Anti-adhesion material and method for manufacturing the same

Abstract

Provided is an anti-adhesive material containing particles, in which the particles comprise a crosslinked product of a gelatin mixture composed of a first gelatin and a second gelatin having such a structure that a hydrocarbon group is introduced into the first gelatin, the ratio of the introduction of hydrocarbon groups in the gelatin mixture is 30 mol% to 50 mol%, and the second gelatin has a structure represented by formula (1). The anti-adhesive material has excellent adhesiveness on a tissue and excellent adhesion stability under an aqueous environment, has an easy operability, and is easy to be produced on an industrially large scale. In formula (1), Gltn represents a residue of gelatin; L represents a single bond or a bivalent linking group; R1 represents an alkyl group having 1 to 20 carbon atoms; and R2 represents a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.

Description

[Technical Field]

[0001] The present invention relates to an anti-adhesion material and a method for producing an anti-adhesion material. [Background technology]

[0002] Postoperative adhesions, in which organs adhere to the abdominal wall due to the healing process or infection at the surgical site, frequently occur after surgery. The incidence rate is high, ranging from 67% to 93%, with 15% to 18% requiring reoperation. Postoperative adhesions cause pain and intestinal obstruction, making reoperation difficult. As a result, it has been reported that reoperation times increase by an average of 18 minutes. Therefore, new strategies to prevent postoperative adhesions are needed.

[0003] Postoperative adhesions are caused by excessive fibrin deposition between the peritoneum and organs due to an intense inflammatory reaction at the wound site, followed by the migration of inflammatory cells, including fibroblasts and macrophages, within 3 to 5 days. Therefore, one promising strategy for preventing postoperative adhesions is to introduce physical barrier materials that prevent fibrin deposition and the migration of inflammatory cells from other wound sites. Reported adhesion barriers (physical barrier materials) can be broadly classified into three types: (1) solution-type, (2) film-type, and (3) hydrogel-type.

[0004] (1) Solution-type adhesion barriers, including hyaluronic acid solutions, have been shown to prevent organ adhesion to the peritoneum, and (2) film-type adhesion barriers are also widely used in clinical settings. For example, films composed of carboxymethylcellulose / sodium hyaluronate (HA / CMC) are often used to prevent postoperative adhesions. (3) Hydrogel-type adhesion barriers are characterized by excellent injectability, rapid in situ gelation, and conformability to tissue shape. Examples of hydrogel-type adhesion barriers that have been reported include carboxymethyldextrin and amino-aldehyde-modified hyaluronic acid.

[0005] Incidentally, the inventor has reported tissue-adhesive microparticles (gelatin derivative microparticles) using decyl-group-modified Alaska pollack gelatin (for example, Patent Document 1). These particles have excellent adhesive strength to biological tissues when applied to wound dressings, etc., and are therefore expected to be used as adhesion barriers. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2020 / 137903 Summary of the Invention [Problem to be solved by the invention]

[0007] However, conventional adhesion barriers have various issues. For example, (1) solution-type adhesion barriers have low stability on tissue and are quickly absorbed. (2) film-type adhesion barriers lack flexibility and stick to surgical gloves. It has been reported that their fragility makes them difficult to introduce into the abdominal cavity, especially in laparoscopic surgery. Furthermore, (3) hydrogel-type adhesion barriers form a hydrogel in situ by mixing two solutions using a dual syringe. The preparation of the hydrogel requires two liquids, a pre-gel solution and a crosslinking solution, and requires dedicated syringes, making the preparation process complicated.

[0008] For this reason, there has been a demand for a new anti-adhesion material that has excellent adhesive properties on tissues, excellent adhesive stability in an aqueous environment, and easy operability. Patent Document 1 discloses that gelatin derivative microparticles have excellent tissue adhesiveness, aqueous stability, etc. However, further improvements were required for application to anti-adhesion materials. Furthermore, anti-adhesion materials were also required to have properties that allow for easy industrial mass production.

[0009] The present invention aims to solve the above-mentioned problems. That is, an object of the present invention is to provide a new anti-adhesion material that has excellent adhesive properties on tissues and excellent adhesive stability in an aqueous environment, is easy to handle, and can be easily mass-produced industrially. [Means for solving the problem]

[0010] As a result of extensive research into achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0011] [1] An anti-adhesion material containing particles, The particles comprise a crosslinked mixture of a first gelatin and a second gelatin in which a hydrocarbon group has been introduced into the first gelatin, the introduction rate of hydrocarbon groups in the gelatin mixture is 30 mol% to 50 mol%, An anti-adhesion material in which the second gelatin has a structure represented by formula (1) described below. [2] The anti-adhesion material according to [1], wherein in formula (1), L is a single bond or —C(O)—. [3] The anti-adhesion material of [2], wherein in formula (1), L is a single bond. [4] In equation (1), R 1 is a straight chain alkyl group, and R 2 The anti-adhesion material according to any one of [1] to [3], wherein is a hydrogen atom. [5] The anti-adhesion material according to any one of [1] to [4], wherein the hydrocarbon group introduction rate of the second gelatin is higher than the hydrocarbon group introduction rate of the gelatin mixture. [6] The anti-adhesion material according to any one of [1] to [5], wherein the hydrocarbon group introduction rate of the second gelatin is 35 mol % to 80 mol %. [7] The anti-adhesion material according to any one of [1] to [6], wherein the introduction rate of hydrocarbon groups in the gelatin mixture is 35 mol % to 45 mol %. [8] The anti-adhesion material according to any one of [1] to [7], wherein the ratio of the mass of the second gelatin to the total mass of the first gelatin and the second gelatin is 35% by mass to 90% by mass. [9] The anti-adhesion material according to any one of [1] to [8], wherein the first gelatin is an alkali-treated gelatin.

[10] The adhesion barrier according to any one of [1] to [9], wherein the first gelatin is a gelatin that has been treated to reduce endotoxin levels.

[11] The anti-adhesion material according to any one of [1] to

[10] , wherein the first gelatin is derived from cold-water fish.

[12] The adhesion barrier according to any one of [1] to

[11] , which can be applied to an affected area of ​​a living body using a spray system.

[13] A method for producing an anti-adhesion material according to any one of [1] to

[12] , dissolving the gelatin mixture of the first gelatin and the second gelatin in a good solvent to prepare a gelatin mixture solution; adding a poor solvent to the gelatin mixture solution to precipitate first intermediate particles containing the gelatin mixture in the gelatin mixture solution; freeze-drying the gelatin mixture solution containing the first intermediate particles to obtain second intermediate particles; A method for producing an adhesion preventing material, comprising cross-linking the gelatin mixture of the second intermediate particles to obtain particles of the cross-linked gelatin mixture.

