WO2010143646A1

WO2010143646A1 - Fiber structure

Info

Publication number: WO2010143646A1
Application number: PCT/JP2010/059745
Authority: WO
Inventors: 浩作大川; 賢大橋; 学北澤
Original assignee: 味の素株式会社
Priority date: 2009-06-09
Filing date: 2010-06-09
Publication date: 2010-12-16
Also published as: WO2010143646A8

Abstract

Disclosed is a fiber structure which is safe for living organisms, has high biocompatibility, and can be used in a medical material (particularly a scaffold material) having a high effect of inhibiting the adhesion of cells or tissues. The fiber structure comprises a specific polyamic acid as the main component.

Description

Fiber structure

The present invention relates to a specific fiber structure, a manufacturing method thereof, and a medical material containing the obtained fiber structure.

In recent years, research and development of structures that function as scaffolds for tissues and cells have been promoted as part of regenerative medicine with the aim of regenerating damaged and deficient biological tissues. As a structure that functions as a scaffold, a fiber structure such as a sponge body, a honeycomb body, or a nonwoven fabric in which fibers are layered is known, and as a fiber among these, a conventionally known fiber diameter is used. In addition to micrometer (μm) order fibers (microfibers), thinner nanometer (nm) order fibers (nanofibers) are being developed and put to practical use. Since nanofibers have a remarkably large specific surface area compared to conventional microfibers, they are advantageous in that a more porous fiber structure can be produced. In addition, nanofibers have an average fiber diameter of several tens to several hundreds of nanometers, and have a fiber diameter comparable to that of an extracellular matrix in vivo, so that they can be easily decomposed in vivo. For this reason, a fiber structure in which nanofibers are integrated is being studied for application as a medical material that is desired to disappear quickly after tissue and cells are repaired and regenerated in vivo.

Nanofibers can be formed by a method called electrospinning (electrospinning). After the polymer compound is dissolved in a solvent to form a solution, the polymer compound solution is discharged while applying a high voltage to form nanofibers, which are then accumulated on the substrate (collector). This is a method for obtaining a fiber structure in which fibers are accumulated. *

In the production of a fiber structure by electrospinning, if a part of the discharged polymer solution is not formed into fibers but is collected in the form of droplets, the nanofibers accumulated on the collector are bound together. . Such accumulation in the droplet state can be avoided by slowing the discharge speed. In this case, however, it takes a long time to discharge the solution. Alternatively, accumulation in a droplet state can be avoided by replacing the solvent that dissolves the polymer compound with a highly volatile solvent. However, the type of solvent that dissolves the polymer compound is usually one. Because it is limited to the part, it is not easy. Therefore, it is often difficult to achieve both the absence of accumulation in the droplet state and the production of the fiber structure without requiring much time.

It is desirable that medical materials such as tissue and cell scaffolds have both biodegradability and biocompatibility. From this viewpoint, electrospinning of collagen, gelatin, cellulose, chitin, chitosan, polylactic acid, etc. Many examples of manufacturing fiber structures by the method are disclosed. However, it is the inventor of the present invention to obtain a fiber structure in which the fiber diameter is controlled and the fibers are not bound to each other, and to produce the fiber structure without requiring much time. As far as we know, they are not compatible. For example, Patent Document 1 discloses a method of electrospinning polysaccharides such as chitosan, chondroitin sulfate, and pectin. However, despite the production on a collector with a very small area of 1 cm square, the resulting fiber structure has a wide and uneven fiber diameter. Patent Document 2 discloses a method of electrospinning after dissolving chitosan in water to which a solubilizing agent such as an organic acid is added. Although the obtained fiber diameter is controlled, since water is volatilized in the stage of forming the fiber, the discharge speed cannot be increased sufficiently. Patent Document 3 discloses a method in which chitosan or cellulose is dissolved in an organic acid solvent, a volatile organic solvent is mixed therein, and then electrospinning is performed. However, since the dissolution of chitosan or cellulose in the solution is not complete, a satisfactory fiber structure has not been obtained.

Non-Patent Document 1 discloses that when poly-γ-benzyl-L-glutamic acid is dissolved in an organic solvent having low polarity, the fiber diameter is controlled by suppressing the interaction between molecules by reducing the concentration thereof. A method for obtaining a fibrous structure is disclosed. However, if the solution concentration is too low, fiber formation becomes difficult, and the fiber diameter has not been reduced to a satisfactory fineness.

Patent Document 4 discloses an adhesion preventing material composed of a fiber structure of biodegradable and absorbable polymer, and specifically uses polylactic acid. However, since polylactic acid lacks flexibility, its compatibility with soft tissues is poor, and it has also been suggested that it causes an inflammatory response to the living body, and a sufficiently safe biodegradable material has not yet been obtained.

JP 2005-290610 A JP 2008-163520 A JP 2008-308780 A International Publication No. 2004/088943 Pamphlet

The problem to be solved by the present invention is a medical material that is safe for the living body, has high biocompatibility, and has a high effect of suppressing adhesion of cells and tissues (the medical material is particularly useful as a scaffold material). ) To provide a fiber structure that can be used.

As a result of intensive studies, the present inventors have found that the above problems can be achieved by incorporating a specific polyamino acid as a main component into a fiber structure, and have completed the present invention.

That is, the present invention is as follows.
[1] A fiber structure containing a polyamino acid as a main component and having an average fiber diameter of 50 nm or more and less than 500 nm.
[2] The fiber structure according to [1], wherein the amino acid is one or more selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.
[3] The fiber structure according to [1] or [2], wherein the amino acid is alanine.
[4] The fiber structure according to any one of [1] to [3], wherein the polyamino acid is composed of one amino acid.
[5] The fiber structure according to [1], wherein the polyamino acid is composed of two kinds of amino acids.
[6] The fiber structure according to [5], wherein the two amino acids are a combination of an acidic amino acid or a basic amino acid and a nonpolar neutral amino acid.
[7] The fiber structure according to [5], wherein the two amino acids are an acidic amino acid or a combination of a basic amino acid and an aromatic amino acid.
[8] The fiber structure according to [6] or [7], wherein the acidic amino acid is glutamic acid.
[9] The fiber structure according to [6] or [7], wherein the basic amino acid is lysine.
[10] The fiber structure according to [6], wherein the nonpolar neutral amino acid is selected from alanine, valine, leucine or isoleucine.
[11] The fiber structure according to [7], wherein the aromatic amino acid is phenylalanine.
[12] The fiber structure according to any one of [1] to [11], wherein a protecting group is bonded to a side chain of an amino acid constituting the polyamino acid.
[13] The protective group is one or more selected from the group consisting of a methyl group, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, and a trifluoroacetyl group, 12].
[14] A method for producing a fiber structure, comprising the following steps;
Step 1) Step of dissolving a polyamino acid in a solvent to form a solution,
Step 2) A step of continuously discharging the solution put in the syringe from a nozzle attached to the tip of the syringe,
Step 3) A step of applying a high voltage between the nozzle and the collector with a high voltage generator in the discharge,
Step 4) changing the discharged solution into a fiber shape between the nozzle and the collector,
Step 5) A step of collecting the fiber on a collector.
[15] The polyamino acid solution concentration in step 1 is 1 to 20% by weight, the polyamino acid solution discharge speed in step 2 is 1 to 20 ml / hour, and is applied between the nozzle and collector in step 3. [14] The method according to [14], wherein the voltage is 11 to 45 kV, and the distance between the nozzle and the collector in step 4 is 10 to 40 cm.
[16] The solvent in Step 1 is trifluoroacetic acid, acetic acid, dichloromethane, chloroform, 1,2-dichloroethane, 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2,2- [14] or [15], wherein the production method is one or more selected from the group consisting of trifluoroethanol, N, N-dimethylformamide and water.
[17] A medical material comprising the fiber structure according to any one of [1] to [13].
[18] The medical material according to [17], which is an adhesion preventing material.

