JP7597315B2 - Three-dimensional structures and their uses - Google Patents

Description

The present invention relates to a three-dimensional structure and its use. More specifically, the present invention relates to a three-dimensional structure for evaluating the skin permeability of a test substance, a method for evaluating the skin permeability of a test substance, and a kit for evaluating the skin permeability of a test substance.

In recent years, the use of nanoparticles has expanded in various fields, including cosmetics, paints, electronic devices, and pharmaceuticals. However, many aspects of the biological response mechanisms of nanoparticles remain unclear, raising safety concerns.

According to the International Organization for Standardization (ISO), nanoparticles are defined as substances with external dimensions in the range of 1 to 100 nm (ISO/TS 27687:2008 Nanotechnologies - Terminology and definitions for nanoobjects-Nanoparticle, nanofibre and nanoplate). Nanoparticles are defined only by the physical quantity of size, so there are many chemical types, with over 100 types in circulation on the market alone. If surface modifications are included, the number is even greater. However, only a small number of nanoparticles have been shown to actually have an effect on living organisms, and the effects of many nanoparticles on living organisms have not been clarified.

In assessing the transdermal toxicity of nanoparticles, drugs, etc. that come into contact with the skin, it is important to evaluate their skin permeability. Human skin, particularly the epithelial layer, has a barrier function that protects the body from the outside world, so the skin permeability of nanoparticles, drugs, etc. may significantly affect the cellular response in the body. As described in Non-Patent Document 1, human skin or the skin of non-human animals such as pigs and mice has traditionally been used for skin permeability tests.

However, particularly in the EU cosmetics industry, the use of new materials whose safety cannot be guaranteed is restricted, while the sale of products containing materials that have been tested on animals is completely prohibited. For this reason, there is an international demand for in vitro methods that can appropriately evaluate the dermal toxicity of nanoparticles outside of living organisms.

Human skin is difficult to obtain, and alternative methods are required for animal testing. In recent years, therefore, the use of artificially created three-dimensional cultured skin using cultured cells has been considered for evaluating the skin permeability of test substances. However, the creation of conventional three-dimensional cultured skin requires a lengthy culture process spanning several weeks, which poses productivity issues. Furthermore, conventional three-dimensional cultured skin can vary in the permeability of test substances, and the quality stability can be low.

In addition, skin tissue detached from humans and animals cannot remain viable for a long period of time. Therefore, even if it is possible to measure the amount of a substance that has temporarily permeated the skin under certain conditions, it is not possible to evaluate the effect of long-term permeation of the substance or release of decomposition products on the physiological activity of cells in the lower layers of the skin.

Against this background, the present invention aims to provide a new technology for evaluating the skin permeability of test substances.

The three-dimensional structure of the present invention is for evaluating the skin permeability of a test substance, and includes a cell layer and a permeation layer containing a hydrogel laminated to the cell layer, the permeation layer having substance permeability equivalent to that of the skin of a living body, and the cell layer containing indicator cells for the test substance.

The present invention provides a new technique for evaluating the skin permeability of a test substance.

FIG. 2 is a schematic cross-sectional view showing an example of a three-dimensional structure. Graphs (a) and (b) show the results of measuring the permeability of Blue Dextran through a hydrogel layer in Experimental Example 1. (c) is an electron microscope photograph showing the results of observing an alginate gel with a scanning electron microscope (SEM). (d) is an electron microscope photograph showing the results of observing a fibrin gel with a SEM. 1 is a graph showing the quantitative results of zinc ions in Experimental Example 2. 1A is a graph showing the quantitative results of zinc ions permeated into wells in Experimental Example 3. FIG. 1B is a fluorescent microscope photograph of GFP fluorescence emitted by IL-8-HaCaT cells in Experimental Example 3. 13( a ) and 13 ( b ) are graphs showing the results of quantitative real-time PCR in Experimental Example 4. 1A is a graph showing the quantitative results of zinc ions in Experimental Example 5. FIG. 1B is a graph showing the results of quantitative real-time PCR in Experimental Example 5.

