JP7628285B2 - Material composition for biological model and biological model - Google Patents

Description

The present disclosure relates to a material composition for a biological model and a biological model.

The functionality (or effects) and usability of cosmetic products are evaluated by expert panels and by methods using tissues derived from humans and non-human animals.
In recent years, in the development of cosmetics such as nail polish and nail paint, the market of which is expanding (see, for example, Non-Patent Document 1), human nail plates and free nails have long been used as materials for evaluating the application, retention, and penetration of nail cosmetics. In particular, human nail plates are difficult to obtain, and alternatives have been considered from the standpoint of ethics and mass production, and cow hooves have been used as evaluation materials as an alternative to human nail plates (see, for example, Non-Patent Document 2).
In recent years, from the standpoint of animal protection, it has become increasingly difficult to conduct in vivo and in vitro evaluations using animals in cosmetic development, and evaluations using biomimetic models (so-called in vivo models) have been attracting attention as an alternative to animal-derived evaluation materials.

For example, there are nail models in the shape of human nails made of polyacrylamide compounds and polymerization agents (see Patent Document 1), models of feet made of resin with nail-shaped parts (see Patent Document 2, for example), a technology to obtain three-dimensional data of human nails and create nail models using a 3D printer (see Patent Document 3, for example), and nail models for nail treatment practice (see Patent Document 4, for example).

By approximating the chemical composition of human nails, models that include not only human nail plate models but also hair have been proposed (see, for example, Patent Documents 5 and 6, and Non-Patent Documents 3 to 7). Specifically, keratin, which has a molecular structure similar to that of human nail plate, is extracted from human hair, wool, etc. without denaturing the molecular structure, and a film is formed through various processes. The processes include a method of adding glycerol as a plasticizer and drying at 110°C to regenerate cysteine bonds to produce a film (see, for example, Non-Patent Documents 3 to 5), a method of mixing a keratin extract with a developing solution containing various salts, and washing and drying the formed precipitate to obtain a film (see, for example, Non-Patent Document 6), and a method of producing a film by a precast method and a postcast method (see, for example, Non-Patent Document 7 and Patent Document 5).

Other examples include a method of extracting keratin from wool or the like, mixing it with polyethylene glycol, and then forming a film (see, for example, Patent Document 7); a method of dissolving wool in 100% thioglycolic acid, forming a film, drying it, volatilizing the thioglycolic acid, and then washing the film with water without heating to remove the remaining thioglycolic acid in the film and obtain a film consisting only of keratin (see, for example, Patent Document 8); and a method of obtaining a keratin extract from hair using an aqueous sodium hydroxide solution, adding various property improvers (sodium salt of carboxymethylcellulose, sodium salt of alginic acid, polyvinyl alcohol, etc.) to produce a film or any molded product (see, for example, Patent Document 9).

JP 2020-152793 A JP 2019-133103 A JP 2017-113176 A Utility Model Registration No. 3168569 JP 2017-72397 A JP 2002-332357 A JP 2011-207858 A Japanese Patent Application Publication No. 4-91138 JP 2016-121271 A

Update on nail cosmetics, Dermatologic Therapy, 25, 481-490 (2012) Nail swelling as a pre-formulation screen for the selection and optimization of ungual penetration enhancers, Pharm. Res. 24 (2007) 2207-2212. Keratin film made of human hair as a nail plate model for studying drug permeation, Euro. J. Pharma. Biopharma., 78, 432-440 (2011) Physicochemical investigations of native nails and synthetic models for a better understanding of surface adhesion of nail lacquers, Euro. J. Pharma. Sci., 131 208-217 (2019) Infected nail plate model made of human hair keratin for evaluating the efficacy of different topical antifungal formulations against Trichophyton rubrum in vitro, Euro. J. Pharma. Biopharma., 84, 599-605 (2013) Preparation of Translucent and Flexible Human Hair Protein Films and Their Properties, Biol. Pharm. Bull., 27(9) 1433-1436 (2004) Convenient Procedures for Human Hair Protein Films and Properties of Alkaline Phosphatase Incorporated in the Film, Biol. Pharm. Bull., 27(1), 89-93 (2004)

However, Non-Patent Document 2 discloses that bovine hooves have a higher degree of swelling in water than human hooves, and that applying the results of permeability evaluation obtained from bovine hooves to human nail plates results in an overestimate.
The nail models described in Patent Documents 1 to 3 are made of materials such as resin and metal, and are significantly different from the physicochemical composition of the human nail plate. Therefore, these nail models are not applicable to the evaluation of the permeability, such as the retention and application properties, of cosmetic ingredients.

It is known that actual human nail plates and human hair have permeation channels for hydrophilic substances and lipophilic substances. The keratin films prepared by the methods described in Patent Documents 5 and 6 and Non-Patent Documents 3 to 7 are composed of hydrophilic substances, and therefore have a problem in that they do not have permeation channels for lipophilic substances contained in actual cosmetics.
All of the mixtures proposed in the methods described in Patent Documents 7 to 9 have permeation channels for hydrophilic substances but not for lipophilic substances, and have not yet been able to produce a keratin film having permeation channels for both hydrophilic and lipophilic substances.
In light of this, there is a demand for the development of a biological model that can permeate both oil and water, just like the human body.

