WO2010147158A1 - Gel enclosing a compound by means of low-molecular weight gelling agent - Google Patents

Abstract

Disclosed is a gel enclosing a compound by means of a low-molecular weight gelling agent. The enclosing gel is characterized by containing a low-molecular weight gelling agent comprising a lipid peptide represented by formula (1) (wherein R₁ through R₅ represent organic groups) or a pharmaceutically usable salt thereof, an aqueous medium gelled by means of said low-molecular weight gelling agent, and at least one type of compound enclosed therein.

Description

Gel in which compound is encapsulated by low molecular gelling agent

The present invention relates to a gel in which a compound is encapsulated by a low molecular gelling agent. More specifically, the present invention relates to a gel in which a hydrophobic compound or a hydrophilic compound, or both compounds, and a protein such as an enzyme are encapsulated as the compound.
The inclusion gel of the present invention is a skin care product, external medicine, wound dressing, anti-adhesion film, drug delivery system, fragrance, deodorant, insect repellent, insecticide, agricultural chemical base, diagnostic base, chemical reaction It can be suitably used as an enzyme reaction solution, a chemical sensor substrate, a biosensor substrate, and the like.

Conventionally, gelling agents that gel various solvents have been studied. Gel agents for gelling aqueous solutions or organic solvents and aqueous solutions include polymers obtained by polymerization cross-linking reactions, natural polymers such as polysaccharides, and low gels that form self-organized low molecular compounds. Molecular gelling agents are known.
By the way, when trying to create a safe and stable sheet-like material without using a polymerization reaction with these gel agents, the solution in which the natural polymer is dissolved is put into a sheet-shaped mold and gelled. However, when the gel sheet prepared in this way is immersed in water, the natural polymer gradually falls from the gel sheet to the water and collapses. As described above, it was extremely difficult to produce a gel sheet using only a physical gel (Non-Patent Document 1). Moreover, preparation of the gel sheet by a low molecular gel was not known.

On the other hand, when a new function is given to the gel by the low molecular gelling agent and the gel is used as a functional material, for example, a gel in which vitamin C and vitamin E are encapsulated simultaneously by the low molecular gelling agent is Although it is expected to be useful as a whitening cosmetic material (Patent Documents 1 and 2), it is extremely difficult to form a gel that encapsulates both a hydrophilic low-molecular compound and a hydrophobic low-molecular compound at the same time. .
In addition, when an enzyme such as cytochrome c is encapsulated by a low molecular gelling agent, the enzyme reaction can proceed in the gel if the enzyme incorporated in the gel can be maintained without impairing its activity. It is considered that the reaction can be observed in the gel, and the gel containing the enzyme can be used as a biosensor, a test / diagnostic agent or the like (Patent Documents 3 to 6).

JP-A-2005-289880 JP2008-222689 Special table 2008-530534 Special table 2007-510155 Special table 2006-516383 JP2007-139730

From the above, until now, including a gel sheet that can encapsulate a hydrophobic low molecular weight compound, or both a hydrophilic low molecular weight compound and a hydrophobic low molecular weight compound at the same time, and a protein such as an enzyme, No gel has been proposed that encapsulates such compounds.

Therefore, the present invention has been made based on the above circumstances, and the problem to be solved is a hydrophilic physiologically active substance such as vitamin C or a hydrophobic physiologically active substance such as vitamin E, or It is an object of the present invention to provide a gel in which these compounds are encapsulated and a gel in which an enzyme such as cytochrome c is encapsulated. It is another object of the present invention to provide a method of using a gel encapsulating the compound of the present invention for a chemical reaction or an enzyme reaction. Moreover, the objective of this invention is providing the method of using this inclusion gel as a biosensor by using the gel which included the compound of this invention for a chemical reaction or an enzyme reaction.

In the gel sheet prepared by the polymerization crosslinking reaction, if an unreacted crosslinking agent or the like remains in the gel, it interferes with the chemical reaction or enzyme reaction performed in the gel. On the other hand, gel sheets made of natural polymers are unstable and dissolve and disintegrate when immersed in water. Providing a stable and safe gel sheet that does not disturb chemical reactions and enzyme reactions in the gel, and does not collapse even when immersed in various water solutions so that the enzyme, substrate, and specimen to be tested can be taken into the gel. It is also a thing.

As a result of intensive studies to solve the above problems, the present inventors have found the present invention.
That is, as a first viewpoint, the formula (1):

　　　　　　　　　　　　　　　　　　

(Wherein R ¹ represents an aliphatic group having 9 to 23 carbon atoms, and R ^{2 to} R ⁵ are each independently a hydrogen atom or a carbon atom that may have a branched chain of 1 to 3 carbon atoms. Represents an alkyl group, a phenylmethyl group, a phenylethyl group, or a — (CH ₂ ) n—X group represented by formulas 1 to 7, and at least one of R ^{2 to} R ⁵ represents a — (CH ₂ ) n—X group. N represents a number of 1 to 4, X is composed of an amino group, a guanidino group, a —CONH ₂ group, or a 5-membered ring or a 5-membered ring and a 6-membered ring which may have 1 to 3 nitrogen atoms. M represents 1 or 2), and a low molecular gelling agent comprising a lipid peptide represented by the following formula: An aqueous medium and at least one compound contained therein. An included gel.
As a second aspect, the first aspect is characterized in that, in the formula (1), R ¹ is a linear aliphatic group having 11 to 23 carbon atoms which may have 0 to 2 unsaturated bonds. The inclusion gel described in 1.
As a third aspect, in the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms that may have a branched chain of 1 or 2 carbon atoms, A phenylmethyl group, or a — (CH ₂ ) n—X group, and one or two of R ^{2 to} R ⁵ represent a — (CH ₂ ) n—X group, and n is a number from 1 to 4; Wherein X represents an amino group, a guanidino group, a -CONH ₂ group, or a 5-membered ring having 1 to 2 nitrogen atoms or a condensed heterocyclic ring composed of a 5-membered ring and a 6-membered ring, The inclusion gel according to the first aspect or the second aspect.
As a fourth aspect, in the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms that may have a branched chain of 1 or 2 carbon atoms, A phenylmethyl group, or a — (CH ₂ ) n—X group, and one or two of R ^{2 to} R ⁵ represent a — (CH ₂ ) n—X group, and n is a number from 1 to 4; X represents an amino group, a guanidino group, a -CONH ₂ group, a pyrrole group, an imidazole group, a pyrazole group, or an indole group according to the third aspect.
As a fifth aspect, in the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i- Butyl, sec-butyl, tert-butyl, phenylmethyl, aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, carbamoylmethyl, 2-carbamoylethyl, 3 -Represents a carbamoylpropyl group, 2-guanidinoethyl group, 3-guanidinopropyl group, pyrrolemethyl group, imidazolemethyl group, pyrazolemethyl group or 3-indolemethyl group, and one or two of R ^{2 to} R ⁵ are Aminomethyl group, 2-aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, carbamoylmethyl group, 2-carbamoylethyl group, The inclusion according to the fourth aspect, characterized in that it represents a 3-carbamoylpropyl group, a 2-guanidinoethyl group, a 3-guanidinopropyl group, a pyrrolemethyl group, an imidazolemethyl group, a pyrazolemethyl group or a 3-indolemethyl group gel.
As a sixth aspect, in the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, a methyl group, an i-propyl group, an i-butyl group, a secondary butyl group, a phenylmethyl group, 4- Represents an aminobutyl group, a carbamoylmethyl group, a 2-carbamoylethyl group, a 3-guanidinopropyl group, an imidazolemethyl group or a 3-indolemethyl group, and one or two of R ^{2 to} R ⁵ is a 4-aminobutyl group Or a carbamoylmethyl group, a 2-carbamoylethyl group, a 3-guanidinopropyl group, an imidazolemethyl group, or a 3-indolemethyl group.
The inclusion gel according to any one of the first to sixth aspects, wherein m represents 1 in the formula (1) as a seventh aspect.
As an eighth aspect, the inclusion gel according to any one of the first to seventh aspects, wherein the compound is a hydrophobic compound, a hydrophilic compound, or a compound of both.
As a ninth aspect, the encapsulated gel according to the first aspect to the seventh aspect, wherein the compound is an enzyme.
As a tenth aspect, the inclusion gel according to the ninth aspect, wherein the compound is cytochrome c.
As an eleventh aspect, a method of using the inclusion gel according to the ninth aspect for an enzyme reaction.
As a twelfth aspect, a method of using the inclusion gel according to the tenth aspect for an oxidation reaction of a target product with H ₂ O ₂ .
As a thirteenth aspect, a method of using the inclusion gel as a biosensor by using the inclusion gel according to the ninth aspect for an enzyme reaction.
As a fourteenth aspect, a method of using the inclusion gel as a biosensor by using the inclusion gel according to the tenth aspect for an oxidation reaction with H ₂ O ₂ .
As a fifteenth aspect, a biosensor comprising the inclusion gel according to the eleventh aspect or the twelfth aspect.