Below, an embodiment of the present invention will be described in detail, with reference to the drawings where necessary. Note that in the drawings, identical or equivalent parts are given the same or corresponding reference numerals, and duplicate explanations will be omitted. Note that the dimensional ratios in each drawing are exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[Three-dimensional structure]
In one aspect of the present invention, a three-dimensional structure for evaluating the skin permeability of a test substance is provided, the structure including a cell layer and a permeable layer containing a hydrogel, the cell layer and the permeable layer being laminated, the permeable layer having the same substance permeability as the skin of a living body, and the cell layer containing an indicator cell for the test substance. The permeable layer containing a hydrogel may be laminated with the cell layer, and a layer other than the cell layer and the permeable layer (e.g., another permeable layer or another cell layer, etc.) may be disposed between the permeable layer and the cell layer, but preferably the permeable layer is laminated in a manner that contacts the cell layer. The test substance applied from the opposite side (hereinafter referred to as the test substance application side) to the side where the cell layer is present (hereinafter referred to as the cell layer side) of the permeable layer permeates the permeable layer and reaches the cell layer, and the physiological properties of the indicator cells change when the test substance comes into contact with the indicator cells contained in the cell layer. By observing and evaluating such changes, the skin permeability of the test substance can be evaluated.

As described later in the Examples, the three-dimensional structure of the present invention allows the skin permeability of a test substance to be evaluated more easily than three-dimensional cultured skin. In addition, the evaluation results of the skin permeability of the test substance are highly reproducible.

Figure 1 is a schematic cross-sectional view showing an example of a three-dimensional structure of the present invention. In this example, the three-dimensional structure 100 includes a cell layer 120 and a permeable layer 110 disposed on the cell layer 120. The permeable layer 110 includes a hydrogel. The permeable layer 110 has substance permeability equivalent to that of living body skin. The cell layer 120 includes indicator cells for a test substance.

As shown in FIG. 1, the three-dimensional structure 100 may be housed in a container or may be immersed in a liquid such as a culture medium. In the example of FIG. 1, the three-dimensional structure 100 is housed in a Transwell insert 130 and immersed in a culture medium 141, but it may also be directly immersed in the culture medium. At least a part of the area of the Transwell insert 130 that contacts the cell layer 120 is made of a highly permeable membrane 131, and as in the example of FIG. 1, the entire area of the area that contacts the cell layer 120 may be made of the membrane 131. Any membrane 131 may be used as long as it is impermeable to cells but permeable to components in the culture medium and secretions from the cells.

A test substance is applied to the surface 112 of the permeable layer 110 opposite the surface 111 facing the cell layer 120. The test substance may be applied in any form that contacts the surface 112, for example, by adding a liquid in which the test substance is dissolved or dispersed, or by applying a gel or cream containing the test substance. In the example of FIG. 1, a culture medium 140 is added on the permeable layer 110, and the test substance is applied in the form of being added to the culture medium 140, as described below. In the example of FIG. 1, the transwell insert 130 is contained in the well 150. The space between the well 150 and the transwell insert 130 contains a culture medium 141, and the test substance that has permeated the permeable layer 110 contacts the cell layer 120, and then permeates the membrane 131 and is released into the culture medium 141. Furthermore, if the test substance secretes any substance upon contact with the indicator cells contained in the cell layer 120, such secretions also permeate the membrane 131 and are released into the medium 141. Skin permeability can also be evaluated by measuring the amount of the test substance or secretions released into the medium 141.

In the present invention, "the permeability layer has substance permeability equivalent to that of the skin of a living body" means that, in the case of a specific test substance, the permeability of the test substance through the skin of a living body is equivalent to the permeability of the test substance through the permeability layer of the three-dimensional structure of the present invention.

In this specification, "the test substance permeates the permeable layer" includes the test substance permeating the permeable layer without being changed, and the test substance changing to produce a substance that permeates the permeable layer. Examples of substances that change to produce a substance include ions that are produced when the test substance dissolves and clusters that are produced when the test substance binds to each other.

In the present invention, the permeable layer contains a hydrogel. Other components may be included as long as they do not affect the cytotoxicity or reactivity with the test substance, but preferably the permeable layer is made of only a hydrogel. The permeable layer preferably does not allow particles such as nanoparticles to pass through, but allows dissolved substances of nanoparticles (low molecular weight substances such as ions) to pass through. In other words, the permeable layer is preferably capable of separating particles from soluble substances (metal ions, etc.). In addition, it is preferable that the permeability of the permeable layer can be adjusted by changing the composition, thickness, etc. of the permeable layer.

The hydrogel contained in the permeable layer is preferably one that is not cytotoxic in itself, at least within the range of concentrations used. In addition, the hydrogel contained in the permeable layer is preferably one that has a structure and density that is similar to that of living skin, with a difference in permeability to particles and polymeric substances and permeability to low molecular weight substances such as ions. Hydrogels are polymeric materials that contain water inside, and examples of such materials include bio-derived polymers (collagen, elastin, gelatin, fibrin, hyaluronic acid, polypeptides, etc.), synthetic polymers such as polylactic acid, polyethylene glycol, and polyacrylamide, polysaccharides such as alginic acid and gellan gum, and polyvalent metal salts.

