Method for collecting skin surface material, method for inspecting skin surface material, and deposit

Abstract

This method for collecting a skin surface material comprises: an adhesion step for applying a deposit containing fibers having an average fiber diameter of 0.01-7 μm to skin and holding the deposit for 0.5 hours or more to adhere, to the deposit, a skin surface material which is an endogenous substance existing in the stratum corneum of the skin; and a removal step for removing the deposit having undergone the adhesion step from the skin. In the method, the deposit after the removal step to which the skin surface material has been adhered is collected.　The fibers preferably contain a thermoplastic resin.　The basis weight of the deposit is preferably 0.1-50 g / m2.<sp / >　Before or during the adhesion step, a liquid agent containing one or more selected from water, a polyol, and an oil agent that is liquid at 20°C is preferably applied to the skin.

Description

Method for collecting skin surface material, method for examining skin surface material, and sediment

[0001] This invention relates to a method for collecting surface skin material, a method for examining surface skin material, and a deposit.

[0002] In recent years, attempts have been made to collect and analyze biomarkers and immune response proteins from skin tissue on the skin surface. For example, Non-Patent Document 1 proposes a method called skin blotting. This document states that "within the skin, there are markers (proteins, etc.) that reflect the overall health status and markers that represent the local condition of the skin in the interstitial fluid (body fluid) that seeps out from the blood vessels (Figure 1). Skin blotting is a technique that extracts these markers simply by moistening a 1 cm square skin patch (a tape that is electrostatically charged and attracts markers) and applying it to the skin for 10 minutes (Figure 2)." According to Non-Patent Document 1, this method allows for monitoring of the body's health status without the need for blood sampling. Patent Document 1 describes that, using the skin blotting method to detect pressure ulcer prediction markers, it was shown that there were differences in the expression of immune response proteins between the skin of mice to which voltage was applied for a long period of time and the skin of control mice.

[0003] Patent Document 2 describes a cosmetic method for protecting the skin, which includes a step (adhesion step) of applying a coating made of a deposit containing fibers with an average fiber diameter of 0.01 to 7 μm to the skin and holding it for 1 to 12 hours. The same document describes that after wiping the skin with cigarette paper before and after the formation of the coating, the sebum attached to the cigarette paper was analyzed (paragraphs

[0094] and

[0096] ).

[0004] Japanese Patent Publication No. US2019 / 0100802A1 2022-074055

[0005] https: / / www.ishikawa-nu.ac.jp / lab / bioengnurs / introduction / (Accessed May 20, 2024)

[0006] The present invention provides a method for collecting surface skin material. In one embodiment, a deposit containing fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less is applied to the skin. In one embodiment, it is preferable to keep the deposit on the skin for 0.5 hours or more to allow surface skin material, which is a substance of internal origin present on the stratum corneum of the skin, to adhere to the deposit. In one embodiment, it is preferable to remove the deposit with the surface skin material attached from the skin. In one embodiment, it is preferable to recover the deposit with the surface skin material attached that has been removed from the skin.

[0007] Figure 1 is a schematic diagram showing the configuration of the electrostatic spraying apparatus used in the present invention. Figure 2 is a schematic diagram showing how the electrostatic spraying method is performed using the electrostatic spraying apparatus. Figures 3(a) to (c) are graphs showing the peak area of ​​albumin in the mass spectrometry of skin samples obtained by the sampling methods of the examples and comparative examples. Figures 4(a) to (c) are the results of the peak area ratio of albumin to keratin-10 (ALB / KRT10) in the mass spectrometry of skin samples obtained by the sampling methods of the examples and comparative examples. Figures 5(a) to (c) are the results of the protein identification number (FDR 5%) in the mass spectrometry of skin samples obtained by the sampling methods of the examples and comparative examples. Detailed description of the invention

[0008] The skin blotting method described in Non-Patent Document 1 involves application using an adhesive sheet, which can easily cause stress to the skin during application and removal. The skin blotting method described in Patent Document 1 shows the application condition under conditions where fluid easily seeps from inside the skin, such as in pressure ulcers. Neither method can be said to demonstrate the effect of routinely collecting internally derived components while reducing stress on the skin. Patent Document 2 does not address the problem of collecting internally derived components as a source after removing deposits of fibers of a specific fiber diameter from the skin.

[0009] The present invention relates to providing a method for efficiently collecting skin surface substances, which are endogenous substances present on the stratum corneum of the skin, using a method that allows for long-term application.

[0010] Preferred embodiments of the present invention will be described below. In the present invention, "surface material" refers to endogenous substances present on the stratum corneum of the skin. The stratum corneum constitutes the outermost surface of the skin. Endogenous components refer to components that originate from the body of the skin owner. In this specification, "endogenous components" is intended to eliminate externally attached components.

[0011] Examples of skin surface materials that can be collected by the present invention include proteins, nucleic acids, lipids, sugars, and amino acids. Furthermore, in the collection method of the present invention, it is preferable to collect one or more selected from proteins, nucleic acids, and lipids. The proteins referred to here preferably include one or more selected from immune response proteins, natural moisturizing factor-producing enzymes, ceramide metabolic enzymes, stratum corneum exfoliating proteases, oxidoreductases, desmosome constituent proteins, and plasma-derived proteins.

[0012] Examples of immune response proteins include antimicrobial peptides, cytokines, chemokines, immunoglobulins, complement system proteins, and fibrinogen. Examples of enzymes that produce natural moisturizing factors include filaggrin-degrading enzymes, aminopeptidases, and Peptidyl Arginine Deiminase 1.

[0013] Examples of ceramide metabolic enzymes include β-glucocerebrosidase, sphingomyelinase, ceramidase, glucosylceramide synthase, ceramide synthase, and serine palmitoyltransferase.

[0014] Examples of oxidoreductases include SOD, peroxidase, catalase, glutathione reductase, thioredoxin reductase, NOX, LOX, COX, aldose reductase, and tyrosinase.

[0015] Desmosome constituent proteins include desmoglein, desmocholine, placoglobin, placofilin, and desmoplakin. Plasma-derived proteins include albumin and transferrin.

[0016] Nucleic acids include DNA and RNA. Lipids include sebum. Sugars and amino acids may be present in the skin surface as metabolites. Metabolites include those of bacteria and other organisms on the skin surface.

[0017] The proteins that can be collected as skin surface material by this invention are thought to have seeped onto the surface of the stratum corneum from interstitial fluid, sweat glands, hair follicles, sebaceous glands, etc. Their origin is thought to be from these organs, as well as from blood and internal skin tissue. Similarly, nucleic acids are thought to have seeped onto the surface of the stratum corneum from interstitial fluid, sweat glands, hair follicles, sebaceous glands, etc. In addition, sebum is secreted onto the surface of the stratum corneum by the sebaceous glands.

[0018] Interstitial fluid is normally released from the human body in small amounts over time. However, there has been no conventional method to capture and store this small amount of released interstitial fluid as it is released. By attaching fibers with a specific fiber diameter as described herein to the skin for an extended period, it becomes possible to retain the small amount of released interstitial fluid between the fibers over a long period. The small amount of released interstitial fluid accumulates between the fibers over a long period, eventually reaching the amount necessary for analysis. As a result, the deposit becomes a sample containing interstitial fluid.

[0019] This accumulated interstitial fluid is composed of internally derived components and is distinct from stratum corneum-derived components immediately wiped away with oil-blotting paper, etc. If these internally derived components are indeed interstitial fluid, then its application as a non-invasive method for collecting interstitial fluid is expected. Furthermore, internally derived components obtained from interstitial fluid can be found to have components equivalent to those found in blood, so applications such as testing interstitial fluid instead of blood tests are expected. The advantage of this alternative test is that, unlike blood tests which use invasive collection methods, this alternative test can be performed using a non-invasive method such as attaching a sample to the skin.

[0020] In this specification, "non-invasive sampling method" means collecting skin tissue without performing procedures that involve incision, puncture, or other tissue damage to the subject's skin. For example, a method of capturing skin tissue by applying a fibrous deposit to the skin surface is included in non-invasive sampling methods.

[0021] The inventors believe that the proteins collected as skin surface material using this invention may have existed on the surface of the stratum corneum all along. However, they believe that their existence was not widely known until now because a suitable collection method had not been developed. The inventors believe that one reason why skin surface material can be efficiently collected using this invention is the effect of capillary force due to the use of fibers of a specific fiber diameter. Because this invention allows for the collection of a certain amount or more of skin surface material, the analytical device for the collected skin surface material is not limited to a high-precision measuring device, but can also be a simple measuring device.