The encapsulated gel of the present invention can encapsulate a hydrophobic compound, a hydrophilic compound, or both compounds in the gel. It is also possible to release the encapsulated compound gradually. Therefore, when applied to a damaged site such as the skin, a therapeutic effect such as a moisturizing effect as a gel and a sustained release of a drug, and a therapeutic effect by capturing a damaging compound or a pathogenic protein can be expected.

In addition, the encapsulated gel of the present invention can encapsulate an enzyme such as cytochrome c as a compound, and when the encapsulated protein is an enzyme-like protein, the enzyme can be incorporated into the gel even if incorporated. Its activity is maintained without loss, rather it is possible to allow the enzymatic reaction to proceed within the gel. Therefore, a gel incorporating such an enzyme can see the reaction in the gel and can be used as a biosensor or a test / diagnostic agent. In addition, the gel encapsulating the compound of the present invention can be subjected not only to the enzyme reaction described above but also to a chemical reaction depending on the compound encapsulated in the gel.

As described above, the encapsulated gel of the present invention is a wound dressing, an adhesion prevention film, a drug delivery system, a base for external medicine, a fragrance / deodorant that is safe for living bodies and the environment, and has an ability to recognize an affected area or damaged site. -It can be widely used for base materials such as insect repellents, insecticides and agricultural chemicals, base materials for biosensors, base materials for environmental analysis, and base materials for capturing contaminants in soil and water.

FIG. 1 is a diagram showing a conceptual diagram of lipid peptide self-assembly and subsequent gelation. FIG. 2 is a diagram showing a comparison of fluorescence intensity of ANS between Pal-GGGH gel and polymer gel. FIG. 3 is a diagram showing a comparison of fluorescence intensity of ANS in a Pal-GGGH gel and an organic solvent. FIG. 4 is a graph showing the ANS release rate depending on the gelling agent and the ANS concentration. FIG. 5 is a diagram showing ANS release rates (left: gel prepared at pH 7.4, right: gel adjusted at pH 9.0) in release solutions having different pHs. FIG. 6 is a graph showing the riboflavin release rate with different pH release solutions in gel pH 7.4. FIG. 7 is a graph showing changes in the ionization state of Pal-GGGH with pH. FIG. 8 is a diagram showing evaluation criteria for the degree of solubilization of hydrophobic substances ((I) is in a dissolved state, (II) is in a slightly cloudy state, and (III) is in a cloudy state without being dissolved). is there. FIG. 9 is a diagram showing the evaluation of solubilized vitamin E concentration by turbidity. FIG. 10 is a view showing a comparison of vitamin E-encapsulated gel between polymer gel and Pal-GGGH. FIG. 11 is a view showing a simultaneous inclusion gel of vitamin E and vitamin C (left: before gel formation, right: after gel formation). FIG. 12 is a diagram showing the release behavior of vitamin E with respect to ethanol having different concentrations. FIG. 13 shows the adsorption of cytochrome c on the gel sheet. FIG. 14 is a diagram showing the amount of cytochrome c adsorbed to the gel sheet under different pH conditions. FIG. 15 is a view showing the reduction of the cytochrome c-immersed gel sheet. FIG. 16 shows the release behavior of cytochrome c with respect to buffers having different pHs. FIG. 17 is a diagram showing the structure of Pal-GGGH. FIG. 18 shows the results of measurement of cytochrome c peroxidase activity. FIG. 19 is a diagram showing the results of CD spectrum measurement. FIG. 20 is a diagram showing the results of UV spectrum measurement. FIG. 21 is a diagram showing a measurement result of the gelling agent concentration dependence of peroxidase activity.

The encapsulated gel of the present invention is an encapsulated gel characterized by encapsulating at least one compound in an aqueous medium gelled by a low molecular gelling agent.

The gel formation mechanism of the low molecular weight gelling agent used for the gel encapsulating the compound of the present invention is that the low molecular weight compound constituting the low molecular weight gelling agent is self-assembled to form a fiber-like form. Forms a network structure, and water, various aqueous solutions, alcohol water, organic solvent water and the like are enclosed in the network structure to form a gel. Here, “self-assembly” means that in a group of substances (molecules) that are initially in a random state, molecules spontaneously associate under non-covalent interactions between molecules under appropriate external conditions. , Refers to growing into a macro functional assembly. Therefore, the inclusion gel of the present invention contains a gel formed of self-assembled fibers and a network structure formed thereby, and if it is a hydrophobic compound, it may be a hydrophilic compound in the fiber in a hydrophobic environment. For example, it can be included in the network structure formed by the fiber.

The encapsulated gel of the present invention is a gel sheet composed of fibers formed by self-assembly of the above-mentioned low molecules. Therefore, unlike the gel sheet obtained by the polymerization crosslinking reaction, the encapsulated gel obstructs or encapsulates the reaction performed in the gel. Providing a safe and stable gel sheet that eliminates the need to remove a reagent that decomposes a compound and that does not cause the natural polymer to fall off from the gel sheet one by one like a gel sheet obtained from a natural polymer. It is possible.
Moreover, it is also possible to provide a film by evaporating the solvent of this gel sheet without freeze-drying.

As the low-molecular gelling agent, lipid peptides or pharmaceutically usable salts thereof (low-molecular compounds having a lipid part which is a hydrophobic part and a peptide part which is a hydrophilic part) can be used.

The lipid peptide or pharmaceutically usable salt thereof includes a lipid part (alkylcarbonyl group) and a peptide part (tetrapeptide having a structure represented by the following formula (1) and having a lipid-soluble long chain. ).

　　　　　　　　　　　　　　　　　　

In the above formula (1), R ¹ represents an aliphatic group having 9 to 23 carbon atoms, and preferably R ¹ is a straight chain having 11 to 23 carbon atoms that may have 0 to 2 unsaturated bonds. It is an aliphatic group.
As a specific example of a lipid part (acyl group) composed of R ¹ and an adjacent carbonyl group,
Examples include myristoyl group, pentadecanoyl group, palmitoyl group, margaroyl group, oleoyl group, eridoyl group, linoleoyl group, stearoyl group, baccenoyl group, octadecylcarbonyl group, arachidoyl group, icosanoyl group, preferably lauroyl. A group, a myristoyl group, a palmitoyl group, a margaroyl group, a margaryl group, a stearoyl group, an elideyl group, a behenoyl group, an oleoyl group, and a cardanoyl group.

In the above formula (1), R ^{2 to} R ⁵ contained in the peptide part are each independently a hydrogen atom or an alkyl group having 1 to 7 carbon atoms that may have a branched chain of 1 to 3 carbon atoms. , A phenylmethyl group, a phenylethyl group, or a — (CH ₂ ) n—X group, and at least one, more preferably one or two of R ^{2 to} R ⁵ is — (CH ₂ ) n—X Represents a group, and m represents 1 or 2.

