JPWO2013021836A1 - Functional hydrogel - Google Patents

Abstract

An object of the present invention is to provide a non-flowable functional hydrogel using keratin as a material and a method for producing the functional hydrogel. Such a problem is solved by a production method of a functional hydrogel including the following steps and a functional hydrogel produced by the production method: 1) In order to convert a disulfide bond into a thiol group in a keratin-containing material. Adding a reducing agent of 2 to extract keratin; 2) adding to the keratin-containing material a protein stabilizer for preventing thiol groups from reforming disulfide bonds; 3) adding a protein stabilizer Step of removing and purifying keratin to obtain a keratin solution: 4) A step of producing a functional hydrogel by concentrating the keratin solution.

Description

  The present invention relates to a method for producing a functional hydrogel, a novel functional hydrogel produced from the production method, and an application of the functional hydrogel.

  This application claims the priority of Japanese Patent Application No. 2011-171678, which is incorporated herein by reference.

  Keratin, a kind of protein, is known to exist in non-vascular tissues of animal tissues such as feathers and hair. Although keratin is used by being blended in hair cosmetics, it is considered as an unused protein when viewed from the industry as a whole. Research and development are underway with the expectation that keratin will be used as an industrial material.

  Keratin can be extracted from feathers and hair. The extraction method of keratin is conventionally known, and an oxidation method, a reduction method, or a combination thereof is used. Keratin contains a large number of cysteine residues and forms multimers by intermolecular and intramolecular disulfide bonds (S-S bonds). In order to extract keratin as an aqueous solution, it is necessary to cleave the S—S bond. In the oxidation method, treatment with peracetic acid or hydrogen peroxide generates sulfonic acid after cleavage of the S—S bond. The sulfonic acid does not return to the S—S bond by being oxidized again. In the reduction method, the S—S bond is cleaved with thioglycolic acid, 2-mercaptoethanol, dithiothreitol, etc., and then converted to a thiol group (SH group). In the combination of the oxidation method and the reduction method, reduction is carried out after converting some S—S bonds to sulfonic acid by light oxidation. In this case, the extracted keratin cysteine residues include those having a sulfonic acid and those having an SH group.

  As applications of the extracted keratin, forms such as water-soluble peptides, sponges and films have been proposed. In addition, gelation of keratin has been studied, and it is known that gelation is possible using a crosslinking agent. Keratin gelled with a cross-linking agent has a problem that its use is limited because the cross-linking agent has unfavorable toxicity to a living body, for example, and does not have re-solubility in an aqueous solution. . Therefore, a method for gelling keratin without using a crosslinking agent is desired.

  It has been reported in Patent Documents 1 to 11 and Non-Patent Document 1 that it is possible to give a viscosity to the keratin-containing composition without using a crosslinking agent. Patent Documents 1 to 3 disclose that a gel-like keratin-containing composition was prepared through a step of converting a keratin disulfide bond into a sulfonic acid using an oxidizing agent. In Patent Documents 4 to 6, a gel-like keratin-containing composition is formed by adding a water-soluble solvent to a powdered keratin material prepared using an oxidizing agent, and water is contained in the gel-like keratin-containing composition. It is disclosed that there are sulfonic acid groups that can be bonded to the benzene. In Patent Document 7, thioglycolic acid is added to human hair, reduction treatment is performed, keratin is extracted, subjected to precipitation with hydrochloric acid, dialysis, derivatization with iodoacetic acid, and then freeze-dried, whereby carboxymethylkeratein is obtained. Is obtained. Thereafter, it is disclosed that a gel-like keratin-containing composition is prepared by dissolving the obtained powder in an aqueous solution. In Non-Patent Document 1, thioglycolic acid is added to human hair to perform reduction treatment, keratin is extracted, dialyzed while adding sodium hydroxide solution for pH adjustment, and concentrated to 20% by weight. Thus, it is disclosed that a gel-like keratin-containing composition was produced.

  Patent Literatures 1 to 11 and Non-Patent Literature 1 are from the same research group represented by Mark Van Dyke. Patent Document 4 and Patent Document 5 disclose that a gel-like keratin-containing composition is used for a breast implant or the like by filling a jacket made of silicon or the like. In Patent Documents 7 to 11, the viscosity (unit: poise) of the gel-like keratin-containing composition is disclosed. Viscosity is a physical constant for a fluid, and the gel-like keratin-containing composition disclosed in Patent Documents 1 to 11 and Non-Patent Document 1 is not a solid or semi-solid, but a viscous fluid having fluidity. It is believed that there is. That is, a non-flowable gel made of keratin has not been reported yet.

U.S. Pat. Special table 2003-501208 US 6270791 Special Table 2003-516775 Publication US Patent 6371984 US Patent 6316598 International Publication No.WO2007 / 050387 Special table 2009-513190 Special table 2009-527274 Special Publication 2009-527481 Special table 2010-524943 gazette

T. Aboushwareb et al., J. Biomed. Mater. Res. Part B: Appl. Biomater., 90B, 45-54 (2009)

  An object of the present invention is to provide a non-flowable functional hydrogel using keratin as a material and a method for producing the functional hydrogel.

  As a result of intensive studies to solve the above problems, the present inventors have added a protein stabilizer to the keratin-containing material to extract keratin, and then extract the keratin from the keratin-containing material. To obtain a purified keratin solution, and concentrating the purified keratin solution to obtain a concentrated keratin solution, and found that a non-flowable functional hydrogel can be produced from the concentrated keratin solution. Was completed.