[0022] Fibers of a specific diameter can adhere to the skin for extended periods. This is because the fine fibers conform to the contours of the skin. In addition, the large specific surface area of ​​the fibers increases adhesion to the skin, which also contributes to long-term adhesion.

[0023] The present invention's method for collecting skin surface material preferably includes an adhesion step. In the adhesion step, it is preferable to apply a deposit containing fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less to the skin. In the adhesion step, after applying the deposit to the skin, it is preferable to maintain the state in which the deposit is in contact with the skin for 0.5 hours or more to allow the skin surface material to adhere to the deposit. In this specification, the adhesion step includes not only applying the deposit to the skin but also subsequent maintenance.

[0024] In the following, "deposits containing fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less" may also be referred to as "fiber deposits" or simply "deposits." The deposits preferably contain 60% by mass or more of fibers with a fiber diameter of 0.01 μm or more and 7 μm or less, more preferably 70% by mass or more, even more preferably 80% by mass or more, and even more preferably 90% by mass or more.

[0025] The skin to which this invention can be applied includes the face, neck, hands, feet, back, shoulders, abdomen, chest, legs, and arms. Among these, the face, hands, and feet are preferred because they are less likely to be covered by clothing during the day, making them convenient for applying the deposit. The face and hands are particularly preferred because they offer both the benefit of collecting surface skin material and the cosmetic effects that have been known to be associated with this fibrous deposit. The face is particularly preferred because it is easy to collect many types of proteins as surface skin material. The cheeks and / or forehead are especially preferred. Oil-blotting films and bandages are noticeable when applied to the face in daily life. On the other hand, the deposit of very fine fibers in this invention is less noticeable, and surface skin material can be collected in that state.

[0026] By applying a deposit containing fibers with an average fiber diameter of 0.01 μm to 7 μm to the skin, a film made of the deposit is formed on the skin. From the viewpoint of efficiently adhering the surface products of the skin to the deposit, the average fiber diameter of the fibers contained in the deposit is preferably 0.05 μm or more, and more preferably 0.1 μm or more. Furthermore, from the viewpoint of maintaining the adhesion of the deposit to the skin, the average fiber diameter is preferably 5 μm or less, and more preferably 3 μm or less. For example, natural cellulose fibers generally used in oil-blotting papers are usually several tens of μm (10 μm or more) thick, and the same is true for the oil-blotting papers used in the comparative example described later.

[0027] The average fiber diameter is the average thickness of the fiber and is the equivalent diameter of a circle. This fiber thickness can be determined, for example, by observing the fiber at a magnification of 10,000 times using a scanning electron microscope. Defects (fiber clumps, fiber intersections, droplets) are removed from the two-dimensional image, and 10 fibers are arbitrarily selected. A line perpendicular to the longitudinal direction of the fiber is drawn, and the fiber diameter can be measured by directly reading it. The average value of the fiber diameters of the 10 fibers is defined as the "average fiber diameter."

[0028] The length of the aforementioned fibers is not particularly limited, but it is preferably 10 times or more the average fiber diameter, more preferably 20 times or more, and even more preferably 50 times or more. There is no particular upper limit to the length of the fibers, but if it is 100 times or more, it is defined as a continuous fiber, and any continuous fiber is acceptable.

[0029] The basis weight of the fiber deposit is preferably 0.1 g / m ２ or more, more preferably 0.4 g / m ２ or more, still more preferably 0.5 g / m ２ or more, even more preferably 0.8 g / m ２ or more, still even more preferably 1 g / m ２ or more, particularly preferably. When the basis weight is not less than the above lower limit, the deposit can be easily peeled off from the skin without being torn. Also, the basis weight of the fiber deposit is preferably 50 g / m ２ or less, more preferably 40 g / m ２ or less, still more preferably 30 g / m ２ or less, even more preferably 10 g / m ２ or less, even still more preferably. By setting the basis weight of the fiber deposit to not more than the above upper limit, it becomes possible to apply the deposit to the skin for a long time.

[0030] The above-mentioned deposit is typically in the form of a film. From the viewpoint of being easily peeled off from the skin without breaking the deposit, its thickness is preferably 1 μm or more, more preferably 3 μm or more. Also, from the viewpoint of being able to apply the deposit to the skin for a long time, the thickness of the above-mentioned deposit is preferably 300 μm or less, more preferably 100 μm or less, still more preferably 20 μm or less. The thickness can be measured with a contact-type film thickness gauge, Mitutoyo Corporation's Lightmatic VL-50, using a measuring head with a super-hard ball for a general dial gauge (7.3 mm). The measuring force applied to the measurement object at regular intervals is 0.01 N. The thickness of the deposit preferably ranges from 1 μm or more to 300 μm or less, more preferably from 1 μm or more to 100 μm or less, still more preferably from 1 μm or more to 20 μm or less, particularly preferably from 3 μm or more to 20 μm or less. The deposits in each of the examples described below are within the range of 1 μm or more to 100 μm or less, and particularly the deposits in Examples 1 and 2 were within the range of 1 μm or more to 20 μm or less in thickness.

[0031] It is preferable that the fiber contains a thermoplastic resin because it is easy to produce a deposit with a desired fiber diameter. Typically, the thermoplastic resin is a synthetic resin, and the fiber contains a synthetic resin as a main component. The main component means that it occupies 60% by mass or more and 100% by mass or less, preferably occupies 70% by mass or more, more preferably occupies 80% by mass or more, and still more preferably occupies 90% by mass or more.

[0032] Thermoplastic resins are roughly classified into water-soluble polymers and water-insoluble polymers. In this specification, the "water-soluble polymer" means that in an environment of 1 atm and 23°C, after weighing 1 g of the polymer, it is immersed in 10 g of deionized water, and after 24 hours, 0.5 g or more of the immersed polymer dissolves in water. On the other hand, the "water-insoluble polymer" means that in an environment of 1 atm and 23°C, after weighing 1 g of the polymer, it is immersed in 10 g of deionized water, and after 24 hours, 0.5 g or more of the immersed polymer does not dissolve.

[0033] The water-soluble thermoplastic resin preferably contains one or more selected from substantially non-crosslinked partial saponified polyvinyl alcohol, water-soluble nylon, polyvinylpyrrolidone, and polyethylene oxide because the burden on the skin is relatively small.

[0034] The water-insoluble thermoplastic resin preferably contains one or more selected from polyvinyl acetal, polyester, acrylic resin, polystyrene resin, polyvinyl butyral (PVB) resin, polyurethane resin, polyamide resin, polyimide resin, polyamideimide resin, olefin resin, fully saponified polyvinyl alcohol, and crosslinked partial saponified polyvinyl alcohol because the burden on the skin is relatively small. More preferably, it contains one or more selected from polyvinyl acetal, polyester, acrylic resin, polystyrene resin, PVB resin, polyurethane resin, polyamide resin, olefin resin, fully saponified polyvinyl alcohol, and crosslinked partial saponified polyvinyl alcohol.

[0035] Substantially non-crosslinked polymers encompass both those manufactured without crosslinking, i.e., without the use of crosslinking agents, and those that are crosslinked under conditions that satisfy the definition of a water-soluble polymer. Crosslinking agents refer to crosslinking agents that are reactive with hydroxyl groups, and examples include monoaldehydes such as formaldehyde, urea, melamine-formaldehyde resin, boric acid, dialdehyde, diacid, urethane, epoxide, etc.

[0036] The polyvinyl acetal preferably contains polyvinyl acetal diethylaminoacetate.

[0037] The polyester preferably contains one or more selected from polylactic acid, polyethylene terephthalate resin, and polybutylene terephthalate resin.

[0038] The acrylic resin preferably contains one or more selected from polyacrylonitrile resin and polymethacrylic acid resin.

[0039] The olefin resin preferably contains one or more selected from polypropylene (PP) and polyethylene.

[0040] In particular, from the viewpoint of being used in contact with the skin for a long period of time, it is more preferable that the fibers include one or more selected from PVB resin and PP.

[0041] The present invention also includes cases in which the fibers are primarily composed of any of the preferred synthetic resins (specifically, each preferred resin group) described herein. For example, this includes cases in which the fibers are primarily composed of one or more selected from the preferred water-soluble synthetic resins, or of one or more selected from the preferred (or more preferred) insoluble synthetic resins.

