose of scouring is to remove sericin, and in order to suppress the decomposition of silk fibroin, the scouring time using an alkaline scouring agent varies depending on the type of silk raw material and alkaline scouring agent, but may be 5 to 60 minutes, preferably 10 to 50 minutes, and more preferably 15 to 45 minutes.

Next, the refined silk fibroin is 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 carry out the hydrolysis of silk fibroin in an 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 (e.g., an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, or ammonia, etc.). 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 may be dissolved and alkali treatment may be carried out at the same time using a mixed solution containing a neutral salt and an alkali treatment agent, or silk fibroin may be dissolved in a neutral salt solution and then an alkali treatment agent may be added to carry out the alkali treatment.

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.

By carrying out 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 less (for example, 5 to 40°C), preferably 10°C to 40°C, and more preferably 15°C to 35°C. The time for carrying out the alkali treatment may be more than 3 hours, preferably 4 hours or more, more preferably 10 hours or more, and even more preferably 15 hours or more. The upper limit of the time for carrying out the alkali treatment is not particularly limited, but may be, for example, 48 hours or less. In this specification, the time for carrying out the alkali treatment means 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, it indicates the time from adding the mixed solution to the silk fibroin (or adding the silk fibroin to the mixed solution), and when an alkali treatment agent is added after dissolving the silk fibroin in a neutral salt solution, it indicates the time from adding the alkali treatment agent.

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).

After the alkaline treatment, the neutral salts and the alkaline treatment agent may be removed by known methods such as dialysis or ultrafiltration. From the viewpoint of using it as a biomaterial, it is preferable to purify and sterilize the silk fibroin before membrane formation (film formation) in order to remove impurities other than the neutral salts and the alkaline treatment agent. Regarding the sterilization, known methods such as autoclave sterilization and filter sterilization can be used, but autoclave sterilization is preferable.

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

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

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.

[Molecular weight]
The measurement sample was 500 μL of a silk fibroin aqueous solution adjusted to a concentration of 1% (w/v). Using this measurement sample, measurements were performed by gel filtration chromatography (GFC) using the following measurement device 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 (Low Molecular Weight; LMW) Gel Filtration Calibration Kits and the high molecular weight (High Molecular Weight; HMW) Gel Filtration Calibration Kits (both manufactured by Cytiva) as standard substances.
Device: Cytiva GFC device "AKTAgo"
Separation column: Cytiva "HiLoad16/600 Superdex 200pg"
Mobile phase: 20 mM sodium phosphate buffer containing 500 mM sodium chloride (pH: 7.4)
・Flow rate: 1mL/min
Temperature: Room temperature (24.0°C) or lower. When simply referring to room temperature, 24.0°C is used as the representative value.

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

[Water solubility of film]
The silk fibroin film stored overnight or longer in an indoor environment was cut out to obtain a sample piece of 0.5 cm length and 0.5 cm width, and the weight of this sample piece was measured (the weight at this time was referred to as 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 tip, and the film was transferred to a 2 mL tube whose weight (the weight at this time was referred to as the "tube weight") had been measured in advance. If the film could not be confirmed at this point, the water solubility was taken as 100%. The tube was then left to stand in a constant temperature dryer (As One, "KM-600V") set at 50°C and incubated overnight to evaporate the moisture retained in the remaining film, and the weight of the tube and the dried silk fibroin film combined was measured (the weight at this time was referred to as the "tube + dried film weight"). The water solubility (%) of the silk fibroin film was then calculated according to the following formula.
Water solubility (%)=100×[film weight before dissolution (mg)−{tube weight+dry film weight (mg)−tube weight (mg)}]/film weight before dissolution (mg)

[Water contact angle]
Using a contact angle meter (manufactured by Excimer Corporation, "SImageAUTO100"), a droplet of about 5 μL of ultrapure water was dropped on the surface of the silk fibroin film at room temperature, referring to the sessile drop method of JIS R 3257:1999, and the change in the contact angle of water measured by the tangent method was measured over time (measurement interval: 1/15 seconds). The time when the droplet was dropped was set to 0 seconds, and measurements were made up to 30 seconds. The contact angle measurement data from 0 to 5 seconds was linearly approximated by the least squares method, and the slope and intercept of the obtained line were calculated. Measurements were made at three points 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 (°) when the droplet was dropped on the film surface (0 seconds).