That is, this invention consists of the following.
1. A method for producing a functional hydrogel comprising the following steps:
1) A step of extracting keratin by adding a reducing agent for converting a disulfide bond to a thiol group to a keratin-containing material;
2) adding a protein stabilizer to the keratin-containing material to prevent thiol groups from re-forming disulfide bonds;
3) Step of removing the protein stabilizer and purifying keratin to obtain a keratin solution:
4) A step of producing a functional hydrogel by concentrating the keratin solution.
2. 2. The method for producing a functional hydrogel according to item 1, wherein the protein stabilizer is sodium dodecyl sulfate.
3. The removal of the protein stabilizer in step 3) is performed at least 5 times by dialysis using 2-5 L of dialysis external solution against 150-250 mL of the keratin extract obtained in steps 1) and 2). The method for producing a functional hydrogel according to 1 or 2 above.
4). 4. The method for producing a functional hydrogel according to any one of items 1 to 3, wherein the concentration of the keratin solution in step 4) is performed under a temperature condition of 40 to 65 ° C.
5. 5. The method for producing a functional hydrogel according to any one of items 1 to 4, wherein a protein denaturant is further added to the keratin-containing material.
6). 6. A functional hydrogel produced by the production method according to any one of 1 to 5 above.
7). 7. The functional hydrogel according to item 6 above, wherein the functional hydrogel has solubility in an aqueous medium.
8). 8. The functional hydrogel according to item 6 or 7, wherein the thiol group in the functional hydrogel is modified with a substituent.
9. 9. A functional hydrogel composition comprising the functional hydrogel according to any one of items 6 to 8, and a physiologically active substance and / or a biocompatible substance.
10. The manufacturing method of the functional hydrogel composition containing a hydroxyapatite by making the functional hydrogel of any one of the preceding clauses 6-8 contact a pseudo-body fluid.
11. A carrier for drug delivery, comprising at least the functional hydrogel according to any one of items 6 to 8 or the functional hydrogel composition according to item 9.
12 12. The drug delivery carrier according to 11 above, wherein the drug delivery carrier is used as a drug delivery system.
13. 13. The drug delivery carrier according to item 11 or 12, wherein the drug delivery carrier is used as a drug sustained release carrier.
14 A regenerative medical material comprising the functional hydrogel according to any one of items 6 to 8 or the functional hydrogel composition according to item 9.

  According to the method for producing a functional hydrogel of the present invention, it is possible to produce a non-flowable functional hydrogel. Since the functional hydrogel obtained by the production method of the present invention is prepared using keratin extracted from non-vascular tissue as a main material, the risk of pathogenicity is extremely low.

  Since the functional hydrogel of the present invention has a thiol group, the physical properties of the hydrogel itself can be obtained by introducing various substituents (for example, acidic residues, basic residues, etc.) and modifying the thiol group. It is possible to modify the function. Various physiologically active substances and biocompatible substances can be introduced into the thiol group or a substituent that modifies the thiol group, and a functional hydrogel composition capable of exhibiting various actions and medicinal effects is prepared and used. It becomes possible to do. Since the functional hydrogel of the present invention is non-flowable, it can be used as a material for regenerative medicine. The functional hydrogel of the present invention can be used as a drug delivery carrier. Since the functional hydrogel of the present invention has re-solubility in an aqueous medium depending on conditions such as pH, it can be used as a drug delivery system (DDS) such as an enteric oral preparation. Moreover, what has the sustained release property of a drug can be used as a carrier for sustained drug release.

  According to the functional hydrogel of the present invention, it is possible to easily produce a functional hydrogel composition containing a physiologically active substance such as a drug or a biocompatible substance (for example, hydroxyapatite (HAP)). .

It is a photograph of the functional hydrogel of the present invention. Example 1 It is the photograph which confirmed HAP on the hydrogel of S-carboxymethylated keratin with the scanning electron microscope. (Example 5) It is the result confirmed by the powder X-ray crystal analysis about HAP on the hydrogel of S-carboxymethylated keratin. (Example 5) It is a figure which shows the sustained release ability of the complex of the hydrogel of S-carboxymethylated keratin and HAP. (Example 6) It is a figure which shows the salicylic acid sustained release ability from a salicylic acid inclusion hydrogel. (Example 8) It is a figure which shows the salicylic acid sustained release ability from a salicylic acid impregnation hydrogel. (Example 8)

(Method for producing functional hydrogel)
The present invention relates to a method for producing a functional hydrogel including the following steps 1) to 4).
1) A step of extracting keratin by adding a reducing agent for converting a disulfide bond to a thiol group to a keratin-containing material.
2) A step of adding a protein stabilizer for preventing the thiol group from reforming the disulfide bond to the keratin-containing material.
3) A step of purifying keratin by removing the protein stabilizer to obtain a keratin solution.
4) A step of producing a functional hydrogel by concentrating the keratin solution.
A non-flowable functional hydrogel can be produced by the production method of the present invention.

  In the present specification, the “keratin-containing material” may contain any material that contains keratin and can extract keratin by the method of the present invention. Examples of keratin-containing materials include animal hairs of mammals such as wool, alpaca, cashmere, mohair, angora, horns, strains, human hair, nails, and feathers of birds. You may use what you did. In addition, when manufacturing clothing such as clothing, carpets, carpets, and knitting from animal hair, a large amount of animal waste is discharged. For example, when manufacturing processes and used items are combined, the amount of waste such as wool reaches about 50,000 tons. Since the waste material of wool can be used as the keratin-containing material of the present invention, it is useful for the reuse of industrial waste. In addition, used clothes, used woven fabrics, knitted fabrics, and the like can be used as the keratin-containing material. The keratin-containing material is preferably used by being finely chopped.

  In the present invention, a reducing agent is used to extract keratin from the keratin-containing material. Any reducing agent may be used as long as it can convert a disulfide bond of keratin (hereinafter referred to as “SS bond”) into a thiol group (hereinafter referred to as “SH group”). Examples include thiol compounds such as glycolic acid, 2-mercaptoethanol, and dithiothreitol, bisulfites such as sodium metabisulfite, and sulfites such as sodium bisulfite. As the reducing agent, a thiol compound is preferable, and 2-mercaptoethanol is more preferable.

  Reduced keratin is produced by step 1). The amount of reducing agent used depends on the type of reducing agent, and it may be added in an amount that can reduce almost all of the S—S bonds of keratin. In order to reduce almost all of the SS bonds of keratin, it is preferable to use the reducing agent in a molar amount of about 100 to 30000 times, preferably about 1000 to 20000 times the cysteine content (mole) of keratin. . If a reducing agent of about 30000 times is used, it is considered sufficient to reduce the S—S bond of keratin. For example, when 2-mercaptoethanol is used as the reducing agent, keratin may be extracted by bringing 10 g of a keratin-containing material into contact with 150 mM or more of 2-mercaptoethanol in 100 ml of an aqueous medium. In Examples described later, 1.66 M of 2-mercaptoethanol is used as a sufficient amount of the reducing agent.