[0042] The aforementioned thermoplastic resins can be used alone or in combination of two or more types.

[0043] The porosity of the aforementioned deposit is preferably 60% or more, more preferably 70% or more, and more preferably 75% or more, from the viewpoint of durability and the like. Furthermore, the porosity of the aforementioned deposit is preferably 90% or less, and more preferably 85% or less, from the viewpoint of making the deposit less noticeable on the skin. The range of the porosity is preferably 60% to 90%, more preferably 70% to 90%, and even more preferably 75% to 85%. The deposits in each of the examples described later had a porosity within the range of 75% to 85%. The porosity is determined by the mercury intrusion method. Specifically, it is determined by the following method. (Method for measuring porosity) The measurement of porosity follows the measurement method (mercury intrusion method) described in "Functionality of Materials (Experimental Chemistry Course 12, 4th Edition), edited by the Chemical Society of Japan, published by Maruzen Co., Ltd., p. 486)" etc. Specifically, the porosity can be measured using a dedicated measuring instrument such as a mercury intrusion type pore size analyzer (Pore Sizer 9320) manufactured by Shimadzu Corporation. The volume of the sediment is measured in advance, and the pore size distribution of the sediment is measured using the mercury intrusion method. The pore size is calculated using the following formula: D = -4γcosθ / P, where D: pore size, γ: surface tension of mercury, θ: contact angle, and P: pressure. The surface tension of mercury is assumed to be 482.536 dyn / cm, the contact angle used is 130°, and the measurement is performed at a mercury pressure of 0 to 30000 psi. The pore size distribution is determined as follows: First, based on the principle of the mercury intrusion method, the pressure applied to the mercury is gradually changed. At that time, the volume of mercury that penetrates into the pores, i.e., the pore volume dV, is measured. The relationship between the pore size D converted according to the above formula and the pore volume dV is plotted. The derivative of this relationship curve, dV / d(logD), is calculated and plotted on the vertical axis, with pore size D on the horizontal axis, to create a graph. The pore size distribution is measured in the range of pore size from 6 nm to 10,000 nm. The unit of pore volume, (mL), indicates the amount of mercury that penetrates the pores, and (g) indicates the mass of the sediment sample used for measurement. At this time, dV / d(logD) is integrated to obtain V. This is divided by the previously determined sediment V1, and V / V1 is taken as the porosity.

[0044] The fibers preferably contain a thermoplastic resin with a glass transition temperature Tg of 150°C or lower, more preferably 120°C or lower, and even more preferably 100°C or lower. Keeping it below this upper limit makes the thermoplastic resin easier to process. The fibers preferably contain a thermoplastic resin with a glass transition temperature Tg of 45°C or higher, and more preferably 50°C or higher. Keeping it above this lower limit improves the handling properties of the thermoplastic resin and the temperature stability of the deposits.

[0045] In particular, when the fibers contain an amorphous resin, the glass transition temperature of the amorphous resin is preferably 150°C or lower, more preferably 120°C or lower, and especially preferably 100°C or lower. Furthermore, the glass transition temperature of the amorphous resin is preferably 45°C or higher, and more preferably 50°C or higher. Examples of amorphous resins include PVB, polyvinyl alcohol, acrylic resin, polystyrene resin, polyvinyl acetal, and polyamide-imide resin.

[0046] To measure the Tg of thermoplastic resins, a PerkinElmer differential scanning calorimetry system, "PYRIS Diamond DSC," is used. First, approximately 5 mg of sample is taken from the fiber. Under a nitrogen atmosphere, the sample is sealed in an aluminum closed cell, and then the cell is set in the instrument holder. This is then heated from 25°C to 300°C at a rate of 10°C / min. The DSC curve is obtained by measuring the heat flow rate at each temperature. In the obtained DSC curve, the "intermediate glass transition temperature (Tmg)" described in JIS K 7121 is considered to be the glass transition temperature.

[0047] In this specification, it is preferable that the deposit used for collecting skin surface material is provided onto the skin from an electrostatic spray device, or is in the form of a single layer sheet, or constitutes a laminated sheet laminated on a substrate.

[0048] The method of providing the material onto the skin from the electrostatic spray device can be any of the methods described later in "Method of spraying and depositing fibers onto the skin using a spray device to form a coating consisting of fiber deposits" and the method described later in (I-1) without limitation.

[0049] In the case of a single-layer sheet or a laminated sheet formed by laminating a substrate, the explanation in "Method for attaching a sheet-like deposit of fibers to the skin" and the explanation in (I-2) below can be adopted without limitation.

[0050] As the base material, a breathable material can be used, such as a mesh sheet, a fiber sheet, or a laminate thereof. Examples of fiber sheets include nonwoven fabrics, woven fabrics, knitted fabrics, and paper.

[0051] When applying fiber deposits to the skin, two methods are typically preferred. The first is "a method of forming a film consisting of fiber deposits by spraying and depositing fibers onto the skin using a spray device." The second is "a method of attaching a sheet of fiber deposits to the skin." The deposits used in this invention adhere to the skin due to capillary force resulting from the small diameter of the constituent fibers. In this invention, due to the aforementioned capillary force, it is minimally invasive, places little burden on the skin, and can be attached for a long period of time. Furthermore, it is not necessary to attach the deposits to the skin using adhesive materials such as adhesive tape or adhesive patches. Examples of fiber deposits in sheet form include the single-layer sheets and laminated sheets of fiber deposits mentioned above.

[0052] The attachment process is not limited to any process that can form a film consisting of the aforementioned fiber deposits on the skin, but it is preferable to include the following steps (I-1) or (I-2). (I-1) A step of electrostatically spraying composition X containing the following volatile substance and polymer onto the skin.

[0053] The aforementioned volatile substance includes one or more volatile substances selected from water, alcohol, and ketones (hereinafter also referred to as "volatile substance A").

[0054] The polymer includes a polymer having the ability to form fibers (hereinafter also simply referred to as "polymer" or "the polymer"). (I-2) A step of applying a deposit of fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less, obtained by electrospinning or melt blowing, to the skin.

[0055] First, the means (I-1) will be described. The method for forming the deposit in means (I-1) typically uses an electrostatic spraying method. The electrostatic spraying method involves applying a positive or negative high voltage to a composition to charge it, and then spraying the charged composition toward an object. The sprayed composition spreads into space while being repeatedly atomized by Coulomb repulsion, and in the process, or after adhering to the object, the solvent, which is a volatile substance, dries, forming a film containing fibrous deposits on the surface of the object. This method (I-1) can be carried out using methods and apparatus described in, for example, WO2018 / 194143

[0007]

[0012]

[0089] , WO2018 / 194140

[0007]

[0012]

[0067] , WO2019 / 103974

[0002]

[0003]

[0004]

[0142]

[0143]

[0144] , etc.

[0056] The volatile substance is typically a substance that is volatile in a liquid state. In composition X, the volatile substance is discharged from the nozzle tip toward the skin after the composition X has been sufficiently charged by being placed in an electric field. As the volatile substance evaporates, the charge density of composition X becomes excessive, and the volatile substance evaporates further while composition X is further atomized by Coulomb repulsion. Finally, a dry film consisting of a deposit of fibers is formed on the skin. For this purpose, the vapor pressure of the volatile substance is preferably 0.01 kPa or more and 106.66 kPa or less at 20°C, more preferably 0.13 kPa or more and 66.66 kPa or less, even more preferably 0.67 kPa or more and 40.00 kPa or less, and even more preferably 1.33 kPa or more and 40.00 kPa or less.

[0057] The volatile substance preferably includes one or more selected from monohydric chain aliphatic alcohols, monohydric cyclic aliphatic alcohols, and monohydric aromatic alcohols.

[0058] Monovalent chain aliphatic alcohols are C １ ~C ６ Preferably, it contains one or more alcohols selected from alcohols.

[0059] Monohydric cyclic aliphatic alcohols are C ４ ~C ６ Preferably, it contains one or more selected cyclic alcohols.

[0060] The monohydric aromatic alcohol preferably contains one or more selected from benzyl alcohol, phenylethyl alcohol, etc. １ ~C ６ The alcohol preferably contains one or more selected from ethanol, isopropyl alcohol, butyl alcohol, n-propanol, and n-pentanol. The monohydric aromatic alcohol preferably contains phenylethyl alcohol.