The fiber structure of the present invention has a nanometer-order fiber diameter that is uniformly controlled and is therefore excellent in degradability in vivo, and the material used as the main component is composed of amino acids widely present in the living body. Therefore, it is safe for the living body. The fiber structure of the present invention is excellent in flexibility in addition to biodegradability because the fibers are not bound to each other, and is highly compatible with living bodies. Furthermore, since the fiber structure of the present invention has low adhesion to cells derived from living tissue, it can be used as a medical material having a high effect of suppressing adhesion of cells and tissues. The medical material is particularly used as a scaffold material. Useful.

FIG. 3 is a photograph of a surface of a fiber structure composed of poly-L-alanine obtained by the operation of Production Example 1 taken with a scanning electron microscope (SEM).

Hereinafter, embodiments of the present invention will be described.

The polyamino acid used in the present invention is composed of an amino acid or a derivative thereof, and is usually prepared using a neutral amino acid, an acidic amino acid, a basic amino acid, an aromatic amino acid, etc. Nonpolar neutral amino acids and polar neutral amino acids are used. As the polyamino acid, one composed of one or more amino acids can be used, but in the present invention, a polyamino acid composed of one or two amino acids is preferable. When the amino acid residue used contains an asymmetric carbon atom, either an optically active substance or a racemic substance may be used.

The amino acid or derivative thereof is not particularly limited as long as it forms a polymer that can constitute the fiber structure of the present invention. Specifically, glycine; sarcosine; alanine; N-methylalanine; β- Alanine; gamma-aminobutyric acid; valine; norvaline; leucine; isoleucine; norleucine; phenylglycine; phenylalanine; methionine; cysteine; cysteine derivatives such as S-benzyl-cysteine; cystine; glutamine; asparagine; glutamic acid; Glutamic acid-α-methyl ester, glutamic acid-γ-ethyl ester, glutamic acid-α-ethyl ester, glutamic acid-γ-benzyl ester, glutamic acid-α-benzyl ester, etc .; aspartic acid; Asparagine such as aspartic acid-β-methyl ester, aspartic acid-α-methyl ester, aspartic acid-β-ethyl ester, aspartic acid-α-ethyl ester, aspartic acid-β-benzyl ester, aspartic acid-α-benzyl ester Acid derivatives; lysine; lysine derivatives such as Nε-benzyloxycarbonyl-lysine and Nα-benzyloxycarbonyl-lysine; ornithine; ornithine derivatives such as Nδ-benzyloxycarbonyl-ornithine and Nα-benzyloxycarbonyl-ornithine; arginine; Nω Arginine derivatives such as methyl-arginine and Nω-nitro-arginine; histidine; histidine derivatives such as N (Im) -methyl-histidine; proline; hydroxyproline, O-benzylhydroxyproline and the like Serine; serine derivatives such as O-benzyl-serine and O-acetyl-serine; threonine; threonine derivatives such as O-benzyl-threonine and O-acetyl-threonine; tryptophan; N (In) -methyl-tryptophan and the like Tryptophan derivatives of tyrosine; tyrosine; tyrosine derivatives such as O-benzyl-tyrosine, O-methyl-tyrosine; dopa and the like.

Amino acid residues with a small number of carbon atoms in the amino acid side chain are likely to form a dense molecular packing structure, and can be expected to give fibers with a small fiber diameter, so that glycine, sarcosine, alanine, N-methylalanine, β-alanine, γ-aminobutyric acid, valine, norvaline, leucine, isoleucine, norleucine, cysteine, glutamine, asparagine, aspartic acid, aspartic acid-β-methyl ester, lysine, proline, hydroxyproline, serine, O-acetyl-serine Threonine is preferable, and glycine, alanine, valine, leucine, isoleucine, proline, glutamine, and lysine are more preferable. These may be used alone or in combination of two or more.

In addition, from the viewpoint that an amino acid residue having an aromatic ring in the amino acid side chain is likely to form a close interaction between aromatic rings and a fiber structure having a small fiber diameter can be expected, phenylglycine, Phenylalanine, aspartic acid-β-benzyl ester, Nε-benzyloxycarbonyl-lysine, Nδ-benzyloxycarbonyl-ornithine, histidine, N (Im) -methyl-histidine, O-benzyl-serine, O-benzyl-threonine, tryptophan N (In) -methyl-tryptophan, tyrosine, O-benzyl-tyrosine, O-methyl-tyrosine, O-acetyl-tyrosine, phenylalanine, aspartic acid-β-benzyl ester, Nε-benzyloxycarbonyl-lysine, Tryptophan, Chiro Syn and O-acetyl-tyrosine are more preferred. These may be used alone or in combination of two or more.

In the present invention, the amino acids exemplified above can be used as appropriate, but when the polyamino acid contained in the fiber structure is composed of one kind of amino acid, such amino acids are glycine, alanine, valine, leucine. , Isoleucine, phenylalanine, proline, aspartic acid-β-benzyl ester, Nε-benzyloxycarbonyl-lysine, tryptophan, O-acetyl-tyrosine are preferred, glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline are more preferred, Alanine, valine and leucine are even more preferred, and alanine is most preferred. In addition, when the polyamino acid is composed of two kinds of amino acids, it is a combination of an acidic amino acid or basic amino acid and a nonpolar neutral amino acid, or a combination of an acidic amino acid or basic amino acid and an aromatic amino acid. Is preferred. At this time, glutamic acid is preferable as the acidic amino acid, and lysine is preferable as the basic amino acid. Moreover, as a nonpolar neutral amino acid, alanine, valine, leucine, and isoleucine are preferable, and as an aromatic amino acid, phenylalanine is preferable.

The polyamino acid used in the present invention may have a structure in which a salt is coordinated to a part or all of the polar groups in the structure. The ratio of the salt to the polar group is such that, when a fiber structure composed of a polyamino acid is produced by an electrospinning method, for example, the solubility of the polyamino acid in a solvent and / or the average fiber diameter of the resulting fiber structure is It is determined from the viewpoint of being optimal. Examples of the salt include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as magnesium and calcium; inorganic bases such as ammonia; monoethanolamine, diethanolamine, triethanolamine, 2-amino-2-methyl-1 Organic amine salts such as propanol, 2-amino-2-methyl-1,3-propanediol, lysine, ornithine, arginine; inorganic acid salts such as hydrochloride, sulfate, carbonate, phosphate; acetate, Examples thereof include organic acid salts such as tartrate, citrate, p-toluenesulfonate, glycolate, malate, lactate, fatty acid salt, acidic amino acid salt and pyroglutamate. These may be used alone or in combination of two or more.

The method for preparing the polyamino acid used in the present invention is not particularly limited as long as it is a method in which an amino acid or a derivative thereof is prepared into a polymerized structure. For example, N-carboxy-α-amino acid anhydride or N-carboxy- An example is a method in which an α-amino acid derivative anhydride is dissolved or suspended in an organic solvent or water, and a polymerization initiator is added thereto if necessary.

Examples of the organic solvent include acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, tetrahydrofuran, diethyl ether, diisopropyl ether, petroleum ether, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, ethyl acetate, butyl acetate. , Trifluoroacetic acid, acetic acid, formic acid, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, trichloroethane, trichloroethylene, trifluoroethane, 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2,2-trifluoroethanol, hexafluoroacetone, methanol, ethanol, 1-propanol, 2-propanol, formamide, N, N-dimethylformamide, N, N-dimethyl Acetamide, N- methylpyrrolidone, dimethyl sulfoxide, pyridine, acetonitrile, may be mentioned trimethylamine, triethylamine, tributylamine and the like. These may be used alone or in combination of two or more.