The alkyl group having 1 to 7 carbon atoms which may have a branched chain having 1 to 3 carbon atoms is preferably a hydrogen atom, or 1 to 4 carbon atoms which may have a branched chain having 1 or 2 carbon atoms. It is an alkyl group.
Examples of the alkyl group having 1 to 4 carbon atoms that may have a branched chain of 1 or 2 carbon atoms include, for example, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i -Butyl group, secondary butyl group, tertiary butyl group and the like can be mentioned, and preferred are methyl group, i-propyl group, i-butyl group, secondary butyl group and the like.

In the — (CH ₂ ) n—X group, n represents a number of 1 to 4, and X is an amino group, a guanidino group, a —CONH ₂ group, or a 5-membered ring that may have 1 to 3 nitrogen atoms or A condensed heterocyclic ring composed of a 5-membered ring and a 6-membered ring is represented.

In the — (CH ₂ ) n—X group, X preferably represents an amino group, a guanidino group, a —CONH ₂ group, a pyrrole group, an imidazole group, a pyrazole group or an indole group.
Accordingly, the — (CH ₂ ) n—X group is preferably an aminomethyl group, 2-aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, carbamoylmethyl group, 2-carbamoylethyl group, 3- A carbamoylpropyl group, a 2-guanidinoethyl group, a 3-guanidinopropyl group, a pyrrolemethyl group, an imidazolemethyl group, a pyrazolemethyl group or a 3-indolemethyl group, more preferably a 4-aminobutyl group, a carbamoylmethyl group, 3-carbamoylpropyl group, imidazolemethyl group or 3-indolemethyl group.

In the compound represented by the above formula (1), a lipid peptide particularly suitable as a low molecular gelling agent is a compound formed from the following lipid part and peptide part (amino acid assembly part).
As abbreviations of amino acids, alanine (Ala), arginine (Arg), glutamine (Gln), glycine (Gly), histidine (His), isorosine (Ile), leucine (Leu), lysine (Lys), tryptophan (Trp) ), Valine (Val). : Lauroyl-Gly-Gly-Gly-His, Lauroyl-Gly-Gly-Gly-Gln, Lauroyl-Gly-Gly-Gly-Asn, Lauroyl-Gly-Gly-Gly-Trp, Lauroyl-Gly-Gly-Gly-Gly-Gly Lauroyl-Gly-Gly-Ala-His, Lauroyl-Gly-Gly-Ala-Gln, Lauroyl-Gly-Gly-Ala-Asn, Lauroyl-Gly-Gly-Ala-Trp, Lauroyl-Gly-Gly-Ala-Lys Lauroyl-Gly-Ala-Gly-His, Lauroyl-Gly-Ala-Gly-Gln, Lauroyl-Gly-Ala-Gly-Asn, Lauroyl-Gly-Ala-Gly-Trp, Lauroyl-Gly-Ala-Gly-Lys Lau Ir-Ala-Gly-Gly-His, Lauroyl-Ala-Gly-Gly-Gln, Lauroyl-Ala-Gly-Gly-Asn, Lauroyl-Ala-Gly-Gly-Trp, Lauroyl-Ala-Gly-Gly-Lys, Lauroyl-Gly-Gly-His-Gly, Lauroyl-Gly-His-Gly-Gly, Lauroyl-His-Gly-Gly-Gly; Myristoyl-Gly-Gly-Gly-His, Myristoyl-Gly-Gly-Gly, Myristoyl-Gly-Gly-Gly-Asn, Myristoyl-Gly-Gly-Gly-Trp, Myristoyl-Gly-Gly-Gly-Lys, Myristoyl-Gly-Gly-Ala-His, Myristoyl-Gly-Gly-Ala-Gl , Myristoyl-Gly-Gly-Ala-Asn, Myristoyl-Gly-Gly-Ala-Trp, Myristoyl-Gly-Gly-Ala-Lys, Myristoyl-Gly-Ala-Gly-His, Myristoyl-Gly-Ala-Gly-Gln , Myristoyl-Gly-Ala-Gly-Asn, Myristoyl-Gly-Ala-Gly-Trp, Myristoyl-Gly-Ala-Gly-Lys, Myristoyl-Ala-Gly-Gly-His, Myristoyl-Ala-Gly-Gly-Gln , Myristoyl-Ala-Gly-Gly-Asn, Myristoyl-Ala-Gly-Gly-Trp, Myristoyl-Ala-Gly-Gly-Lys, Myristoyl-Gly-Gly-His-Gly, Myristoyl-Gly -His-Gly-Gly, Myristoyl-His-Gly-Gly-Gly; Palmitoyl-Gly-Gly-Gly-His, Palmitoyl-Gly-Gly-Gly-Gln, Palmitoyl-Gly-Gly-Gly-Asn, Palmitoyl-Gly-Asn -Gly-Gly-Trp, Palmitoyl-Gly-Gly-Gly-Lys, Palmitoyl-Gly-Gly-Ala-His, Palmitoyl-Gly-Gly-Ala-Gln, Palmitoyl-Gly-Gly-Ala-Asn, Palmytoyl-Asl -Gly-Ala-Trp, Palmitoyl-Gly-Gly-Ala-Lys, Palmitoyl-Gly-Ala-Gly-His, Palmitoyl-Gly-Ala-Gly-Gln, Palmitoyl-Gly-Ala-Gly-As Palmitoyl-Gly-Ala-Gly-Trp, Palmitoyl-Gly-Ala-Gly-Lys, Palmitoyl-Ala-Gly-Gly-His, Palmitoyl-Ala-Gly-Gly-Gln, Palmitoyl-Ala-Gly-Gly-Gly-Gly-Gly Palmitoyl-Ala-Gly-Gly-Trp, Palmitoyl-Ala-Gly-Gly-Lys, Palmitoyl-Gly-Gly-His-Gly, Palmitoyl-Gly-His-Gly-Gly, Palmitoyl-His-Gly-Gly-Gly Stearoyl-Gly-Gly-Gly-His, stearoyl-Gly-Gly-Gly-Gln, stearoyl-Gly-Gly-Gly-Asn, stearoyl-Gly-Gly-Gly-Trp, stearoyl-Gl -Gly-Gly-Lys, stearoyl-Gly-Gly-Ala-His, stearoyl-Gly-Gly-Ala-Gln, stearoyl-Gly-Gly-Ala-Asn, stearoyl-Gly-Gly-Ala-Trp, stearoyl-Gly -Gly-Ala-Lys, stearoyl-Gly-Ala-Gly-His, stearoyl-Gly-Ala-Gly-Gln, stearoyl-Gly-Ala-Gly-Asn, stearoyl-Gly-Ala-Gly-Trp, stearoyl-Gly -Ala-Gly-Lys, stearoyl-Ala-Gly-Gly-His, stearoyl-Ala-Gly-Gly-Gln, stearoyl-Ala-Gly-Gly-Asn, stearoyl-Ala-Gly-Gly-Tr p, stearoyl-Ala-Gly-Gly-Lys, stearoyl-Gly-Gly-His-Gly, stearoyl-Gly-His-Gly-Gly, stearoyl-His-Gly-Gly-Gly.

Most preferred are lauroyl-Gly-Gly-Gly-His, myristoyl-Gly-Gly-Gly-His, palmitoyl-Gly-Gly-Gly-His, palmitoyl-Gly-Gly-His-Gly, palmitoyl-Gly-His -Gly-Gly, palmitoyl-His-Gly-Gly-Gly, stearoyl-Gly-Gly-Gly-His and the like.

The gelled aqueous medium used for the inclusion gel of the present invention is formed containing the low molecular weight gelling agent and the aqueous medium (solvent).
The solvent is not particularly limited as long as it does not prevent fiber formation or hydrogelation of the low-molecular gelling agent. Preferably, water, alcohol, a mixed solvent of water and alcohol, or a mixed solution of water and water-soluble solvent is used. Can be used. More preferred is water or a mixed solvent of water and alcohol, and further preferred is water.
The alcohol is preferably a water-soluble alcohol that is freely soluble in water, more preferably an alcohol having 1 to 6 carbon atoms, still more preferably methanol, ethanol, 2-propanol, or i-butanol, Further particularly preferred is ethanol or 2-propanol.
The water-soluble organic solvent means an organic solvent other than alcohol and that dissolves in water at an arbitrary ratio. Examples of the water-soluble organic solvent to be used include acetone or dioxane.