[14] The method for producing an anti-adhesion material according to

[13] , wherein the second intermediate particles are heated to crosslink the gelatin mixture. [Effects of the Invention]

[0012] The anti-adhesion material of the present invention has excellent adhesive properties on tissues and adhesive stability in an aqueous environment, is easy to handle, and can be easily mass-produced industrially. [Brief explanation of the drawings]

[0013] [Figure 1] 1A and 1B are schematic diagrams illustrating the configuration and manufacturing method of the anti-adhesion material (particles) of the present embodiment. [Figure 2] FIG. 2 is a schematic diagram illustrating the function of the anti-adhesion material (particles) of the present embodiment. [Figure 3]FIG. 1 is a schematic diagram illustrating the function of the anti-adhesion material (particles) of the present embodiment in a living body (rat). [Figure 4] FIG. 2 is a schematic diagram illustrating application of the anti-adhesion material (particles) of this embodiment using a spray system. [Figure 5A] 1 shows SEM (Scanning Electron Microscope) images of particles prepared in Examples. The scale bar indicates 10 μm. [Figure 5B] FIG. 1 is a diagram showing the particle size distribution of particles produced in the examples. [Figure 6] 1 is a photograph showing particles prepared in the examples being sprayed using an endoscopic spray device. [Figure 7A] 1 is a graph showing the time dependence of the water content of colloidal gels of particles prepared in Examples (the relationship between hydration time and water content). [Figure 7B] 1 is a bright-field micrograph showing the aggregation behavior of particles prepared in the examples, with the scale bar indicating 20 μm. [Figure 8A] FIG. 1 is a schematic diagram illustrating the procedure of an in vitro tissue adhesion / non-adhesion test in an example. [Figure 8B] 1 is a graph showing the relationship between hydration time and adhesive strength in an in vitro tissue adhesion / non-adhesion test in an example. [Figure 8C] 1 shows hematoxylin and eosin (HE) stained images of the tissue and colloid gel fixed to the upper and lower jigs after an in vitro tissue adhesion / non-adhesion test in an example. [Figure 9A] 1 shows hematoxylin and eosin (HE) stained images of tissue and colloidal gel after an aqueous stability test in an example. [Figure 9B] 1 is a graph showing the area of ​​colloid gel remaining on tissue after an aqueous stability test in an example. [Figure 10A]Photographs illustrating an experiment in which an anti-adhesion material (particles) was applied to an SD rat cecum-abdominal wall adhesion model in an example: (a) Photograph of a normal cecum and peritoneum, (b) Photograph of a cecum abrasion and an abdominal wall defect (labeled "Defect" in the figure) formed, (c) Photograph of particles (MPs) prepared in the example applied to the entire defect area, (d) Photograph of particles prepared in the example hydrated with saline to form a colloidal gel on the defect. [Figure 10B] 1 shows photographs illustrating the results of an experiment in which an anti-adhesion material (particles) was applied to an SD rat cecum-abdominal wall adhesion model in an example. [Figure 10C] 1 is a graph showing the results of an experiment in which an adhesion barrier (particles) was applied to an SD rat cecum-abdominal wall adhesion model (adhesion score one week after application (W1, 1 week)) in an example. [Figure 10D] 1 is a graph showing the results of an experiment in which an adhesion barrier (particles) was applied to an SD rat cecum-abdominal wall adhesion model (adhesion score 2 weeks after application (W2, 2 weeks)) in an example. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will be described in detail below. The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0015] In the description of groups (atomic groups) in this specification, when a notation does not specify whether the group is substituted or unsubstituted, it encompasses both unsubstituted and substituted groups within the scope of the effects of the present invention. For example, the term "alkyl group" encompasses not only alkyl groups without a substituent (unsubstituted alkyl groups) but also alkyl groups with a substituent (substituted alkyl groups). This also applies to each compound.

[0016] The anti-adhesion material of this embodiment contains particles, which contain a cross-linked gelatin mixture. The gelatin mixture contains raw gelatin (an example of a "first gelatin") and hydrophobized gelatin (an example of a "second gelatin") in which hydrocarbon groups have been introduced into the raw gelatin.

[0017] <Raw material gelatin (first gelatin)> The raw material gelatin may be naturally derived or synthetic (including fermented and genetically modified). Alternatively, it may be natural or synthetic gelatin that has been subjected to some kind of processing. More specifically, examples include naturally derived gelatin obtained from the skin, bones, tendons, etc. of mammals, birds, fish, etc.; and processed gelatin obtained by treating naturally derived gelatin with acid or alkali (and optionally heat-extracting). Among these, alkali-treated gelatin is preferred because it can produce particles that have better effects of the present invention.

[0018] The raw material gelatin is preferably endotoxin-reduced gelatin with a reduced endotoxin content. There are no particular limitations on the type of endotoxin-reduced gelatin, and any known gelatin can be used. For example, the gelatin described in JP 2007-231225 A can be mentioned. The contents of this document are incorporated herein by reference.

[0019] Examples of mammal-derived gelatin include porcine and bovine gelatin. Fish-derived gelatin is not particularly limited, but gelatin derived from cold-water fish (cold-water fish) such as salmon, trout, cod, Alaska pollock, sea bream, tilapia, and tuna (hereinafter also referred to as "cold-water fish-derived gelatin") is preferred.

[0020] Cold-water fish-derived gelatin is a polymer in which two or more amino acids are linked in a linear chain. Cold-water fish-derived gelatin has 190 or fewer imino acids per 1,000 constituent amino acids. More specifically, it has 80 or fewer hydroxyprolines and 110 or fewer prolines. The room-temperature fluidity of cold-water fish-derived gelatin is thought to be due to the number of hydroxyprolines being 80 or fewer, or the number of prolines being 110 or fewer. If either of these conditions is met, the denaturation temperature is thought to be below room temperature, resulting in room-temperature fluidity.

[0021] Thai gelatin has 73 hydroxyprolines, 108 prolines, and a denaturation temperature of 302.5 K. Tilapia gelatin has 82 hydroxyprolines, 110 prolines, and a denaturation temperature of 309 K. In comparison, porcine gelatin has 95 hydroxyprolines, 121 prolines, and a denaturation temperature of 316 K.

[0022] Furthermore, the amino acid sequence of gelatin derived from cold-water fish is similar to that of gelatin derived from animals, and it can be easily decomposed by enzymes. It also has high biocompatibility.

[0023] The molecular weight of the starting gelatin is not particularly limited, but the weight average molecular weight (Mw) is preferably 5,000 to 100,000, more preferably 10,000 to 50,000, and even more preferably 20,000 to 40,000. In this specification, the weight average molecular weight means the weight average molecular weight determined by gel permeation chromatography (GPC).

[0024] The raw material gelatin may consist of only one type of gelatin, or may be a mixture of two or more types of gelatin.

[0025] <Hydrophobic gelatin (secondary gelatin)> In this specification, "hydrophobicized gelatin" means a gelatin derivative in which a hydrocarbon group has been introduced into raw gelatin. Hydrophobicized gelatin has a structure represented by the following formula (1).

[0026] [ka] In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, and R 1 is an alkyl group having 1 to 20 carbon atoms, and R 2 is a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.

[0027] In formula (1), GltnNH- is a structure derived from the raw gelatin described above. Therefore, raw gelatin is represented by GltnNH2. -CHR 1 R 2 is a hydrocarbon group introduced into the starting gelatin, and is introduced into the starting gelatin via L (a single bond or a divalent linking group).

[0028] The divalent linking group for L is not particularly limited, but examples thereof include -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -N(R)- (wherein R represents a hydrogen atom or a monovalent organic group (preferably a hydrocarbon group having 1 to 20 carbon atoms)), an alkylene group (preferably an alkylene group having 2 to 10 carbon atoms), an alkenylene group (preferably an alkenylene group having 2 to 10 carbon atoms), and combinations thereof. Of these, -C(O)- is preferred. L is preferably a single bond or -C(O)-.

[0029] In formula (1), -CHR 1 R 2 The (hydrocarbon group) is preferably bonded to the ε-amino group of the starting gelatin, and more preferably to the ε-amino group of lysine (Lys) in the starting gelatin. The (hydrocarbon group) is bonded to an amino group, preferably the amino group of lysine, with or without a linking group (in other words, directly) by *-CHR 1 R 2Examples of methods for binding include the so-called reductive amination reaction (a method using an aldehyde or ketone) and the Schotten-Baumann reaction (a method using an acid chloride).

[0030] The -NH- structure (secondary amino group) of formula (1) is, for example, expressed at 3300 cm in an FT-IR (Fourier transform infrared) spectrum. -1 It can be detected by nearby bands.

[0031] In formula (1), R 1 and R 2 The hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, and examples thereof include a chain hydrocarbon group having 1 to 20 carbon atoms, an alicyclic hydrocarbon group having 3 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 14 carbon atoms, and groups formed by combining these.