Examples of the polymerization initiator include ethylenediamine, propylenediamine, hexamethylenediamine, 1,4-cyclohexanediamine, 1,2-cyclohexanediamine, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and toluene-2. Primary amines such as methylamine, ethylamine and 1-propylamine; Alcohol amines such as methanolamine, ethanolamine and diethanolamine; Dimethylamine and diethylamine Secondary amines such as dipropylamine; primary tertiary diamines such as N, N-dimethylethylenediamine and N, N-dimethyl-1,3-propanediamine; trimethylamine and triethylamine Tertiary amines such as tributylamine; amino group-containing polymers such as polyether diamine and polyester diamine; primary alcohols such as methanol and ethanol; secondary alcohols such as isopropanol; ethylene glycol, propylene glycol, 1,4-butanediol, hexa Examples include glycols such as methylene glycol; hydroxyl group-containing polymers such as polyether diol and polyester diol; thiols. These may be used alone or in combination of two or more.

When preparing polyamino acids using two or more amino acids, N-carboxy-α-amino acids (including derivatives) anhydrides corresponding to various amino acids are mixed appropriately and dissolved or suspended in an organic solvent. Depending on this, a polymerization initiator can be added to this to form a copolymer. The mixing ratio of amino acids is, for example, 95: 5 to 5:95, preferably 90:10 to 10:90, more preferably 70:30 to 30:70 in terms of molar ratio for two types of amino acids. it can. The mixing ratio can be calculated based on the molar ratio of the N-carboxy-α-amino acid anhydride to be used.

An N-carboxy-α-aliphatic amino acid anhydride which is different from any of the N-carboxy-α-aliphatic amino acid anhydride and the N-carboxy-α-aromatic amino acid anhydride, By mixing and / or polymerizing with an N-carboxy-α-aromatic amino acid anhydride to form a copolymer, the solubility in an organic solvent can be increased. Such N-carboxy-α-amino acid anhydrides include aspartic acid-β-methyl ester, aspartic acid-β-benzyl ester, glutamic acid-γ-methyl ester, Nε-benzyloxycarbonyl-lysine, Nδ-benzyloxycarbonyl- N-carboxy-α-amino acid (including derivatives) anhydrides corresponding to ornithine, O-benzyl-serine or O-benzyl-threonine are preferred, corresponding to glutamic acid-γ-methyl ester or Nε-benzyloxycarbonyl-lysine N-carboxy-α-amino acid anhydride is more preferred. These may be used alone or in combination of two or more.

In addition to the method using the above-mentioned N-carboxy-α-amino acid anhydride or N-carboxy-α-amino acid derivative anhydride, a method for preparing the polyamino acid used in the present invention includes γ-polyglutamic acid using a microorganism. Examples thereof include a method for preparing (γ-PGA) and a method for preparing ε-polylysine using a microorganism.

In the polyamino acid used in the present invention, a protecting group can be bonded to the side chain of an amino acid residue serving as a structural unit. In the present specification, the protecting group refers to an atomic group used for the purpose of protecting a highly reactive characteristic group in the side chain of an amino acid so as not to react with other compounds. The protecting group may be bonded during the preparation of the polyamino acid as described above, or may be bonded after the preparation. Further, the protecting group may be bonded to the side chains of all amino acid residues constituting the polyamino acid, or may be bonded only to the side chains of some amino acid residues. In the present invention, it is preferable that a protective group is bonded to the polyamino acid because the adhesion suppressing effect of living tissue or cells in the fiber structure can be enhanced.

The protecting group is not particularly limited as long as it can convert a highly reactive characteristic group in the side chain of an amino acid residue into an inactive functional group. For example, amino-protecting groups include substituted benzyloxycarbonyl groups such as benzyloxycarbonyl group and p-methoxybenzyloxycarbonyl group; tert-butoxycarbonyl group, p-biphenylisopropyloxycarbonyl group, 9-fluorenylmethyl Urethane-type protecting groups such as oxycarbonyl group; acyl-type protecting groups such as formyl group, phthaloyl group, trifluoroacetyl group, p-toluenesulfonyl group, o-nitrophenylsulfenyl group; trityl group, benzyl group, 2-benzoyl Examples thereof include alkyl-type protecting groups such as -1-methylvinyl group; and arylidene-type protecting groups such as 2-hydroxyarylidene group. Examples of the protecting group for the carboxyl group include substituted benzyl esters such as methyl group, ethyl group, benzyl group, tert-butyl group, and p-nitrobenzyl group; phenacyl group; trichloroethyl group; cyclohexyl group and the like. Protected in the form of Examples of protecting groups for guanidino groups include nitro groups, p-toluenesulfonyl groups, benzyloxycarbonyl groups, and the like. Examples of the protecting group for the imidazolyl group include a benzyl group, a benzyloxycarbonyl group, a p-toluenesulfonyl group, a trityl group, a diphenylmethyl group, a dinitrofluorobenzene group, and a tert-butoxycarbonyl group. Examples of the protecting group for the carbamide group include a xanthyl group, a bis-2,4-dimethoxybenzyl group, and a 4,4'-dimethoxybenzhydryl group. Examples of the protective group for the hydroxy group include ether-type protective groups such as a benzyl group, a substituted benzyl group, and a tert-butyl group; and acyl-type protective groups such as an acetyl group, a trifluoroacetyl group, and a benzyloxycarbonyl group. Examples of the protecting group for the mercapto group include substituted benzyl groups such as a benzyl group and a p-methoxybenzyl group; a trityl group; a benzhydryl group; an acetamidomethyl group; and a carbomethoxysulfenyl group. Examples of the protecting group for the indolyl group include a formyl group. In the present invention, among these protecting groups, a methyl group, a benzyloxycarbonyl group, a 9-fluorenylmethylcarbonyl group, and a trifluoroacetyl group are preferable.

The polyamino acid used in the present invention can be further chemically modified after being prepared as described above. For example, in the case of poly-γ-methyl-glutamic acid, it can be converted to a polyamino acid having a structure in which part or all of the methyl ester group is saponified by allowing alkali to act on this, or reactive. Can be made into a polyamino acid having a structure in which a part or all of the methyl ester group is transesterified by introducing a high substituent group by transesterification. Alternatively, a polyamino acid having a structure in which a nucleophilic functional group such as an amino compound is reacted to convert part or all of the methyl ester group into an amide group can be obtained. In the case of polyglutamic acid, a polyamino acid having a structure in which an alkylamine is reacted with this to convert a part or all of the carboxyl group into an alkylamide group can be obtained.

The weight average molecular weight of the polyamino acid in the present invention is not particularly limited, but is usually 1,000 or more. In the present invention, it is preferably 10,000 or more, more preferably 100,000 or more, from the viewpoint of increasing the degree of entanglement between polymers in the solution and improving the productivity of the nanofiber.

The polyamino acid used in the present invention may have a structure in which a compound other than an amino acid or a derivative thereof is polymerized in addition to the amino acid or a derivative thereof. For example, N-carboxy-α-amino acid anhydride and urethane Examples thereof include a copolymer obtained by polymerizing a prepolymer.