The gelled aqueous medium may contain a salt. Such a salt may be added at any stage leading to the formation of the hydrogel, but it is preferable to add a solvent to the solution before adding the hydrogelator.
A plurality of types of salts may be added, but one or two types are preferable. It is also desirable that the solution has buffer capacity by adding two salts.
The salt is an inorganic salt or an organic salt. Examples of preferred inorganic salts include carbonates, inorganic sulfates or inorganic phosphates. More preferred are calcium carbonate, sodium carbonate, potassium carbonate, sodium sulfate, potassium sulfate, sodium sulfate, magnesium sulfate, potassium phosphate, sodium phosphate, disodium hydrogen phosphate or sodium dihydrogen phosphate. Examples of preferred organic salts include organic amine hydrochlorides or organic amine acetates. More preferred are ethylenediamine hydrochloride, ethylenediaminetetraacetate, and trishydroxymethylaminomethane hydrochloride.

Further, examples of the compound encapsulated in the gel of the present invention include a hydrophobic compound, a hydrophilic compound, an enzyme, and pyrene.

Examples of the hydrophobic compound include vitamin E (tocopherol), pyrene, azelaic acid derivatives, retinol (vitamin A alcohol), retinoic acid, hydroxycinnamic acid, caffeine, hinokitiol, carotenoid, astaxanthin, steroid, indomethacin, and Ketoprofen and the like can be mentioned.

Examples of the hydrophilic compound include vitamin C (ascorbic acid), vitamin B2 (riboflavin), kojic acid, glucosamine, azelaic acid, pyridoxine (vitamin B6), pantothenic acid (vitamin B5), arbutin, and chitosan. It is done.

Examples of the enzyme include cytochrome c.

Furthermore, the encapsulated gel of the present invention includes, for example, ascorbic acid and derivatives thereof, kojic acid and derivatives thereof, glucosamine and derivatives thereof, azelain and derivatives thereof, retinoic acid and derivatives thereof, pyridoxine and derivatives thereof, pantothenic acid and Whitening effect can be achieved by encapsulating its derivatives, arbutin and its derivatives, tocopherol and its derivatives, hydroxycinnamic acid and its derivatives, chitosan, chitosan degradation products, caffeine derivatives, hinokitiol, carotenoid, astaxanthin, etc. .

[Gel formation mechanism]
When the low molecular weight compound (lipid peptide) represented by the above formula (1), which is a low molecular gelling agent used in the present invention, is introduced into an aqueous solution or an alcohol solution system, the peptide portion in the formula (1) is hydrogen. An intermolecular non-covalent bond is formed by the bond, while the lipid part in the formula (1) is self-assembled so as to be hydrophobically packed (also referred to as self-assembly), and a fiber is formed. Although the shape of a fiber is not limited, A cylindrical shape or plate shape is mentioned.
For reference, FIG. 1 shows an example of a conceptual diagram of lipid peptide self-assembly and gelation (however, in the inclusion gel of the present invention, all lipid peptides take the form of self-assembly and gelation shown in FIG. Not necessarily). The lipid peptide molecule (a) assembles around a lipid part which is a hydrophobic site (b), and forms a fiber (c) by self-assembly.
In forming the fiber, one kind of the low molecular gelling agent may be used, or two or more kinds may be used in combination. Preferably, one type or two types are used, and more preferably one type is used. However, when two types are used, it can be expected to obtain different properties from the case of one type.

The low-molecular gelling agent used in the present invention can also form a fiber by mixing with a surfactant and self-assembling. Examples of such surfactants include anionic surfactants, nonionic surfactants, and cationic surfactants.

When the fiber is formed in an aqueous solution or an alcohol aqueous solution, the fiber forms a three-dimensional network structure (for example, see (d) in FIG. 1), and further, a hydrophilic portion (peptide portion) on the fiber surface. By forming a non-covalent bond between the aqueous solvent and the aqueous solvent to swell, the entire aqueous system or aqueous alcohol solution gels, and the aqueous medium gels.

In the inclusion gel of the present invention, the hydrophobic compound is taken into the self-assembled fiber, while the hydrophilic compound or protein is taken into the network structure formed by the fiber, and the compound is included in the gel. It will be.

In the case of the encapsulated gel of the present invention, when the encapsulated protein is an enzyme-like protein, the enzyme is maintained without losing its activity even if it is incorporated into the gel. It is possible to proceed a redox reaction. Therefore, a gel containing such an enzyme can be used as a biosensor or a test / diagnosis agent.

In addition, cytochrome c has heme and histidine as an axial ligand, but usually methionine is coordinated as an axial ligand and hydrogen peroxide is difficult to come into contact with heme. Since there is no amino acid responsible for the Push-Pull mechanism such as peroxidase from horseradish or horseradish, cytochrome c alone has very low peroxidase activity, but it interacts with biological membranes, mainly with cytochrome c. It is thought that the structure near the heme of cytochrome c is changed and the peroxidase activity is enhanced mainly by electrostatic interaction between the positively charged lysine residue on the surface of cytochrome c and the negative charge of the biological membrane. Yes.
Thus, cytochrome c encapsulated in the Pal-GGGH gel is predicted to have an interaction with cytochrome c similar to that of the biological membrane, and the cytochrome c encapsulated in the Pal-GGGH gel Enhancement of peroxidase activity and stability of cytochrome c are expected.

Furthermore, in this invention, even if it coat | covers a skin surface etc., it can be used safely and safely by using the low molecular gelatinizer comprised by the natural origin raw materials, such as a fatty acid and an amino acid.
For this reason, the encapsulated gel of the present invention is a wound dressing having an ability to recognize an affected area or damaged site, an adhesion preventing film, a drug delivery system, a skin care product, a hair care product, an external medicine, an fragrance, a deodorant, an insecticide, an insecticide. It can be widely used as an agent, a substrate for agricultural chemicals, a cleaning agent, a paint, an antiseptic, a substrate for capturing environmental pollutants.

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

[Abbreviations used in Examples]
The meanings of the abbreviations used in the following examples are as follows.
Gly: glycine His: histidine HBTU: 2- (1-H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium-hexafluorophosphate (Watanabe Chemical Co., Ltd.)
HOBt: 1-hydroxy-benzotriazole (Peptide Institute, Inc.)
DMF: Dimethylformamide DCM: Dichloromethane DIEA: N, N-diisopropylethylamine (Tokyo Chemical Industry Co., Ltd.)
TFA: trifluoroacetic acid (Watanabe Chemical Co., Ltd.)
TIS: Triisopropylsilane (Watanabe Chemical Co., Ltd.)
Pal-GGGH: Palmitoyl-Gly-Gly-Gly-His
cyt c: cytochrome c

[Synthesis of lipid peptides]
The lipid peptide was synthesized according to the procedure of Fmoc solid phase peptide synthesis method shown below. The resin was mainly an amino acid-Barlos Resin. The synthesis scale was 0.3 mmol.

<Synthesis Example 1: Synthesis of trifluoroacetate salt of N-palmitoyl-Gly-Gly-Gly-His>
390 mg of histidine Barlos Resin (Watanabe Chemical Co., Ltd.) was added to the PD-10 column, and washed 3 times with 5 ml of DCM and 3 times with 5 ml of DMF. Next, 270 mg of Fmoc-Gly-OH (Watanabe Chemical Co., Ltd.) and 2.1 ml of condensing agent solution 1 (3.05 g of HBTU and 1.25 g of HOBt dissolved in 16 ml of DMF) were added to the above column. Furthermore, 2.1 ml of a condensing agent solution 2 (DIEA 2.75 ml dissolved in DMF 14.25 ml) was added. This was stirred with a vibrator for 30 minutes, then washed 5 times with 5 ml of DMF, 3 times with 5 ml of DCM, and further 3 times with 5 ml of DMF.
Next, 5 ml of 20% piperidine / DMF solution was added and stirred for 1 minute, then the solution was discarded, 5 ml of 20% piperidine / DMF solution was added again, stirred for 45 minutes, and washed 5 times with 5 ml of DMF.