Hydrogels may be used alone or in combination of two or more types.

As described later in the Examples, the inventors have clarified that, for example, an alginate gel with low porosity has too low permeability to the test substance to allow any of it to pass through, whereas a fibrin gel with higher porosity allows the permeability of the test substance to be adjusted by adjusting the thickness of the permeable layer.

For this reason, when a fibrin gel or the like is used as the hydrogel contained in the permeable layer, the permeability of the test substance can be set to the same level as that of living skin by adjusting the thickness of the permeable layer.

The three-dimensional structure of the present invention makes it possible to construct a system that can more accurately screen the dermal toxicity of nanoparticle materials, other chemical substances, and biological substances, as well as the skin permeability and efficacy of pharmaceuticals in drug delivery systems.

The three-dimensional structure of the present invention has a cell layer containing indicator cells for a test substance under a permeable layer. In this specification, indicator cells refer to cells whose physiological properties change in response to contact with a certain substance. Examples of physiological properties include gene expression profiles, expression levels of proteins including reporter proteins such as fluorescent proteins, cell phenotypes, survival rates, vitality, etc. The indicator cells used in the three-dimensional structure of the present invention are cells whose physiological properties change in response to contact with a test substance.

In the three-dimensional structure of this embodiment, the indicator cells contained in the cell layer can be primary cultured cells derived from mammalian living organisms such as humans, subcultured cells, stem cells, cells induced to differentiate from stem cells, established cell lines, cells that have been subjected to various gene editing processes, etc.

In terms of evaluating skin permeability, epidermal keratinocytes that constitute the skin and are involved in inflammatory reactions or epidermal keratinocyte cell lines such as HaCaT cells are preferably used as indicator cells. In addition to these, fibroblasts, vascular endothelial cells, Langerhans cells, peripheral nerve cells, etc. can also be used as appropriate depending on the purpose.

As a means for non-invasively evaluating the responsiveness of indicator cells in real time, for example, cells into which a construct in which a specific foreign gene (hereinafter referred to as a reporter gene) is linked downstream of the response sequence of a target gene has been introduced can be suitably used. There are no particular limitations on the reporter gene, and it can be selected appropriately depending on the purpose. Examples of the reporter gene include the luciferase gene, the green fluorescent protein (GFP) gene, and derivatives thereof.

This allows the transcription activity of the target gene to be detected by fluorescence or the like. In other words, the indicator cells may be cells that express a fluorescent protein, or cells that express an enzyme that reacts with a substrate to produce fluorescence. Furthermore, the cells that express a fluorescent protein may be cells into which a reporter gene has been introduced.

The reporter gene is linked downstream of the response element of the target gene whose expression level is to be measured. There are no particular limitations on the response element of the target gene, but the NF-kB response element of the IL-8 gene promoter can be preferably used to evaluate the induction of inflammation in the skin.

Other examples of response elements include the ARE sequence of the HO-1 gene that responds to oxidative stress, the p53 response element that responds to DNA damage, the ATF6 response element that responds to endoplasmic reticulum stress, and the MRE element of MT2A that responds to metal stress.

The NCBI accession numbers for the human IL-8 gene mRNA are NM_000584.4, NM_001354840.2, etc. The NCBI accession numbers for the human HO-1 gene mRNA are NM_002133.3, etc. The NCBI accession numbers for the human p53 gene mRNA are NM_000546.5, NM_001126112.2, etc. The NCBI accession numbers for the human ATF6 gene mRNA are NM_007348.4, etc. The NCBI accession numbers for the human MT2A gene mRNA are NM_005953.5, etc.

As shown in FIG. 1, the three-dimensional structure may be housed in a container. The container is preferably a culture container made of a substrate suitable for cell adhesion and proliferation. In addition, from the viewpoint of measuring the response of the indicator cells by fluorescence or the like, it is preferable to use a multi-well type culture plate or the like that has high light transmittance and does not self-illuminate as the container.

In particular, it is preferable to use a transwell insert such as Transwell (Corning) that includes a membrane, in order to allow recovery and evaluation of the test substance after it has passed through the three-dimensional tissue structure without interfering with the supply of nutrients, oxygen, etc. to the cells from below.

The test substance may be any substance for which skin permeability is to be evaluated. More specifically, examples include nanoparticles, synthetic compounds, natural compounds, biological substances, medicines, etc.