The fiber structure of the present invention containing the polyamino acid is composed of nanofibers having an average fiber diameter (diameter) of 5 nm or more and less than 1,500 nm, preferably 10 nm or more and less than 1,000 nm, more preferably 50 nm or more and less than 500 nm. The The average fiber diameter of the fiber structure can be measured using a method known to those skilled in the art. Specifically, from a photograph of the surface of the fiber structure taken at random with a scanning electron microscope, 10 positions are arbitrarily selected, the fiber diameter is measured, and the average value is obtained as the average fiber diameter. be able to. For the photograph of the fiber structure, a photograph taken at a magnification of 500 to 200,000 times can be used. In the present invention, more specifically, the average fiber diameter of the fiber structure can be measured according to Examples described later.

In this specification, the fiber structure refers to a structure formed of one or a plurality of fibers, and examples of the form thereof include a filament, a stable, a filament yarn, a spun yarn, a woven fabric, a knitted fabric, Nonwoven fabric, paper, sheet-like material, tube, mesh, thread-like material and the like can be mentioned. A preferred form of the fiber structure in the present invention is a nonwoven fabric.

The fiber structure of the present invention is manufactured by electrospinning using an electrospinning apparatus. Examples of the electrospinning apparatus include a nanofiber electrospinning unit (NEU) manufactured by Kato Tech Co., Ltd., which includes a syringe, a nozzle (needle), a syringe pump, a high voltage generator, and a collector.

The manufacturing method of the fiber structure containing the polyamino acid of the present invention comprises the following steps.
Step 1) Step of dissolving a polyamino acid in a solvent to form a solution,
Step 2) A step of continuously discharging the solution put in the syringe from a nozzle attached to the tip of the syringe,
Step 3) A step of applying a high voltage between the nozzle and the collector with a high voltage generator in the discharge,
Step 4) changing the discharged solution into a fiber shape between the nozzle and the collector,
Step 5) A step of collecting the fiber on a collector.

The concentration of the polyamino acid in the solution in Step 1 is appropriately set depending on the type of polyamino acid to be used, the type of solvent for dissolving the polyamino acid, etc., but is generally 0.1 to 60% by weight, preferably 1 It can be made -45 wt%, more preferably 1-20 wt%. If the polyamino acid concentration is higher than the above range, the viscosity of the solution tends to increase and the spinnability tends to be poor. If the concentration is lower than the above range, it takes a long time to produce the fiber structure. When two or more polyamino acids are used, a solution in which various polyamino acids are appropriately mixed and dissolved may be prepared. The mixing ratio of the polyamino acids can be, for example, 95: 5 to 5:95, preferably 90:10 to 10:90 as a weight ratio in the case of two kinds of polyamino acids.

The solvent for dissolving the polyamino acid used in the present invention in Step 1 is not particularly limited as long as it is a solvent that dissolves the polyamino acid and is easily removed by evaporation or the like at the stage of forming the fiber. , Methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, methanol, ethanol, 2-propanol, tetrahydrofuran, diethyl ether, petroleum ether, 1,4-dioxane, benzene, toluene, xylene, hexane, cyclohexane, ethyl acetate, butyl acetate, trifluoroacetic acid Acetic acid, formic acid, dichloromethane, chloroform, carbon tetrachloride, dichloroethane, trichloroethane, trichloroethylene, trifluoroethane, 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2,2-to Trifluoroethanol, hexafluoroacetone, formamide, N, N- dimethylformamide, N, N- dimethylacetamide, N- methylpyrrolidone, pyridine, acetonitrile, water and the like.

From the viewpoint of controlling the average fiber diameter of the resulting fiber structure, trifluoroacetic acid, acetic acid, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, trichloroethane, trichloroethylene, trifluoroethane, 1,1,1,1 3,3,3-hexafluoro-2-propanol, 2,2,2-trifluoroethanol, N, N-dimethylformamide, water are preferred, trifluoroacetic acid, acetic acid, dichloromethane, chloroform, 1,2-dichloroethane, 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2,2-trifluoroethanol, N, N-dimethylformamide, and water are more preferable. These may be used alone or in combination of two or more.

For dissolution of the polyamino acid in Step 1, methods such as heating, use of an ultrasonic device, addition of a solubilizing agent, etc. can be used as necessary. Specific examples of solubilizing agents include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, inorganic bases such as ammonia, monoethanolamine, diethanolamine, and triethanolamine. Organic amines such as 2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol, lysine, ornithine and arginine, inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, tartaric acid And organic acids such as citric acid, p-toluenesulfonic acid, glycolic acid, malic acid, lactic acid, fatty acid, acidic amino acid, pyroglutamic acid. These may be used alone or in combination of two or more.

The rate at which the polyamino acid solution is discharged in step 2 is appropriately set according to the viscosity of the solution to be discharged, the scale of the electrospinning apparatus, etc., but is generally 0.1 to 60 ml / hour, preferably 1 to 20 ml / hour. It can be. When the discharge speed is made faster than the above range, the discharged solution tends to reach the collector in the form of droplets and cause fusion of fibers and the like. Moreover, if the discharge speed is slower than the above range, it takes a long time to produce the fiber structure.

In step 2, the number of nozzles from which the solution is discharged may be one or two or more. Therefore, the discharge of the solution may be performed at one or two or more locations. In addition, if the discharge of the nozzle in the step 2 is blocked by the adhesion of the fibers formed in the step 4, the precipitation of solids from the discharged solution in the step 2, etc., it is appropriate. By discharging while removing these deposits, precipitates, etc., by the method, it is possible to prevent the discharge of the solution from being hindered.

The voltage applied between the nozzle and the collector in step 3 is appropriately set depending on the viscosity of the solution to be discharged, the scale of the electrospinning apparatus, etc., but is generally 5 to 50 kV, preferably 11 to 45 kV. it can. In the present invention, the voltage can be 11 kV or more, preferably 13 kV or more, more preferably 15 kV or more, further preferably 17 kV or more, particularly preferably 19 kV or more, and the voltage can be 45 kV or less, preferably 40 kV or less, More preferably, it can be 35 kV or less, more preferably 30 kV or less, and particularly preferably 25 kV or less. If the applied voltage is higher than the above range, there is a risk of discharge between the nozzle and the collector. On the other hand, when the applied voltage is lower than the above range, the fiber diameter is not reduced and the spinnability tends to be poor.

The interval between the nozzle and the collector in step 4 is appropriately set depending on the scale of the electrospinning apparatus, but can be generally 5 to 50 cm, preferably 10 to 40 cm. In the present invention, the interval can be 10 cm or more, preferably 12 cm or more, more preferably 14 cm or more, still more preferably 16 cm or more, and particularly preferably 18 cm or more, and the interval can be 40 cm or less, preferably 35 cm or less, More preferably, it is 30 cm or less, More preferably, it is 25 cm or less, Most preferably, it can be 20 cm or less. When the interval is longer than the above range, the formed fiber does not reach the collector and adheres to the portion between the nozzle and the collector. If the interval is shorter than the above range, the discharged solution tends to reach the collector in the form of droplets and cause fusion of fibers.

The fiber structure formed on the collector in step 5 can be subjected to operations such as neutralization and drying as necessary. For example, when an organic acid is used as a solvent or a solubilizing agent for dissolving the polyamino acid used in the present invention, an alkaline aqueous solution immersed in an alkaline aqueous solution such as sodium hydroxide, potassium hydroxide, or sodium bicarbonate is used. The organic acid remaining in the fiber structure can be neutralized by a method such as spraying or leaving it in ammonia saturated vapor. Further, when the polyamino acid used in the present invention is dissolved in a solvent having low volatility, it remains in the fiber structure by a method such as heating, blowing dry air, or leaving it under vacuum or reduced pressure. Solvent to be removed.

The thickness of the fiber structure formed on the collector in step 5 is arbitrary depending on the application. For example, it is about 1 to 30 μm when used as a scaffold for tissue or cells, and 50 when used as artificial skin. About 100 μm.