In view of the above, the problem to be solved by one embodiment of the present disclosure is to provide a material composition for a biological model having permeation channels for both hydrophilic and lipophilic substances in the resulting biological model.
Another problem to be solved by another embodiment of the present disclosure is to provide a biological model formed from the above-mentioned biological model material composition.

Specific means for solving the above problems include the following aspects.
<1> A material composition for a biological model, comprising keratin and a lipid.
<2> The material composition for a biological model according to <1>, wherein the content of the lipid is 1% by mass to 30% by mass based on the total mass of the keratin.
<3> The material composition for a biological model according to <1> or <2>, wherein the lipid contains a liquid crystal compound.
<4> The material composition for a biological model according to any one of <1> to <3>, wherein the lipid includes a compound capable of forming a cholesteric liquid crystal.
<5> The material composition for a biological model according to any one of <1> to <4>, wherein the lipid contains a compound represented by the following general formula (1):

In formula (1), L represents a single bond, -O(C=O)-, or -O(C=O)O-, and ^R1 represents a monovalent substituent or a hydrogen atom.

<6> The material composition for a biological model according to any one of <1> to <5>, wherein the keratin includes a water-soluble keratin.
<7> The material composition for a biological model according to <6>, wherein the water-soluble keratin is keratin derived from human hair or keratin derived from wool.
<8> The material composition for a biological model according to any one of <1> to <7>, which is used for a human nail plate model.

<9> A biological model formed from the material composition for a biological model according to any one of <1> to <8>.
<10> The biological model according to <9>, which is a human nail plate model or a hair model.

According to one embodiment of the present disclosure, a material composition for a biological model is provided that has permeation channels for both hydrophilic and lipophilic materials in the resulting biological model.
According to another embodiment of the present disclosure, there is provided a biological model formed from the above-mentioned biological model material composition.

FIG. 1 is a diagram showing an example of a cross-section of a biological model stained with rhodamine B or oil red, the biological model being prepared using a material composition for a biological model according to the present disclosure. FIG. 2 is a diagram showing an example of a cross-section stained with rhodamine B of a biological model prepared using a biological model material composition according to the present disclosure. FIG. 3 is a diagram showing an example of a cross-section stained with rhodamine B of a biological model prepared using a biological model material composition according to the present disclosure. FIG. 4 is a diagram showing an example of a cross-section of a section stained with oil red of a biological model prepared using a biological model material composition according to the present disclosure. FIG. 5 is a diagram showing an example of a cross-section of a section stained with oil red of a biological model prepared using a biological model material composition according to the present disclosure. FIG. 6 is a diagram showing an example of a cross-section of a section stained with oil red of a biological model prepared using a biological model material composition according to the present disclosure. FIG. 7 is a diagram showing an example of a cross section of a slice of Comparative Example 1 stained with Rhodamine B or Oil Red. FIG. 8 is a diagram showing an example of a cross section of a slice of Comparative Example 2 stained with Rhodamine B or Oil Red. FIG. 9 shows the results of infrared spectroscopic analysis (ATR-IR) of the examples and comparative examples.

The present disclosure will be described in detail below. The following description of the components may be based on a representative embodiment of the present disclosure, but the present disclosure is not limited to such an embodiment.
In the present disclosure, a numerical range indicated using "to" means a range including the numerical values described before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described in stages in the present disclosure, the upper limit or lower limit described in a certain numerical range may be replaced with the upper limit or lower limit of another numerical range described in stages. In addition, in the numerical ranges described in the present disclosure, the upper limit or lower limit described in a certain numerical range may be replaced with a value shown in the examples.
In the present disclosure, combinations of two or more preferred aspects are more preferred aspects.

In the present disclosure, when a plurality of substances corresponding to each component are present in the composition, the amount of each component in the composition means the total amount of the plurality of substances present in the composition, unless otherwise specified.
In the present disclosure, the total solid content refers to the total mass of the components excluding the solvent from the entire composition of the composition. In the present disclosure, the solid content is the solid content at 25°C.
In the present disclosure, the term "process" includes not only independent processes, but also processes that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.