[0061] The aforementioned volatile substance is diC １ -C ４ Preferably, it contains one or more selected alkyl ketones. １ -C ４ The alkyl ketone preferably contains one or more selected from acetone, methyl ethyl ketone, and methyl isobutyl ketone.

[0062] From the viewpoint of continuous use on the skin, the volatile substance more preferably contains one or more selected from ethanol, isopropyl alcohol, butyl alcohol, and water, even more preferably contains one or more selected from ethanol and butyl alcohol, and even more preferably contains ethanol.

[0063] From the viewpoint of fiber-forming properties, the content of volatile substances in composition X is preferably 30% by mass or more, more preferably 55% by mass or more, and even more preferably 60% by mass or more. In particular, the content of volatile substance A in composition X is preferably 30% by mass or more, more preferably 55% by mass or more, and even more preferably 60% by mass or more. Furthermore, from the viewpoint of fiber-forming properties, the content of volatile substances in composition X is preferably 98% by mass or less, more preferably 96% by mass or less, and even more preferably 94% by mass or less. Furthermore, the content of volatile substance A in composition X is preferably 30% by mass or more, more preferably 55% by mass or more, and even more preferably 60% by mass or more. From the above viewpoint, the content of volatile substances in composition X is preferably 30% by mass or more and 98% by mass or less, more preferably 55% by mass or more and even more preferably 60% by mass or more. Furthermore, the content of volatile substance A in composition X is preferably 30% by mass or more and 98% by mass or less, more preferably 55% by mass or more and 96% by mass or less, and even more preferably 60% by mass or more and 94% by mass or less.

[0064] Composition X preferably contains one or more selected from oils and polyols. In this case, the content of volatile substances in composition X is preferably 30% by mass or more and 90% by mass or less, more preferably 55% by mass or more and 90% by mass or less, and even more preferably 60% by mass or more and 90% by mass or less. Furthermore, the content of volatile substance A in composition X is preferably 30% by mass or more and 90% by mass or less, more preferably 55% by mass or more and 90% by mass or less, and even more preferably 60% by mass or more and 90% by mass or less. By including volatile substances in composition X in these proportions, the volatility of the volatile substances is improved when performing the electrostatic spray method.

[0065] If composition X contains ethanol, the amount of ethanol is preferably 50% by mass or more and 100% by mass or less, more preferably 65% ​​by mass or more and 100% by mass or less, and even more preferably 80% by mass or more and 100% by mass or less, based on the total amount of volatile substances. Furthermore, the amount of ethanol is preferably 50% by mass or more and 100% by mass or less, more preferably 65% ​​by mass or more and 100% by mass or less, and even more preferably 80% by mass or more and 100% by mass or less, based on the total amount of volatile substance A.

[0066] Polymers having fiber-forming ability are typically substances that can dissolve in the aforementioned volatile substances. Here, dissolution means that the substance is dispersed at 20°C and that the dispersion is uniform to the naked eye, preferably transparent or translucent to the naked eye. Examples of polymers having fiber-forming ability include the aforementioned thermoplastic resins, and preferred examples include preferred resins among thermoplastic resins.

[0067] The polymer content in composition X is preferably 2% by mass or more and 50% by mass or less, more preferably 4% by mass or more and 45% by mass or less, and even more preferably 6% by mass or more and 40% by mass or less. By incorporating the polymer into composition X in this proportion, a film consisting of fiber deposits is formed, and the skin protection effect is achieved by this film.

[0068] The ratio of the volatile substance to the polymer content in composition X (volatile substance / polymer) is preferably 0.5 to 40, more preferably 1 to 30, and even more preferably 1.3 to 25, from the viewpoint of ensuring sufficient volatilization of the volatile substance when performing the electrostatic spray method.

[0069] Composition X may contain glycol. The glycol may include one or more selected from ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, dipropylene glycol, polyethylene glycol, and polypropylene glycol, etc.

[0070] From the viewpoint of ensuring sufficient volatilization of volatile substances when performing the electrostatic spraying method, the glycol content in composition X is preferably 0% by mass or more and 10% by mass or less, and more preferably 8% by mass or less. When composition X contains water, from the viewpoint of fiber-forming properties and conductivity, the water content is preferably more than 0% by mass and less than 50% by mass, more preferably 45% by mass or less, even more preferably 10% by mass or less, and even more preferably 5% by mass or less, relative to the total amount of volatile substances. For similar reasons, the water content is preferably more than 0% by mass and less than 50% by mass, more preferably 45% by mass or less, even more preferably 10% by mass or less, and even more preferably 5% by mass or less, relative to the total amount of volatile substance A.

[0071] When performing the electrostatic spray method, it is preferable to use a composition X whose viscosity at 25°C is preferably 1 mPa·s to 5000 mPa·s, more preferably 10 mPa·s to 2000 mPa·s, and even more preferably 50 mPa·s to 1500 mPa·s. By using a composition X with a viscosity in this range, a porous film containing fiber deposits can be successfully formed by the electrostatic spray method. The viscosity of the composition X used in each of the examples described later is in the range of 50 mPa·s to 1500 mPa·s. The viscosity is measured at 25°C using an E-type viscometer. An E-type viscometer manufactured by Tokyo Keiki Co., Ltd. can be used as the E-type viscometer. In that case, rotor No. 43 can be used.

[0072] An electrostatic spray device typically comprises a container 14, a nozzle 15, a supply device, a power supply 12 (high-voltage power supply), a low-voltage power supply 11, and an auxiliary electrical circuit 13. The container 14 contains the composition X. The nozzle 15 dispenses the composition X. The supply device supplies the composition X contained in the container 14 to the nozzle 15. The supply device typically comprises a microgear pump 15A, a conduit 15B, and a flexible conduit 15C. The power supply 12 applies voltage to the nozzle 15. The low-voltage power supply 11 typically consists of one or two batteries. The auxiliary electrical circuit 13 adjusts the voltages of the high-voltage power supply 12 and the low-voltage power supply 11. In addition, the auxiliary electrical circuit 13 controls the rotational speed of the motor provided in the microgear pump 15A. Figure 1 shows a schematic diagram of the configuration of an electrostatic spray device preferably used in the present invention. Figure 2 shows electrostatic spraying on one's skin.

[0073] Next, method (I-2) will be described. Method (I-2) is a step of applying a deposit of fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less, obtained by electrospinning or melt blowing, to the skin. An example of applying the fiber deposit to the skin in method (I-2) is a method of attaching a pre-formed film-like fiber deposit to the skin.

[0074] The deposit of fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less used in this method (I-2) is preferably obtained by electrospinning or melt-blowing on a substrate using the composition X. The electrospinning method can be carried out in the same manner as the electrostatic spraying method. However, the voltage during electrospinning may be high since it is not performed on the skin. Examples of substrates include metals, resins, and the various substrate films mentioned above. The melt-blowing method melts the resin at a temperature above its melting point and discharges it while also discharging hot air around the discharge port of the molten resin to form a deposit of fibers with the aforementioned average fiber diameter.

[0075] While not limited to this, when applying a fibrous deposit to the skin, the outer surface area of ​​the skin portion in contact with the deposit during a single application is, for example, 0.5 cm².２ More than 400cm ２ The following is preferable: 0.7 cm ２ 225cm or more ２ The following is more preferable: When applying the fibrous deposit to the skin, it may be applied separately to multiple locations on the skin, or it may be applied continuously to one location.

[0076] When employing means (I-1) or (I-2), it is preferable to apply liquid agent Y to the skin either “before the adhesion process” or to the deposit “during the adhesion process.” Liquid agent Y preferably contains one or more components selected from water, polyol, and an oil that is liquid at 20°C. This provides good transparency to the deposit formed by the adhesion process, improving its appearance. It also enhances the adhesion between the skin and the deposit, making it easier to adhere the skin surface material to the deposit. An example of “before the adhesion process” is before applying the deposit to the skin. An example of “during the adhesion process” (while the skin surface material is attached to the deposit) is after applying the deposit to the skin. In this case, it is preferable to apply the deposit to the skin, then apply liquid agent Y, and then maintain the adhesion for the preferred holding time described above. In the present invention, when the deposit is used in combination with liquid agent Y, even if the skin is covered with the deposit in daily life, it is less likely to cause a change in appearance and is easy to wear for long periods of time.