The temperature and humidity at which the electrospinning in Steps 1 to 5 is performed are appropriately set depending on the type of the solvent that dissolves the polyamino acid used in the present invention, but generally 5 to 30 ° C. and relative humidity 10 to 80. % Adjusted.

The nozzle used in steps 1 to 5 may have any shape and size as long as it can discharge a solution in step 2 and can serve as an electrode when a high voltage is applied in step 3. For example, an injection needle can be mentioned. The diameter of the injection needle used for electrospinning including the steps 1 to 5 is generally 0.01 to 2.0 mm, preferably 0.1 to 1.5 mm. When the diameter of the injection needle is larger than the above range, the discharged solution tends to reach the collector in the form of droplets. On the other hand, if the diameter is smaller than the above range, the amount of discharge per hour is small, so that it takes a lot of time to manufacture the fiber structure.

The collector used in steps 1 to 5 may have any shape and size as long as it comprises a metal having good conductivity such as copper, aluminum, or stainless steel and can serve as an electrode when a high voltage is applied in step 3. . For example, a membrane-like, tubular, or hollow fiber structure can be obtained by using a collector as a cylindrical rotating body. Furthermore, in order to easily remove the fiber structure of the present invention from the collector, a material that does not impair the function of the collector electrode, such as aluminum foil, spunbond nonwoven fabric, gauze, micro-nanofiber sheet, etc., is laid on the collector. The fiber structure of the present invention may be formed on these.

The fiber structure of the present invention is characterized by containing a polyamino acid as a main component. In the present specification, the main component means a main component contained as a raw material of the fiber structure, and the content thereof is usually 80% by weight or more, preferably 90% by weight with respect to the total weight of the fiber structure. As mentioned above, More preferably, it is 95 weight% or more.

The fiber structure containing the polyamino acid of the present invention can further contain a polymer compound other than the polyamino acid in addition to the polyamino acid. Examples of such a polymer compound include polyvinyl chloride, polyacrylonitrile, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, polybutylene succinate, polyethylene succinate, polystyrene, polycarbonate, Polyhexamethylene carbonate, polyarylate, polyvinyl isocyanate, polybutyl isocyanate, polymethyl methacrylate, polyethyl methacrylate, polypropyl methacrylate, polybutyl methacrylate, polymethyl acrylate, polyethyl acrylate, polybutyl acrylate, polyethylene terephthalate, polytrimethylene terephthalate , Polyethylene naphthalate, polyparaphenylene terephthalamide, polyparaphenylene terephthal Luamide-3,4'-oxydiphenylene terephthalamide copolymer, polymetaphenylene isophthalamide, cellulose diacetate, cellulose triacetate, methylcellulose, propylcellulose, benzylcellulose, carboxycellulose, carboxymethylcellulose, oxidized regenerated cellulose, hyaluronic acid, Sodium hyaluronate, fibroin, natural rubber, polyvinyl alcohol, polyvinyl acetate, polyvinyl methyl ether, polyvinyl ethyl ether, polyvinyl propyl ether, polyvinyl butyl ether, polyvinylidene chloride, poly-N-vinyl pyrrolidone, poly-N-vinyl carbazole, poly -N-vinyl pyridine, polyvinyl methyl ketone, polyvinyl isopropenyl ketone, polyethylene Synthetic polymer compounds such as xoxide, polypropylene oxide, polycyclopentene oxide, polystyrene sulfone, nylon 6, nylon 66, nylon 11, nylon 12, nylon 610, nylon 612, and copolymers thereof; collagen, atelocollagen, gelatin, laminin And proteins such as fibronectin and sericin; polysaccharides such as chitin, chitosan and cellulose. These may be used alone or in combination of two or more.

Of the polymer compounds exemplified above, polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer are preferable from the viewpoint of maintaining or improving the strength or / and shape of the fiber structure containing a polyamino acid. , Polycaprolactone, carboxymethylcellulose, oxidized regenerated cellulose, hyaluronic acid, sodium hyaluronate, polyvinyl alcohol, collagen, atelocollagen, gelatin, laminin, fibronectin, sericin, chitin, chitosan, cellulose, more preferably polylactic acid Polyglycolic acid, polylactic acid-glycolic acid copolymer, carboxymethyl cellulose, oxidized regenerated cellulose, sodium hyaluronate, polyvinyl alcohol, collagen, and gelatin can be used. These may be used alone or in combination of two or more.

The ratio of the polymer compound to the polyamino acid is appropriately set depending on the function of the fiber structure to be obtained, and can be generally 1 to 80% by weight, preferably 5 to 50% by weight. When the ratio with respect to the polyamino acid is higher than the upper limit range, the function of the fiber structure based on the polyamino acid tends to be impaired, and when the ratio with respect to the polyamino acid is lower than the lower limit range, the effect of containing the polymer compound is obtained. It becomes difficult to be recognized.

The polymer compound may be electrospun in a state of being mixed with a polyamino acid to form a fiber structure, or may be combined after electrospinning separately from the polyamino acid to form a fiber structure.

The fiber structure of the present invention can be widely used for medical materials. Specifically, it can be used for medical materials such as scaffolds for regenerative medicine, surgical sutures, wound dressings, artificial blood vessels, and drug-supporting substrates. In addition, the fiber structure of the present invention is used in various applications such as a cell-supporting substrate in a cell culture apparatus, a supporting substrate such as a living organism in a bioreactor, various filters, a catalyst-supporting substrate, a clothing substrate, and a cosmetic substrate. Can also be used.

In the present specification, the scaffold or scaffold is a biological tissue culture substrate in the field of regenerative medicine, and refers to a molded body that functions as a biological material for the purpose of defect, repair, regeneration, and treatment of biological tissue, For example, a three-dimensional cell culture substrate, a cell aggregate (spheroid) formation substrate, an adhesion prevention material, and the like are included in this.

Since the fiber structure of the present invention has a fiber diameter comparable to that of an extracellular matrix in vivo, it can be easily decomposed in vivo. Therefore, the fibrous structure is suitable for a scaffold material that is desired to disappear quickly after a living tissue or cell is repaired and regenerated in a living body.

Further, the fiber structure of the present invention is particularly suitable as a scaffold material that is required to have an effect of suppressing the adhesion of cells and living tissues, and is useful as, for example, a cell aggregate formation base (spheroid) formation material or an adhesion prevention material. In addition, it can be used for wound dressings, hemostatic materials, etc. due to such effects, as well as artificial organs (artificial blood vessels, artificial lungs, artificial kidneys, artificial hearts) that need to avoid thrombus formation, or medical devices (catheters, It is also useful as a surface covering material for cannulas, artificial dialyzers, dialysis membranes, injection needles, syringes, blood storage containers, shunts, blood circuits).

The fiber structure of the present invention is formed into a shape and structure corresponding to a part of a living tissue, and the defect, repair, regeneration, etc. of living tissue such as skin, blood vessel, nerve, bone, cartilage, esophagus, valve, other organs, It can be used directly or indirectly for treatment. Moreover, various agents, such as an anti-inflammatory agent, an antiallergic agent, an antitumor agent, vitamins, can also be contained in a fiber structure or its fiber.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Production Example 1] Poly-L-alanine fiber structure 1.4% by weight of N-carboxy-L-alanine anhydride was added to benzene and stirred for 2 to 3 days to obtain poly-L-alanine. This was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (50:50 (v / v)) to obtain a 4% by weight poly-L-alanine solution. This solution is put into a syringe (Terumo) connected with a nozzle (needle) having a diameter of 940 μm, the distance between the nozzle and the collector is set to 10 to 15 cm, and a voltage of 20 to 38 kV is applied between them to discharge the solution. Thus, a fiber structure composed of poly-L-alanine was obtained on the collector. A scanning electron microscope (SEM) photograph of the surface of the obtained fiber structure composed of poly-L-alanine is shown in FIG.