[0032] R 2 is 1 to 20 hydrocarbon groups, R 2 is R 1 may be the same as or different from R 1 , and R 2 The alkyl group may be linear or branched.

[0033] The chain hydrocarbon group having 1 to 20 carbon atoms is not particularly limited, but examples thereof include methyl, ethyl, propyl, butyl, hexyl, octyl (or capryl), nonyl (or pelargoryl), decyl, dodecyl (or lauryl), and tetradecyl (or myristyl). Among these, R 1 is preferably an alkyl group having 1 to 13 carbon atoms, more preferably an alkyl group having 7 to 12 carbon atoms, even more preferably an alkyl group having 8 to 11 carbon atoms, and particularly preferably an alkyl group having 9 to 11 carbon atoms. 2 Although there are no particular limitations on the group, it is preferably a hydrogen atom.

[0034] Examples of the alicyclic hydrocarbon group having 3 to 20 carbon atoms include a cyclopropyl group, a cyclopentyl group, a cyclohexyl group, an adamantyl group, and a norbornyl group.

[0035] The aromatic hydrocarbon group having 6 to 14 carbon atoms is not particularly limited, but examples thereof include a phenyl group, a tolyl group, and a naphthyl group.

[0036] The group obtained by combining the above is not particularly limited, but examples thereof include aralkyl groups having 6 to 12 carbon atoms such as a benzyl group, a phenethyl group, a naphthylmethyl group, and a naphthylethyl group.

[0037] In addition, hydrocarbon groups (-CHR 1 R 2 The total number of carbon atoms in the hydrocarbon group (-CHR) is preferably 9 to 20, 9 to 18, or 9 to 14. 1 R 2 ) in R 1 is a straight chain alkyl group, and R 2 is preferably a hydrogen atom or a linear alkyl group, and R 1 is a straight chain alkyl group, and R 2 is more preferably a hydrogen atom.

[0038] The hydrophobized gelatin represented by formula (1) is preferably at least one selected from the group consisting of the following formulas (2) and (3), and more preferably a gelatin derivative represented by formula (2).

[0039] [ka] [ka]

[0040] Formula (2) is the case where L is a single bond in Formula (1). In Formula (2), 1 R 2) is introduced to the gelatin residue Gltn of the raw gelatin via an imino bond (-NH-). Formula (3) is the case where L is -C(O)- in Formula (1). In Formula (3), the hydrocarbon group (-CHR 1 R 2 ) is introduced to the gelatin residue Gltn of the raw gelatin via an amide bond (-NHCO-). In formulas (2) and (3), the meanings of the symbols are the same as those in formula (1) already explained, and the preferred embodiments are also the same.

[0041] The hydrophobized gelatin may consist of only one type of gelatin derivative, or may be a mixture of two or more types of gelatin derivatives.

[0042] Here, the ratio of the content of imino groups (-NH-CHR) in hydrophobicized gelatin to the content of amino groups (-NH) in gelatin (raw gelatin) before hydrophobicization is 1 R 2 The molar ratio of the content of each of the above is defined as the "hydrocarbon group introduction rate."

[0043] The hydrocarbon group introduction rate of the hydrophobized gelatin is not particularly limited, as long as it is higher than the hydrocarbon group introduction rate of the gelatin mixture described below. The hydrocarbon group introduction rate of the hydrophobized gelatin may be, for example, 35 mol% to 80 mol%, 40 mol% to 70 mol%, or 45 mol% to 60 mol%. In other words, the imino group / amino group (molar ratio) in the hydrophobized gelatin may be 35 / 65 to 80 / 20, 40 / 60 to 70 / 30, or 45 / 55 to 60 / 40. When the hydrocarbon group introduction rate of the hydrophobized gelatin is within the above range, it becomes easier to adjust the hydrocarbon group introduction rate of the gelatin mixture described below to a specific range. As a result, an appropriate level of hydrophobicity is imparted to the anti-adhesion material (particles) of this embodiment.

[0044] In the embodiment, the hydrocarbon group introduction rate is calculated from the values ​​obtained by quantifying the number of amino groups in the raw gelatin and the number of amino groups in the hydrophobized gelatin using the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method) using the following formula: Hydrocarbon group introduction rate (mol%) of hydrophobicized gelatin = [Number of amino groups in raw gelatin - Number of amino groups in hydrophobicized gelatin] / [Number of amino groups in raw gelatin] x 100

[0045] Hydrocarbon groups (-CHR) introduced into one molecule of hydrophobic gelatin 1 R 2 The number of hydrocarbon groups is not particularly limited. The number of hydrocarbon groups can be appropriately adjusted based on the molecular weight of the starting gelatin so that the hydrocarbon group introduction rate is a predetermined value. For example, the number of hydrocarbon groups in one molecule of hydrophobized gelatin may be 5 to 10, 6 to 9, or 6 to 8.

[0046] The molecular weight of hydrophobized gelatin is not particularly limited. The molecular weight of hydrophobized gelatin is determined by the molecular weight of the raw material gelatin and the type and amount (number) of hydrocarbon groups introduced. Therefore, the range of the weight average molecular weight (Mw) of hydrophobized gelatin is almost the same as the range of the weight average molecular weight of the raw material gelatin mentioned above.

[0047] The hydrophobic gelatin described above may be a commercially available product or may be a self-synthesized product. Hydrophobic gelatin may be synthesized by introducing a hydrophobic group (hydrocarbon group) into raw material gelatin, for example, by the synthesis method disclosed in Patent Document 1. The content of the synthesis method for hydrophobic gelatin (gelatin derivative) disclosed in Patent Document 1 is incorporated herein by reference.

[0048] <Particle> The particles of this embodiment contain a cross-linked product of a gelatin mixture. The gelatin mixture is a mixture of the above-mentioned raw gelatin (first gelatin) and hydrophobized gelatin (second gelatin) (see FIG. 1). As described above, the raw gelatin and hydrophobized gelatin have the same basic skeleton. Therefore, the cross-linking reaction proceeds uniformly in this mixture, and the composition of the resulting particles (cross-linked product) is also uniform, making it easier to achieve the effects of this embodiment described below.

[0049] The hydrocarbon group introduction rate of a gelatin mixture can be calculated by the following formula (I).

[0050]

number

[0051] The hydrocarbon group introduction rate of the gelatin mixture may be 30 mol% to 50 mol%, or 35 mol% to 45 mol%. When the hydrocarbon group introduction rate of the gelatin mixture is within this range, the anti-adhesion material (particles) can be imparted with appropriate hydrophobicity, making it easier to achieve the effects of this embodiment described below.

[0052] The mixing ratio between the starting gelatin and the hydrophobized gelatin is not particularly limited. The mixing ratio can be adjusted based on the hydrocarbon group introduction rate of the hydrophobized gelatin. In one embodiment, the mixing ratio can be appropriately adjusted so that the hydrocarbon group introduction rate of the gelatin mixture is a specific value. For example, the mass ratio (Xc) of the second gelatin relative to the total mass of the starting gelatin and the hydrophobized gelatin can be 35% to 90% by mass, 35% to 70% by mass, or 35% to 50% by mass.

[0053] The particles of this embodiment contain a crosslinked gelatin mixture (a crosslinked gelatin mixture). In this specification, "crosslinked" does not include a reversible physical crosslinked structure, but refers to a crosslinked structure obtained by an irreversible crosslinking reaction. Therefore, a "crosslinked gelatin mixture (a crosslinked gelatin mixture)" has an irreversible crosslinked structure obtained by a crosslinking reaction. The crosslinking reaction occurs when energy is applied to the gelatin mixture by heat, light, energy rays, or the like. The crosslinking reaction can also occur with a crosslinking agent. The crosslinking reaction in a crosslinked gelatin mixture can be caused by one or both of the above methods.