(Material composition for biological models)
The material composition for a biological model according to the present disclosure contains keratin and lipids.
As mentioned above, conventional alternative models for human nails, human hair, etc., have been limited to imitations using keratin, which is their main component. Because keratin is a hydrophilic polymer compound, nail models and hair models based on keratin exhibit good permeability to hydrophilic substances, but are almost impermeable to lipophilic substances, and improvements in permeability of lipophilic substances have been required.
On the other hand, it has been reported that human nail plate, human hair, etc. have some internal permeation channels for lipophilic substances, and show good permeability not only to hydrophilic substances but also to lipophilic substances. In terms of permeability to both hydrophilic and lipophilic substances, the difference between conventional nail plate models and hair models based on keratin and human-derived biological models is very large.
For example, known emulsification techniques are used to improve the permeability of lipophilic substances in keratin substrates, which appears to improve the permeability of both hydrophilic and lipophilic substances in nail plate and hair models. By mixing a surfactant with oil and water, the interfacial energy between the oil and water is reduced, forming a state in which oil particles are suspended (dispersed) in a water matrix (i.e., the continuous phase of water) (O/W emulsion) or vice versa, a state in which water particles are dispersed in an oil matrix (continuous phase of oil) (W/O emulsion), resulting in a macroscopically uniform mixed state. However, since only the matrix (i.e., the continuous phase of water or oil) side is connected at the microscopic level, when the matrix is oil, the water droplets are independent, and when the matrix is water, the oil droplets are independent.
For this reason, it was difficult to achieve both hydrophilicity and lipophilicity. For these reasons, a keratin-based nail plate and hair model with a matrix of hydrophilic keratin and channels that allow lipophilic substances to permeate was eagerly awaited.
As a result of intensive research, the inventors have found that the material composition for a biological model according to the present disclosure has the above-mentioned configuration, and thus has permeation channels for both hydrophilic and lipophilic substances in the obtained biological model. It is presumed that the above-mentioned configuration causes lipids to be chemically bound to keratin molecules to form a membrane of keratin, thereby forming lipophilic channels in the matrix of keratin, which is a hydrophilic polymer.
In addition, since the biological model material composition according to the present disclosure has the above-mentioned configuration, it is presumed that when the obtained biological model is used for permeability evaluation, the uniformity of penetration is also excellent.
Each component of the biological model material composition according to the present disclosure will be described below.

<Fats>
The material composition for a biological model according to the present disclosure contains a lipid. Examples of the lipid include fatty acids and their ester compounds, phospholipids, and glycolipids.
Examples of the fatty acid and its ester compound include saturated fatty acid and unsaturated fatty acid and their ester compound.
Examples of phospholipids include glycerophospholipids such as phosphatidic acid, bisphophatidic acid, lecithin (phosphatidylcholine), phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerin, and diphosphatidylglycerin (cardiolipin).
Examples of glycolipids include sphingoglycolipids such as glucosylceramide and galactosylceramide.
The lipid preferably contains a fatty acid ester compound, which promotes the formation of a permeation channel, and from the viewpoint of excellent uniformity of penetration, it is more preferable that the lipid contains a liquid crystal compound, and further preferably is a liquid crystal compound.
A liquid crystal compound means a compound having a liquid crystal structure. Liquid crystal is an intermediate state between a crystalline state and a liquid state, in which the molecular orientation maintains some order, but the three-dimensional positional order is lost compared to the crystalline state.
X-ray diffraction can be used to confirm liquid crystals (i.e., liquid crystal structure). Liquid crystals are classified into nematic phase, cholesteric phase, smectic phase, etc. based on the arrangement of their structure, and a unique X-ray diffraction pattern can be obtained for each structure. Therefore, the liquid crystal structure can be confirmed from the obtained X-ray diffraction pattern.

Examples of the liquid crystal compound include liquid crystal compounds that form a nematic structure, a smectic structure, a cholesteric structure, a discotic structure, etc. When the lipid is a liquid crystal compound, from the viewpoint of promoting the formation of a transmission channel and excellent uniformity of penetration, it is preferable that the lipid is a compound having a liquid crystal structure at around room temperature (25°C), more preferably a liquid crystal compound that can form a cholesteric structure (cholesteric liquid crystal compound), and even more preferably a compound represented by the following general formula (1).
When the lipid has a liquid crystal structure at around room temperature, its compatibility with keratin, which will be described later, is improved, and the formation of permeation channels for both hydrophilic and lipophilic substances can be further promoted.

In this specification, "around room temperature" refers to a temperature within a range of error (°C) ±30°C, which is an error generally acceptable in the technical field to which the technology of the present disclosure belongs and does not deviate from the spirit of the technology of the present disclosure, and is preferably any temperature within the range of 25 ±25°C.
The cholesteric structure can be confirmed by the same method as the above-mentioned method for confirming the liquid crystal structure.
In this specification, the term "cholesteric structure" refers to a structure (phase state) in which rod-shaped or discotic liquid crystal molecules are arranged in a spiral shape.
The shape of the cholesteric liquid crystal compound is not particularly limited, and examples thereof include rod-shaped cholesteric liquid crystal compounds and disc-shaped cholesteric liquid crystal compounds.

In formula (1), L represents a single bond, -O(C=O)-, or -O(C=O)O-, and ^R1 represents a monovalent substituent or a hydrogen atom.

The monovalent substituent may be a halogen atom, an aliphatic hydrocarbon group, an aromatic hydrocarbon group, or a group represented by a combination thereof. The aliphatic hydrocarbon group, the aromatic hydrocarbon group, or a group represented by a combination thereof may have a substituent. The substituent may be a halogen atom, a carboxy group, or an alkoxy group.
Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Among these, the halogen atom is preferably a chlorine atom or a bromine atom.