[Production Example 2] Poly-L-leucine fiber structure After adding 11.1 wt% of N-carboxy-L-leucine anhydride to 1,2-dichloroethane, N, N-dimethyl-1, 3-Propanediamine was added. The mixture was stirred for 2 to 3 days to obtain poly-L-leucine. This was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (30:70 (v / v)) to obtain a 4% by weight poly-L-leucine solution. From this solution, a fiber structure composed of poly-L-leucine was obtained in the same manner as in Production Example 1.

[Production Examples 3 to 7] Various polyamino acid fiber structures In the same manner as in Production Example 1 or 2, various N-carboxy-L-amino acid anhydrides were added to the polymerization solvent, and a polymerization initiator was added as necessary. The mixture was stirred for 2 to 3 days to obtain various polyamino acids. The polymerization solvent and polymerization initiator used are as shown in Table 1. Various polyamino acids were made into solutions using the solvents shown in Table 1, and fiber structures composed of various polyamino acids were obtained from these solutions by the same method as in Production Example 1.

[Production Example 8] Fiber structure of poly-L-alanine / γ-methyl-L-glutamic acid copolymer (9/1) N-carboxy-L-alanine anhydride and N-carboxy-γ-methyl- A poly-L-alanine / γ-methyl-L-glutamic acid copolymer was prepared by adding L-glutamic anhydride in a molar ratio of 9: 1 to a total of 1.4% by weight and stirring for 2 to 3 days. (9/1) was obtained. This was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (50:50 (v / v)), and an 8% by weight poly-L-alanine / γ-methyl-L-glutamic acid copolymer (9/1) solution was obtained. did. From this solution, a fiber structure comprising a poly-L-alanine / γ-methyl-L-glutamic acid copolymer (9/1) was obtained in the same manner as in Production Example 1.

[Production Examples 9 to 22] Fiber structures of various polyamino acid copolymers In the same manner as in Production Example 8, two types of N-carboxy-L-amino acid anhydrides were added to the polymerization solvent, and polymerization was started as necessary. The agent was added and stirred for 2 to 3 days to obtain various polyamino acid copolymers. Each was carried out under the materials and conditions shown in Table 1. Various polyamino acid copolymers were made into solutions using the solvents shown in Table 1, and fiber structures composed of various polyamino acid copolymers were obtained from these solutions by the same method as in Production Example 1.

[Production Example 23] Fiber structure of poly-L-glutamine / γ-methyl-L-glutamic acid copolymer 1,6.6% by weight of 1,2-dichloroethane with N-carboxy-γ-methyl-L-glutamic anhydride After the addition, N, N-dimethyl-1,3-propanediamine was added as a polymerization initiator. The mixture was stirred for 2 to 3 days to obtain poly-γ-methyl-L-glutamic acid. 2,2,2-trichloro-1-ethanol (Tokyo Kasei Kogyo) and p-toluenesulfonic acid monohydrate (Tokyo Kasei Kogyo) were added to N-carboxy-γ-methyl-L-glutamic anhydride. 4 equivalents and 0.8 equivalents were added to the number of moles, respectively, and reacted at 80 ° C. to obtain a poly-γ-methyl / (2,2,2-trichloroethyl) -L-glutamic acid copolymer. A large excess of ammonia was allowed to act on the moles of N-carboxy-γ-methyl-L-glutamic anhydride in tetrahydrofuran to obtain poly-L-glutamine / γ-methyl-L-glutamic acid. This was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (1: 3 (v / v)) to obtain a 14 to 15 wt% poly-L-glutamine / γ-methyl-L-glutamic acid copolymer solution. From this solution, a fiber structure comprising a poly-L-glutamine / γ-methyl-L-glutamic acid copolymer was obtained in the same manner as in Production Example 1.

[Production Example 24] Poly-γ-methyl / (polyethylene glycol) -L-glutamic acid copolymer fiber structure Polyethylene glycol methyl ether (Sigma-Aldrich) was added to poly-γ-methyl-L-glutamic acid obtained in Production Example 23. , Average molecular weight 350) and p-toluenesulfonic acid monohydrate are added in an amount of 2 equivalents and 0.2 equivalents relative to the number of moles of N-carboxy-γ-methyl-L-glutamic anhydride and reacted at 75 ° C. A -γ-methyl / (polyethylene glycol) -L-glutamic acid copolymer was obtained. The weight average molecular weight measured by the method described in Measurement 1 was 1.4 × 10 ⁶ . This was dissolved in 2,2,2-trifluoroethanol to obtain a 10% by weight poly-γ-methyl / (polyethylene glycol) -L-glutamic acid copolymer solution. From this solution, a fiber structure comprising a poly-γ-methyl / (polyethylene glycol) -L-glutamic acid copolymer was obtained in the same manner as in Production Example 1.

[Production Example 25] Poly-L-alanine / L-lysine copolymer fiber structure Poly-L-alanine / ε-benzyloxycarbonyl-L-lysine copolymer (9/1) obtained in Production Example 16 Trifluoroacetic acid (Tokyo Chemical Industry Co., Ltd.) and thioanisole (Tokyo Chemical Industry Co., Ltd.) were added at 270 equivalents and 5 equivalents, respectively, with respect to the number of moles of N-carboxy-ε-benzyloxycarbonyl-L-lysine anhydride. To obtain a poly-L-alanine / L-lysine copolymer. This was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (50:70 (v / v)) to obtain a 4.4% by weight poly-L-alanine / L-lysine copolymer solution. From this solution, a fiber structure comprising a poly-L-alanine / L-lysine copolymer was obtained in the same manner as in Production Example 1.

[Production Example 26] Fiber structure of poly-L-alanine-poly-L-lysine mixture Poly-L-alanine and poly-L-lysine hydrobromide obtained in Production Example 1 (Sigma Aldrich, weight average molecular weight) 300,000) in a ratio of 9: 1 by weight in a dichloromethane-trifluoroacetic acid-N, N-dimethylformamide mixed solvent (50: 50: 12.5 (v / v)). An L-alanine-L-lysine mixture solution was obtained. From this solution, a fiber structure comprising a poly-L-alanine-L-lysine mixture was obtained in the same manner as in Production Example 1.

[Production Example 27] Fiber structure of poly-L-alanine-poly-ε-benzyloxycarbonyl-L-lysine mixture (9/1) Poly-L-alanine obtained in Production Example 1 and obtained in Production Example 5 The poly-ε-benzyloxycarbonyl-L-lysine was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (50:50 (v / v)) at a molar ratio of 9: 1 in terms of monomer, and 5.3 wt. % Poly-L-alanine-poly-ε-benzyloxycarbonyl-L-lysine mixture (9/1) solution. From this solution, a fiber structure comprising a poly-L-alanine-poly-ε-benzyloxycarbonyl-L-lysine mixture (9/1) was obtained in the same manner as in Production Example 1.

[Production Example 28] Fiber structure of poly-L-alanine-poly-ε-benzyloxycarbonyl-L-lysine mixture (6/4) Poly-L-alanine obtained in Production Example 1 and obtained in Production Example 5 Poly-ε-benzyloxycarbonyl-L-lysine was dissolved in a dichloromethane-trifluoroacetic acid mixed solvent (50:50 (v / v)) at a molar ratio of 6: 4 in terms of monomer, and 5.3 wt. % Poly-L-alanine-poly-ε-benzyloxycarbonyl-L-lysine mixture (6/4) solution. From this solution, a fiber structure comprising a poly-L-alanine-poly-ε-benzyloxycarbonyl-L-lysine mixture (6/4) was obtained in the same manner as in Production Example 1.