  In the above step 1), a reducing agent may be added to the aqueous medium in which the keratin-containing material is suspended to extract keratin. Or you may add a keratin containing material in the aqueous medium which added the reducing agent previously. The aqueous medium used in this step may be an aqueous medium having any component and composition as long as the extracted keratin can produce a functional hydrogel. For example, distilled water, phosphate , Carbonates, acetates, borates, citrates, tris buffers and the like. The aqueous medium in the present invention may be distilled water or a buffer solution containing alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide.

  In step 2), a protein stabilizer is added to prevent the SH group from reforming the S—S bond. Protein stabilizers can also be referred to as S—S bond formation inhibitors or protein dissociators. The protein stabilizer may be any as long as it can prevent the SH group from reforming the S—S bond and can solubilize keratin. Examples of protein stabilizers include surfactants such as nonionic surfactants, amphoteric surfactants, and anionic surfactants, preferably anionic surfactants, and more Alkyl sulfates are preferable, and sodium dodecyl sulfate (hereinafter referred to as “SDS”) is more preferable.

  The protein stabilizer in step 2) may be added separately from the reducing agent in step 1) (for example, after addition of the reducing agent), or may be added simultaneously with the reducing agent. The protein stabilizer is preferably added to the aqueous medium in the same manner as the reducing agent. The amount of protein stabilizer used varies depending on the type of protein stabilizer, and it can maintain a solubilized state in solution without precipitating keratin by preventing the SH group from reforming the SS bond. Any amount can be used. For example, when SDS is used as a protein stabilizer, 14 g of SDS is considered to bind to 1 g of protein. When half the weight of 10 g of keratin-containing material corresponds to protein, SDS is used at about 0.25 M, so that a saturating amount of SDS is used relative to the amount of protein. SDS does not necessarily require a saturation amount, and can be used by appropriately adjusting the amount capable of preventing the SH group from reforming the S—S bond. Specifically, 10 g of the keratin-containing material may be contacted with 0.1 to 0.5 M SDS, preferably 0.2 to 0.3 M SDS, in 100 ml of an aqueous medium.

  In steps 1) and 2), a protein denaturant may be added to an aqueous medium in which a keratin-containing material is suspended to extract keratin. The order of adding the protein denaturant is not particularly limited, and may be the same as that of the reducing agent or the protein stabilizer, or may be separate. Any protein denaturant may be used as long as it can reduce the stability of the higher-order structure due to hydrogen bonding by interrupting the hydrogen bonding of the protein in the keratin-containing material. Examples of protein denaturing agents include urea, thiourea, guanidine hydrochloride and the like, but it is preferable to use urea. The amount of the protein denaturing agent used varies depending on the type of the protein denaturing agent, but may be any amount as long as it reduces the stability of the protein higher-order structure. For example, when urea is used as a protein denaturant, 10 g of keratin-containing material may be contacted with 4 to 10 M urea, preferably 6 to 8 M urea, in 100 ml of an aqueous medium. In protein denaturation treatment, urea of about 8 M is usually used, but it is considered that protein can be denatured by adjusting the reaction time even at about 4 M. Further, considering the solubility of urea, it is considered that the upper limit is about 10 M.

  By steps 1) and 2), a keratin extract solution obtained by extracting keratin from the keratin-containing material can be obtained. In steps 1) and 2), the extraction reaction is carried out by bringing the keratin-containing material into contact with a reducing agent and a protein stabilizer (optionally a protein denaturing agent) and incubating in an aqueous medium. What is necessary is just to perform for about 12 to 24 hours on the temperature conditions of -60 degreeC. After the incubation, the residue of the keratin-containing material after extracting the keratin is removed from the solution by filtration or the like as a precipitate. Thus, a keratin extraction solution containing no residue can be obtained.

  In step 3), the protein stabilizer is removed from the keratin extraction solution to obtain a purified keratin solution (purified keratin solution). Any method for removing the protein stabilizer may be used, and it may be appropriately selected depending on the type of the protein stabilizer. For example, dialysis using a dialysis membrane, ultrafiltration, dialysis using a desalting column, gel filtration, methods using charges such as ion exchange chromatography, methods using specific affinity such as affinity chromatography, reversed-phase high-performance liquid Examples include, but are not limited to, a method using a difference in hydrophobicity such as chromatography and a method using a difference in isoelectric point such as isoelectric focusing. When the protein stabilizer is SDS, since SDS forms a complex with the protein, it cannot be easily removed. When removing SDS by dialysis using a dialysis membrane, in order to remove SDS that has formed a complex with protein, the keratin extract solution obtained in steps 1) and 2) is not dialyzed. It is preferred to perform dialysis at least 5 times using 2-5 L of liquid (preferably about 3 L). One dialysis is preferably performed for 2 hours or more. By any of these techniques, the protein stabilizer can be substantially removed to obtain a purified keratin solution.

  In the present invention, the purified keratin solution is more preferably one in which not only the protein stabilizer but also the reducing agent and protein denaturant are substantially removed from the keratin extract solution. The reducing agent and the protein denaturant may be removed at the same time by the above-described protein stabilizer removing step, or may be removed by a separate step by a known method or a newly found method.

  The protein concentration in the purified keratin solution is not particularly limited, but is, for example, about 10 to 50 mg / ml, preferably about 30 to 50 mg / ml.

  In step 4), a concentrated keratin solution (concentrated keratin solution) is obtained by concentrating the purified keratin solution obtained in step 3), and a functional hydrogel is prepared from the concentrated keratin solution. Concentration of the purified keratin solution may be performed by any technique, but it is necessary to perform it under temperature conditions where the protein is not denatured. The concentration step is performed under a temperature condition of 30 to 65 ° C, preferably 45 to 60 ° C, more preferably 50 to 60 ° C.

  Concentration of the purified keratin solution may be performed by a known concentration method such as dialysis, ultrafiltration, atmospheric distillation, vacuum distillation, a method using a vacuum evaporator, a newly found concentration method, or a combination thereof. For example, a precipitation method using an organic solvent or the like, or concentration of a keratin solution by freeze-drying is not preferable in the present invention because keratin is not in a solution state but in a precipitate or powder state. The concentration of the keratin solution in the present invention is preferably performed using the concentration method as described above, which can be concentrated in the solution.