[0077] Liquid Y preferably contains water and a polar oil, as this improves the adhesion of the deposit to the skin. The total proportion of water and polar oil in liquid Y is preferably 40% by mass or more and 100% by mass or less. The polar oil preferably contains an ester oil, as described later.

[0078] The polyol preferably contains one or more selected from alkylene glycols, polyalkylene glycols, and glycerin compounds. The alkylene glycols preferably contain one or more selected from ethylene glycol, propylene glycol, 1,3-propanediol, and 1,3-butanediol. The polyalkylene glycols preferably contain one or more selected from diethylene glycol, dipropylene glycol, polyethylene glycol with a molecular weight of 1000 or less, and polypropylene glycol. The glycerin compounds preferably contain one or more selected from glycerin, diglycerin, and triglycerin. Of these, from the viewpoint of excellent adhesion to the skin, abrasion resistance, stretchability, and transparency of the coating, it is preferable to include one or more selected from ethylene glycol, propylene glycol, 1,3-butanediol, dipropylene glycol, polyethylene glycol with a molecular weight of 1000 or less, glycerin, and diglycerin. It is even more preferable to include one or more selected from propylene glycol, 1,3-butanediol, and glycerin, and even more preferable to include glycerin.

[0079] The polyol content in liquid Y is preferably 1% by mass or more and 40% by mass or less, more preferably 1% by mass or more and 30% by mass or less, even more preferably 3% by mass or more and 25% by mass or less, even more preferably 5% by mass or more and 20% by mass or less, and particularly preferably 10% by mass or more and 20% by mass or less, from the viewpoint of excellent abrasion resistance, stretchability and transparency of the coating.

[0080] Liquid agent Y preferably contains an oil that is liquid at 20°C, from the viewpoint of excellent abrasion resistance, spreadability, and transparency. The oil that is liquid at 20°C also includes semi-solid substances that are fluid. The oil that is liquid at 20°C preferably contains one or more selected from hydrocarbon oils, ester oils, higher alcohols, silicone oils, and fatty acids. Of these, it is preferable to include one or more selected from hydrocarbon oils, ester oils, and silicone oils from the viewpoint of smoothness during application, abrasion resistance, and spreadability of the coating.

[0081] The hydrocarbon oil preferably contains one or more selected from liquid paraffin, squalane, squalene, n-octane, n-heptane, cyclohexane, light isoparaffin, liquid isoparaffin, hydrogenated polyisobutene, polybutene, polyisobutene, hydrogenated polydecene, and α-olefin oligomers. In particular, from the viewpoint of usability, it is preferable to contain one or more selected from liquid paraffin, light isoparaffin, liquid isoparaffin, squalane, squalene, n-octane, n-heptane, cyclohexane, hydrogenated polydecene, and α-olefin oligomers.

[0082] The ester oil preferably contains an ester consisting of a straight-chain or branched-chain fatty acid and a straight-chain or branched-chain alcohol or polyhydric alcohol.Such esters include isopropyl myristate, cetyl isooctanoate, isocetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, decyl oleate, octyldodecyl oleate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, ethylhexyl isononanoate, isononyl isononanoate, isotridecyl isononanoate, isostearyl isostearate, and 12-hydroxystearyl Cholesteryl ethyl acid, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, N-alkyl glycol monoisostearate, propylene glycol dicaprylate, propylene glycol diisostearate, neopentyl glycol dicaprate, diisostearyl malate, glyceryl di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentaerythritol tetra-2-ethylhexanoate, glyceryl tri-2-ethylhexanoate, triisos Trimethylolpropane thearate, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, diethylhexyl naphthalenedicarboxylate, alkyl benzoate (C12-C15), cetearyl isononanoate, caprylic / capric triglyceride, dicaprylic / capric butylene glycol, dicaprylic / capric propylene glycol, glyceryl triisostearate, glyceryl tri-heptyl undecanoate, glyceryl coconut oil fatty acid, methyl castor oil fatty acid ester, oleyl oleate, palmitate It is preferable to include one or more selected from 2-heptyl undecyl tinate, diisobutyl adipate, 2-octyldodecyl N-lauroyl-L-glutamic acid, di-2-heptyl undecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, di-2-ethylhexyl succinate, triethyl citrate, 2-ethylhexyl paramethoxycinnamate, and tripropylene glycol dipivalate.Among these, it is preferable to include one or more selected from octyldodecyl myristate, myristyl myristate, isocetyl stearate, isononyl isononanoate, isocetyl isostearate, cetearyl isononanoate, diisobutyl adipate, di-2-ethylhexyl sebacate, isopropyl myristate, isopropyl palmitate, diisostearyl malate, neopentyl glycol dicaprate, and tri(caprylic / capric acid)glycerin, from the viewpoint of excellent abrasion resistance, stretchability, and transparency.

[0083] The ester oil may contain one or more selected from vegetable oils and animal oils. The vegetable oil may contain one or more selected from olive oil, jojoba oil, macadamia nut oil, meadowfoam oil, castor oil, safflower oil, sunflower oil, avocado oil, canola oil, apricot kernel oil, rice germ oil, and rice bran oil, etc.

[0084] The aforementioned higher alcohol preferably contains one or more liquid higher alcohols having 12 to 20 carbon atoms. The liquid higher alcohol having 12 to 20 carbon atoms preferably contains one or more liquid higher alcohols having branched fatty acids as components, and more alcohols, and more preferably contains one or more liquid higher alcohols having branched fatty alcohols, and more preferably contains one or more liquid higher alcohols having 12 to 20 carbon atoms.

[0085] The silicone oil preferably contains one or more selected from linear silicones, cyclic silicones, and modified silicones, and more preferably contains one or more selected from dimethylpolysiloxane, dimethylcyclopolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, phenyl-modified silicones, and higher alcohol-modified organopolysiloxanes.

[0086] The content of the liquid oil in liquid Y, which is liquid at 20°C, is preferably 1% by mass or more and 20% by mass or less, more preferably 2% by mass or more and 18% by mass or less, and even more preferably 3% by mass or more and 16% by mass or less, from the viewpoint of excellent adhesion to the skin, abrasion resistance, stretchability and transparency of the coating.

[0087] The step of applying liquid Y to the skin may occur before or during the adhesion step, specifically while or after the application of the deposit to the skin. Means for applying liquid Y to the skin include using an applicator. Note that composition X and liquid Y have different compositions.

[0088] The fiber deposit and liquid agent Y used in the present invention may be adhesive-free. Here, adhesive refers to a component that exhibits adhesiveness on its own, and examples include various adhesives such as natural rubber-based, synthetic rubber-based, acrylic-based, silicone-based, and hot-melt-based adhesives. In the case of the fiber deposit, being adhesive-free means that the adhesive content in the fiber deposit is 0% by mass or more and 5% by mass or less, with 3% by mass or less being preferable. In the case of liquid agent Y, being adhesive-free means that the adhesive content in liquid agent Y is 0% by mass or more and 1% by mass or less, with 0.5% by mass or less being preferable.

[0089] The adhesion process of the present invention typically involves applying a deposit containing fibers with an average fiber diameter of 0.01 μm to 7 μm onto the skin, and then holding the deposit in place. From the viewpoint of adhering more skin surface material to the deposit, the holding time of the deposit is preferably 0.5 hours or more, more preferably 2 hours or more, even more preferably 3 hours or more, and even more preferably 4 hours or more. However, since the amount of skin surface material in the deposit does not increase significantly even if it is held for a certain period of time or longer, a holding time of 12 hours or less is practical.

[0090] After retaining the deposit on the skin for a predetermined period of time, the film is typically removed from the skin. The removal method is preferably done by wearing gloves and peeling the deposit off the skin with tweezers or similar tools. The removed deposit is then collected. This step allows for the collection of the deposit along with any skin surface material that had adhered to it during the retention period.

[0091] The recovered fiber deposits preferably contain hydrophilic and hydrophobic components as surface substances. Examples of hydrophilic components include proteins, nucleic acids, sugars, and amino acids. These may or may not be metabolites. Examples of hydrophobic components include sebum (lipids derived from sebaceous glands) and lipids derived from keratinocytes. Examples of lipids include triglycerides, diglycerides, monoglycerides, free fatty acids, squalene, waxes, and cholesterol. Albumin, an immune response-related protein, is a hydrophilic component, while keratin, a structural protein of the stratum corneum, is a hydrophobic component.