[Comparative Example 1] Poly-γ-benzyl-L-glutamic acid fiber structure N-carboxy-γ-benzyl-L-glutamic acid anhydride was polymerized into poly-γ-benzyl-L-glutamic acid, and then dichloromethane-tri It was dissolved in a mixed solvent of fluoroacetic acid to obtain a 4% by weight poly-γ-benzyl-L-glutamic acid solution. From this solution, a fiber structure composed of poly-γ-benzyl-L-glutamic acid was obtained in the same manner as in Production Example 1.

Comparative Example 2 Chitosan Fiber Structure Chitosan powder (Wako Pure Chemical Industries, Ltd.) was dissolved in an acetic acid-trifluoroacetic acid mixed solvent (10:90 (v / v)) to obtain a 4% by weight chitosan solution. From this solution, a fiber structure made of chitosan was obtained in the same manner as in Production Example 1.

[Comparative Example 3] Cellulose fiber structure Cellulose powder (Wako Pure Chemical Industries, Ltd.) was dissolved in an acetic acid-trifluoroacetic acid mixed solvent (10:90 (v / v)) to obtain a 4% by weight cellulose solution. From this solution, a fiber structure made of cellulose was obtained in the same manner as in Production Example 1.

[Comparative Example 4] Gelatin fiber structure Gelatin powder was dissolved in trifluoroacetic acid to obtain a 9 wt% gelatin solution. From this solution, a fiber structure made of gelatin was obtained in the same manner as in Production Example 1.

[Measurement 1] Method for measuring weight average molecular weight The weight average molecular weight is measured by attaching an analytical column (Showa Denko, Shodex K-802 and K-806M) to a gel permeation chromatography apparatus (GPC, Hitachi, LaChrom Elite). I went. The measurement solution was prepared by dissolving in chloroform so that the polyamino acid concentration was 0.25 to 1.0% (w / v), followed by filter filtration. 10 to 80 μl of a measurement solution was injected, and measurement was performed under the conditions of an eluent flow rate of 1 ml / min and a column holding temperature of 40 ° C. Calibration polystyrene was used to calculate the weight average molecular weight.

[Evaluation 1] Evaluation Method of Discharge Rate in Electrospinning In the production of a fiber structure by the electrospinning method including steps 1 to 5, between the start and end of solution discharge from the nozzle in step 2 The value obtained by dividing the total amount of the discharged solution by the time from the start to the end was taken as the discharge speed. The evaluation criteria are as follows.
○: Discharge speed is 10 ml / hour or more ×: Discharge speed is less than 10 ml / hour

[Evaluation 2] Evaluation method of average fiber diameter After visually confirming that the surface of the obtained fiber structure is uniform, a part of the surface was sampled and a circular cover glass or cover slip having a diameter of 12 to 13 mm. (Thermo Scientific, Nunc Thermanox Plastic Coverslips). The surface of the collected fiber structure was observed with a stereo microscope (Nikon, SMZ800) (magnification 10 times), and after confirming that the surface was uniform, a scanning electron microscope (SEM, Hitachi, S-4800) It was used for surface observation at. First, the whole measurement sample was looked down at a magnification of 500 times, and after confirming that the surface was uniform, the magnification was switched to 5,000 times and photographs were taken at random. The obtained photograph was placed horizontally and divided into 10 equal area sections by dividing the horizontal direction and the vertical direction into 5 equal parts and 2 equal parts, respectively. The fibers in focus at the center of each section or the fibers closest to the center were selected and their diameters were measured, and the average value of them was obtained to obtain the average fiber diameter. The evaluation criteria are as follows.
A: Average fiber diameter is less than 150 nm B: Average fiber diameter is 150 nm or more and less than 500 nm Δ: Average fiber diameter is 500 nm or more and less than 1 μm X: Average fiber diameter is 1 μm or more

[Evaluation 3] Evaluation Method of Fiber Diameter Uniformity The standard deviation value of the average fiber diameter measured in Evaluation 2 was obtained. The evaluation criteria are as follows.
◎: Standard deviation value is less than 30 nm ○: Standard deviation value is 30 nm or more and less than 90 nm Δ: Standard deviation value is 90 nm or more and less than 200 nm ×: Standard deviation value is 200 nm or more

[Evaluation 4] Evaluation method for the presence or absence of droplets Among photographs (1,000x magnification) obtained by randomly photographing the surface of the obtained fiber structure with a scanning electron microscope (SEM, Hitachi, S-4800) In addition, the number of circular objects having a maximum diameter of 5 μm or more (traces indicating that they were collected on the collector in a droplet state) was measured. The evaluation criteria are as follows.
◎: The number of circular objects is 3 or less ○: The number of circular objects is 4 or more and 7 or less △: The number of circular objects is 8 or more and 15 or less ×: The number of circular objects is 16 or more

[Evaluation 5] Evaluation method of cell adhesion inhibitory effect (1)
The obtained fiber structure was attached to a circular cover glass or coverslip (Thermo Scientific, Nunc Thermanox Plastic Coverslips) having a diameter of 12 to 13 mm, and set in a 24-well culture plate (Nippon Becton Deckonson, Falcon culture plate). . After immersing in 70% ethanol aqueous solution for sterilization, it was washed with phosphate buffered saline (Takara Bio, PBS (-)). Human bone marrow mesenchymal stem cells (Lonza, Human Mesenchymal Stem Cell) or mesenchymal stem cell medium (Lonza, MSCGM), or inactivated fetal bovine serum (Invitrogen, GIBCO FBS) and penicillin-streptomycin (Sigma- Aldrich Co.) laden Dulbecco's modified Eagle's medium (Invitrogen Corp., was suspended in GIBCO DMEM), was seeded by 20,000Cell / well, 5% CO _2/37 ℃ conditions incubator (Thermo Scientific, Inc., former incubator ) For 3 days. After the culture, the medium was removed, and the cells were lysed with a citrate (Nacalai Tesque) -sodium chloride (Nacalai Tesque) buffer containing sodium lauryl sulfate (Nacalai Tesque). Bisbenzimide H33258 fluorochrome trihydrochloride DMSO solution (Nacalai Tesque) was added thereto, and the amount of intracellular DNA was quantified to determine the number of cells adhered and grown on the fiber structure. For comparison, cells were seeded and cultured without setting the fiber structure in a 24-well culture plate, and the amount of intracellular DNA was quantified. The evaluation criteria are as follows.
A: The amount of intracellular DNA is less than 15% of that of the comparison control. ○: The amount of intracellular DNA is 15% or more and less than 30% of that of the comparison control. Less than 50% ×: The amount of intracellular DNA is 50% or more of that of the control

[Evaluation 6] Evaluation method of cell adhesion inhibitory effect (2)
As in Evaluation 5, the obtained fiber structure was set on a culture plate, and cells were seeded and cultured. After seeding and culturing for 3 days, a viable cell count measuring reagent (Nacalai Tesque, Cell Count Reagent SF) was added to the medium. Again, after 4-5 hours in the incubator, the absorbance (measurement wavelength: 450 nm) was measured. For comparison, cells were seeded and cultured without setting the fiber structure in a 24-well culture plate, and the addition of a reagent for measuring the number of viable cells and the measurement of absorbance were performed in the same manner. The evaluation criteria are as follows.
A: Absorbance is less than 15% relative to that of the comparison control. O: Absorbance is not less than 15% and less than 30% relative to that of the comparison control. More than 50% of that of the control

The evaluation methods shown in Evaluations 5 and 6 are all general-purpose methods for measuring the number of cells from the viewpoint of ease of operation, measurement accuracy, and the like.