Again, 270 mg of Fmoc-Gly-OH and 2.1 ml of the condensing agents 1 and 2 were added to the column. This was stirred for 20 minutes with a vibrator, and then washed 5 times with 5 ml of DMF, 3 times with 5 ml of DCM, and further 3 times with 5 ml of DMF. Next, 5 ml of 20% piperidine / DMF solution was added and stirred for 1 minute, then the solution was discarded, 5 ml of 20% piperidine / DMF solution was added again, stirred for 45 minutes, and washed 5 times with 5 ml of DMF.

Again, 270 mg of Fmoc-Gly-OH and 2.1 ml of the condensing agents 1 and 2 were added to the above column and stirred for 20 minutes with a vibrator, then 5 times with 5 ml of DMF and 3 times with 5 ml of DCM. And further washed 3 times with 5 ml of DMF. Next, 5 ml of 20% piperidine / DMF solution was added and stirred for 1 minute, then the solution was discarded, 5 ml of 20% piperidine / DMF solution was added again, stirred for 45 minutes, and washed 5 times with 5 ml of DMF.

About 230 mg of palmitic acid (manufactured by Aldrich) was added to the column, 2.1 ml of the condensing agents 1 and 2 were added, and the mixture was stirred for 90 minutes with a vibrator. After the reaction, the resin was washed 5 times with 5 ml of DMF, 5 times with 5 ml of DCM, and 5 times with 5 ml of methanol, and then the resin was vacuum-dried overnight.
After drying, 3.8 ml of TFA and 0.1 ml of TIS were added to the column and stirred for 1 hour.
Water is added to the collected mixed solution to precipitate a solid, and then suction filtration is performed to collect the product. After freeze-drying, the target compound is washed with 4 ml of acetonitrile three times. Obtained.

FT-MS + m / z calc. For C28H49N606 [M + H] + 565.37140, found 5655.3572

<Synthesis Example 2: Neutralization of N-palmitoyl-Gly-Gly-Gly-His trifluoroacetate>
To 0.5 g of trifluoroacetate of N-palmitoyl-Gly-Gly-Gly-His, 50 ml of saturated NaHCO ₃ water was added and dispersed, and centrifuged (3500 rpm, 10 minutes, 8 ° C.) (Tomy, SRX -201 high speed cooling centrifuge). 40 ml of EtOH was added to the obtained pellet and centrifuged (3500 rpm, 10 minutes, 8 ° C.). Further, 2% NaCl water was added to the pellet and centrifuged (3500 rpm, 10 minutes, 8 ° C.). Thereafter, 50 ml of pure water was added to the obtained pellets and washed by centrifugation (3500 rpm, 10 minutes, 8 ° C.). This washing operation was repeated twice, and the pellet was lyophilized (VFD-SP, manufactured by Ikeda Rika Co., Ltd., vacuum lyophilizer) to obtain 450 mg of a white solid.

The following examples were carried out using palmitoyl-Gly-Gly-Gly-His synthesized by the above method as a low-molecular gelling agent.
In addition, the low molecular gelling agent used in the present invention can easily form a gel into various shapes including a sheet shape by using only the low molecular gelling agent without using a base material such as a nonwoven fabric or a polymer compound. However, in Examples 1 to 7, the gel is used in the form of a sheet for convenience.

<Example 1: Gel sheet of palmitoyl-Gly-Gly-Gly-His by acidic, neutral, alkaline solution>
40.8 mg of palmitoyl-Gly-Gly-Gly-His is placed in a screw tube (Marem NO7) and 0.2% (w / v) (w means mass (g), v means volume (mL)). Phosphate buffer (made by Wako Pure Chemical Industries, Ltd., phosphate buffer powder, 1/15 mol / L, pH = 7.4, composition: Na ₂ HPO ₄ 7.6 g, KH ₂ PO ₄ 1 .8 g / L) was added, and heated (100 ° C., 10 minutes) with a dry bath incubator (manufactured by First Gene). 3 ml of the resulting solution was added to a styrene non-charged square case (36 mm × 36 mm × 14 mm) and cooled to room temperature. After the solution was solidified and gelled, 3 ml of Japanese Pharmacopoeia water was added dropwise and allowed to stand at room temperature. One day later, a sheet-like gel slice (1 cm × 0.5 cm) was cut out, placed in a screw tube (Marem NO5), Japanese Pharmacopoeia water, and phosphate buffer (pH = 2, pH = 7.4, pH = 11, pH was prepared by adding NaOH or HCl dropwise) 6 ml was added, and it was objectively observed for 3 months whether or not the section of the sheet gel melted and decomposed for 3 months.

As a result, the sheet-like slice gel of palmitoyl-Gly-Gly-Gly-His was observed as a sheet-like slice gel for 3 months without melting / degrading in acidic, neutral or alkaline water immersion liquid. .

[Adsorption evaluation of albumin on palmitoyl-Gly-Gly-Gly-His gel sheet]
Palmitoyl-Gly-Gly-Gly-His 81.6 mg and 60.1 mg were added to ultrapure water (Kurita Kogyo Co., Ltd.), phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.) phosphate buffer powder, 1/15 mol / L, pH = 7.4, composition: Na ₂ HPO ₄ 7.6 g, KH ₂ PO ₄ 1.8 g / L), 2 hours sonication and heating at 90 ° C. for 3 minutes. 3 ml of the solution was transferred to a styrene non-charged square case (36 mm × 36 mm × 14 mm), and a palmitoyl-Gly-Gly-Gly-His gel having a concentration of 0.3% (w / v) was prepared by standing at room temperature. 1 g of this gel was cut out and placed in a screw tube (Marume NO7) containing 5 ml of ultrapure water (manufactured by Kurita Kogyo Co., Ltd.) and allowed to stand at room temperature for 24 hours to form a sheet. Albumin from bovine serum Fraction V (manufactured by Sigma, ≧ 98%, BSA) dissolved in ultrapure water was added to this gel sheet immersed in water to a final concentration of 1 mg / ml. 500 μl of the immersion liquid was collected, the amount of BSA in the immersion liquid of the external liquid was measured by HPLC, and the amount of protein adsorbed on the gel was calculated.

[HPLC measurement conditions]
Column: Inertsil WP300 C8 5 μm, 2.1 mm i. d. × 150mm
Eluent: A; 0.1% TFA-MeCN B; 0.1% TFA-Water
B: 70% (0 min) -20% (10 min) (Analysis time: 10 min)
Flow rate: 0.3 ml / min
Column temperature: 60 ° C
Detection: UV214nm (BW10nm)
Injection volume: 2 μl
Post conditioning: 7 min

　　　　　　　　　　　　　　　　　　

From the results shown in Table 1, palmitoyl-Gly-Gly-Gly-His gel sheet prepared using water and PBS as a medium adsorbed BSA.
From the above results, palmitoyl-Gly-Gly-Gly-His gel sheet has an albumin adsorption effect.

<Example 3: Palmitoyl-Gly-Gly-Gly-His gel sheet containing riboflavin>
Add 128.2 mg of palmitoyl-Gly-Gly-Gly-His to a screw tube (Marem NO7) and make phosphate buffer (Wako Pure Chemical Industries, Ltd.) to a concentration of 0.3% (w / v) Phosphate buffer powder, 1/15 mol / L, pH = 7.4, composition: Na ₂ HPO ₄ 7.6 g, KH ₂ PO ₄ 1.8 g / L), dry bath incubator (manufactured by First Gene) Then, heating (100 ° C., 10 minutes) and 3 ml of the obtained solution was placed in an aluminum cylinder (diameter 2.5 cm, height 4 cm) placed in a glass petri dish (diameter 6 cm, height 4 cm). Transfer and cool to room temperature. After confirming that the solution was solidified, the aluminum cylinder was removed, and 6 ml of Japanese Pharmacopoeia water was added to the glass petri dish. After 1 hour, 250 μL of aqueous riboflavin (manufactured by Wako Pure Chemical Industries, Ltd.) (2 mg / mL) was added to the glass petri dish, and after 14 hours, the gel was colored yellow, and the riboflavin was encapsulated in the gel to form a sheet. did.