[0092] One method for analyzing sediment after the removal process is to examine the amount and / or type of compounds constituting the surface material contained in the collected sediment. The examination of the amount and / or type of compounds preferably includes one or more selected from genome analysis, epigenetics (e.g., methylation), transcriptome analysis (RNA expression), proteome analysis (proteomics), etc. Genomic analysis can be performed, for example, by a DNA sequencer. Methylation analysis can be performed, for example, by methylated base conversion followed by DNA sequencing. RNA expression analysis can be performed, for example, by RT-PCR analysis or RNA-seq analysis. Proteomics analysis can be performed, for example, by mass spectrometry (e.g., tandem mass spectrometry (MS / MS) or by methods using LC-MS / MS, which combines MS / MS with liquid chromatography (LC) (e.g., sequence tagging). Multi-omics analysis, which analyzes these simultaneously, may also be employed. Proteome analysis includes the analysis of post-translational modifications. Analysis of post-translational modifications includes the analysis of phosphorylation, methylation, glycosylation (including glycosylation), ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis.

[0093] Analysis may be performed using methods other than those described above. For example, for protein analysis, the Edman method or methods using peptide sequencers or protein sequencers that automate these methods may be employed, or methods that detect proteins using antigen-antibody reactions of specific proteins may be used. In addition, various component analyses can be used for sebum analysis.

[0094] In particular, in the present invention, it is preferable to include the quantity and / or type of protein as the target of analysis, in order to take advantage of the excellent capture ability of the capillary force of the deposits according to the present invention for proteins that have seeped from inside the skin to the surface. Examples of protein types include those listed above. In particular, in the present invention, it is more preferable to include the quantity and / or type of immune response-related proteins as the target of analysis.

[0095] In the present invention, the sediment recovered by the above collection method (sediment removed from the skin) may be directly subjected to analysis of the amount and / or types of compounds constituting the skin surface material. Alternatively, the skin surface material may be separated from the sediment before being subjected to analysis of the amount and / or types of compounds constituting the skin surface material. From the viewpoint of applicability to various analytical methods, it is preferable to separate the skin surface material from the recovered sediment.

[0096] When separating surface material from recovered sediment, it is preferable to dissolve the components to be analyzed in the surface material in a test solution capable of dissolving them. Any liquid used in the art can be used as the test solution. The test solution is often an aqueous solution. In this specification, an aqueous solution means a liquid containing more than 50% by mass of water, for example, 60% by mass or more is preferred, 70% by mass or more is more preferred, 80% by mass or more is even more preferred, and 90% by mass or more is particularly preferred. As the test solution, for example, an aqueous solution containing various inorganic salts, sugars, surfactants, amino acids, etc. may be used. Extracts from commercially available proteome extraction kits may also be used.

[0097] When performing mass spectrometry on proteins, proteins are generally broken down with proteolytic enzymes. However, to suppress autolysis and improve the efficiency of the breakdown, the proteins are usually thermally denatured before enzymatic decomposition. First, a deposit containing thermoplastic resin fibers is immersed in the test solution. After dissolving the proteins attached to the deposit in the test solution, the deposit and the test solution are heated to a temperature above the protein denaturation temperature. In this case, the heating temperature can be easily set above the glass transition temperature of the thermoplastic resin contained in the deposit. Therefore, heating at this temperature causes the film-like deposit to shrink, making it easier to remove only the deposit from the test solution. Thus, heating the recovered deposit before mass spectrometry of the protein allows for simultaneous protein denaturation and deformation of the deposit, which is preferable because it improves work efficiency. From the viewpoint of keeping the temperature above the protein denaturation temperature and facilitating deformation of the thermoplastic resin, a heating temperature of 60°C or higher is preferable, 70°C or higher is more preferable, and 80°C or higher is particularly preferable.

[0098] As described above, the heating temperature of the deposit is preferably above the Tg of the thermoplastic resin, from the viewpoint of shrinking the film and improving handling. For example, when the thermoplastic resin is PVB, the glass transition temperature Tg is usually 65°C or higher and less than 70°C, so the heating temperature is preferably 70°C or higher, and more preferably 80°C or higher. For example, when the thermoplastic resin is PP, the heating temperature is preferably 60°C or higher, and more preferably 70°C or higher, due to its ease of deformation. Furthermore, from the viewpoint of preventing excessive denaturation such as protein decomposition, the heating temperature is preferably 160°C or lower, more preferably 130°C or lower, and particularly preferably 100°C or lower.

[0099] When heating to a temperature (Tg) or higher, the heating time is preferably 5 seconds or more from the viewpoint of improving handling. In practice, when heating to a temperature (Tg) or higher, the heating time is 120 minutes or less.

[0100] The present invention's method for inspecting skin surface substances is preferably applied to continuously collecting skin surface substances from a subject's skin at predetermined intervals, inspecting the collected substances, and accumulating the analysis results. This allows the minimally invasive and simple nature of the present invention's inspection method to be utilized. Furthermore, it enables daily monitoring of the subject's health status in a way that places minimal physical burden on the subject. By covering the skin with a deposit of fibers, the present invention's method for inspecting skin surface substances can prevent unconsciously touching the face or other parts of the body and wiping off substances, or rubbing off substances with clothing or masks during daily life. This is effective for accumulating trace amounts of substances released from the body over a long period of time.

[0101] The method for collecting skin material of the present invention may be applied to the skin of mammals other than humans. Application to humans is preferable because it eliminates the need for pretreatment such as hair removal and is inconspicuous when applied to the skin. It is also preferable because it offers high industrial applicability due to the cosmetic effects that conventional fiber deposits have had. The person who applies the fiber deposit to the skin and collects the deposit may be the same person as the person from whom the skin material is collected, or a different person. For example, the same person may use this collection method and examination method as a means of self-diagnosis. An example of a different person is when a nurse, caregiver, etc., applies the fiber deposit to the skin of a subject and collects the deposit when they want to measure the health status of the subject.

[0102] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. Unless otherwise specified, "%" and "parts" mean "mass percent" and "parts by mass," respectively. In Experimental Example 1, the coating formed by electrostatic spraying was a deposit containing fibers. Its average fiber diameter was 1 μm, and its basis weight was 4 g / m². ２ This has been confirmed. The resin used is PVB with a Tg of 65°C.

[0103] Experimental Example 1 (Examples 1-1 to 1-3, Comparative Examples 1-1 to 1-3) Skin samples were taken from the cheeks and forehead of one subject (a woman in her 30s). On the right cheek and right forehead, (1) a film was formed consisting of fibrous deposits, and on the left cheek and left forehead, (2) an oil-absorbing film was used to attempt to collect skin surface material. In Figures 3 to 5, film-1 corresponds to the left forehead, film-2 to the left cheek, deposit-1 to the right forehead, and deposit-2 to the right cheek. The sampling from the forehead and the sampling from the cheeks were performed on different days. On the first day, the forehead was sampled using deposits and an oil-absorbing film, and on the second day, the cheeks were sampled using deposits and an oil-absorbing film. The procedure for Example 1 described below also explains the sampling from the forehead and cheeks. (1) For the formation of a film consisting of fibrous deposits, steps (a) to (c) below were performed consecutively in this order. (a) A film was formed on the skin at 6:30 and maintained for 6.5 hours until 13:00. (b) A film was formed on the skin at 13:00 and maintained for 7 hours until 20:00. (c) A film was formed on the skin at 20:00 and maintained for 9.5 hours until the next morning. In (a) to (c), after washing the face, a film consisting of fiber-containing deposits was formed on the target area (right forehead or right cheek). The film consisting of fiber-containing deposits was obtained by electrostatic spraying the target area with the composition shown in Table 1 under the following conditions. After electrostatic spraying, it was gently pressed with a puff to blend it in. On the right forehead, the area where the film consisting of fiber-containing deposits was formed covered almost the entire area of ​​half of the forehead. Similarly, on the right cheek, the area where the film consisting of fiber-containing deposits was formed covered almost the entire area of ​​the right cheek. <Electrostatic spray conditions> Voltage: 25kV Flow rate: 60mL / min Distance from nozzle to skin: 16cm Spray time: 15 seconds Room temperature 23℃, humidity 35%RH

[0104] The composition of the electrostatic spray is as follows:

[0105] At each of the following points in time, after the holding time in (a) (before (b)), after the holding time in (b) (before (c)), and after the holding time in (c), the film consisting of fibrous deposits was collected. The collector wore gloves, grasped the edge of the film with tweezers, and slowly peeled it off. This operation was repeated until the entire formed film was collected. The collected film was placed in a screw tube and stored in a deep freezer until it was to be used for pretreatment.