[0054] The particles may be a gelatin mixture crosslinked with a crosslinking agent (a compound different from the raw gelatin and hydrophobized gelatin). Using no crosslinking agent is preferred because it is safe and does not generate impurities derived from the crosslinking agent. In this case, crosslinking occurs, for example, through a reaction between functional groups (-NH, -OH, -SH, -COOH, etc.) on the side chains of gelatin. Particles containing a crosslinked gelatin mixture have increased adhesive strength and are therefore more suitable for use as adhesion barriers.

[0055] In an embodiment of the present invention, the particles may be composed solely of a cross-linked gelatin mixture, or may contain other components as long as the effects of the present invention are achieved. The proportion (content) of the crosslinked gelatin mixture in the particles is not particularly limited, and the proportion of the crosslinked gelatin may be 100% by mass, 98% by mass or more, or 90% by mass or more, in order to easily obtain particles having better effects of the present invention. Other components that the particles may contain include, but are not limited to, solvents, buffers, colorants, preservatives, excipients, and drugs (antithrombotic drugs, antibacterial agents, growth factors, etc.).

[0056] The average particle size of the particles of this embodiment is not particularly limited, but is, for example, 0.5 μm to 50 μm, preferably 1 μm to 30 μm, and more preferably 1 μm to 10 μm. In this specification, the "average particle size" is a value determined by measuring the particle sizes (major axes) of 100 particles randomly selected using an electron microscope and averaging the measured values.

[0057] <Anti-adhesion material> The particles described above can be used as an adhesion barrier. An adhesion barrier is defined as a bioabsorbable synthetic material that is used directly at the site of surgery for the purpose of reducing postoperative adhesions (Ministry of Health, Labour and Welfare Notification No. 402 of 2016, "Specific Medical Materials and Their Material Prices (Material Price Standards)"). Adhesion barriers are generally called "absorbable adhesion barriers."

[0058] The anti-adhesion material of this embodiment has the following advantages. First, the adhesion preventing material of this embodiment is in the form of particles, and therefore is easier to handle than sheet-type adhesion preventing materials. Furthermore, the adhesion preventing material (powder) of this embodiment is a one-component type and does not require a crosslinking agent or the like. Therefore, it is less expensive and easier to handle than two-component types. The adhesion preventing material of this embodiment can be applied to the affected area (defective area) using a general-purpose spray system. This makes it easy to use not only in laparoscopic surgery but also in endoscopic surgery.

[0059] 4 is a schematic diagram illustrating application of the anti-adhesion material (particles) of this embodiment using a spray system. A defect 37 exists in tissue 41 within a living body. Particles 10 (e.g., "C10-MPs"), which are the anti-adhesion material, are applied (sprayed) to this defect using a spray device ("Spray" in the figure). That is, the anti-adhesion material (particles) of this embodiment has the feature of being "sprayable" onto the defect area 37.

[0060] The anti-adhesion material (particles) of this embodiment contains a cross-linked gelatin mixture. As described above, the gelatin mixture has a hydrocarbon group introduction rate within a specific range. As a result, the particles of this embodiment have appropriate hydrophobicity. Due to this moderate hydrophobicity, for example, when the particles are applied to the affected tissue during surgery, they hydrate and fuse together through hydrophobic interactions to form a colloidal gel layer, which acts as a physical barrier and exhibits adhesion prevention properties.

[0061] FIG. 2 is a schematic diagram illustrating the function of the anti-adhesion material (particles). First, particles 10, which are an adhesion barrier, adhere to tissue (A). The tissue has a layered structure of a serous membrane layer 22, a muscular layer 23, a submucosal layer 24, and a mucosal layer 25, and a water layer 26 exists on the surface. When particles 10 adhere to tissue, they are gradually hydrated by a water layer 26 (B). Particles 10 that have adhered to the tissue surface form a layer of colloidal gel 38 (colloidal gel layer) on the serous membrane layer 22 (C). The colloidal gel 38 layer strongly adheres to the serous membrane layer 22 due to hydrophobic interactions 21. On the other hand, colloidal gel 38 exhibits non-adhesive properties 20 with other tissues.

[0062] 3 is a schematic diagram illustrating the function of the anti-adhesion material (particles) of this embodiment in a living body (rat). Defects 37 (37a, 37b) are present inside the abdomen 31 (peritoneal cavity 36) and in the cecum 39 of a rat 30. Image 32 shows the state before treatment with the anti-adhesion material (particles 10). Next, anti-adhesion material (particles 10) is applied to the defect 37 (arrow 44). Image 33 shows the state after the anti-adhesion material (particles 10) has been applied. Particles 10 are applied to the defect 37a, 37b, respectively, and hydrated. This results in the formation of layers of colloidal gels 38a, 38b on the defect 37a, 37b.

[0063] Image 34 is a schematic diagram of a tissue cross section at portion AB. A layer of colloidal gel 38b made of hydrated particles 10 is formed on tissue 41. The colloidal gel 38b layer acts as a physical barrier to other tissues (arrow 42). On the other hand, it exhibits tissue adhesiveness on the tissue 41 side (arrow 43) due to hydrophobic interactions between particles 10 and tissue 41. This prevents adhesion between tissue 41 and other tissues.

[0064] The case where no anti-adhesion material (particles 10) is used (arrow 45) will be described. Image 35 shows the state where no anti-adhesion material (particles 10) is used. In this case, defective areas 37a and 37b are adhered 40 to each other.

[0065] The colloidal gel layer of the particles of this embodiment, which have moderate hydrophobicity, absorbs moisture from the surface of the affected tissue and forms hydrophobic interactions between the particles and the affected tissue, resulting in strong adhesion to the affected surface. However, after complete hydration, the colloidal gel layer no longer exhibits adhesiveness to other tissues due to the presence of moisture on the surface of the colloidal gel layer and on the tissue surface (the surface of other tissues different from the tissue on which the colloidal gel layer is formed). Thus, the adhesion barrier (particles) of this embodiment possesses both adhesive properties to the affected tissue and non-adhesive properties (anti-adhesion ability) to other tissues (see Figures 2 and 3). Conventional adhesion barriers function as a physical barrier, but have the problem of low adhesiveness to soft tissues in a wet state, resulting in easy detachment from the tissue defect (affected area). The adhesion barrier of this embodiment can solve this problem as described above.

[0066] Anti-adhesion materials are required to remain stable at the affected area in a moist state for a certain period of time. If the anti-adhesion material is not stably adhered to the affected area under physiological conditions (moist conditions), there is a risk that postoperative adhesions will be induced due to fibrin deposition and fibroblast migration. The anti-adhesion material of this embodiment, which has moderate hydrophobicity, forms hydrophobic interactions between the colloidal gel layer and the affected tissue, thereby exhibiting high stability even in a moist state.

[0067] Digestive organs such as the stomach, duodenum, and large intestine frequently contract due to peristaltic movement. Therefore, the ability of an anti-adhesion material to conform to changing shapes on tissues is an important property. In the anti-adhesion material of this embodiment, the colloidal gel layer firmly adheres to the affected tissue and exhibits excellent conformability to tissue deformation. Furthermore, the anti-adhesion material of this embodiment has the advantage that after the colloidal gel layer exerts its adhesion prevention effect, the colloidal gel layer is rapidly degraded and absorbed.