Nanoparticles are materials with external dimensions in the range of 1-100 nm. Specific examples of nanoparticles include zinc oxide (ZnO), SiO2 _- coated ZnO (ZnO-Si), copper oxide (CuO), nickel oxide (NiO), antimony trioxide (Sb2O3), cobalt monoxide (CoO), _molybdenum trioxide ( _MoO3 ), yttrium oxide ( _Y2O3 ), magnesium oxide (MgO), _gadolinium oxide ( _Gd2O3 ), tin oxide ( _SnO2 ), tungsten trioxide _{( WO3} ), _zirconium oxide ( _ZrO2 ), ferric oxide ( _Fe2O3 ), titanium dioxide ( _TiO2 ), cerium oxide ( _CeO2 ), aluminum oxide ( _Al2O3 ), bismuth trioxide ( _Bi2O3 ), lanthanum oxide ( _La2O3 ), indium tin oxide (ITO, _In2O3 ), _{and the like . 3} /SnO ₂ ), cobalt blue pigment (Al ₂ O ₃ /CoO), gold nanoparticles, and the like, but are not limited to these.

When these test substances are applied to the skin of a living body or to the three-dimensional structure of this embodiment, soluble substances may be released due to decomposition, etc. For example, as described later in the Examples, metal nanoparticles such as zinc oxide dissolve and produce metal ions.

As a result, certain areas are exposed to high concentrations of metal ions. Previously, in order to evaluate biological responses under such conditions, it was necessary to use an in vivo analysis system, and it was difficult to analyze using an in vitro model.

In contrast, as described later in the Examples, the three-dimensional structure of the present invention makes it possible to analyze the biological response under conditions in which a certain location is exposed to a high concentration of metal ions. In addition, as described later in the Examples, for example, it is possible to simultaneously evaluate the permeability of dissolved zinc and the inflammatory properties of the cell layer, making it possible to perform an evaluation close to that in vivo.

[Method for evaluating skin permeability of test substance]
In one aspect, the present invention provides a method for evaluating the skin permeability of a test substance, the method comprising: a step of contacting a test substance with a surface (test substance application side) of the permeation layer opposite a surface (cell layer side) of the permeation layer facing the cell layer of a three-dimensional structure comprising a cell layer and a permeation layer containing a hydrogel, the cell layer and the permeation layer being laminated, the permeation layer having substance permeability equivalent to that of living body skin, and the cell layer containing indicator cells for the test substance; and a step of measuring physiological characteristics of the indicator cells.

In the evaluation method of the present invention, the three-dimensional structure described above can be used as the three-dimensional structure. As an example, a method for evaluating the skin permeability of a test substance using the three-dimensional structure shown in Figure 1 will be described.

In the evaluation method of the present invention, first, a test substance is brought into contact with the surface 112 (test substance application side) of the permeable layer 110 of the three-dimensional structure 100, which is opposite to the surface 111 (cell layer side) facing the cell layer 120. This can be carried out by adding the test substance to the culture medium 140 in the Transwell insert 130, for example. The test substance can be the same as that described above.

The test substance added to the culture medium 140 may dissolve and produce low molecular weight substances such as ions. The test substance or low molecular weight substances produced from the test substance permeate the permeable layer 110, come into contact with the cell layer 120, and further permeate the membrane 131 to be released into the culture medium 141.

The test substance or a low molecular weight substance generated from the test substance permeates the permeation layer 110 and contacts the indicator cells contained in the cell layer 120, affecting the physiological properties of the indicator cells. The physiological properties of the indicator cells contained in the cell layer 120 are then measured.

The indicator cells are cells into which a reporter gene has been introduced, and the physiological characteristics may be measured based on the fluorescence intensity corresponding to the expression level of the reporter gene. Alternatively, the physiological characteristics of the indicator cells may be measured by measuring the expression level of the target gene by quantitative real-time PCR, comprehensively measuring the expression level of genes using a DNA microarray, measuring the expression level of a target protein by ELISA or Western blotting, or analyzing the metabolites of the indicator cells by liquid chromatography (LC), LC-mass spectrometry (MS), or the like. The skin permeability of the test substance can be evaluated in correspondence with the physiological characteristics of the indicator cells.

The evaluation method of the present invention may further include a step of quantifying the test substance released into the medium 141 contained in the space between the well 150 and the Transwell insert 130, or the low molecular weight substance generated from the test substance. The test substance or the low molecular weight substance generated from the test substance can be quantified using LC-MS, ICP optical emission spectrometry (ICP-OES), or the like. By quantifying the test substance released into the medium 141, or the low molecular weight substance generated from the test substance, the skin permeability of the test substance can be evaluated.