The protein concentration in the concentrated keratin solution is, for example, about 110 to 160 mg / ml in the case of a keratin solution containing keratin not modified with an SH group. When the protein concentration is less than 110 mg / ml, only a fluid gel-like composition can be obtained even when cooled and incubated in step 4), and the functional hydrogel of the present invention can be obtained. Can not. When the protein concentration is higher than 160 mg / ml, fluidity is lost and gelation occurs, so that the concentrated keratin solution cannot be poured into a desired container as a template.
In addition, in the case of adding a physiologically active substance or the like to a solution containing a keratin having an SH group modified with various substituents or a physiologically active substance as described later, or a purified keratin solution, it is concentrated. The protein concentration in the keratin solution is not limited to 120 to 160 mg / ml. For example, when the SH group is modified with a carboxymethyl group, an ethylamino group, an acetamide group or the like, the protein concentration in the concentrated keratin solution is 140 to 180 mg / ml.

  In step 4), the concentrated keratin solution is placed in a suitable container and incubated to produce a functional hydrogel. The incubation time can be adjusted depending on the temperature conditions and the hardness (elasticity, etc.) of the functional hydrogel to be produced, and is, for example, about 3 to 16 hours. Incubation may be performed at room temperature or under cooling conditions of 4 to 20 ° C. lower than room temperature. What is necessary is just to produce hydrogel under room temperature or cooling conditions according to the protein concentration of the concentrated keratin solution, the kind of SH group modification of protein, etc. For example, when the protein concentration is relatively high, the hydrogel can be easily prepared even at room temperature, and when the concentration is relatively low, the hydrogel can be easily prepared by cooling.

  The protein in the purified or concentrated keratin solution obtained by the above steps 1) to 4) or the produced functional hydrogel has an unmodified SH group. Such SH groups can be modified with various substituents such as acidic residues, basic residues, and neutral residues by known methods. Modification of the SH group can be performed in a purified keratin solution or a concentrated keratin solution, but can also be performed after gelling keratin to prepare a functional hydrogel. In the present specification, modification of the SH group with a carboxymethyl group may be expressed as “carboxymethylation” or “S-carboxymethylation”. The same applies to other modifications other than carboxymethylation.

Examples of the acidic residue include a carboxy group (—COOH), a hydroxamic acid group (—CONHOH), an acyl cyanamide group (—CONHCN), a sulfo group (—SO ₃ H), a sulfonamide group (—SO ₂ NH ₂ or — NRSO ₃ H), acylsulfonamide groups (—CONHSO ₂ R or —SO ₂ NHCOR), phenol groups (—C ₆ H ₄ OH), hydrocarbon groups substituted with these acidic residues (eg, carboxymethyl group) ) And the like. R in the acidic residue represents a hydrogen atom or an optionally substituted hydrocarbon group. The acidic residue is protected by a protecting group such as an optionally substituted hydrocarbon group, a C _1-6 alkoxy group, an optionally protected amino group, a 1-piperidinyl group or a 4-morpholinyl group. It may be. Examples of the “hydrocarbon group” include C _1-15 alkyl group, C _3-8 cycloalkyl group, C _2-10 alkenyl group, C _2-10 alkynyl group, C _3-10 cycloalkenyl group, C _6-14 aryl. Groups and the like. Examples of the substituent in R include, for example, nitro group, hydroxyl group, oxo group, thioxo group, cyano group, carbamoyl group, aminocarbonyl group substituted with C _1-8 hydrocarbon, carboxy group, C _1-6 Illustrative are halogen atoms such as alkoxycarbonyl group, sulfo group, fluorine, chlorine, bromine and iodine.

Examples of the neutral residue include a hydroxyl group, a hydrocarbon group substituted with a hydroxyl group, a cyclic group substituted with a hydroxyl group, a cyclic ether group, or a cyclic thioether group. Furthermore, the hydroxyl group may be protected. Moreover, the cyclic ether group or the cyclic thioether group may have a substituent. Examples of protecting groups for protecting hydroxyl groups include C _1-8 alkyl groups, C _2-8 alkenyl groups, C _2-8 alkynyl groups, C _7-15 aralkyl groups, C _3-8 cycloalkyl groups, phenyl groups, naphthyl groups. , C _1-8 acyl group, C _1-8 alkylsulfonyl group and the like, and these groups may be further substituted with 1 to 6 halogen atoms (which have the same meaning as described above) and the like.

Basic residues include, for example, guanidino group, amino group, C _1-6 alkylcarbonyl-amino group (eg, acetamide), hydrocarbon group (eg, aminoethyl group) substituted with these basic residues, etc. Is mentioned. These basic residues may have a substituent, and examples of the substituent include a C _1-6 alkyl group and a C _1-6 acyl group.

  The modification of the SH group with the acidic residue or the like can be performed using various known reagents or newly discovered reagents, that is, SH group chemical modifiers capable of modifying the SH group. As the SH group chemical modifier, for example, an alkylating agent can be used. A functional hydrogel having an alkylated SH group can be prepared with a known alkylating agent. When iodoacetic acid is used as the alkylating agent, iodoacetic acid is added to the purified keratin solution obtained in step 3) and reacted by a known method. After the reaction, the functional hydrogel can be prepared by performing the concentration step of step 4).

(Functional hydrogel)
The present invention relates to a functional hydrogel obtained from steps 1) to 4), and further to a functional hydrogel obtained through an SH group modification step.

  The functional hydrogel of the present invention has the property of being redissolved in an aqueous medium. "Re-dissolution" means that once a semi-solid hydrogel is placed in an aqueous medium, the hydrogel swells with the aqueous medium and then dissolves. The aqueous medium after the functional hydrogel of the present invention is redissolved has a viscosity equivalent to that of the aqueous medium before the functional hydrogel is redissolved. Among functional hydrogels, those that have not undergone the SH group modification step are soluble in basic solutions and neutral solutions, but are particularly highly soluble in basic solutions. Here, the basic solution has a pH of 8 to 12, preferably a pH of 9.5 to 11. The neutral solution has a pH of 6 to 8.5, preferably pH 6.5 to 8.0. In the functional hydrogel of the present invention, it is considered that the solubility of the functional hydrogel can be modified by changing the substituent introduced into the SH group.