[0068] Furthermore, when anti-adhesion materials (particles) are produced from only one type of hydrophobicized gelatin, the procedure for adjusting the hydrocarbon group introduction rate to a specific value is complicated, as it is necessary to adjust the hydrocarbon group introduction rate while taking into account yield and other factors, and also to carry out measurements to confirm the adjustment. The anti-adhesion material (particles) of this embodiment is advantageous in that it uses a mixture of raw gelatin (first gelatin) and hydrophobized gelatin (second gelatin). The anti-adhesion material (particles) of this embodiment can be prepared by simply adjusting the mixing ratio (A) of raw gelatin and hydrophobized gelatin based on the hydrocarbon group introduction rate (Xc) of the hydrophobized gelatin, which makes it easy to adjust the hydrocarbon group introduction rate (Xm) of the gelatin mixture, i.e., the hydrophobicity of the anti-adhesion material (particles) (see formula (I) above).

[0069] Furthermore, by changing the mixing ratio (A), it is possible to produce multiple types of adhesion preventing materials with different degrees of hydrophobicity from one type of hydrophobicized gelatin. Furthermore, compared to when an adhesion preventing material is produced from only one type of hydrophobicized gelatin, the amount of hydrophobicized gelatin used can be reduced. This is because the adhesion preventing material of this embodiment is produced by mixing raw material gelatin with hydrophobicized gelatin. Because hydrophobicized gelatin is synthesized from raw gelatin, the synthesis is time-consuming and the production costs are higher than those of raw gelatin. The anti-adhesion material of this embodiment is also superior in that the amount of hydrophobicized gelatin required to produce particles with a predetermined hydrophobicity is relatively small. This is because the synthesis time and production costs depend largely not on the amount of hydrophobic groups introduced into the raw gelatin, but on the amount of hydrophobicized gelatin to be synthesized and the number of times it is synthesized. As a result, the synthesis time and effort can be saved and production costs can be reduced. The anti-adhesion material of this embodiment, which has the advantages described above, is suitable for industrial mass production, and mass production makes it possible to further reduce manufacturing costs.

[0070] The particles of this embodiment are used as an adhesion inhibitor, but can also be used as a wound dressing. That is, they can be used as a component having two functions: wound dressing and adhesion prevention. For example, when applied to a postoperative injury, a film having both wound dressing and adhesion prevention effects is formed. Compared to the conventional method of applying a wound dressing and an adhesion inhibitor separately, both can be achieved more easily. Furthermore, the particles according to the embodiment of the present invention have excellent blood clotting ability and can also be used as a hemostatic material. In addition to the functions of wound dressing and adhesion prevention, they can also be used to form a component having blood clotting ability.

[0071] <Particle manufacturing method> The method for producing the particles of this embodiment is not particularly limited. For example, they may be produced using the coacervation method disclosed in Patent Document 1. The content of the particle production method disclosed in Patent Document 1 is incorporated herein by reference. An example of the method for producing the particles of this embodiment will be described below.

[0072] The manufacturing method of this embodiment includes, for example, the following steps. Step 1: A step of dissolving a gelatin mixture of raw material gelatin and hydrophobized gelatin in a good solvent to prepare a gelatin mixture solution; Step 2: adding a poor solvent to the gelatin mixture solution to precipitate first intermediate particles containing the gelatin mixture in the gelatin mixture solution; Step 3: freeze-drying the gelatin mixture solution containing the first intermediate particles to obtain second intermediate particles; Step 4: Crosslinking the gelatin mixture of the second intermediate particles to obtain particles of a crosslinked gelatin mixture.

[0073] Step 1: Step 1 is a step of dissolving the gelatin mixture in a good solvent as already explained to obtain a gelatin mixture solution. In this specification, a good solvent means a solvent that can easily dissolve the gelatin mixture. The type of good solvent is not particularly limited, but examples include water, glycerin, acetic acid, and mixtures thereof, and among these, it is preferable that the good solvent contains water. In addition, the good solvent may be heated. The temperature during heating is not particularly limited, but 50°C to 70°C is preferred.

[0074] The method for dissolving a gelatin mixture in a good solvent is not particularly limited, and known methods can be used. For example, a method in which a good solvent at a low temperature (for example, room temperature) is added to a gelatin mixture to swell the gelatin mixture, and the resulting swollen body is heated to obtain a gelatin mixture solution (swelling dissolution method), and a method in which a gelatin mixture is poured into the above-mentioned good solvent that has been preheated to obtain a gelatin mixture solution (direct dissolution method) can be used.

[0075] The content of the gelatin mixture in the gelatin mixture solution is not particularly limited, but the content (final concentration) of the gelatin mixture relative to the total volume of the gelatin mixture solution is preferably 0.01 mass / volume % to 30 mass / volume %, more preferably 1 mass / volume % to 25 mass / volume %, even more preferably 5 mass / volume % to 20 mass / volume %, and particularly preferably 5 mass / volume % to 15 mass / volume %.

[0076] Step 2: Step 2 is a step of adding a poor solvent to the gelatin mixture solution to precipitate first intermediate particles containing the gelatin mixture in the gelatin mixture solution (coacervation). In this specification, a poor solvent means a solvent that is more difficult to dissolve the gelatin mixture in compared to the good solvent used in step 1. That is, in this specification, a good solvent and a poor solvent are not defined by the absolute amount of solubility of the gelatin mixture, but are defined relatively in relation to the poor solvent and the good solvent, respectively.

[0077] The poor solvent is not particularly limited, but examples thereof include organic solvents, and among these, water-soluble organic solvents are preferred, and alcohols such as methanol, ethanol, propanol, isopropanol, butanol, and t-butyl alcohol are more preferred.

[0078] When a poor solvent is added to the gelatin mixture solution, first intermediate particles are precipitated in the gelatin mixture solution. These first intermediate particles are particulate matter containing the above-mentioned gelatin mixture. The particle size of the first intermediate particles precipitated in this step is not particularly limited, but is preferably 0.1 μm to 100 μm, more preferably 1 μm to 50 μm, and even more preferably 1 μm to 10 μm. When the particle size is within the above range, the intermediate particles precipitated in the gelatin mixture solution are less likely to settle. As a result, aggregation of the intermediate particles is more likely to be suppressed in the freeze-drying process in step 3 described below.

[0079] The temperature at which the poor solvent is added is not particularly limited, but is generally preferably 10° C. to 30° C., more preferably 15° C. to 25° C. When the solvent is heated to dissolve the gelatin mixture in step 1, it is preferable to further include a step of cooling the gelatin mixture solution between steps 1 and 2.

[0080] When the poor solvent is added dropwise, it is preferable to stir the gelatin mixture solution. The stirring method is not particularly limited, and any known method can be used. By adding the poor solvent while stirring the gelatin mixture solution, the precipitated particles are less likely to aggregate and less likely to settle.

[0081] Step 3: Step 3 is a step of freeze-drying the dispersion solution of uncrosslinked gelatin particles (first intermediate particles) precipitated by the above coacervation to obtain second intermediate particles. The method for freezing the gelatin mixture solution is not particularly limited, but it is preferable to freeze it more quickly. This makes it more difficult for particles containing the uncrosslinked gelatin mixture to aggregate during freezing. The ambient temperature during freezing is not particularly limited, but is preferably -20°C or lower, more preferably -30°C or lower. The freeze-drying method is also not particularly limited, and any known method can be used. The second intermediate particles are particles containing the first intermediate particles. The second intermediate particles may contain components other than the first intermediate particles. Examples of such components include the good solvent and poor solvent described above.

[0082] Step 4: Step 4 is a step of crosslinking the gelatin mixture of the second intermediate particles to obtain particles containing a crosslinked gelatin mixture (crosslinked product). Through this step, the gelatin mixture of the particles is irreversibly crosslinked between molecules and / or intramolecularly. As a result, particles containing a crosslinked product of the gelatin mixture are obtained.