<Comparative Example 1: Effect of cellulose gel, carboxyvinyl polymer gel, xanthan gum gel, curdlan gel on water immersion>
The cellulose gel was prepared by adding 166 g of Japanese pharmacopoeia water to 100 g of Serodine 4M (NanoWope, cellulose gel 4 wt%, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.). K. Using a mixing analyzer MA2500 (Primics Co., Ltd.), the mixture was stirred at 5000 rpm for 240 minutes and allowed to stand at room temperature until gelation was observed to obtain a cellulose gel (1.5 wt% cellulose aqueous dispersion).
The 2% carboxyvinyl polymer was gelled by adding Japanese Pharmacopoeia water to 0.252 g of Carbopol 940 (manufactured by IT Corporation) and then heating in a water bath until dissolved.
5% xanthan gum was gelled by adding Japanese pharmacopoeia water to 0.725 g of xanthan gum (manufactured by Tokyo Chemical Industry Co., Ltd.) and then heating in a water bath until dissolved.
1 g of carboxyvinyl polymer (2 wt%, carbopol), 1 g of cellulose gel (1.5 wt%, cellodine), 1 g of xanthan gum (1.5 wt%) and curdlan (1.5 wt%) were added to a plastic petri dish (diameter 8.5 cm × When the sample was placed in a depth of 1.4 cm and immersed in 6 ml of water, the gel disappeared after 72 days and no sheet was formed.

[Evaluation of hydrophobic environment in gel fiber]
A hydrophobic environmentally responsive probe, 8-anilino-1-naphthalene sulfonic acid (ANS), was used. It is known that ANS, which is a hydrophobic environmentally responsive probe, decreases in polarity in the vicinity thereof and shifts the fluorescence maximum wavelength to the short wavelength side, thereby increasing the fluorescence intensity. Therefore, ANS was dissolved in a phosphate buffer (pH 7.5) so that the final concentration was 5 μM, and each gel (0.5 or 0.02 wt% Pal-GGGH, 0.5 wt% agarose, 3 wt% Of sodium alginate, 5 wt% polyacrylamide).
In addition, ANS was dissolved in each organic solvent (ethanol, 1-propanol, acetone, dioxane) so as to be 5 μM. The fluorescence intensity of each gel and organic solvent was measured (ex 350 nm, em 480 nm).
As a result, it was revealed that only Pal-GGGH had an increase in fluorescence intensity and had a hydrophobic environment in the gel (FIG. 2). On the other hand, since there is almost no change in the fluorescence intensity in other polymer gels, the formation of the hydrophobic region accompanying the self-assembly of Pal-GGGH has an effect on the increase in fluorescence intensity, not the gel formation. It is thought to have been given. Further, since Pal-GGGH showed fluorescence even at a gel formation concentration or less (0.02 wt%), it is considered that a hydrophobic region is formed by self-organization even at a low concentration that is not gelled.

Further, when the fluorescence maximum wavelengths of ANS in an organic solvent and a Pal-GGGH gel were compared, the hydrophobic regions of the gel fiber were ethanol, propanol because ethanol and 1-propanol were close to the fluorescence maximum wavelength of the Pal-GGGH gel. (Fig. 3).

[Evaluation of release from gel sheet]
The release of the encapsulated substance from the gel sheet was evaluated. ANS and vitamin B2 (riboflavin) used in the previous experiment were used as hydrophilic inclusion substances. As the buffer solution, pH 3: citrate buffer solution, pH 7.4: phosphate buffer solution, pH 9.0: Tris-HCl buffer solution were used, respectively. ANS or riboflavin was dissolved in a buffer so as to be 100 or 300 μM, and a gelling agent (gelling agent concentration of 0.3 or 0.6 wt%) was added to 1 ml of each solution to prepare a gel sheet. Thereto was added 10 ml of an extraction buffer, and the inclusion substance was extracted by shaking at 40 rpm. Samples were sampled over time, and the release concentration at each time was calculated from the absorbance (ANS: 350 nm, riboflavin: 445 nm).

The sustained release from the gel sheet was compared by changing the concentration of the gelling agent and the pH of the gel and the release liquid. As a result, it was shown that the release rate of ANS decreased when the concentration of the gelling agent was increased (FIG. 4). The reason for this is thought to be that the amount of encapsulated ANS increased because the gel fiber increased as the gelling agent concentration increased. In ANS, the release rate increased as the pH increased (FIG. 5), but riboflavin did not differ depending on the pH (FIG. 6). The charge state of the gelling agent molecule at each pH of Pal-GGGH (FIG. 7) is based on the pKa of the amino acid residue. It is considered that the ionized state of (II) is taken at 6.0 and (III) is taken at pH 6.0 or higher. When the pH is 9.0, more Pal-GGGH molecules are considered to be in the ionized state of (III) and are expected to have a negative charge. Since ANS is an anion, it is considered that the ANS was released into the release liquid repelling the negative charge on the surface of the gel fiber. Since riboflavin has no charge in aqueous solution, it is considered that it was not affected by pH.

<Example 4: Encapsulation of hydrophilic substance by immersion in gel sheet>
An experiment was conducted to determine whether riboflavin can be incorporated into the gel sheet by immersing the gel sheet in a solution of riboflavin. A gel sheet was prepared by adding a gelling agent (gelling agent concentration: 0.3 wt%) to 1 ml of phosphate buffer. The gel sheet was immersed in 5 ml of a riboflavin solution of each concentration (10 μg / ml to 200 μg / ml) for 68 hours, and the concentration of the soaking solution was calculated from the absorbance (445 nm) to determine the amount of riboflavin encapsulated in the gel sheet.

From the results in Table 2, riboflavin was encapsulated in the gel sheet by immersing the gel sheet in the riboflavin solution. In addition, the amount of inclusion in the gel sheet increased in proportion to the increase in the concentration of the riboflavin immersion liquid.

[Evaluation of Solubilization of Hydrophobic Substance by Gelation of Pal-GGGH]
To what extent a hydrophobic substance can be solubilized by using a gel with a gelling agent, Pal-GGGH, was evaluated for its solubility. Pyrene and vitamin E (α-tocopherol) were used as model hydrophobic substances. First, a hydrophobic material was dissolved in DMSO or ethanol to prepare a stock solution. This stock solution was diluted with a phosphate buffer (pH 7.5) to prepare a sample solution having a final concentration of 100 μM to 900 μM of a hydrophobic substance (containing DMSO and ethanol 5% or 1%). A gelling agent (final concentration of 0.1 wt% to 0.4 wt%) was added to each sample solution to cause gelation.
Using the standard of the degree of solubilization of the hydrophobic substance shown in FIG. 8, the solubility of the hydrophobic substance by gelation was evaluated visually. Here, the criteria of the degree of solubilization of the hydrophobic substance shown in FIG. 8 are as follows: (I) shows a dissolved state, (II) shows a slightly cloudy state, and (III) shows no dissolution. It shows a cloudy state.

As a result of the evaluation of the hydrophobic environment in the gel fiber described above, it has been clarified that the hydrophobic region exists in the gel fiber. However, the hydrophobic substance is encapsulated in the hydrophobic region, and apparently in the gel fiber. It was considered that solubilization in an aqueous solvent was possible, and solubilization of hydrophobic substances was evaluated (FIG. 9). As a result, it was revealed that both pyrene and vitamin E can be solubilized in the gel in a state containing DMSO and ethanol (Table 3).

<Example 5: Preparation of vitamin E-containing gel>
The aforementioned stock solution was diluted with MOPS buffer (pH 7.5) to prepare a 500 μM vitamin E aqueous solution (containing 5% ethanol). Using this, each gel (0.5 wt% Pal-GGGH, 0.5 wt% agarose, 5% polyacrylamide) was prepared, and a vitamin E-encapsulating gel was prepared.