  The degree of swelling of the functional hydrogel of the present invention will be described. Functional hydrogels not modified with SH groups have a degree of swelling of 100-500%, preferably 100-300% for acidic solutions and 1500-3500%, preferably 2000 for neutral solutions. Has a degree of swelling of ~ 3000%. A functional hydrogel having no SH group modification dissolves in a relatively short time with a basic solution, and thus the degree of swelling is not specified. The swelling degree with respect to the acidic solution is a value when measured using an acetic acid buffer solution at pH 3.5, and the swelling degree with respect to a neutral solution is a value when measured with a phosphate buffer solution at pH 7.5. The degree of swelling with respect to a basic solution is a value measured using a carbonate-bicarbonate buffer having a pH of 10.4. The degree of swelling is calculated by weight after swelling (g) / dry weight (g) × 100. The dry weight is the weight of a substance obtained by freeze-drying an equal amount of concentrated keratin solution as that for producing a functional hydrogel.

(Functional hydrogel composition and production method thereof)
Furthermore, the present invention includes a functional hydrogel composition comprising a functional hydrogel and a bioactive substance and / or a biocompatible substance (also simply referred to as “bioactive substance etc.”). The physiologically active substance and / or biocompatible substance may be contained in the functional hydrogel, and the form of inclusion is not limited. For example, a physiologically active substance or the like may be contained in a form that modifies the SH group, may be contained in a form that is bonded to a residue other than the SH group, or simply contained in the functional hydrogel. It may be dispersed or dissolved. Modification of SH group with a physiologically active substance, etc. can be performed in purified keratin solution or concentrated keratin solution, similar to the above-described modification with substituents, but keratin is gelled to produce a functional hydrogel. It is possible even after.

  The physiologically active substance used in the present invention is not particularly limited as long as it is pharmacologically useful, and may be a non-peptide compound, or a protein or peptide compound. Examples of non-peptide compounds include antibiotics, antitumor agents, anti-inflammatory agents, and various genes that can exert pharmacologically useful actions. Examples of peptide compounds include insulin, somatostatin, growth hormone, growth hormone releasing hormone, prolactin, erythropoietin, corticosteroid, tumor necrosis factor and other cytokines, nerve growth factor, cell growth factor, and endothelin antagonistic activity. And the like, and derivatives thereof, as well as bioactive peptides such as these fragments or fragment derivatives.

  The physiologically active substance used in the present invention may be a pharmacologically acceptable salt. As such salts, when the physiologically active substance has a basic group such as an amino group, an inorganic acid (also referred to as an inorganic free acid) (eg, carbonic acid, bicarbonate, hydrochloric acid, sulfuric acid, nitric acid, boric acid, etc.) ), Organic acids (also referred to as organic free acids) (eg, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.) and the like. When the physiologically active substance has an acidic group such as a carboxyl group, an inorganic base (also referred to as an inorganic free base) (eg, an alkali metal such as sodium or potassium, an alkaline earth metal such as calcium or magnesium) or an organic base ( Salts (for example, organic amines such as triethylamine, basic amino acids such as arginine) and the like. In addition, the physiologically active peptide may form a metal complex compound (eg, copper complex, zinc complex, etc.).

  Biocompatible substances include calcium phosphates such as hydroxyapatite, synthetic polymers such as polyacrylamide, polylactic acid and polyglycolic acid, polysialic acid, alginic acid and alginates, chitin, chitosan, hyaluronic acid, starch and pectin. Polysaccharides such as sugars, poly-γ-glutamic acid, copolymers thereof, derivatives thereof, proteins such as gelatin, collagen and derivatives thereof, citric acid, aspartic acid, glutamic acid, malic acid, tartaric acid, succinic acid, oxalic acid, Including low molecular weight compounds such as lactic acid, glycolic acid, glucose, mannose, sucrose, and maltose.

  A functional hydrogel composition can be prepared by adding a physiologically active substance and / or a biocompatible substance to a keratin solution that has been purified or concentrated in advance to prepare a hydrogel. When a physiologically active substance or the like is added to a purified or concentrated keratin solution, the physiologically active substance or the like may be dispersed or dissolved in the keratin solution, or a state in which a residue such as an SH group is modified It may be. A product prepared by such a method can be referred to as a functional hydrogel composition containing a physiologically active substance or the like.

  Moreover, after producing a functional hydrogel, a functional hydrogel composition containing a physiologically active substance or the like is obtained by bringing the functional hydrogel into contact with a physiologically active substance or a material that is a raw material such as a physiologically active substance. Can be produced. The contact with the functional hydrogel of a physiologically active substance or a raw material such as a physiologically active substance may be performed by immersing the functional hydrogel in a solution containing these substances. A product prepared by such a method can be referred to as a functional hydrogel composition impregnated with a physiologically active substance or the like.

  Examples of the biocompatible substance include hydroxyapatite. A method for producing a functional hydrogel composition containing hydroxyapatite will be described. A functional hydrogel is obtained by steps 1) to 4) (optionally including a modification step of an SH group), and the functional hydrogel is immersed in a simulated body fluid that is a raw material for hydroxyapatite. A functional hydrogel composition in which hydroxyapatite crystals are bonded to the surface can be prepared. In the present invention, it is preferable to prepare a functional hydrogel composition in which hydroxyapatite crystals are bonded by immersing a functional hydrogel in which an SH group is modified with a carboxymethyl group in a simulated body fluid.

Here, the simulated body fluid is a simulated body fluid containing at least calcium ions and phosphate ions. Preferably, the simulated body fluid is a solution that gives calcium phosphates such as tricalcium phosphate (Ca ₃ (PO ₄ ) ₂ ), and Na ⁺ , K ⁺ , Mg ²⁺ , Ca ²⁺ , Cl ⁻ , HCO ³⁻ , HPO ₄ ²⁻ , SO ₄ ²⁻ is approximately equal to human plasma. The simulated body fluid can be prepared, for example, by dissolving NaCl, NaHCO ₃ , KCl, K ₂ HPO _{4 ·} 3H ₂ O, MgCl _{2 ·} 6H ₂ O, CaCl ₂ , Na ₂ SO ₄ or NaF in water. it can. Moreover, it is preferable to adjust pH to 7-8. The simulated body fluid used here preferably has a Ca ²⁺ ion concentration of 2 to 5 mM and a phosphate ion concentration of 0.5 to 2 mM.