The fiber structures obtained in the above Production Examples 1 to 28 were referred to as Examples 1 to 28, respectively, and Comparative Examples 1 to 4 were combined with them to perform the above Evaluations 1 to 6. The results are shown in Tables 2 and 3.

As shown in Table 2, with respect to poly-γ-benzyl-L-glutamic acid, a fiber structure having a small average fiber diameter could not be obtained even when the ejection speed was adjusted, and the cell adhesion inhibitory effect was not sufficient. (Comparative Example 1). In addition, with regard to chitosan, cellulose, and gelatin, all of the droplets remained and the uniformity of the average fiber diameter was found to be uneven, so that a fiber structure with sufficient quality could not be obtained (Comparative Example 2, 3, 4). On the other hand, with respect to Examples 1 to 28, the droplet problem was solved, and preferable results were obtained that satisfied the fiber diameter. Of these, poly-L-alanine, poly-L-leucine, poly-β-benzyl-L-aspartic acid, poly-ε-benzyloxycarbonyl-L-lysine, poly-L-alanine / γ-methyl-L -Glutamic acid copolymer (9/1), poly-L-alanine / γ-methyl-L-glutamic acid copolymer (6/4), poly-L-valine / γ-methyl-L-glutamic acid copolymer ( 6/4), poly-L-isoleucine / γ-methyl-L-glutamic acid copolymer (6/4), poly-L-alanine / ε-benzyloxycarbonyl-L-lysine copolymer (9/1) Poly-L-valine / ε-benzyloxycarbonyl-L-lysine copolymer (6/4), poly-L-alanine / L-lysine copolymer (9/1), poly-L-alanine-poly -L-lysine mixture, poly-L-alanine-po The -ε-benzyloxycarbonyl-L-lysine mixture (9/1) showed a high cell adhesion inhibitory effect (Examples 1, 2, 4, 5, 8, 9, 12, 13, 16, 19 , 25, 26, 27). In particular, poly-L-alanine was a favorable result in all of the average fiber diameter, uniformity, droplet problem, and cell adhesion suppression.

[Evaluation 7] Influence of manufacturing conditions (setting of applied voltage and distance between nozzle and collector)

A poly-L-valine was prepared in the same manner as in Production Example 22 except that the voltage applied to the poly-L-valine / L-phenylalanine (1/1) solution shown in Production Example 22 was changed to 19 to 20 kV. An attempt was made to produce a fiber structure composed of / L-phenylalanine (1/1).
Further, poly-L-valine was produced in the same manner as in Production Example 22 except that the voltage applied to the poly-L-valine / L-phenylalanine (1/1) solution shown in Production Example 22 was changed to 15 kV. An attempt was made to produce a fiber structure composed of / L-phenylalanine (1/1).
Further, poly-L-valine was produced in the same manner as in Production Example 22 except that the voltage applied to the poly-L-valine / L-phenylalanine (1/1) solution shown in Production Example 22 was changed to 10 kV. An attempt was made to produce a fiber structure composed of / L-phenylalanine (1/1).
Further, the voltage applied to the poly-L-valine / L-phenylalanine (1/1) solution shown in Production Example 22 was changed to 19 to 20 kV, and the distance between the nozzle containing the solution and the collector was changed to 5 cm. Except for this, an attempt was made to produce a fiber structure composed of poly-L-valine / L-phenylalanine (1/1) by the same method as in Production Example 22.
The surface of the obtained fiber structure was visually evaluated. Specifically, the number of visible granular materials was measured. The evaluation criteria for the granular materials are as follows, and the results are shown in Table 4.
◎: The number of granular materials is 0 per 1 cm square ○: The number of granular materials is 1 or more and 9 or less per 1 cm square △: The number of granular materials is 10 or more and 49 or less per 1 cm square ×: Number of granular materials 50 or more per 1 cm square-: Impossible to measure

In the production of a poly-L-valine / L-phenylalanine (1/1) fiber structure, when the voltage applied between the nozzle and the collector is lowered, particularly at 10 kV, the spinnability becomes poor and a fiber structure is obtained. I couldn't. In addition, when the voltage was 15 kV, the fiber structure could be manufactured, but most of the discharged solution reached the collector in the form of droplets, and 10 or more granular particles were formed on the surface of the fiber structure. Things were visible. Moreover, when the distance between the nozzle and the collector was shortened to 5 cm in particular, most of the discharged solution reached the collector in a droplet state.

By containing a specific polyamino acid as a main component, fibers that can be used for medical materials (particularly scaffold materials) that are safe for the living body, high in biocompatibility, and highly effective in suppressing adhesion of cells and tissues, It is significant that we can now provide medical materials or scaffolds.

This application is based on Japanese Patent Application No. 2009-138599 filed on Jun. 9, 2009, the entire contents of which are included in this specification.

Claims

A fiber structure comprising polyamino acid as a main component and having an average fiber diameter of 50 nm or more and less than 500 nm.
The fiber structure according to claim 1, wherein the amino acid is one or more selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, phenylalanine and proline.
The fiber structure according to claim 1 or 2, wherein the amino acid is alanine.
The fiber structure according to any one of claims 1 to 3, wherein the polyamino acid is composed of one kind of amino acid.
The fiber structure according to claim 1, wherein the polyamino acid is composed of two kinds of amino acids.
The fiber structure according to claim 5, wherein the two types of amino acids are a combination of an acidic amino acid or a basic amino acid and a nonpolar neutral amino acid.
The fiber structure according to claim 5, wherein the two kinds of amino acids are acidic amino acids or a combination of basic amino acids and aromatic amino acids.
The fiber structure according to claim 6 or 7, wherein the acidic amino acid is glutamic acid.
The fiber structure according to claim 6 or 7, wherein the basic amino acid is lysine.
The fiber structure according to claim 6, wherein the nonpolar neutral amino acid is selected from alanine, valine, leucine or isoleucine.
The fiber structure according to claim 7, wherein the aromatic amino acid is phenylalanine.
The fiber structure according to any one of claims 1 to 11, wherein a protective group is bonded to a side chain of an amino acid constituting the polyamino acid.
The protective group is one or more selected from the group consisting of a methyl group, a benzyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, and a trifluoroacetyl group. The fiber structure described.
A method for producing a fiber structure, comprising the following steps;
Step 1) Step of dissolving a polyamino acid in a solvent to form a solution,
Step 2) A step of continuously discharging the solution put in the syringe from a nozzle attached to the tip of the syringe,
Step 3) A step of applying a high voltage between the nozzle and the collector with a high voltage generator in the discharge,
Step 4) changing the discharged solution into a fiber shape between the nozzle and the collector,
Step 5) A step of collecting the fiber on a collector.
The polyamino acid solution concentration in step 1 is 1 to 20% by weight, the polyamino acid solution discharge speed in step 2 is 1 to 20 ml / hour, and the voltage applied between the nozzle and collector in step 3 is 11 15. The manufacturing method according to claim 14, wherein the manufacturing method is characterized in that it is ˜45 kV, and the distance between the nozzle and the collector in step 4 is 10 to 40 cm.
The solvent in Step 1 is trifluoroacetic acid, acetic acid, dichloromethane, chloroform, 1,2-dichloroethane, 1,1,1,3,3,3-hexafluoro-2-propanol, 2,2,2-trifluoroethanol. The production method according to claim 14 or 15, wherein the production method is one or more selected from the group consisting of N, N-dimethylformamide and water.
A medical material comprising the fiber structure according to any one of claims 1 to 13.
The medical material according to claim 17, which is an adhesion preventing material.