As a result, in the Pal-GGGH gel, the turbidity of the gel was not changed regardless of the presence or absence of vitamin E (FIG. 10). This is probably because vitamin E was apparently dissolved because it was encapsulated in a hydrophobic region in the gel fiber. On the other hand, when an attempt was made to encapsulate vitamin E in polyacrylamide gel and agarose gel, vitamin E was precipitated because it was highly hydrophobic and insoluble in water, and white turbidity of the gel was observed. It can be seen that the polymer gel cannot solubilize vitamin E.
From the above, it was shown that Pal-GGGH gel solubilizes vitamin E.

<Example 6: Preparation of simultaneous inclusion gel of vitamin E and vitamin C>
Vitamin C (sodium ascorbate phosphate) was dissolved in MOPS buffer (pH 7.5) to a concentration of 1 mg / ml. Thereto, 0.5 wt% of Pal-GGGH is added, and a stock solution is added so as to become 500 μM vitamin E (containing 5% ethanol), the solution is gelled, and the hydrophobic substance vitamin E is added. And a simultaneous inclusion gel of vitamin C which is a hydrophilic substance.

As a result, even in the presence of vitamin C derivative, vitamin E was dissolved in the hydrophobic region of the gel fiber, and no white turbidity of the gel accompanying the precipitation of vitamin E was observed (FIG. 11).
From the above, it was shown that by using Pal-GGGH, it is possible to prepare a gel in which vitamin E and vitamin C having different solubilities are dissolved and encapsulated at the same time.

[Evaluation of Vitamin E Release from Gel Sheet]
The vitamin E stock solution was diluted with MOPS buffer (pH 7.5) to prepare a 500 μM vitamin E aqueous solution (containing 5% ethanol). Here, Pal-GGGH was added so that it might become 0.3 wt%, and it heated and allowed to cool and created the gel sheet. The gel sheet was immersed in 5 ml of an aqueous ethanol solution (50, 70, 100%) and shaken at 45 rpm to release vitamin E. The release solution was sampled over time, and the release concentration of vitamin E at each time was calculated from the absorbance (292 nm).

In order to evaluate the release of hydrophobic substances, the release behavior of vitamin E from gel sheets was evaluated and compared with aqueous ethanol solutions having different concentrations (FIG. 12). It was shown that vitamin E is released from the gel when the ethanol concentration is 50% or more. The release rate tended to increase with increasing ethanol concentration. Since the hydrophobic region in the gel fiber is considered to be about the same as ethanol (see FIG. 3), the release rate may vary depending on the ethanol concentration.

<Example 7: Examination of adsorptivity of cytochrome c to gel sheet>
In order to examine the use of the Pal-GGGH gel sheet as a protein adsorption material, an adsorption experiment of cytochrome c on the gel sheet was conducted. As the buffer solutions, phosphate buffer solution: pH 7.4 and Tris-HCl buffer solution: pH 9.0 were used, respectively. A gel sheet was prepared by adding a gelling agent (gelling agent concentration: 0.3 wt%) to 1 ml of each buffer solution. The gel sheet was immersed in a 0.3 mg / ml cytochrome c solution, and the concentration of the immersion liquid was calculated over time by absorbance (407 nm) to determine the amount of cytochrome c adsorbed on the gel sheet.
Next, in order to confirm that the adsorbed cytochrome c was not denatured, 10 μl of 1M DTT was dropped onto the cytochrome c adsorption gel sheet to reduce the cytochrome c.

As a result, when the gel sheet (pH 7.4 and pH 9.0) was immersed in the cytochrome c solution, cytochrome c was adsorbed inside the gel sheet (FIG. 13). The adsorption rate was found to be faster at pH 9.0 than at pH 7.4 (FIG. 14).
Furthermore, when 1M DTT was dropped onto the gel sheet adsorbed with cytochrome c, cytochrome c that was oxidized and orange red changed to pink, so it can be said that cytochrome c adsorbed on the gel sheet changed to reduced form. (FIG. 15). That is, it was suggested that it was not denatured even when adsorbed on the sheet. In addition, cytochrome c is a protein having an isoelectric point near pI10 and has a positive charge under the present conditions. Therefore, the adsorption amount in a gel of pH 9.0 having more negative charges (FIG. 7) is large. It is thought that it rose.

[Evaluation of cytochrome c release from gel sheet]
Dissolve cytochrome c in phosphate buffer solution (pH 7.4) to 0.5 mg / ml, add Pal-GGGH to 0.3 wt%, heat and cool, and cytochrome c-encapsulated gel sheet It was created. The gel sheet is immersed in 10 ml of each buffer solution (pH 5.0 sodium citrate buffer solution, pH 7.4 phosphate buffer solution, pH 11.0 phosphate / sodium hydroxide buffer solution), shaken and stirred at 40 rpm, and cytochrome. c was released. The release solution was sampled over time, and the release concentration of cytochrome c at each time was calculated from the absorbance (407 nm).

As described above, since the pH affected the adsorptivity, the release behavior of cytochrome c from the gel sheet was evaluated with respect to buffers having different pHs (FIG. 16). As a result, release of cytochrome c was observed only in the release solution having a pH of 11.0. Considering the pKa of histidine in Pal-GGGH (imidazole group: pKa about 6.0, carboxyl group: pKa about 1.8) (FIG. 17), the gel fiber surface is negative at pH 11.0 and pH 7.4. It is presumed that the pH is both positive and negative at pH 5.0. Here, cytochrome c is a protein having an isoelectric point near pI10, and is considered to have a negative charge only at pH 11.0 under the present conditions. Therefore, it is considered that only at pH 11.0, cytochrome c and gel fiber were repelled by the negative charge of each other and released.

<Example 8: Measurement of peroxidase activity of cytochrome c encapsulated in Pal-GGGH gel (oxidation reaction of 2,6-dimethoxyphenol with H ₂ O ₂ using Pal-GGGH gel encapsulating cytochrome c)>
In order to evaluate the peroxidase activity of cytochrome c encapsulated in Pal-GGGH gel, the oxidation reaction of 2,6-dimethoxyphenol with H ₂ O ₂ using Pal-GGGH gel encapsulating cytochrome c shown below Went.
Here, for the Pal-GGGH gel encapsulating cytochrome c, cytochrome c is added to a 50 mM phosphate buffer solution containing Pal-GGGH powder as a gelling agent, and the buffer solution is heated at 95 ° C. It was obtained by dissolving and allowing to cool to room temperature.

Pal obtained by adding cytochrome c to each well of a 96-well microplate at 3 μM in 50 mM phosphate buffer (pH 7) containing a gelling agent at a concentration of 0.2 wt% and heating at 95 ° C. -GGGH gel, 50 mM phosphate buffer (pH 7) containing 3 μM of cytochrome c and heat-treated at 95 ° C. as in the gel preparation, and 50 mM not containing heat-treated cytochrome c at a concentration of 3 μM 50 μL each of phosphate buffer (pH 7) was prepared, and 100 μL of a mixed solution containing 2,6-dimethoxyphenol (7.5 mM) and H ₂ O ₂ (1.5 mM) was added thereto. When the mixed solution was added to the Pal-GGGH gel, the mixed solution was added onto the gel. Comparison of peroxidase activity was performed by following the absorbance at 469 nm derived from the product. The heat treatment time was 30 minutes, and the measurement was performed after leaving for about 30 minutes thereafter, and the temperature of the apparatus was set to 35 ° C.

As shown in FIG. 18, the reaction using the cytochrome c-encapsulated Pal-GGGH gel has a larger amount of product than the reaction with the cytochrome c solution not subjected to heat treatment. It was confirmed that per-oxidase activity was increased in the Pal-GGGH gel encapsulating c. Moreover, in the reaction with the cytochrome c solution that had been subjected to the heat treatment, the amount of the product was large, and it was confirmed that the peroxidase activity was similarly increased. However, since the amount of product is larger in the reaction using the cytochrome c-encapsulated Pal-GGGH gel than in the reaction with the heat-treated cytochrome c solution, the Pal-GGGH gel encapsulating the cytochrome c However, it is considered that the stability of the catalytic action of cytochrome c is enhanced in the Pal-GGGH gel.