The aliphatic hydrocarbon group may be a saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group, and may be a straight-chain or branched-chain aliphatic hydrocarbon group.
The number of unsaturated bonds contained in the unsaturated aliphatic hydrocarbon group may be one or two or more. When the number of unsaturated bonds is two or more, the number of unsaturated bonds may be only double bonds, only triple bonds, or a mixture of these bonds. From the viewpoint of promoting the formation of permeation channels and achieving excellent uniformity of permeation, the unsaturated aliphatic hydrocarbon group is preferably an unsaturated aliphatic hydrocarbon group having an unsaturated double bond. The number of unsaturated double bonds is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1.

Examples of the aromatic hydrocarbon group include a substituted or unsubstituted phenyl group and a substituted or unsubstituted aralkyl group.
Examples of the aralkyl group include a substituted or unsubstituted phenylalkyl group (excluding a benzyl group) and a substituted or unsubstituted benzyl group, with a substituted or unsubstituted benzyl group being preferred.

From the viewpoint of promoting the formation of permeation channels and achieving excellent uniformity of penetration, the aliphatic hydrocarbon group is preferably a substituted or unsubstituted aliphatic hydrocarbon group having 1 to 20 carbon atoms or a substituted or unsubstituted aromatic hydrocarbon group having 6 to 20 carbon atoms, more preferably an unsubstituted saturated aliphatic hydrocarbon group having 1 to 20 carbon atoms or an unsubstituted unsaturated aliphatic hydrocarbon group having 10 to 20 carbon atoms, even more preferably an unsubstituted unsaturated hydrocarbon group having 12 to 18 carbon atoms, and particularly preferably an unsubstituted unsaturated hydrocarbon group having 12 to 18 carbon atoms and having 1 to 3 (preferably 1 or 2, and even more preferably 1) unsaturated double bonds.

From the viewpoint of promoting the formation of permeation channels and achieving excellent uniformity of penetration, it is more preferable that the compound represented by general formula (1) is a compound represented by general formula (1A).

In formula (1A), ^R2 represents an aliphatic hydrocarbon group, an aromatic hydrocarbon group, or a group represented by a combination thereof.
The aliphatic hydrocarbon group and aromatic hydrocarbon group represented by ^R2 have the same meaning as the aliphatic hydrocarbon group and aromatic hydrocarbon group described above, and preferred embodiments are also the same.
From the viewpoint of excellent permeability and uniformity of penetration, ^R2 is preferably an aliphatic hydrocarbon group, more preferably an unsubstituted aliphatic hydrocarbon group having 1 to 20 carbon atoms, even more preferably an unsubstituted saturated aliphatic hydrocarbon group having 1 to 20 carbon atoms or an unsaturated aliphatic hydrocarbon group having 10 to 20 carbon atoms, and particularly preferably an unsubstituted unsaturated hydrocarbon group having 12 to 18 carbon atoms and having 1 to 3 (preferably 1 or 2, more preferably 1) unsaturated double bonds.

Specific examples of lipids contained in the biological model material composition according to the present disclosure are shown below, but the present disclosure is not limited to these specific examples.

From the viewpoint of promoting the formation of permeation channels and achieving excellent uniformity of penetration, the lipid content is preferably 0.5% by mass to 30% by mass, more preferably 1% by mass to 30% by mass, more preferably 1% by mass to 20% by mass, and even more preferably 1% by mass to 10% by mass, relative to the total mass of the keratin described below.
The material composition for biological models according to the present disclosure contains a specific amount of a specific lipid (lipophilic compound) relative to a keratin molecule, thereby enabling the formation of permeation channels for both hydrophilic and lipophilic substances to be further improved.

The material composition for a biological model according to the present disclosure may contain one type of lipid alone, or may contain two or more types of lipids.
When two or more types of lipids are contained, the total content of the lipids is preferably within the above range.

The lipids contained in the biological model material composition according to the present disclosure may contain a liquid crystal compound and a compound that does not have a liquid crystal structure (a non-liquid crystal compound). From the viewpoint of promoting the formation of a transmission channel and achieving excellent uniformity of penetration, the content of the non-liquid crystal compound is preferably 5% by mass or less, more preferably 3% by mass or less, and even more preferably 0% by mass or more and 1% by mass or less, based on the total mass of the composition.