[Kit for evaluating skin permeability of test substance]
In one aspect, the present invention provides a kit for evaluating the skin permeability of a test substance, comprising a hydrogel. The kit of this embodiment can produce the above-mentioned three-dimensional structure. Furthermore, the three-dimensional structure can be used to evaluate the skin permeability of the test substance.

In the kit of the present invention, the hydrogel may be the same as that described above. The hydrogel may be included in the kit in the form of a hydrogel, or may be included in the kit in the form of a material that forms a hydrogel.

The kit of the present invention may further include a container. As the container, a Transwell insert, or a combination of a Transwell insert and a well plate, etc., can be preferably used.

The kit of the present invention may further include indicator cells. As the indicator cells, the same ones as those described above can be used. In addition, the kit may further include a medium, a buffer solution, etc. for culturing the indicator cells and preparing a cell layer.

In the preparation of the three-dimensional structure, first, indicator cells are seeded in the Transwell insert. The indicator cells may be cells prepared by the user according to the purpose. Alternatively, the kit of the present invention may further contain indicator cells, and the indicator cells may be seeded. As a result, a cell layer is formed. Next, a hydrogel is layered on the cell layer. This forms a three-dimensional structure. The formed three-dimensional structure can be suitably used in the above-mentioned method for evaluating the skin permeability of a test substance.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Study on hydrogel permeability 1)
The permeability of the test substance was examined by contacting the test substance with the permeable layer formed of hydrogel. Alginate gel and fibrin gel were used as the hydrogel. A 1% by mass aqueous solution of blue dextran (molecular weight approximately 2,000 kDa) was used as the test substance.

The alginate gel permeable layer was formed by placing 50 μL, 100 μL, or 150 μL of 10 mg/mL alginate aqueous solution in the Transwell insert of a 24-well plate, and then adding 100 mM calcium chloride as a crosslinking agent to each solution to form a gel.

The fibrin gel permeation layer was prepared as follows: First, a thrombin solution was prepared by dissolving 50 units/mL thrombin (Sigma-Aldrich) and 80 mM calcium chloride in phosphate buffered saline (PBS), and a fibrinogen solution was prepared by dissolving 60 mg/mL fibrinogen (Sigma-Aldrich) in PBS. Next, 50 μL, 100 μL, or 150 μL of the solution, which was mixed so that the thrombin solution:fibrinogen solution was 1:5 (volume ratio), was placed in a Transwell insert of a 24-well plate, and gelled for 30 minutes at 37°C in a 5% _CO2 environment.

Each of the above-mentioned Transwell inserts was placed in a 24-well plate with 300 μL of water added to each well. Next, 100 μL of a 1% by mass aqueous solution of Blue Dextran (molecular weight approximately 2,000 kDa) was added to each Transwell insert, and the inserts were incubated for one week.

The amount of Blue Dextran that penetrated into the 24 wells was then evaluated by measuring the absorbance at a wavelength of 620 nm. Figures 2(a) and (b) are graphs showing the results of measuring the transmittance of Blue Dextran. Figure 2(a) shows the results when alginate gel was used, and Figure 2(b) shows the results when fibrin gel was used. As a control, a Transwell insert in which 100 μL of a 1% by mass aqueous solution of Blue Dextran was added to a non-gel-formed insert was used. In Figures 2(a) and (b), the vertical axis shows the absorbance (%) with the absorbance at a wavelength of 620 nm in the control taken as 100%, and the horizontal axis shows the volume (μL) of the hydrogel.

As a result, it was found that fibrin gel exhibited permeability to the test substance depending on the thickness of the permeable layer. On the other hand, alginate gel showed low permeability to the test substance regardless of the thickness of the permeable layer.

Figure 2(c) is an electron microscope photograph showing the results of observing an alginate gel with a scanning electron microscope (SEM). Figure 2(d) is an electron microscope photograph showing the results of observing a fibrin gel with a SEM.

[Experimental Example 2]
(Study of hydrogel permeability 2)
The permeability of a test substance was examined by contacting the test substance with the permeable layer formed of a hydrogel. Fibrin gel was used as the hydrogel. 1 mM zinc chloride aqueous solution was used as the test substance.

The fibrin gel was prepared by mixing the thrombin solution and the fibrinogen solution prepared in the same manner as in Experimental Example 1 at a volume ratio of thrombin solution:fibrinogen solution = 1:5, placing 200 µL of the solution in a Transwell insert of a 24-well plate, and gelling for 30 minutes at 37°C in a 5% _CO2 environment.