<Example 9: Measurement of CD spectrum and UV spectrum of cytochrome c encapsulated in Pal-GGGH gel>
Pal-GGGH gel (gelling agent 0.2 wt%) containing cytochrome c at a concentration of 10 μM, 50 mM phosphate buffer solution containing cytochrome c at a concentration of 10 μM and heat-treated at 95 ° C. in the same manner as the gel preparation ( A 50 mM phosphate buffer (pH 7) containing pH 7) and cytochrome c at a concentration of 20 μM and not subjected to the heat treatment was prepared in 1 mL cells, and CD spectra at wavelengths of 250 nm to 450 nm were measured. The heat treatment time was 26 minutes, and the temperature of the apparatus was set at 25 ° C.

In the CD spectrum measurement result shown in FIG. 19, the peaks of 280 nm and 380 nm observed in the CD spectrum of cytochrome c in the solution in the spectrum of cytochrome c included in the Pal-GGGH gel could not be confirmed.

In addition, a Pal-GGGH gel (gelator 0.1 wt%) containing cytochrome c at a concentration of 20 μM, and a 50 mM phosphate buffer containing cytochrome c at a concentration of 20 μM and subjected to the same 95 ° C. heat treatment as in the gel preparation. A 50 mM phosphate buffer solution (pH 7) containing a solution (pH 7) and cytochrome c at a concentration of 20 μM and not subjected to the heat treatment was prepared in 1 mL cells, and UV spectra at wavelengths of 350 nm to 800 nm were measured. The heat treatment time was 15 minutes, and the temperature of the apparatus was set to 25 ° C.

The UV spectrum measurement results shown in FIG. 20 are based on the Soret band near 400 nm caused by the π-π ^* transition of the porphyrin ring of cytochrome c and the transition conjugated to the molecular vibration appearing near 480-650 nm in both gel and solution. Q band was confirmed. However, a charge transfer absorption band (S → Fe: LMCT band) derived from a coordination bond between heme iron and Met80, which is the sixth ligand, in the vicinity of 700 nm could not be confirmed with cytochrome c in the gel.

From the results of CD spectrum and UV spectrum, it was recognized that cytochrome c was structurally changed in the Pal-GGGH gel, which was related to the increase in peroxidase activity of the Pal-GGGH gel containing cytochrome c. It seems that there is.

<Example 10: Gelling agent concentration dependence of peroxidase activity>
The same experiment as in Example 8 was performed when the gelling agent concentrations were 0.1 wt%, 0.2 wt%, and 0.3 wt%.
From FIG. 21, it was found that the initial reaction rate and the amount of product increased as the gelling agent concentration increased.

In the encapsulated gel of the present invention, it becomes possible to encapsulate a hydrophobic compound, a hydrophilic compound, or both compounds in the gel. The encapsulated gel of the present invention can also release the encapsulated compound gradually. Furthermore, the gel in which the compound of the present invention is encapsulated can also encapsulate an enzyme such as cytochrome c as a compound, and if the encapsulated protein is an enzyme-like protein, Once incorporated, the enzyme maintains its activity without loss, but rather allows the enzymatic reaction to proceed within the gel. Therefore, a gel incorporating such an enzyme can see the reaction in the gel and can be used as a biosensor or a test / diagnostic agent.
Therefore, the encapsulated gel of the present invention is a wound dressing having an ability to recognize a damaged site, an adhesion-preventing film, a drug delivery system, a base for external medicine, a base such as a fragrance, a deodorant, an insecticide, an insecticide and an agrochemical. It can be widely used for materials, inspection / diagnosis, biosensors, base materials for environmental analysis, and base materials for capturing contaminants in soil and water.

Claims (15)

1. 

   
   (Wherein R ¹ represents an aliphatic group having 9 to 23 carbon atoms, and R ^{2 to} R ⁵ are each independently a hydrogen atom or a carbon atom that may have a branched chain of 1 to 3 carbon atoms. Represents an alkyl group, a phenylmethyl group, a phenylethyl group, or a — (CH ₂ ) n—X group represented by formulas 1 to 7, and at least one of R ^{2 to} R ⁵ represents a — (CH ₂ ) n—X group. N represents a number of 1 to 4, X is composed of an amino group, a guanidino group, a —CONH ₂ group, or a 5-membered ring or a 5-membered ring and a 6-membered ring which may have 1 to 3 nitrogen atoms. M represents 1 or 2), and a low molecular gelling agent comprising a lipid peptide represented by the following formula: An aqueous medium and at least one compound contained therein. An included gel.
2. The inclusion gel according to claim 1, wherein, in the formula (1), R ¹ is a linear aliphatic group having 11 to 23 carbon atoms which may have 0 to 2 unsaturated bonds. .
3. In the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a branched chain of 1 or 2 carbon atoms, a phenylmethyl group, or Represents a — (CH ₂ ) n—X group, and one or two of R ^{2 to} R ⁵ represent a — (CH ₂ ) n—X group,
   n represents a number from 1 to 4, and X is an amino group, guanidino group, —CONH ₂ group, or a 5-membered ring or a 5-membered ring and a 6-membered ring which may have 1 or 2 nitrogen atoms The inclusion gel according to claim 1 or 2, wherein the inclusion gel represents a heterocyclic ring.
4. In the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms which may have a branched chain of 1 or 2 carbon atoms, a phenylmethyl group, or Represents a — (CH ₂ ) n—X group, and one or two of R ^{2 to} R ⁵ represent a — (CH ₂ ) n—X group,
   The encapsulated gel according to claim 3, wherein n represents a number from 1 to 4, and X represents an amino group, a guanidino group, a -CONH ₂ group, a pyrrole group, an imidazole group, a pyrazole group, or an indole group. .
5. In the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, second Butyl group, tert-butyl group, phenylmethyl group, aminomethyl group, 2-aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, carbamoylmethyl group, 2-carbamoylethyl group, 3-carbamoylpropyl group, A 2-guanidinoethyl group, a 3-guanidinopropyl group, a pyrrolemethyl group, an imidazolemethyl group, a pyrazolemethyl group or a 3-indolemethyl group, and one or two of R ^{2 to} R ⁵ are an aminomethyl group, 2 -Aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, carbamoylmethyl group, 2-carbamoylethyl group, 3-carbamoyl The inclusion gel according to claim 4, which represents a propyl group, a 2-guanidinoethyl group, a 3-guanidinopropyl group, a pyrrolemethyl group, an imidazolemethyl group, a pyrazolemethyl group or a 3-indolemethyl group.
6. In the formula (1), R ^{2 to} R ⁵ are each independently a hydrogen atom, methyl group, i-propyl group, i-butyl group, sec-butyl group, phenylmethyl group, 4-aminobutyl group, carbamoyl. Represents a methyl group, 2-carbamoylethyl group, 3-guanidinopropyl group, imidazolemethyl group or 3-indolemethyl group, and one or two of R ^{2 to} R ⁵ are a 4-aminobutyl group, a carbamoylmethyl group, The inclusion gel according to claim 5, which represents a 2-carbamoylethyl group, a 3-guanidinopropyl group, an imidazolemethyl group, or a 3-indolemethyl group.
7. The encapsulated gel according to any one of claims 1 to 6, wherein m represents 1 in the formula (1).
8. The inclusion gel according to any one of claims 1 to 7, wherein the compound is a hydrophobic compound, a hydrophilic compound, or a compound of both.
9. The inclusion gel according to any one of claims 1 to 7, wherein the compound is an enzyme.
10. The inclusion gel according to claim 9, wherein the compound is cytochrome c.
11. A method of using the inclusion gel according to claim 9 for an enzyme reaction.
12. A method of using the encapsulated gel according to the oxidation reaction of the desired product by H ₂ O ₂ in claim 10.
13. A method of using the inclusion gel as a biosensor by using the inclusion gel according to claim 9 in an enzyme reaction.
14. By using encapsulated gel according to oxidation reaction with H ₂ O ₂ in claim 10, methods of using the inner hull gel as a biosensor.
15. A biosensor comprising the inclusion gel according to claim 11 or 12.

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