<Keratin>
The material composition for a biological model according to the present disclosure contains keratin.
From the viewpoints of accelerating the formation of permeation channels and achieving excellent uniformity of penetration, the keratin is preferably a water-soluble keratin.
In this specification, water-soluble means that keratin dissolves in water at 25°C at a concentration of 1% by mass or more.
As a method for obtaining water-soluble keratin, any known method for solubilizing keratin can be used. Specifically, the method is not particularly limited as long as it is a method for breaking hydrogen bonds (>NH...0=C<) and S-S bonds present between and within keratin molecules.
The above bond may be cleaved by, for example, an oxidizing agent, a reducing agent, an enzyme, or the like.
From the viewpoint that keratin that retains the structure it has when present in the living body can be obtained more easily, the method for obtaining water-soluble keratin preferably includes an oxidation or reduction treatment, and more preferably includes a reduction treatment.
Furthermore, from the viewpoint of easily obtaining water-soluble keratin by an inexpensive and simple extraction method, oxidation or reduction treatment is preferred, and from the viewpoint of regenerating the S—S bonds that are characteristic of keratin, it is more preferable to include a reduction treatment.

Examples of the oxidizing agent used in the oxidation treatment include peracetic acid and performic acid.
As the reducing agent used in the reduction treatment, a reducing agent for disulfide bonds (S—S bonds) can be suitably used, and specific examples thereof include dithiothreitol, glutathione, thioglycolic acid, phosphine compounds, etc. Among the reducing agents, phosphine compounds are preferred from the viewpoint of ease of handling, and trialkylphosphine is more preferred.

In the method for obtaining water-soluble keratin, it is preferable to use a chaotropic agent (denaturant) in combination with the reducing agent in order to further promote the cleavage of hydrogen bonds.
Examples of chaotropic agents include urea, formamide, guanidine salts, etc. Among these, urea and thiourea are preferred.
In the above oxidation or reduction treatment, by using a chaotropic agent such as urea in combination, hydrogen bonds between keratin molecules are easily broken, and the water solubility of keratin can be promoted.

In the method for obtaining water-soluble keratin, a heat treatment may be further carried out. The temperature of the heat treatment may be appropriately set as long as the keratin is not denatured.

The pH in the method for obtaining water-soluble keratin can be set appropriately, and is preferably, for example, pH 8 to pH 10.

The method for obtaining water-soluble keratin may include pretreating the keratin prior to the oxidation or reduction treatment.
The pretreatment of the keratin preferably involves washing with a neutral detergent, rinsing with water, and air drying. If necessary, the keratin may be cut or crushed.

The raw material of keratin is not particularly limited, and examples thereof include hair, skin, hooves, horns, scales, nails, etc. Among these, hair is preferred as the raw material of keratin.
The hair may be human hair or animal hair. Examples of animal hair include animal hair from sheep, birds, etc., and among these, wool is preferable because it has a composition similar to that of human hair. There is no particular limit to the type of wool, but examples include wool from merino and Lincoln breeds.
From the viewpoints of promoting the formation of permeation channels and achieving excellent uniformity of penetration, the keratin is preferably keratin derived from human hair or keratin derived from wool, and more preferably keratin derived from wool.

From the viewpoint of promoting the formation of permeation channels and achieving excellent uniformity of penetration, the keratin is preferably water-soluble keratin, more preferably water-soluble keratin derived from human hair or wool, and even more preferably water-soluble keratin derived from wool.

The molecular weight of keratin can be appropriately set depending on the conditions for producing the water-soluble keratin. For example, the molecular weight of the water-soluble keratin obtained by reduction treatment is preferably 20 kDa to 100 kDa, and more preferably 40 kDa to 60 kDa.
The molecular weight of keratin is determined by SDS-PAGE (SDS-polyamide gel electrophoresis).

The keratin content is preferably from 85% by mass to 99.5% by mass, and more preferably from 90% by mass to 99% by mass, based on the total mass of the composition.
The material composition for a biological model according to the present disclosure may contain one type of keratin alone, or may contain two or more types of keratin.

<<Other ingredients>>
The biological model material composition according to the present disclosure may further contain components (other components) other than the above lipids and keratin.
Other ingredients include inorganic substances, excipients, stabilizers, etc.
The content of other components is preferably 10 mass% or less, more preferably 5 mass% or less, even more preferably 3 mass% or less, and particularly preferably 0 mass% or more and 1 mass% or less, relative to the total mass of the composition.

The method for preparing the biological model material composition according to the present disclosure includes, for example, a method of mixing lipids and keratin in any ratio. The mixing method is not limited, and any known method can be used.
The lipids may be dissolved in an organic solvent, such as ethanol or acetone.

The material composition for biological models according to the present disclosure can be suitably used for preparing biological models, and from the viewpoint of promoting the formation of permeation channels and excellent uniformity of penetration, it is preferably used for a human nail plate model and a hair model, and more preferably for a human nail plate model.

<Biomodel>
The biological model according to the present disclosure includes the lipid and the keratin. From the viewpoint of promoting the formation of permeation channels and achieving excellent uniformity of penetration, the biological model according to the present disclosure is preferably a biological model formed from the material composition for biological models, more preferably a human nail plate model or a hair model, and even more preferably a human nail plate model.

The shape of the biological model can be appropriately shaped as desired. Methods for shaping the biological model include known shaping methods.

For example, the shape of the human nail plate model may be a plate or film.
The biological model according to the present disclosure may be prepared in the form of a plate or film, for example, by applying the above-mentioned biological model material composition onto a substrate, and then peeling it off from the substrate as necessary.