The above-mentioned Transwell inserts were then placed in a 24-well plate with 1,000 μL of water added to each well. Then, 100 μL of 1 mM zinc chloride aqueous solution was added to each Transwell insert and incubated.

After 24 and 48 hours, the amount of zinc ions that had permeated into each of the 24 wells was quantified using an ICP optical emission spectrometer (Inductivity Coupled Plasma Optical Emission Spectrometer: ICP-OES, model number "5110VDV", Agilent Technologies).

Figure 3 is a graph showing the quantitative results of zinc ions. The results revealed that the concentration of zinc ions released into the lower layer of the Transwell insert increased over time.

[Experimental Example 3]
(Assessment of cell layer response to zinc chloride)
The thickness of the permeable layer was varied to evaluate the effect of the test substance on the indicator cells. Fibrin gels of different thicknesses were used as the permeable layer. A zinc chloride solution was used as the test substance. IL-8-HaCaT cells, which will be described later, were used as the indicator cells.

<Preparation of reporter genes>
A reporter gene construct was prepared in which a gene encoding green fluorescent protein (GFP) was linked downstream of the promoter of the IL-8 gene, an inflammation-inducing marker. Specifically, a DNA fragment of the promoter region of the IL-8 gene was first amplified by PCR using human genomic DNA as a template and a sense primer (SEQ ID NO: 1) and an antisense primer (SEQ ID NO: 2).

The amplified DNA fragment was then digested with the restriction enzymes XhoI and BamHI, and ligated with pAcGFP1-1 vector (Clontech) similarly digested with the restriction enzymes XhoI and BamHI using T4 DNA ligase (Takara Bio). The resulting vector was then introduced into competent cells (DH5 alpha, Nippon Gene) for cloning, yielding a reporter gene construct.

Preparation of indicator cells
The above-mentioned reporter gene was introduced into HaCaT cells, a human epidermal keratinocyte cell line, using a gene introduction reagent (product name: Lipofectamine 3000, Thermo Fisher Scientific). The cells were then subcultured for 4 weeks in a medium containing 800 μg/mL G418 (Nacalai Tesque), to obtain a stable reporter gene-introduced line. Hereinafter, these cells may be referred to as IL-8-HaCaT cells. IL-8-HaCaT cells emit GFP fluorescence in accordance with the expression level of the IL-8 gene.

Evaluation of cell layer response to zinc chloride
IL-8-HaCaT cells were seeded into a Transwell insert of a 24-well plate at a cell density of 5× ¹⁰⁴ cells/well, and then incubated for 24 hours before overlaying fibrin gel on top of the IL-8-HaCaT cells.

The fibrin gels were prepared by adding 0, 50, 75, 100, 125, 150, 175, 200, and 225 μL each of a solution prepared by mixing thrombin solution and fibrinogen solution in the same manner as in Experimental Example 1 at a volume ratio of thrombin solution:fibrinogen solution = 1:5, and gelling the solution at 37°C and in a 5% _CO2 environment for 30 minutes.

Next, 100 μL of 5 mM zinc chloride aqueous solution was added to each Transwell insert and incubated for 48 hours. Note that no zinc chloride aqueous solution was added to the Transwell inserts to which no fibrin gel had been added.

The amount of zinc ions that had permeated into each of the 24 wells was then quantified using an ICP optical emission spectrometer (ICP-OES, model number "5110VDV", Agilent Technologies). In addition, the GFP fluorescence emitted by the IL-8-HaCaT cells on each Transwell insert was photographed using a fluorescence microscope.

Figure 4(a) is a graph showing the quantitative results of zinc ions that permeated into each of the 24 wells. Figure 4(b) is a fluorescence microscope photograph of the GFP fluorescence emitted by IL-8-HaCaT cells. In Figure 4(b), "w/o fibrin" indicates the results for IL-8-HaCaT cells to which no fibrin gel was added, "175 μL" indicates the results when 175 μL of fibrin gel was layered, "200 μL" indicates the results when 200 μL of fibrin gel was layered, and "225 μL" indicates the results when 225 μL of fibrin gel was layered.

As a result, it was found that the amount of zinc chloride permeated decreased as the thickness of the permeable layer increased. It was also found that the expression level of the IL-8 gene decreased in proportion to the amount of zinc chloride permeated.

[Experimental Example 4]
(Comparison between hydrogel and 3D cultured skin)
The hydrogel was compared with a commercially available three-dimensional cultured skin (catalog number "EPI-200", MatTek). Fibrin gel was used as the hydrogel.

First, IL-8-HaCaT cells were seeded into the Transwell insert of a 24-well plate at a cell density of 5 x ¹⁰⁴ cells/well. After incubation for 24 hours, fibrin gel was layered on top of the IL-8-HaCaT cells.