  Conditions such as time and temperature for immersing the functional hydrogel in the simulated body fluid are not particularly limited, and may be performed, for example, for about 70 to 200 hours and about 36 to 38 ° C.

(Use of functional hydrogel and functional hydrogel composition)
The functional hydrogel or functional hydrogel composition obtained in the present invention can be used for various applications. For example, a functional hydrogel or a functional hydrogel composition can be used as a drug delivery carrier that is a carrier for carrying a drug and administering it to a living body. The drug is an example of the aforementioned physiologically active substance.

  The drug delivery carrier of the present invention can also be used as a carrier for delivering a drug only to a target site such as a living tissue, organ or lesion site. A carrier for delivering a drug only to such a target site can be used as a drug delivery system (DDS) and has a target directivity. Examples of DDS include oral agents, and particularly enteric oral agents. For example, functional hydrogels with no SH group modification are expected to be applied to enteric oral agents because they do not dissolve when acidic and dissolve at neutrality or higher. The functional hydrogel or functional hydrogel composition may contain a substance that specifically recognizes the target site and directs to the target site. Thus, the drug delivery carrier of the present invention can deliver a drug only to the target site. A substance that specifically recognizes a target site can be contained in a functional hydrogel or a functional hydrogel composition by modifying the SH group of keratin. The substance that specifically recognizes the target site is, for example, a substance that specifically binds to a molecule present in the target site. The substance may be any molecule such as protein or peptide.

  Some of the drug delivery carriers of the present invention have sustained release characteristics, and can be used as drug sustained release carriers. When the functional hydrogel or functional hydrogel composition of the present invention is used as a drug sustained-release carrier, the hydrogel does not easily diffuse from the implantation site, and the pharmacological action of the drug is locally and continuously maintained. It is thought that it can be exhibited. Since the functional hydrogel of the present invention is slowly re-dissolved in an aqueous medium such as a neutral aqueous solution, the release of the drug by exchanging the drug retained by the gel and the external solution, and the release of the drug by dissolution of the gel. It is thought that sustained release of heavy will be possible.

  Moreover, in the functional hydrogel or functional hydrogel composition of the present invention, the properties of the hydrogel can be changed by applying various modifications to the SH group to make it adaptable to a wide range of drugs. In the functional hydrogel or functional hydrogel composition of the present invention, the sustained release pattern of the drug can be controlled by selecting the modification mode of the SH group and the type of the drug.

  The drug delivery carrier of the present invention may be provided with both target directivity and sustained release characteristics.

  The functional hydrogel or functional hydrogel composition of the present invention is considered to be applicable not only to pharmaceuticals but also to health foods.

  Further, the functional hydrogel or functional hydrogel composition of the present invention can be used as a bone substitute material, a regenerative medical material such as a scaffold capable of inducing / promoting regeneration of a living tissue, a cell culture substrate, and the like. It is. It is considered that the functional hydrogel composition containing the hydroxyapatite of the present invention can be effectively used as a bone substitute material by further containing a drug such as an antibiotic.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. In the following examples, the protein concentration was measured by the Lowry method.

(Example 1) Preparation of hydrogel 27 g of wool that was chopped after washing with distilled water was added to an aqueous solution containing 0.26 M sodium dodecyl sulfate (SDS), 1.66 M 2-mercaptoethanol (2-ME), and 8 M urea. Mix with 300 ml and shake at 60 ° C. overnight. The solid residue was removed with a stainless steel mesh to obtain a keratin extract solution. Next, in order to remove SDS, 2-ME, and urea, the extract is transferred to a dialysis tube (Dialysis Membrane size36, Wako) and purified by dialysis using about 3000 ml of distilled water as an external dialysis solution. A keratin solution was obtained. Exchange of the external dialysis solution was performed 6 times in 2 days. The precipitate produced during dialysis was filtered off to obtain about 700 ml of solution. At this stage, the protein concentration was measured by the Lowry method. As a result, the protein concentration was 20 to 40 mg / ml.

  After concentrating the purified keratin solution to a protein concentration of 60-70 mg / ml using an ultra filter (P0200, ADVANTEC), the protein concentration is about 110-120 mg while stirring in a hot water bath at 60 ° C. The solution was further concentrated to / ml to obtain a concentrated keratin solution. This was stored in a suitable container at 4 ° C. to obtain a hydrogel.

  A photograph of the resulting hydrogel is shown in FIG. The hydrogel obtained in this example was non-fluid and semi-solid.

(Example 2) Production of hydrogel of keratin modified with SH group In the same manner as in Example 1, keratin was extracted and purified from wool. However, dialysis is performed using 0.5 M Tris-HCl (pH 8.5) as an external dialysis solution to obtain a purified keratin solution (protein concentration 20-40 mg / ml, 0.5 M Tris-HCl (pH 8.5)). It was.

Iodoacetic acid was added to a final concentration of 0.12 M, and the mixture was stirred at room temperature for about 2 hours. As the reaction proceeded, the pH decreased, so trishydroxymethylaminomethane was added in a timely manner to maintain the pH at 8.5. After the reaction, dialysis was performed by exchanging the external dialysis solution twice a day against distilled water. After dialysis, the protein concentration was concentrated to about 70-80 mg / ml by ultrafiltration. When the residue SH content was examined using the Elman reagent, the SH content was reduced to about 20% before the reaction, and 80% was considered to be carboxymethylated. A concentrated keratin solution was obtained by concentrating to a protein concentration of 150 to 160 mg / ml in a 60 ° C. water bath, and the concentrated keratin solution was transferred to a petri dish and refrigerated at 4 ° C. to obtain a hydrogel.
Further, using 2-bromoethylamine or iodoacetamide instead of iodoacetic acid, keratins in which the SH group was ethylaminated and acetamidated were obtained, and these hydrogels were prepared.