[0106] (2) When using oil-absorbing film, instead of forming a film consisting of fibrous deposits as in (1), after the holding time in (a) to (c) had elapsed, skin surface material was collected using an oil-absorbing film (5 cm x 8 cm, manufactured by 3M) in the following manner. The collector wore gloves. The target area (left forehead or left cheek) was rubbed evenly with one oil-absorbing film to collect skin surface material. The oil-absorbing film was placed in a screw-top tube and stored in a deep freezer until ready for pretreatment. The area rubbed with the oil-absorbing film on the left cheek and left forehead was equivalent to the area where a film consisting of fibrous deposits formed on the right cheek and right forehead, respectively. The subjects did not have any restrictions on their activities, including skincare, makeup, or mask-wearing, and lived their lives as usual.

[0107] (Protein extraction (albumin / keratin area ratio, number of identified proteins)) Proteins were extracted from the obtained coatings and oil-absorbing films by the following method. For each of the obtained coatings and oil-absorbing films, the peeled portion from the cheek or forehead for the coatings, and the film used to rub the cheek or forehead for the oil-absorbing films, were placed in 0.3 mL of the extraction solution (12 mmol / L Sodium deoxycholate, 12 mmol / L Sodium lauroyl sarcosinate, 100 mmol / L Tris-HCl (pH 9.0) aqueous solution) and heated at 95°C for 5 minutes. After that, sonication was performed in ice water for 20 minutes. 3000 g was centrifuged at 4°C for 5 minutes, and the supernatant was collected to separate the coatings that had shrunk due to heating. 0.2 mL of the supernatant was transferred to another tube, and 8.7 μL of TCEP (Tris(2-carboxyethyl)phosphine) and 8.7 μL of S-Methyl Methanethiosusponate were added for reductive alkylation treatment. Then, a 27.5% aqueous phosphoric acid solution was added to achieve a final concentration of 2.5%. Each sample was then treated with S-Trap. TM The samples were loaded onto micro columns (ProtiFi). After loading, the samples were washed with 90% methanol / 10% 1 mol / L TEAB (Triethylammonium bicarbonate) aqueous solution and 50% chloroform / 50% methanol. Next, trypsin and Lys-C were added, and the samples were enzymatically digested by incubation at 37°C overnight. 40 μL of 50 mmol / L TEAB aqueous solution, 40 μL of 0.2% formic acid aqueous solution, and 40 μL of 50% acetonitrile aqueous solution were added sequentially to elute the digested peptides. The eluates from each stage were pooled together and dried by vacuum centrifugation. After desalting using GL-Tip SDB (GL Sciences Co., Ltd.), the samples were dried by vacuum centrifugation. The samples were kept at -80°C until further analysis.

[0108] The digested peptides described above were dissolved in 30 μL of a 2% acetonitrile aqueous solution containing 0.1% formic acid. The peptide concentration was calculated using liquid chromatography with UV detection (LC-UV, measurement conditions the same as in Experimental Example 2), and the amount of recovered protein was measured.

[0109] Liquid chromatography-mass spectrometry (LC-MS) was used for peptide analysis. The liquid chromatography conditions were as follows: Solvent of sample solution: 2% acetonitrile aqueous solution containing 0.1% formic acid Mobile phase: A) 0.1% formic acid aqueous solution, B) 80% acetonitrile aqueous solution containing 0.1% formic acid Desalting column: Acclaim PepMap 100 Nano Trap C18 nano Viper (inner diameter 75 μm, length 20 mm, particle size 3 μm) manufactured by Thermo Fisher Scientific Separation column: Acclaim PepMap 100 RSLC, C18 (inner diameter 75 μm, length 150 mm, particle size 2 μm) manufactured by Thermo Fisher Scientific Elution rate: 0.3 μL / min Solvent gradient conditions: B 5% (0-5 min) → B50% (125 min) → B95% (126-150 min) → auto-calibration B95% (155 min) → B5% (156-180 min) Column temperature: 40°C Peptide injection volume: 0.3 μg Peptides were eluted from the column to a mass spectrometer by liquid chromatography under the above conditions.

[0110] The following equipment and conditions were set up for mass spectrometry: Emitter: New Objective Pico Tip NanoSpray Emitter FS360-50-15-N Mass Spectrometer: Thermo Fisher Scientific Orbitrap Q Exactive Plus Mode: nano ESI

[0111] Software for processing and analyzing mass spectrometry data: Proteome Discoverer (Thermo Fisher Scientific) Analysis database: Swiss-prot (Homo sapience) (Other settings: Number of cleavage errors allowed: 2, Variable modification: Oxidation (M), Fixed modification: Methylchio (C), Digestive enzyme: Trypsin, Search engine: Mascot (Matrix Science), Peptide identification threshold: FDR < 5%) Under the above conditions, a database search was performed, and the peak area values ​​of each identified protein were normalized to the total peak area value of the identified protein. Using the normalized peak area values, the albumin / keratin-10 ratio was calculated.

[0112] The results are shown in Figures 3 to 5.

[0113] (a) is designated as Example 1-1 and Comparative Example 1-1, (b) as Example 1-2 and Comparative Example 1-2, and (c) as Example 1-3 and Comparative Example 1-3, and they are summarized in Table 2 below. The long-term adhesion, peelability, and appearance of the samples in Table 2 were evaluated according to the following criteria.

[0114] (Long-lasting adhesion) Indicates the duration of adhesion. When the long-lasting adhesion time (adhesion duration) is equal to the retention time, it indicates that the patch adheres to the skin for the set retention time. If it is 0, it indicates that the patch does not adhere at all.

[0115] (Peelability) Peelability was evaluated by whether or not the sheet tore when peeled, and if so, how many pieces it broke into. A value of 1 indicates that there is only one sheet, no splitting, and the best peelability.

[0116] (Appearance) Examples 1-1 to 1-3 were prepared by forming deposits on commercially available nylon film using electrospinning. Comparative Examples 1-1 to 1-3 were prepared by layering oil-absorbing film onto commercially available nylon film. The transmittance (%T) of the prepared samples was measured using a spectrophotometer (U-3310, Hitachi, Ltd., wavelength 600 nm). The measurement was performed by cutting the deposit or oil-absorbing film (object) of the above sample into 20 mm x 40 mm pieces and setting it in the cell holder of the spectrophotometer so that the light from the light source was perpendicular to the object. A transmittance of 20% or more was evaluated as transparent, and a transmittance of less than 20% was evaluated as opaque.

[0117]

[0118] Figure 3 shows the total peak area of ​​albumin-derived peptides extracted from sediment or oil-absorbing film. Albumin is an example of an immune response protein present in interstitial fluid, and it can be seen that more albumin can be obtained from fibrous sediment than when using oil-absorbing film. Furthermore, as shown in Figure 4 and Table 2, the peak area ratio of keratin-10 to albumin (ALB / KRT10) was also higher when forming fibrous sediment than when using oil-absorbing film. Keratin-10 is a protein that makes up the stratum corneum and is not a skin surface substance that exists on top of the stratum corneum. Therefore, it can be seen that according to the present invention, albumin, which is a skin surface substance, can be selectively obtained from stratum corneum constituent proteins such as keratin-10, and non-invasively from the skin. Figure 5 and Table 2 show the measurement results of the number of identified proteins. It can be seen that a larger number of types of proteins can be obtained by the present invention than when using oil-absorbing film. In other words, according to the present invention, since albumin can be selectively obtained and a large number of types of proteins can be obtained, skin surface materials can be efficiently collected. Furthermore, as can be seen from Table 2, the present invention has excellent long-term adhesion properties and excellent appearance when applied by using a deposit of specific fibers.

[0119] Experimental Example 2 (Examples 2-1 to 2-6, Comparative Examples 2-1, 2-2) Experimental Example 2 was conducted with one different subject (one female in her 30s) from Experimental Example 1.