The fibrin gel was prepared by adding 200 or 225 μL of a solution prepared by mixing thrombin solution and fibrinogen solution in the same manner as in Experimental Example 1 at a volume ratio of thrombin solution:fibrinogen solution = 1:5, and gelling the solution at 37°C and in a 5% _CO2 environment for 30 minutes.

Next, 100 μL of 5 mM zinc chloride aqueous solution was added to each Transwell insert and incubated for 48 hours. Next, the expression of the IL-8 gene by the IL-8-HaCaT cells on each Transwell insert was quantified by quantitative real-time PCR.

A commercially available kit (product name: "RNeasy Mini Kit", Qiagen) was used to prepare total RNA. A commercially available kit (product name: "High Capacity cDNA Reverse Transcription Kit", Applied Biosystems) was used to synthesize cDNA.

A commercially available kit (product name: TaqMan Gene Expression Assay (ID: Hs00174103_m1), Applied Biosystems) was used for quantitative real-time PCR of IL-8 gene cDNA. In addition, cDNA of the housekeeping gene β-actin was quantified as an internal standard by quantitative real-time PCR using a commercially available kit (product name: TaqMan Gene Expression Assay (ID: Hs99999903_m1), Applied Biosystems). A commercially available device (product name: 7300 Real-Time PCR System, Applied Biosystems) was used for quantitative real-time PCR.

Meanwhile, 0, 2, and 5 mM zinc chloride aqueous solutions were added to each well of a commercially available three-dimensional cultured skin (catalog number "EPI-200", MatTek) and incubated for 48 hours. Next, the expression of the IL-8 gene by the three-dimensional cultured skin in each well was quantified by quantitative real-time PCR in the same manner as for the IL-8-HaCaT cells described above.

Figures 5(a) and (b) are graphs showing the results of quantitative real-time PCR. Figure 5(a) shows the results for IL-8-HaCaT cells, and Figure 5(b) shows the results for commercially available three-dimensional cultured skin (catalog number "EPI-200", MatTek).

5(a) and (b), the vertical axis of the graph indicates the expression level (relative value) of IL-8 gene, and "ZnCl ₂ conc." indicates the concentration of the added aqueous zinc chloride solution. Furthermore, "*" indicates that there is a significant difference at P<0.05 compared to the group to which zinc chloride was not added, and "**" indicates that there is a significant difference at P<0.01 compared to the group to which zinc chloride was not added. Furthermore, in FIG. 5(a), "Fibrin volume" indicates the volume (μL) of fibrin gel added to the Transwell insert.

As a result, it was revealed that IL-8-HaCaT cells layered with 200-225 μL of fibrin gel showed the same reactivity to a 5 mM zinc chloride aqueous solution as commercially available three-dimensional cultured skin (catalog number "EPI-200", MatTek). This result indicates that by adjusting the porosity and thickness of the hydrogel layer, it is possible to reproduce permeability equivalent to that of three-dimensional cultured skin.

[Experimental Example 5]
(Study on hydrogel permeability 3)
The permeability of the test substance was examined by contacting the test substance with a permeable layer formed of a hydrogel. Fibrin gel was used as the hydrogel. Metal nanoparticles were used as the test substance. As the metal nanoparticles, zinc oxide (ZnO) particles with a diameter of 20 to 50 nm and _SiO2- coated ZnO (ZnO-Si) particles with a diameter of 10 to 100 nm were used.

First, IL-8-HaCaT cells were seeded into the Transwell insert of a 24-well plate at a cell density of 5 x ¹⁰⁴ cells/well. After incubation for 24 hours, fibrin gel was layered on top of the IL-8-HaCaT cells.

The fibrin gel was prepared by adding 200 μL of a solution prepared by mixing thrombin solution and fibrinogen solution in the same manner as in Experimental Example 1 at a volume ratio of thrombin solution:fibrinogen solution = 1:5 to each well and gelling for 30 minutes at 37°C in a 5% _CO2 environment.

Next, 100 μL of 1 mg/mL ZnO particles or 1 mg/mL ZnO-Si particles were added to each Transwell insert and incubated. As a control, wells to which no metal nanoparticles were added were also prepared.

After 48 hours, the amount of zinc ions that had permeated into each of the 24 wells was quantified using an ICP optical emission spectrometer (ICP-OES, model number "5110VDV", Agilent Technologies).

Figure 6(a) is a graph showing the quantitative results of zinc ions. In Figure 6(a), "Control" indicates a control to which no metal nanoparticles were added, and "**" indicates a significant difference from the control at P<0.01.