[0083] The crosslinking method is not particularly limited, and examples thereof include a method of applying thermal energy to a gelatin mixture or irradiating it with actinic rays or radiation (e.g., electron beams, etc.). Among these, a method of applying thermal energy (in other words, heating) (thermal crosslinking) is preferred because it allows a crosslinked product of the gelatin mixture to be obtained more easily and is safer without generating impurities derived from the crosslinking agent. In this method, for example, amino groups in the gelatin mixture react with other reactive groups (e.g., carboxy groups, mercapto groups, etc.) to form a crosslinked structure.

[0084] The thermal crosslinking method is not particularly limited and any known method can be used. For example, the thermal crosslinking method may include placing the container containing the second intermediate particles in a heated atmosphere (for example, in an oven) and maintaining the container for a predetermined period of time.

[0085] The heating temperature during thermal crosslinking is not particularly limited, but is generally preferably 80°C to 200°C, and more preferably 100°C to 200°C. The heating time during thermal crosslinking is not particularly limited, but is generally preferably 0.1 to 20 hours, more preferably 0.5 to 10 hours, even more preferably 1 to 6 hours, even more preferably 2 to 5 hours, and particularly preferably 2.5 to 4 hours. When the heating time is within the above numerical range, the resulting particles tend to have better adhesiveness.

[0086] The crosslinked gelatin mixture may be obtained by reacting the gelatin mixture with a crosslinking agent. Examples of crosslinking agents include, but are not limited to, genipin, N-hydroxysuccinimide, polybasic acids activated with N-sulfoxysuccinimide, aldehyde compounds, acid anhydrides, dithiocarbonates, and diisothiocyanates. Examples of crosslinking agents that can be used include compounds described in paragraphs 0021 to 0024 of International Publication No. 2018 / 079538, the contents of which are incorporated herein by reference. [Example]

[0087] The present invention will be described in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the examples shown below.

[0088] 1. Particle Preparation [Synthesis of hydrophobic gelatin] Alaska pollack-derived gelatin (hereinafter referred to as "Org-ApGltn" or simply "Org") was used as the raw gelatin. A gelatin derivative in which a decyl group was introduced into Org-ApGltn (hereinafter referred to as "C10-ApGltn") was used as the hydrophobized gelatin. The hydrophobized gelatin was obtained by reacting decanal with the amino groups of the raw gelatin to form a Schiff base, and then reducing the resulting Schiff base to a stable secondary amine using a reducing agent. The synthesis of the hydrophobized gelatin is described in detail below.

[0089] One hundred grams of Alaska pollack-derived gelatin (Org-ApGltn) (Nitta Gelatin Co., Ltd., weight-average molecular weight (Mw): 38,552 Da, amino group content: 339 μmol / g) was dissolved in 105 mL of ultrapure water. While the solution was heated to 50°C and stirred, decanal (Tokyo Chemical Industry Co., Ltd., 67.8 mmol) in an amount twice the amino group content of Org-ApGltn (339 μmol / g) was added together with ethanol (Junsei Kagaku Co., Ltd.). This resulted in the formation of imine bonds between the decanal and the amino groups of Org-ApGltn.

[0090] After stirring the solution at the same temperature (50°C) for 1 hour, 2-picoline borane (Junsei Kagaku Co., Ltd., 50.85 mmol) was added together with ethanol to reduce the imine. The resulting mixed solution (Org-ApGltn concentration: 20% by mass / volume, water:ethanol = 105:45 mL) was stirred at 50°C for 17 hours to allow the reaction to proceed. The resulting mixture (150 mL) was added dropwise to 1500 mL of cold ethanol (-7 to 4°C) to purify C10-ApGltn. The resulting precipitate was washed with 1500 mL of ethanol (1 hour x 3 times) to remove unreacted decanal and 2-picoline borane. The precipitate was then dried in vacuo for 3 days to obtain C10-ApGltn in a yield of 92.9% by mass.

[0091] The introduction of a decyl group into the obtained C10-ApGltn was confirmed by Fourier transform infrared spectroscopy and 1 This was confirmed by H-NMR. The hydrocarbon group introduction rate of C10-ApGltn (hereinafter referred to as "decyl group introduction rate DS" or simply "DS") was calculated. DS was calculated from the number of amino groups in the raw gelatin, which was determined by the 2,4,6-trinitrobenzenesulfonic acid method (TNBS method), and the number of amino groups in the hydrophobized gelatin.

[0092] The calculated DS of C10-ApGltn was 49 mol%. This means that 6.4 decyl groups were introduced to the amino groups in one molecule of C10-ApGltn. Hereinafter, hydrophobized gelatin with a DS of 49 mol% may be referred to as 49C10-ApGltn (or simply "49C10"). The DS value and other information for the obtained 49C10-ApGltn are summarized in Table 1.

[0093] [Table 1]

[0094] [Particle preparation] Eight types of particles with different hydrophobicity were prepared by the coacervation method using ethanol, a poor solvent for hydrophobicized gelatin, using raw gelatin and / or hydrophobicized gelatin in the ratios shown in Table 2 (see Figure 1). Details of particle preparation are described below.

[0095] <Sample 1 (49C10-MPs)> First, the synthesized 49C10-ApGltn was dissolved in ultrapure water at 50°C to obtain a 5% (wt / v) C10-ApGltn aqueous solution. Next, an equal volume of ethanol was added dropwise to the solution while stirring (400 rpm) at room temperature (25°C). The resulting solution was temporarily frozen at -30°C for 24 hours and then lyophilized. The resulting dried particles were heated at 150°C for 3 hours under vacuum (less than 3 mbar) to thermally crosslink 49C10-ApGltn by forming an amide bond between the carboxyl and amino groups. This resulted in particles (49C10-MPs) that were crosslinked solely with 49C10-ApGltn.

[0096] <Sample 2 (Organo-MPs)> Sample 2 was prepared in the same manner as Sample 1, except that raw gelatin (Org-ApGltn) was used instead of hydrophobized gelatin (49C10-ApGltn).

[0097] <Samples 3-8> Sample 1 used only hydrophobized gelatin (49C10-ApGltn), but instead, samples 3 to 8 used a mixture of raw gelatin and hydrophobized gelatin in the mixing ratios shown in Table 2. Other than that, samples 3 to 8 (10C10-MPs to 45C10-MPs) were prepared in the same manner as sample 1.

[0098] The yield of each of Samples 1 to 8 (particles) and the DS of the raw material of the particles (raw gelatin, hydrophobized gelatin, or gelatin mixture) are also shown in Table 2. The DS was calculated according to the above-mentioned formula (I).

[0099] [Table 2]

[0100] 2. Evaluation [SEM observation of particles, particle size distribution] The prepared samples 1 to 8 were observed under SEM, and the particle size distribution was analyzed using "Image J." As shown in Figure 5A, all samples 1 to 8 were spherical particles with microscale particle diameters (microparticles). Furthermore, as shown in Figure 5B, the average particle diameter of samples 1 to 8 was 2 to 3 μm, and the particle size distribution of each sample did not change with DS. 5A and 5B, "Org" represents "Org-MPs" of sample 2. Furthermore, "10C10" represents "10C10-MPs" of sample 3. Furthermore, "20C10," "30C10," "35C10," "40C10," "45C10," and "49C10" represent samples 4, 5, 6, 7, 8, and 1, respectively.

[0101] [Spray test] A spray test was conducted using an endoscopic spray device (spray system) for the prepared samples 1 to 8. As shown in Figure 6, it was confirmed that samples 1 to 8 could be sprayed with the endoscopic spray device, and could be sprayed directly onto the surface of internal organ tissue during surgery, for example.