(Example 3) Measurement of swelling degree of hydrogel The hydrogel prepared in Example 1 was prepared by using 1 M acetate buffer (pH 3.5), 1 M phosphate buffer (pH 7.5), or 1 M carbonate-heavy. It was immersed in a carbonate buffer (pH 10.4) and the degree of swelling was measured.

The results are shown in Table 1 below.
In 1 M acetate buffer (pH 3.5 acidic region), the gel contracted over time. This is thought to be because the gel contracted as the charge disappeared because the isoelectric point of keratin was on the acidic side. In 1 M phosphate buffer (neutral region of pH 7.5), the gel swelled, but the swelling stopped after about 48 hours. In 1 M carbonate-bicarbonate buffer (basic region at pH 10.4) the gel swelled vigorously and dissolved within 24 hours. The dry weight is the weight of the substance obtained by freeze-drying the same amount of keratin solution and concentrated keratin solution as used in preparing the hydrogel in Example 1.

(Example 4) Evaluation of sustained release ability of hydrogel Bromophenol blue (BPB) was supported on the hydrogel, and the sustained release ability under various conditions was examined.
A concentrated keratin solution was obtained in the same manner as in Example 1, and BPB was added to the concentrated keratin solution, followed by cooling to prepare a hydrogel. Slow release of BPB in aqueous solutions of 1 M acetate buffer (pH 4.4), 1 M phosphate buffer (pH 7.5), 1 M carbonate-bicarbonate buffer (pH 10.2) at 37 ° C, 590 nm It was calculated from the absorbance.

In the aqueous solution at pH 4.4, the BPB-supported hydrogel contracted, but sustained release of BPB was not observed.
The results of confirming the sustained release ability at pH 7.5 and pH 10.2 are shown in Table 2 below.
At pH 7.5, sustained release of BPB was observed up to 72 hours, and about 80% of the drug was released in 72 hours. On the other hand, a high initial burst was seen at pH 10.4, but the release stopped when 55% of the drug was released in 24 hours.
An attempt was also made to immerse and carry the hydrogel in a BPB solution, and showed a similar release pattern after a higher initial burst.

(Example 5) Preparation of hydrogel composition of keratin containing hydroxyapatite and SH group modified with carboxymethyl group Keratin (S- the hydrogel 37 ° C. carboxymethylation keratin), simulated body fluid ^{(Na +, K +, Mg} 2+, Ca 2+, Cl -, HCO 3-, HPO 4 2-, almost SO ₄ ^2-a human plasma Soaked for 1 to 7 days. After the hydrogel was covered with white crystals, it was washed with ethanol. The precipitated crystals were subjected to scanning electron microscope and powder X-ray crystal analysis to confirm that the precipitated crystals were hydroxyapatite (HAP).

  A scanning electron micrograph of HAP on keratin gel is shown in FIG. It is the photograph of the 1st day (upper figure) and the 7th day (lower figure) after immersing hydrogel in a simulated body fluid. From FIG. 2, it can be confirmed that HAP is deposited. The results of powder X-ray crystal analysis are shown in FIG. In FIG. 3, 1d, 3d, and 7d show the results of the first day, the third day, and the seventh day, respectively, immersed in the simulated body fluid. A broad peak attributed to low crystalline hydroxyapatite was observed at around 32 ° from the first day of immersion in the simulated body fluid. It was confirmed that the peak intensity increased as the number of days of immersion in the simulated body fluid increased. From these results, it was found that as the immersion time in the simulated body fluid increases, the crystallinity of hydroxyapatite increases.

(Example 6) Evaluation of sustained release ability of hydrogel of S-carboxymethylated keratin containing hydroxyapatite (HAP) Hydrogel composition of S-carboxymethylated keratin containing HAP prepared in Example 5 Was immersed in an aqueous BPB solution (5 mg / ml) under reduced pressure for 1 hour to carry BPB. Slow release of BPB in PBS at 37 ° C. was observed in the same manner as in Example 5.

  The results are shown in FIG. In about 10 hours, 25% of the total supported amount was released, but when observed up to 150 hours thereafter, sustained release was observed up to 50%. Furthermore, it was suggested that sustained release continues.

(Example 7) Measurement of degree of swelling of hydrogel In the same manner as in Example 3, various hydrogels prepared in Example 1 and Example 2 were prepared using 1 M acetate buffer (pH 3.5), 1 M phosphate buffer. The sample was immersed in a solution (pH 7.5) or 1 M carbonate-bicarbonate buffer (pH 10.4), and the degree of swelling was measured.

The results are shown in Table 3 below. Various hydrogels were immersed in an aqueous solution of each pH, and the degree of swelling (weight after swelling / dry weight × 100) at the time when the volume did not change (48 to 72 hours) was shown.

(Example 8) Confirmation of salicylic acid sustained release ability of various hydrogels (1) Salicylic acid-encapsulated hydrogel 10 mL of 10% of concentrated keratin (100 to 120 mg / ml) obtained in the same manner as in Example 1 After adding mM salicylic acid in Tris-HCl (pH 7.4), the mixture was concentrated to 140-160 mg / ml at 60 ° C. This was allowed to cool and stand to obtain an unmodified hydrogel encapsulating salicylic acid. After being cut into a cylindrical shape having a diameter of 1 cm and a height of 0.5 cm, it was immersed in 50 ml PBS, and the released salicylic acid was detected by absorbance at 296 nm.
Carboxymethylation, ethylamination, and acetamidation were carried out by adding iodoacetic acid, 2-bromoethylamine, and iodoacetamide, respectively, to a purified keratin solution (protein concentration of 20 to 40 mg / ml) as in Example 2. Was done. Then, it concentrated similarly to the unmodified hydrogel, added salicylic acid, and further performing concentration to obtain various modified hydrogels encapsulating salicylic acid.

(2) Salicylic acid-impregnated hydrogel The keratin hydrogel obtained in the same manner as in the method of Example 1 was cut into a cylindrical shape having a diameter of 1 cm and a height of 0.5 cm, and 10 mL of 10 mM salicylic acid in Tris-HCl. It was immersed in (pH 7.4) and impregnated with salicylic acid. Thereafter, it was immersed in 50 ml PBS, and the released salicylic acid was detected by absorbance at 296 nm.