The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited to these examples as long as they do not deviate from the gist of the disclosure. Unless otherwise specified, "parts" and "%" are by mass.

<<Preparation of water-soluble keratin>>
600 parts by mass of urea, 396 parts by mass of thiourea, 280 parts by mass of a 36.5% to 38.4% by mass aqueous solution of tris(3-hydroxypropyl)phosphine (trade name: Hishicolin P540, Nippon Chemical Industry Co., Ltd.), and 6.16 parts by mass of tris(hydroxymethyl)aminomethane hydrochloride were added to 600 mL of ion-exchanged water and dissolved, and the total volume was then increased to 2 L. Furthermore, the pH was adjusted to 8.5 with a 1 N (mol/L, the same applies hereinafter) aqueous hydrochloric acid solution to obtain an extract solution.
14.4 parts by mass of 100% wool (trade name: Canadian 3S, Hamanaka Co., Ltd.) was added to 315 mL of the extraction solution, and then heated and stirred at 50 ° C. for 4 days to extract reduced keratin. After the extraction was completed, the residue was removed from the obtained crude extract by centrifugation (17,000 rpm (revolutions per minute, the same applies below), 30 minutes, 25 ° C.). Furthermore, using a dialysis tube (product name: Spectra Por7, MWCO 8 kDa, manufactured by REPLIGEN Co., Ltd.), the mixture was dialyzed for 2 to 3 days with 2 L and then 10 L of ion-exchanged water, and the precipitated insoluble matter was removed by centrifugation (17,000 rpm, 30 minutes, 25 ° C.), and 484 mL of reduced keratin extract (water-soluble keratin) with a solution concentration of 21.2 mg / mL was obtained. The molecular weight of the keratin in the extract thus obtained was measured by SDS-PAGE and found to be 40 kDa to 60 kDa.

<Evaluation of the presence or absence of transmission channels>
The keratin films (human nail plate models) obtained in Examples 1 to 6 and Comparative Examples 1 and 2 were subjected to the following permeability test for hydrophilic substances and/or lipophilic substances to evaluate the presence or absence of permeation channels.
When permeability of both hydrophilic substances and lipophilic substances is confirmed, it can be said that the human nail plate model has permeation channels for both hydrophilic substances and lipophilic substances.
In addition, even if a human nail plate model has not been subjected to either one of permeability tests for hydrophilic substances or lipophilic substances, if the permeability of hydrophilic or lipophilic substances has been confirmed in a human nail plate model containing less lipid than the lipid content contained in the human nail plate model, it can be determined that the keratin film has permeation channels for both hydrophilic substances and lipophilic substances.

<<Permeability test of hydrophilic substances>>
The keratin films (approximately 1 cm square) prepared in the following Examples 1 to 3 and Comparative Examples 1 and 2 were immersed for 10 minutes in a 5 mL screw tube into which 2 mL of an aqueous solution of rhodamine B (250 μg/mL) was poured as a hydrophilic substance. After completion of the immersion, the aqueous solution of rhodamine B adhering to the film was wiped off with a Kimwipe, and the film was embedded in a freezing embedding agent, 4% CMC (sodium carboxymethylcellulose), and then a 10 μm section of the keratin film was prepared with a freezing microtome and placed on a slide glass. The cross section of the keratin film was observed with an upright microscope (magnification: ×40), and the penetration depth of rhodamine B from the surface of the keratin film was measured with ImageJ.
It can be said that the greater the penetration depth of Rhodamine B from the surface of the keratin film, the better the permeability of hydrophilic substances and the more permeation channels there are for hydrophilic substances.

<<Lipophilic substance permeability test>>
The keratin films (approximately 1 cm square) prepared in the following Examples 1, 4 to 6 and Comparative Examples 1 and 2 were immersed for 2 hours in a 5 mL screw tube containing 1.5 mL to 2.0 mL of an oil red/isopropanol aqueous solution (oil red isopropanol solution with a concentration of 30 mg/10 mL: ion-exchanged water = volume ratio 3:2) as a lipophilic substance. After completion of the immersion, the oil red/isopropanol aqueous solution attached to the keratin film was wiped off with a Kimwipe, and the keratin film was embedded in a freezing embedding agent 4% CMC (sodium carboxymethylcellulose), and then a 10 μm keratin film slice was prepared using a freezing microtome, and the cross section of the film was observed on a slide glass using an upright microscope (magnification: ×60 times), and the penetration depth of the oil red from the keratin film surface was measured using ImageJ.
It can be said that the greater the penetration depth of Oil Red from the surface of the keratin film, the better the permeability of lipophilic substances and the more permeation channels there are for lipophilic substances.

<Evaluation of uniformity of penetration>
The permeability test of the above hydrophilic and/or lipophilic substances was carried out, the depth to which Rhodamine B and/or Oil Red had penetrated was measured at several points, and the average value and standard deviation of the values were calculated.
The smaller the standard deviation, the more uniform the penetration.