[0102] [Preparation and evaluation of colloidal gels] <Water content of colloidal gel> 50 mg of sample 8 (45C10-MPs) was packed into a 1 mm thick, 10 mm diameter silicone mold and leveled using a spatula. 300 μL of saline (Otsuka Pharmaceutical Co., Ltd.) was added dropwise and hydrated for a specified time (hydration time: 1 to 30 minutes) to produce a colloidal gel. The excess water on the surface of the colloidal gel was then removed, and the weight (Ww) of the resulting colloidal gel was measured and then freeze-dried. Finally, the weight (Wd) of the dried gel was measured, and the water content of each colloidal gel was calculated using the following formula:

[0103]

number

[0104] Figure 7A shows the time dependence of the water content of the colloidal gel (relationship between hydration time and water content) for sample 8 (45C10-MPs). The time dependence of the water content of the colloidal gel was also evaluated for sample 2 (Org-MPs). The results are also shown in Figure 7A.

[0105] As can be seen from Figure 7A, Sample 2 (Org-MPs) and Sample 8 (45C10-MPs) gradually absorbed water to form colloidal gels, which reached equilibrium swelling after 30 minutes. Furthermore, after 5 minutes, the water content of Sample 8 (45C10-MP), which had a higher DS, was lower (53-69%) than that of Sample 2 (Org-MPs) (64-82%), confirming its higher hydrophobicity. The test was performed with n=3, and in FIG. 7A, "**" means P<0.01, and "***" means P<0.001.

[0106] <Observation of particle fusion> The aggregation behavior of particles in a humid environment was observed. Ten mg of each of Sample 2 (Org-MPs) and Sample 8 (45C10-MPs) was added to 200 μL of physiological saline to prepare a suspension. After incubation for up to 2 hours, the aggregation behavior of the particles was observed using a bright-field microscope (Keyence Corporation, BZ-X710). A bright-field micrograph is shown in Figure 7B.

[0107] The Org-MPs remained isolated from each other at all hydration times, and no particle fusion was observed. On the other hand, the 45C10-MPs aggregated with increasing hydration time and formed colloidal gels through hydrophobic interactions. After 30 minutes of hydration, the 45C10-MPs exhibited a densely packed structure. After 60 minutes of hydration, almost all of the 45C10-MPs fused with each other to form large aggregates. This result also suggests that hydrophobic interactions between 45C10-MPs are the driving force for colloidal gel formation under humid conditions.

[0108] [In vitro tissue adhesion / non-adhesion test] The effect of hydration time on the adhesive strength of the colloidal gel to tissue was evaluated according to the American Society for Testing and Materials standard (ASTM F-2258-05) (see Figure 8A). First, a fresh porcine stomach (purchased from Tokyo Shibaura Organs) was opened, and the mucosal layer was removed to expose the submucosal serous tissue (81). The resulting serous tissue (81) was cut into 2.5 cm square tissue pieces and fixed to the upper and lower stages of the jig (80) of a testing device (Texture Analyzer TA-XT2i, manufactured by Stable Micro Systems) using cyanoacrylate (manufactured by Henkel Japan Co., Ltd.). The temperature of the porcine stomach lining tissue was maintained at 37°C during measurement using a hot plate (83).

[0109] To remove excess moisture from the tissue surface, an industrial paper cloth (82) (trade name "Kimwipes") was pressed against the tissue at 80 kPa for 3 minutes. Then, 100 mg of sample 8 (45C10-MPs) was sprinkled over the tissue on the stage of the lower jig and allowed to hydrate in a humid environment for a predetermined period (hydration time: 1 to 30 minutes). This resulted in the formation of a colloidal gel (38) layer. Specifically, to bring the humidity around the tissue surface and sample 8 (45C10-MPs) closer to that of the intestinal environment, the area around the tissue was filled with 7 mL of saline (84) and the tissue was covered with a plastic cover (85). After hydration for a predetermined period of time, the upper jig was placed on top of sample 8 (colloid gel 38) in the lower jig and pressed together under a pressure of 80 kPa for 3 minutes. The upper jig was then raised at 10 mm / min, and the adhesive strength of sample 8 (45C10-MPs) was measured. Figure 8B shows the relationship between hydration time and adhesive strength. A similar evaluation was performed on sample 2 (Org-MPs), and the results are shown in Figure 8B. As a control, the adhesive strength between two tissues without particle application is also shown in Figure 8B.

[0110] After measuring the adhesive strength, the tissue sections were fixed in 10% formalin buffer (Wako Pure Chemical Industries, Ltd.) and stained with hematoxylin and eosin (HE). The interface between the colloid gel and the tissue was then observed. An optical microscope photograph is shown in Figure 8C.

[0111] As shown in Figure 8B, the adhesive strength of the colloidal gel of sample 2 (Org-MPs) did not change significantly with increasing hydration time (maximum strength: 2.73 kPa, minimum strength: 1.48 kPa). On the other hand, the adhesive strength of the colloidal gel of sample 8 (45C10-MPs) decreased from 5.24 kPa to 2.41 kPa with increasing hydration time, and after 30 minutes of hydration, it had decreased to a level close to that of the colloidal gel of sample 2 (Org-MPs).

[0112] As can be seen from the HE staining observations in Figure 8C, the colloidal gel of sample 8 (45C10-MPs) adhered to both the upper and lower tissue surfaces after 1 and 3 minutes of hydration, but the amount of colloidal gel adhered to the upper tissue surface decreased after 30 minutes of hydration. This result indicates that sample 8 (45C10-MPs) is fully hydrated after 30 minutes of hydration to form a colloidal gel and become non-adhesive to other tissues. During the particle hydration process, sample 8 (45C10-MPs) absorbs water from the tissue surface and forms hydrophobic interactions between the particles and the tissue, enabling strong adhesion. After complete hydration, the colloidal gel layer no longer exhibits adhesive properties (exhibiting an anti-adhesion effect) due to the presence of water on the colloidal gel surface and the tissue surface (see Figure 2).

[0113] [Stability test in water] The aqueous stability of the colloidal gel particles was evaluated by adhering the colloidal gel to gastric tissue (submucosa) and then immersing it in saline. First, porcine gastric tissue was cut into 2.5 × 2.5 cm pieces. 50 mg of sample 8 (45C10-MPs) was dispersed in a 10 mm diameter circle on the tissue and hydrated with 300 μL of saline for 30 minutes to form a colloidal gel. The colloidal gel adhered to the gastric tissue was immersed in saline containing 0.05 wt / vol% sodium azide (Wako Pure Chemical Industries, Ltd.) and incubated at 37 °C for 2 days. The colloidal gel adhered to the gastric tissue was then fixed in 10% formalin buffer, and the interface between the colloidal gel and the tissue was observed after HE staining. Optical micrographs are shown in Figure 9A. Similar evaluations were performed on samples 1 to 7, and the results are shown in Figure 9A.

[0114] Furthermore, the area of ​​remaining colloid gel on the stomach tissue was quantified by image analysis of HE-stained images. The results for Samples 1 to 8 are shown in Figure 9B. Note that in Figures 9A and 9B, each sample is indicated by the first half of the particle name (the part before -MPs). For example, particle 45C10-MPs (Sample 8) is indicated as "45C10."

[0115] As shown in Figures 9A and 9B, for samples with a DS of less than 30 mol%, almost no colloidal gel was observed on the tissue surface after the aqueous stability test. On the other hand, colloidal gels from samples with a DS of 30 mol% or more, and more preferably from samples with a DS of 35 mol% or more, exhibited high aqueous stability even after immersion in physiological saline for two days. The area of ​​colloidal gel (38) remaining on the tissue (41) surface tended to increase with increasing DS, suggesting that the increased hydrophobicity of the particles improved the stability of the colloidal gel on the tissue. Previous research by the inventors (e.g., Acta Biomaterialia 99 (2019) 387-396) has revealed that hydrophobic groups introduced into gelatin interact with extracellular proteins such as fibronectin and cells through hydrophobic interactions. Furthermore, gastric tissue is known to be primarily composed of collagen, and it is speculated that the decyl groups interact with the hydrophobic amino acid residues of collagen. From the above results and findings, it is presumed that the particles prepared in the examples interact with extracellular proteins and cells on the stomach tissue, thereby exhibiting high stability in water.