As a result, it was found that when ZnO particles or ZnO-Si particles were added to the permeable layer, these particles dissolved and became zinc ions, which were then released into the lower layer of the Transwell insert.

In addition, the expression of the IL-8 gene by IL-8-HaCaT cells on each Transwell insert was quantified by quantitative real-time PCR in the same manner as in Experimental Example 4.

Figure 6(b) is a graph showing the results of quantitative real-time PCR. In Figure 6(b), the vertical axis of the graph shows the expression level (relative value) of the IL-8 gene, "Control" shows the control to which no metal nanoparticles were added, and "**" indicates a significant difference from the control at P<0.01.

As a result, IL-8 gene expression was observed in proportion to the amount of zinc ions released through the permeable layer. This result indicates that this experimental system can be applied to evaluate the effects of nanoparticles on the lower layers of the skin.

The present invention includes the following aspects.
[1] A three-dimensional structure for evaluating skin permeability of a test substance, comprising a cell layer and a permeation layer containing a hydrogel, the cell layer and the permeation layer being laminated, the permeation layer having substance permeability equivalent to that of living skin, and the cell layer containing indicator cells for the test substance.
[2] The three-dimensional structure described in [1], wherein the transparent layer does not allow nanoparticles to pass through.
[3] The three-dimensional structure described in [1] or [2], wherein the permeability of the permeable layer is adjustable by changing the thickness.
[4] The three-dimensional structure according to any one of [1] to [3], wherein the indicator cells are cells that express a fluorescent protein.
[5] The three-dimensional structure described in [4], wherein the cell expressing the fluorescent protein is a cell into which a reporter gene has been introduced.
[6] The three-dimensional structure described in any one of [1] to [5], wherein the hydrogel contains fibrin.
[7] A method for evaluating the skin permeability of a test substance, comprising: a step of contacting a test substance with a surface of a three-dimensional structure opposite to a surface of the permeation layer facing the cell layer, the surface of the three-dimensional structure comprising a cell layer and a permeation layer containing a hydrogel, the cell layer and the permeation layer being laminated, the permeation layer having substance permeability equivalent to that of living skin, and the cell layer containing indicator cells for the test substance; and a step of measuring physiological characteristics of the indicator cells.
[8] The evaluation method according to [7], wherein the three-dimensional structure is the three-dimensional structure according to any one of [1] to [6].
[9] The evaluation method described in [7] or [8], wherein the physiological characteristic is measured by fluorescence intensity.
[10] A kit for evaluating the skin permeability of a test substance, comprising a hydrogel.

100...three-dimensional structure, 110...permeable layer, 111, 112...surface, 120...cell layer, 130...Transwell insert, 131...membrane, 140, 141...culture medium, 150...well.

JP 2017-209103 A

Godin B., and Touitou E., Transdermal skin delivery: predictions for humans from in vivo, ex vivo and animal models, Adv Drug Deliv Rev., 59 (11), 1152-1161, 2007.

Claims (7)

A cell layer;
a permeable layer comprising a hydrogel;
Including,
The cell layer and the permeable layer are laminated together,
The permeable layer has substance permeability equivalent to that of the skin of a living body,
the cell layer comprises indicator cells for a test substance;
the transparent layer is impermeable to nanoparticles;
The hydrogel comprises fibrin.
A three-dimensional structure for evaluating the skin permeability of a test substance. The three-dimensional structure of claim 1, wherein the material permeability of the permeable layer can be adjusted by changing the thickness. The three-dimensional structure according to claim 1 or 2, wherein the indicator cells are cells that express a fluorescent protein. The three-dimensional structure according to claim 3, wherein the cells expressing the fluorescent protein are cells into which a reporter gene has been introduced. a permeable layer including a cell layer and a hydrogel;
Including,
The cell layer and the permeable layer are laminated together,
The permeable layer has substance permeability equivalent to that of the skin of a living body,
the cell layer comprises indicator cells for a test substance;
the transparent layer is impermeable to nanoparticles;
The hydrogel is a three-dimensional structure containing fibrin .
contacting a test substance with a surface of the permeation layer opposite to a surface facing the cell layer;
measuring a physiological property of the indicator cells;
A method for evaluating the skin permeability of a test substance, comprising: The evaluation method according to claim 5 , wherein the physiological characteristic is measured by fluorescence intensity. A kit for evaluating the skin permeability of a test substance, comprising the three-dimensional structure according to any one of claims 1 to 4 , for use in the evaluation method according to claim 5 or 6 .

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