[Infrared Spectroscopic Analysis]
The keratin film containing cholesterol oleate prepared in the examples described below and Comparative Example 1 (additive-free keratin film) were subjected to infrared spectroscopy (ATR-IR, model number: FT/IR-6100, manufactured by JASCO Corporation). Comparative Example 2 (nail plate free edge) was used as a control sample.

(Example 1) Preparation of keratin film containing 1% by mass of lipid and permeability test - Preparation of material composition for biological model -
To 5 mL of the reduced keratin extract, 0.21 mL of a cholesterol oleate (a liquid crystal compound in which L in the general formula (1) is -O(C=O)- and ^R1 is an unsubstituted unsaturated hydrocarbon group having 17 carbon atoms and having one saturated double bond)/ethanol solution with a concentration of 5 mg/mL was added as a lipid component, and the mixture was then shaken in a water bath at 60°C for 10 to 15 minutes to obtain a film preparation solution (material composition for a biological model). The content of cholesterol oleate (lipid) was 1% by mass relative to the total mass of the keratin.
- Creation of a human nail plate model -
The obtained film preparation solution was poured into a 3.5 cmφ polystyrene petri dish, dried to dryness at 25° C. and 50% rh, and then allowed to stand for 2 days at 60° C. Then, a heat treatment was performed at 110° C. for 3 hours to obtain a keratin film (human nail plate model) containing 1% by mass of cholesterol oleate.
The obtained keratin film was subjected to the above-mentioned permeability test for hydrophilic substances and lipophilic substances. The results are shown in Figure 1. Figure 1(a) shows the results of the permeability test for hydrophilic substances, and Figure 1(b) shows the results of the permeability test for lipophilic substances.
The penetration depths of Rhodamine B and Oil Red were 19.6±1.9 μm and 10.6±0.7 μm, respectively, indicating that both hydrophilic and lipophilic substances penetrated.
It was confirmed that the human nail plate model prepared from the material composition for biological models of Example 1 had permeation channels for both hydrophilic substances and lipophilic substances.

(Example 2) Preparation of keratin film containing 2% by mass of lipid and permeability test A film preparation solution (material composition for biological models) was prepared in the same manner as in Example 1, except that the amount of cholesterol oleate/ethanol solution with a concentration of 5 mg/mL was changed to 0.41 mL. The content of cholesterol oleate (lipid) was 2% by mass relative to the total mass of keratin.
Using this film preparation solution, a keratin film (human nail plate model) containing 2% by mass of cholesterol oleate was obtained. The obtained keratin film was subjected to a permeability test of a hydrophilic substance. The results are shown in FIG. 2.
It can be seen that the penetration depth of Rhodamine B was 22.4±0.9 μm, which is approximately the same as that of Example 1.

(Example 3) Preparation of keratin film containing 4% by mass of lipid and permeability test A film preparation solution (material composition for biological models) was prepared in the same manner as in Example 1, except that the amount of cholesterol oleate/ethanol solution with a concentration of 5 mg/mL was changed to 0.84 mL. The content of cholesterol oleate (lipid) was 4% by mass relative to the total mass of keratin. This film preparation solution was used to obtain a keratin film (human nail plate model) containing 4% by mass of cholesterol oleate. The obtained keratin film was subjected to a permeability test for hydrophilic substances. The results are shown in FIG. 3.
It can be seen that the penetration depth of Rhodamine B was 20.8±1.1 μm, which is similar to that of Examples 1 and 2. Furthermore, from the results of Examples 1 to 3, it can be seen that the amount of cholesterol oleate added does not affect the penetration of Rhodamine B.

(Example 4) Preparation of keratin film containing 3% by mass of lipid and permeability test A film preparation solution (material composition for biological models) was prepared in the same manner as in Example 1, except that the amount of cholesterol oleate/ethanol solution with a concentration of 5 mg/mL was changed to 0.63 mL. The content of cholesterol oleate (lipid) was 3% by mass relative to the total mass of keratin. This film preparation solution was used to obtain a keratin film (human nail plate model) containing 3% by mass of cholesterol oleate. The obtained keratin film was subjected to a permeability test for lipophilic substances. The results are shown in FIG. 4.
The penetration depth of Oil Red was 17.5±2.7 μm, and it was found that the penetration of Oil Red was improved compared to Example 1, promoting the formation of permeation channels for lipophilic substances.

(Example 5) Preparation of keratin film containing 5% by mass of lipid and permeability test A film preparation solution (material composition for biological models) was prepared in the same manner as in Example 1, except that the amount of 5 mg cholesterol oleate/ethanol solution added was changed to 1.32 mL. The content of cholesterol oleate (lipid) was 5% by mass relative to the total mass of keratin. This film preparation solution was used to obtain a keratin film (human nail plate model) containing 5% by mass of cholesterol oleate. The obtained keratin film was subjected to a permeability test of lipophilic substances. The results are shown in FIG. 5. It can be seen that the penetration depth of oil red was deeper than 110 μm, and the permeability of oil red was improved compared to Examples 1 and 4, promoting the formation of a permeation channel for lipophilic substances.