[0116] [In vivo adhesion prevention test] First, an abdominal wall-cecal defect model was created in SD (Sprague-Dawley) rats (7-week-old males, purchased from Jackson Laboratory Japan) as follows. The rats were anesthetized with 2.5% isoflurane, the abdominal hair was shaved, and infection was prevented with ethanol. A 5-cm abdominal incision was made to expose the abdominal wall and cecum (Figure 10A(a)). A cecal abrasion (cecal defect) was created by rubbing the cecum with sterile gauze. An abdominal wall defect was created by excising the abdominal wall tissue (1 cm x 2 cm, including the mesothelial layer) adjacent to the cecal defect with a surgical knife (Figure 10A(b)).

[0117] Next, 100 mg of sample 8 (45C10-MPs), heat-sterilized at 150°C for 3 hours, was sprayed onto each defect to cover the wound (Fig. 10A (c)). The wound was then hydrated with saline for 15 minutes to form a colloidal gel (Fig. 10A (d)). Amikamycin (1.0 mg / kg) was injected intraperitoneally to prevent infection, and the abdomen was sutured closed.

[0118] After a predetermined time (1-2 weeks), the abdomen was reopened, and postoperative adhesions were evaluated using the peritoneal wall adhesion score (5-point scale) shown in Table 3. The results are shown in Figures 10C and 10D. A lower score indicates less adhesion. Figure 10B shows photographs of the cecum and peritoneum immediately before abdominal closure (W0), 1 week (W1), and 2 weeks (W2) after abdominal closure. The black arrows in the photographs indicate the adhesions between the abdominal wall and cecum. Sample 8 (45C10-MPs) is labeled "C10" in Figure 10B and "C10-MPs" in Figures 10C and 10D. The test was performed with n = 5, and *** indicates P < 0.001.

[0119] [Table 3]

[0120] Similar evaluations were performed using Sample 2 (Org-MPs) and a hyaluronic acid / carboxymethylcellulose sheet (HA / CMC) (a commercially available adhesion barrier, manufactured by Kaken Pharmaceutical Co., Ltd., Seprafilm®) instead of Sample 8 (45C10-MPs). Similar evaluations were also performed on untreated rats (Untreated) that were not given an adhesion barrier. These evaluation results are shown in Figures 10B to 10D. Sample 2 (Org-MPs) is indicated as "Org" in Figure 10B.

[0121] One week later, severe adhesions (mean score: 2.80) were observed in the untreated rat group (Untreated) (Figure 10C), and an adhesion layer was formed between the cecum and abdominal wall (Figure 10B). This is thought to be due to the lack of a physical barrier between the tissues, which led to excessive fibrin deposition and migration of macrophages and fibroblasts, resulting in the formation of fibrous tissue (FT) between the cecum and peritoneum, resulting in postoperative adhesions. In contrast, no adhesions were observed in the groups administered Sample 8 (45C10-MPs), Sample 2 (Org-MPs), or the commercially available HA / CMC sheet (Figure 10B, mean score: 0 for all groups). This result is thought to be due to the colloid gel and the commercially available sheet effectively preventing the migration of fibrin and inflammatory cells.

[0122] After two weeks, severe adhesions were confirmed in the untreated rat group (Untreated) with scores higher than those after one week (Fig. 10C, Fig. 10D). A slight increase in adhesion scores was observed in the other groups compared to one week after treatment. The HA / CMC-treated group showed an increase in adhesion scores (scores: 2 and 3), consistent with previous findings. Furthermore, adhesions (score: 2) were observed in one out of five rats in the group treated with Sample 2 (Org-MPs), while only mild adhesions were observed in one rat in the group treated with Sample 8 (45C10-MPs).

[0123] From the results of the adhesion prevention test described above, it was confirmed that sample 8 (45C10-MPs) adheres to the cecal abrasion and peritoneal defect area upon hydration, forming a colloidal gel layer, and that this colloidal gel layer functions as a physical barrier to prevent adhesions after hydration, thereby preventing postoperative adhesions. [Industrial Applicability]

[0124] The present invention provides a new anti-adhesion material that has excellent adhesive properties on tissues and excellent adhesive stability in an aqueous environment, is easy to handle, and can be easily mass-produced industrially. [Explanation of symbols]

[0125] 10 Particles, 22 Serosa layer, 23 Muscle layer, 24 Submucosa layer, 25 Mucosa layer, 26 Water layer, 30 Rat, 31 Abdomen, 36 Abdominal cavity, 37, 37a, 37b Defect, 38, 38a, 38b Colloid gel, 39 Cecum, 40 Adhesion, 41 Tissue, 80 Jig, 81 Serosa tissue, 82 Industrial paper rag, 83 Hot plate, 84 Saline, 85 Plastic cover

Claims

1. An anti-adhesion material containing particles, The particles comprise a crosslinked mixture of a first gelatin and a second gelatin in which a hydrocarbon group has been introduced into the first gelatin, the introduction rate of hydrocarbon groups in the gelatin mixture is 30 mol % to 50 mol %; An anti-adhesion material, wherein the second gelatin has a structure represented by the following formula (1): 【Chemistry 1】 In formula (1), Gltn represents a residue of gelatin, L represents a single bond or a divalent linking group, and R １ is an alkyl group having 1 to 20 carbon atoms, and R ２ is a hydrogen atom or an alkyl group having 1 to 20 carbon atoms.

2. The adhesion preventing material according to claim 1, wherein in formula (1), L is a single bond or —C(O)—.

3. The adhesion preventing material according to claim 2, wherein in formula (1), L is a single bond.

4. In formula (1), R １ is a straight chain alkyl group, and R ２ The adhesion preventing material according to claim 1 , wherein is a hydrogen atom.

5. The adhesion preventing material according to claim 1 , wherein the hydrocarbon group introduction rate of the second gelatin is higher than the hydrocarbon group introduction rate of the gelatin mixture.

6. The adhesion preventing material according to claim 1, wherein the hydrocarbon group introduction rate of the second gelatin is 35 mol % to 80 mol %.

7. The adhesion preventing material according to claim 1, wherein the introduction rate of hydrocarbon groups in the gelatin mixture is 35 mol % to 45 mol %.

8. The adhesion preventing material according to claim 1, wherein the ratio of the mass of the second gelatin to the total mass of the first gelatin and the second gelatin is 35% by mass to 90% by mass.

9. The adhesion preventing material according to claim 1 , wherein the first gelatin is an alkali-treated gelatin.

10. The adhesion barrier according to claim 1, wherein the first gelatin is a gelatin that has been treated to reduce endotoxin levels.

11. The adhesion preventing material according to claim 1 , wherein the first gelatin is derived from cold-water fish.

12. The adhesion preventing material according to any one of claims 1 to 11, which can be applied to an affected area of ​​a living body using a spray system.

13. A method for producing an anti-adhesion material according to any one of claims 1 to 11, comprising: dissolving the gelatin mixture of the first gelatin and the second gelatin in a good solvent to prepare a gelatin mixture solution; adding a poor solvent to the gelatin mixture solution to precipitate first intermediate particles containing the gelatin mixture in the gelatin mixture solution; freeze-drying the gelatin mixture solution containing the first intermediate particles to obtain second intermediate particles; A method for producing an adhesion preventing material, comprising crosslinking the gelatin mixture of the second intermediate particles to obtain particles of the crosslinked gelatin mixture.

14. The method for producing an anti-adhesion material according to claim 13, wherein the second intermediate particles are heated to crosslink the gelatin mixture.

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