[Moisture content]
The moisture content (%) of the silk fibroin film 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 in an indoor environment were placed in an aluminum pan, and nitrogen was passed through them at a flow rate of 200 ml/min. The weight change was measured when the temperature was raised from 30°C at 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 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. In the same manner, measurements were performed on each of the three individually prepared samples, and the average value of these values was calculated as the moisture content (%) of the silk fibroin film.
Moisture content (%) = 100 x water weight (mg) / sample weight (mg)

[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 pedestal. Then, the silk fibroin film sample stored overnight or longer in an indoor environment was placed on the pedestal, 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 the three individually prepared samples, and the average value of these measurements was calculated as the thickness (μm) of the silk fibroin film.

[Puncture strength]
The silk fibroin film samples stored overnight or longer in an indoor environment were used to measure the puncture strength (N) using the following measuring device and conditions. Similarly, measurements were performed on three individually prepared samples, and the average value was calculated as the puncture strength (N) of the silk fibroin film.
Equipment: Small tabletop tester (Shimadzu Corporation, "EZTest")
・Load cell capacity: 10N
- Piercing tool: diameter φ3mm, tip angle 60°
Piercing speed: 50 mm/min
Temperature: Room temperature

[Example 1]
Silkworm cocoons were cut into pieces and degummed in a boiling 0.02 M aqueous solution of sodium carbonate for 30 minutes to obtain silk fibroin.

3 g of the refined silk fibroin was immersed in a mixed aqueous solution (50 mL) containing 9 M lithium bromide and 0.1 M sodium hydroxide (NaOH) and left at room temperature for 1 to 6 hours. Next, this mixed aqueous solution was stirred at room temperature for 17 hours, dissolving the silk fibroin in the mixed aqueous solution and subjecting it to an alkali treatment to obtain an aqueous silk fibroin solution.

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

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

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.

The silk fibroin aqueous solution after autoclave sterilization was centrifuged (40,000 x g, 20°C, 30 minutes) using a centrifuge (Beckman Coulter, "Avanti 20I") to remove precipitated insoluble matter.

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

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 at room temperature for at least 2 days to evaporate the water, yielding a silk fibroin film.

[Example 2]
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 3 mL of the silk fibroin aqueous solution was spread in a polystyrene petri dish.

[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 in a polystyrene petri dish.

[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 in a polystyrene petri dish.

[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 in a polystyrene petri dish.

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

[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 lithium bromide aqueous solution (50 mL) that did not contain NaOH.

[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 lithium bromide aqueous solution (50 mL) that did not contain NaOH.

[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 lithium bromide aqueous solution (50 mL) that did not contain NaOH.

[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 lithium bromide aqueous solution (50 mL) that did not contain NaOH.

The results of various measurements performed on each Example and Comparative Example are shown in Table 1.

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 immediately after and about 3 minutes after a sample piece of silk fibroin film of Example 4 was floated on water. Figure 2 is a photograph showing the state immediately after and about 8 minutes after a sample piece of silk fibroin film of Comparative Example 4 was floated on water. In Comparative Examples 1 to 5, six straight lines drawn with an oil-based pen were not broken even after more than 7 days.

In addition, 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.

[Evaluation of adhesion prevention effect in a rat cecal abrasion model]
A rat (SD, 7-week-old male, Japan SLC) was anesthetized with 2-2.5% isoflurane inhalation, and the rat's cecum was exposed by laparotomy. The surface of the exposed cecum was rubbed 50 times with dry gauze to form an abrasion wound of about 1 cm x 2 cm. Next, the surface of the abdominal wall was scooped with tweezers to form a laceration wound of about 1 cm x 1 cm. The cecum was then returned to the abdominal cavity, and the wounds on the cecum surface and abdominal wall surface were aligned. Then, one film-type adhesion inhibitor (2 cm x 2 cm) was attached to each of the wounds on the cecum surface and abdominal wall surface, and the wound was closed. The film-type adhesion inhibitors used were the silk fibroin film of Example 4, the silk fibroin film of Comparative Example 4, Seprafilm (manufactured by Baxter), and Tenalef (manufactured by Gunze Co., Ltd.). Four rats were prepared each with the silk fibroin film of Example 4, the silk fibroin film of Comparative Example 4, and Tenaleaf, and five rats were prepared with Seprafilm. Four rats were also prepared with 3 mL of saline administered intraperitoneally. Each rat was given 0.6 μg of buprenorphine intramuscularly after applying the adhesion inhibitor and closing the wound, and then awakened. Two weeks after the above surgery, the rats were euthanized and re-opened to evaluate adhesion.