(Example 6) Preparation of keratin film containing 10% by mass of lipid and permeability test A film preparation solution (material composition for biological models) was prepared in the same manner as in Example 1, except that the amount of cholesterol oleate/ethanol solution with a concentration of 5 mg/mL was changed to 2.63 mL. The content of cholesterol oleate (lipid) was 10% by mass relative to the total mass of keratin. This film preparation solution was used to obtain a keratin film containing 10% by mass of cholesterol oleate (human nail plate model). The obtained keratin film was subjected to a permeability test for lipophilic substances. The results are shown in FIG. 6.
The penetration depth of Oil Red was greater than 110 μm, similar to the keratin film containing 5% by mass cholesterol oleate, and it is clear that the permeability of Oil Red is improved compared to Examples 1 and 4, promoting the formation of permeation channels for lipophilic substances.

The results of Examples 1, 4, 5, and 6 show that it is possible to adjust the permeability of lipophilic substances, i.e., the formation of permeation channels for lipophilic substances, by adjusting the amount of cholesterol oleate added.

The above infrared spectroscopic analysis was also carried out using the keratin film obtained in Example 6. Comparison of the characteristics of these spectra revealed that the keratin film containing lipid (cholesterol oleate) showed an absorption spectrum similar to that of the free edge of the nail plate in the lipid-derived absorption around 3,000 cm ⁻¹ (C—H stretching).

Comparative Example 1: Preparation of additive-free keratin film and permeability test A film preparation solution (material composition for biological models) was prepared in the same manner as in Example 1, except that no cholesterol oleate/ethanol solution was added to the reduced keratin extract, and an additive-free keratin film (human nail plate model) was obtained using this film preparation solution. The obtained keratin film was subjected to a permeability test for hydrophilic and lipophilic substances. The results are shown in FIG. 7.
The penetration depth of Rhodamine B was 19.4±1.8 μm, which was similar to that of Examples 1 to 3, whereas no penetration of Oil Red was observed. This result indicates that no lipophilic substance (lipid) permeation channel was formed in the additive-free keratin film to which no cholesterol oleate/ethanol solution was added.

The above-mentioned infrared spectroscopic analysis was carried out using the keratin film obtained in the above-mentioned Comparative Example 1. The results are shown in Figure 9. It can be seen that the additive-free keratin film does not show any absorption at around 3,000 cm ^-1 (C-H stretching) derived from lipids.

Comparative Example 2: Permeability test of free nail edge (human nail) A free nail edge with a width of about 3 mm was subjected to a permeability test of hydrophilic and lipophilic substances in the same manner as in Examples 1 to 5, except that the immersion time for the free nail edge was set to one week. The results are shown in FIG.
Since the permeability of Rhodamine B was significantly different between the upper and lower sides of the free nail edge, the penetration depth was measured for the upper and lower sides. As a result, the penetration depth of the free nail edge after immersion for one week was 42.5±7.8 μm on the upper side and 23.8±2.3 μm on the lower side. Not only was the depth significantly different between the upper and lower sides, but the error (standard deviation) was also larger than in Examples 1 to 3.
The penetration depth of Oil Red was 24.9±7.8 μm on the upper side and 17.6±3.0 μm on the lower side, with the error (standard deviation) on the upper side being larger than in Examples 4 to 6. From these results, it can be seen that the data for human nails has a large variance (standard deviation value) and poor penetration uniformity.

The above results show that the biological models prepared using the biological model material compositions of Examples 1 to 6 according to the present disclosure have permeation channels for both hydrophilic and lipophilic substances and have superior permeation uniformity compared to the biological models of Comparative Examples 1 and 2.

Claims (7)

A material composition for a biological model comprising water-soluble keratin and a lipid , the lipid comprising a liquid crystal compound represented by the following general formula (1) :

In formula (1), L represents a single bond, -O(C=O)-, or -O(C=O)O-, and R1 ^represents a monovalent substituent or a hydrogen atom. The material composition for a biological model according to claim 1, wherein in general formula (1), the monovalent substituent is a substituted or unsubstituted aliphatic hydrocarbon group having 1 to 20 carbon atoms, or an unsubstituted unsaturated aliphatic hydrocarbon group having 10 to 20 carbon atoms and having 1 to 3 unsaturated double bonds. 2. The material composition for a biological model according to claim 1, wherein the content of said lipid is 1% by mass to 30% by mass based on the total mass of said water-soluble keratin. 2. The material composition for a biological model according to claim 1 , wherein the water-soluble keratin is keratin derived from human hair or keratin derived from wool. The material composition for a biological model according to any one of claims 1 to 4 , which is used for a human nail plate model. A biological model formed from the material composition for a biological model according to any one of claims 1 to 5 . The biological model according to claim 6 , which is a human nail plate model or a hair model.