The results of the salicylic acid sustained release ability of the salicylic acid-encapsulating hydrogel are shown in Table 4 and FIG. “Keratin” in Table 4 and “keratin hydrogel” in FIG. 5 represent unmodified keratin. The total supported amount of salicylic acid was determined by dissolving salicylic acid-encapsulating hydrogel with 1 M hydrochloric acid.
Since the carboxymethylated keratin hydrogel dissolved in about 5 hours, the release of salicylic acid was completed in about 5 hours. The aminoethylated keratin hydrogel was also dissolved in 24 hours and the salicylic acid release was completed in 24 hours. Unmodified keratin and acetamidated keratin hydrogel did not dissolve and sustained release was observed for 72 hours.
The hydrogel containing salicylic acid tended to be more easily dissolved.

The results of the salicylic acid sustained release ability of the salicylic acid-impregnated hydrogel are shown in Table 5 and FIG. “Keratin” in Table 5 and “keratin hydrogel” in FIG. 6 represent unmodified keratin. The total supported amount of salicylic acid was determined by dissolving salicylic acid-impregnated hydrogel with 1 M hydrochloric acid.
In all hydrogels, a higher initial burst was seen compared to the salicylic acid-encapsulating hydrogel. In the method of immersing the gel in the chemical solution, it seems that the amount of the drug that entered the gel surface layer increased and the initial burst increased. Carboxymethylated keratin and aminoethylated keratin hydrogel dissolved in 24 hours and drug release was complete at that time, but sustained release was seen over 5-24 hours. Unmodified keratin was observed for sustained release up to 72 hours after an initial burst of 3 hours. The acetamidated keratin hydrogel had a relatively small initial burst amount, and sustained release occurred until 72 hours after the initial burst of 1 hour.

The total amount of drug supported was approximately the same amount of salicylic acid regardless of the loading method and the type of gel.
Since salicylic acid is an acidic substance, it is considered that the isoelectric point does not electrostatically bind to keratin having a pH of 5 to 6 and is repelled, and salicylic acid is considered to be dispersed in the gel. In the unmodified keratin hydrogel and the acetamidated keratin hydrogel, the sustained release property of the acidic substance could be confirmed. Moreover, since the carboxymethylated keratin gel was neutral and dissolved, rapid release of acidic substances was confirmed. It was found that aminoethylated keratin can dissolve even in neutrality by supporting an acidic substance, and shows a release pattern similar to carboxymethylated keratin.

(Example 9) Confirmation of BPB sustained release ability of hydrogel
Instead of 1 M acetate buffer (pH 4.4), 1 M phosphate buffer (pH 7.5), 1 M carbonate-bicarbonate buffer (pH 10.2), 0.2 M hydrochloric acid-potassium chloride buffer (pH 1.2), The sustained release ability of bromophenol blue (BPB) from the unmodified hydrogel was confirmed in the same manner as in Example 4 except that 0.2 M phosphate buffer (pH 5.8) was used.

The results are shown in Table 6 below.
This result strongly suggested that when the drug was supported on the hydrogel, release did not occur at the stomach pH but occurred at the intestinal pH.

  The present invention provides a novel non-flowable functional hydrogel. Such functional hydrogels contain SH groups, and it is possible to modify physical properties and functions by modifying SH groups with various substituents, bioactive substances, biocompatible substances, etc. according to the application. . The functional hydrogel and functional hydrogel composition of the present invention include a drug delivery carrier, a drug delivery system, a sustained drug release carrier, a bone substitute material, a regenerative medical material such as a transplant material, and a cell culture substrate. It can be applied in a wide range of fields such as medicine and food.

  Furthermore, the method for producing the functional hydrogel of the present invention comprises a functional hydrogel made from wastes from the production of clothing, carpets, carpets and other woven fabrics and knitted fabrics, and used clothing, woven fabrics and knitted fabrics as raw materials. It is possible to make a gel. The present invention can make effective use of resources, considers the environment, and is an excellent technology from the viewpoint of social contribution.

Claims (14)

A method for producing a functional hydrogel comprising the following steps:
1) A step of extracting keratin by adding a reducing agent for converting a disulfide bond to a thiol group to a keratin-containing material;
2) adding a protein stabilizer to the keratin-containing material to prevent thiol groups from re-forming disulfide bonds;
3) Step of removing the protein stabilizer and purifying keratin to obtain a keratin solution:
4) A step of producing a functional hydrogel by concentrating the keratin solution. The method for producing a functional hydrogel according to claim 1, wherein the protein stabilizer is sodium dodecyl sulfate. The removal of the protein stabilizer in step 3) is performed at least 5 times by dialysis using 2-5 L of dialysis external solution against 150-250 mL of the keratin extract obtained in steps 1) and 2). The method for producing a functional hydrogel according to claim 1 or 2. The method for producing a functional hydrogel according to any one of claims 1 to 3, wherein the concentration of the keratin solution in step 4) is performed under a temperature condition of 40 to 65 ° C. The method for producing a functional hydrogel according to any one of claims 1 to 4, wherein a protein denaturant is further added to the keratin-containing material. The functional hydrogel produced by the manufacturing method of any one of Claims 1-5. The functional hydrogel according to claim 6, wherein the functional hydrogel has solubility in an aqueous medium. The functional hydrogel according to claim 6 or 7, wherein a thiol group in the functional hydrogel is modified with a substituent. A functional hydrogel composition comprising the functional hydrogel according to any one of claims 6 to 8, and a physiologically active substance and / or a biocompatible substance. The manufacturing method of the functional hydrogel composition containing a hydroxyapatite by making the functional hydrogel of any one of Claims 6-8 contact a pseudo-body fluid. A carrier for drug delivery comprising at least the functional hydrogel according to any one of claims 6 to 8, or the functional hydrogel composition according to claim 9. The drug delivery carrier according to claim 11, wherein the drug delivery carrier is used as a drug delivery system. 13. The drug delivery carrier according to claim 11 or 12, wherein the drug delivery carrier is used as a drug sustained release carrier. A regenerative medical material comprising the functional hydrogel according to any one of claims 6 to 8 or the functional hydrogel composition according to claim 9.