The adhesions were evaluated by visual observation and scored according to the following adhesion score of 0 to 5, which is a 6-level scale.
0: no adhesion; 1: thin membrane-like adhesion; 2: multiple thin membrane-like adhesions; 3: thick adhesions with spots; 4: thick adhesions with surface attachment; 5: very thick adhesions with neovascularization or multiple surface attachments

After scoring, the abraded parts of the cecum and the abdominal wall parts with lacerations were collected and washed with saline. The collected tissues were immersed in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Ltd.) and fixed by leaving them at room temperature for more than 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 tissues were immersed in 95% (v/v) ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) at 4°C for 10 minutes and dried at room temperature. Thereafter, the sections were 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 to perform 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").

Figure 4 is a box plot showing the adhesion score results for each adhesion prevention material. Specifically, for saline, the 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, the 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, the adhesion scores for four rats were 0, 0, 0, and 4, with an average adhesion score of 1.00. For Seprafilm, two of the six rats died within three days after implantation, and the adhesion scores for four rats were 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 of implantation, and the adhesion scores of three rats were 0, 3, 3, and 0, with an average adhesion score of 1.50.

In the adhesion score evaluation results of the adhesion prevention material, the silk fibroin film of Example 4 (LMW) showed a better average adhesion score than the silk fibroin film of Comparative Example 4 (HMW), demonstrating the 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 found to remain after two weeks, indicating that the film had been decomposed 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 area. When the silk fibroin film of Comparative Example 4 was applied, a lump was formed at the adhesion area, and the film was found to remain inside, indicating that the film had not been decomposed and absorbed by the body (Figure 7).

In addition, the silk fibroin film of Comparative Example 4 had a lower average adhesion score than the physiological saline solution used as a reference example, indicating that simply containing silk fibroin is not enough to achieve adhesion prevention effects.

Furthermore, the silk fibroin film of Example 4 showed a better average adhesion score than the commercially available film-type adhesion prevention materials Seprafilm and Tenaleaf. Also, although the cause is unknown, some rats that received Seprafilm and Tenaleaf died within three days after transplantation, whereas no rats died after receiving the silk fibroin film of Example 4, suggesting that it is also safe.

Figures 8 and 9 are stained photographs showing the condition of the abdominal wall (AW) and cecum (C) two weeks after applying saline (Saline) of Comparative Example 1 and silk fibroin film (HMW) of 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.

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

Figures 11 and 12 are stained photographs showing the condition of the abdominal wall (AW) and cecum (C) two weeks after the application of commercially available Seprafilm and Tenaleaf, respectively, to the laceration. As shown in Figures 11 and 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.

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

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

The silk fibroin film of the present invention can be used as an adhesion prevention 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).

Although the preferred embodiments of the present invention have been described above, those skilled in the art will easily imagine various changes and modifications within the obvious scope upon reading this specification.
Therefore, such changes and modifications are intended to be within the scope of the invention as defined by the claims.

Claims (11)

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. A silk fibroin film in which, when the contact angle of water on the film surface is measured, the change in contact angle is -5°/sec or less within 5 seconds after a 5μL droplet of ultrapure water is dropped onto the film surface. The 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. The 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. The 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. The 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. 　A silk fibroin film according to any one of claims 1 to 6, the silk fibroin film having a moisture content of 30% or less. A silk fibroin film containing silk fibroin having a weight-average molecular weight of 40-150 kDa and a molecular weight distribution (PDI) of 1-10. An adhesion prevention material comprising the silk fibroin film according to any one of claims 1 to 8. A method for manufacturing 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. 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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