[0120] The sample extraction and liquid chromatography conditions were as follows: <Sample Extraction Conditions> Proteins were extracted from the obtained coatings and oil-blotting films using the following method. For the obtained coatings and oil-blotting films, the peeled portion from the cheek for the coatings and the film used to rub the cheek for the oil-blotting films were combined and placed in 0.5 mL of the test extraction solution (5% Sodium n-Dodecyl Sulfate, 1 mol / L TEAB (pH 8.5) aqueous solution) and heated at 95°C for 5 minutes. After that, sonication was performed in ice water for 20 minutes. 3000 g was centrifuged at 4°C for 5 minutes, and the supernatant was collected to separate the coatings that had shrunk due to heating. 0.2 mL of the supernatant was transferred to another tube, 8.7 μL of TCEP and 8.7 μL of iodoacetamide were added for reductive alkylation, and then methanol, chloroform, and ultrapure water were added in that order, stirred and mixed, and then centrifuged. Each sample was placed in an S-Trap TM The samples were loaded onto micro columns (ProtiFi). After loading, the samples were washed with 90% methanol / 10% 1 mol / L TEAB aqueous solution and 50% chloroform / 50% methanol. Next, trypsin and Lys-C were added, and the samples were enzymatically digested by incubation at 37°C overnight. 40 μL of 50 mmol / L TEAB aqueous solution, 40 μL of 0.2% formic acid aqueous solution, and 40 μL of 50% acetonitrile aqueous solution were added sequentially to elute the digested peptides. The eluates from each stage were pooled together and dried by vacuum centrifugation. After desalting using GL-Tip SDB (GL Sciences Co., Ltd.), the samples were dried by vacuum centrifugation. Finally, 30 μL of 2% acetonitrile aqueous solution containing 0.1% formic acid was added to dissolve the samples, and the peptide concentration was measured.

[0121] <Liquid Chromatography Conditions> The following conditions were used for LC-UV analysis. Mobile phase: A) 0.1% formic acid aqueous solution, B) 80% acetonitrile aqueous solution containing 0.1% formic acid Desalting column: Thermo Fisher Scientific Acclaim PepMap 100 Nano Trap C18 nano Viper (inner diameter 75 μm, length 20 mm, particle size 3 μm) Separation column: Thermo Fisher Scientific Acclaim PepMap 100 RSLC, C18 (inner diameter 75 μm, length 150 mm, particle size 2 μm) Elution rate: 0.3 μL / min Solvent gradient conditions: B5% (0-5 min) → B50% (20 min) → B95% (21-45 min) → B5% (46-60 min) Column temperature: 40°C Sample injection volume: 1 μL Detection wavelength: UV 214 nm

[0122] (Example 2-1) Instead of spraying the electrostatic spray directly onto the skin as in Example 1-1, 100% rayon, basis weight 80 g / m² was applied to a metal collection plate. ２ A nonwoven fabric (manufactured by Ikeda Paper Industry Co., Ltd.) was placed as a base material, and a coating consisting of fiber deposits was formed on one surface. The electrostatic spraying conditions were changed as follows, and the basis weight of the resulting coating was 2.5 g / m². ２ The method was changed to the following. The obtained film was cut into a circle with a diameter of 2.5 cm. Instead of forming a film by electrostatic spraying the composition, this film was applied to the cheek of the subject. After applying the film, a cosmetic emulsion (Est Biomimesis Veil Effector, manufactured by Kao Corporation) was applied over the film. This cosmetic emulsion contained water, polyol, and an oil that is liquid at 20°C. The retention time was 2 hours. Except for these points, the procedure was the same as in Example 1-1, and the deposited film, along with the skin surface material, was collected non-invasively from the skin. <Electrostatic spraying conditions> Voltage: 25 kV Flow rate: 60 mL / min Distance from nozzle to base nonwoven fabric: 16 cm Room temperature: 23°C, humidity: 35% RH In Experimental Example 2-1, 5.4 μg of protein was obtained from one film. Therefore, it was possible to inject 0.5 μg of peptide, which is necessary for LC-MS under the above conditions.

[0123] (Example 2-2) In the same test as in Example 2-1, the basis weight of the coating was 0.4 g / m². ２ The method was changed to non-invasively collect the deposit, which is a film on the skin, along with the surface material.

[0124] (Example 2-3) In the same test as in Example 2-1, the holding time in the state where the coating was formed was changed to 1 hour. The deposit, which was the coating, was collected along with the skin surface material in a non-invasive manner.

[0125] (Example 2-4) In the same test as in Example 2-1, instead of forming a film by electrostatic spraying of the composition, a meltblown nonwoven fabric was attached to the forehead of the subject. The basis weight of the meltblown nonwoven fabric was 3 g / m². ２ The average fiber diameter of the constituent fibers was 1 μm. Meltblown nonwoven fabric was used, cut into circles with a diameter of 2.5 cm. The retention time for the coating was set to 5 hours. The coating, which was a deposit, was collected along with the skin surface material in a non-invasive manner.

[0126] (Example 2-5) In the same test as in Example 2-1, the retention time of the coating was set to 6.5 hours. The coating, which was a deposit, was collected along with the skin surface material in a non-invasive manner.

[0127] (Comparative Examples 2-1, 2-2) The same tests as in Experimental Example 1 (2) using an oil-absorbing film were conducted. That is, in the same manner as in Experimental Example 1 (2), after washing the face, the left cheek of the subject was rubbed evenly with one oil-absorbing film to collect surface skin material. The obtained oil-absorbing film was collected in the same manner as in Experimental Example 1 (2), and protein extraction and analysis were performed. However, in this example, the time from washing the face to wiping with the oil-absorbing film was set to 2 hours and 6.5 hours. In Comparative Example 2-1, only 0.54 μg of protein was obtained from the film used in one collection, and in Comparative Example 2-2, only 0.39 μg of protein was obtained. Therefore, it was difficult to inject the same amount of peptide as the coating using LC-MS under the above conditions. The adhesion, peelability, and appearance of the deposits in Examples 2-1 to 2-5 and Comparative Examples 2-1 and 2-2 were evaluated using the above method. Furthermore, the amount of recovered protein was determined for Examples 2-1 to 2-5 and Comparative Examples 2-1 and 2-2 using the method described above. The results are shown in Table 3. From Table 3, it can be seen that the amount of recovered protein was high in the examples, and that more protein can be obtained by the present invention than when using an oil-absorbing film.

[0128]

[0129] (Experimental Example 3) (Comparative Example 3-1) A spunbond nonwoven fabric made of PP was used. The basis weight of the spunbond nonwoven fabric was 17 g / m². ２ The average fiber diameter of the constituent fibers was 15 μm. The spunbond nonwoven fabric was cut to 3 cm x 3 cm. In a test similar to that of Example 1-1, instead of forming a film by electrostatic spraying of the above composition, this spunbond nonwoven fabric was attached to the right cheek of the subject, and the same test was performed. The nonwoven fabric fell off the skin after about 5 minutes, indicating that it could not be attached for a long period of time.

[0130] (Example 3-1) In the same test as in Example 2-1, the basis weight of the coating was 50 g / m². ２The method was changed to the following. The capsule was also applied to the forehead of the subject. The peptide injection amount was 0.055 μg. The capsule, which is a deposit, was collected along with the skin surface material in a non-invasive manner. Table 4 shows that protein can be collected from the forehead, that the present invention has excellent long-term adhesion properties by using a deposit of specific fibers, and that it also has excellent appearance when applied.

[0131]

[0132] According to the present invention, a method for efficiently collecting skin surface substances, which are endogenous substances present on the stratum corneum of the skin, can be provided that allows for long-term application. By analyzing the skin surface substances obtained using the present invention, it becomes possible to monitor the health status.

Claims

1. A method for collecting surface skin matter, comprising: an adhesion step of applying a deposit containing fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less to the skin and holding it for 0.5 hours or more to cause surface skin matter, which is a substance of internal origin present on the stratum corneum of the skin, to adhere to the deposit; and a removal step of removing the deposit from the skin after the adhesion step, wherein the deposit with the surface skin matter attached is recovered after the removal step.

2. The method for collecting a skin surface material according to claim 1, wherein the fibers include a thermoplastic resin.

3. The basis weight of the aforementioned sediment is 0.1 g / m². ２ 50g / m or more ２ The method for collecting skin surface material according to claim 1 or 2, which is as follows:

4. A method for collecting skin surface material according to any one of claims 1 to 3, wherein a liquid agent containing one or more selected from water, polyol, and oils that are liquid at 20°C is applied to the skin before or during the adhesion process.

5. A method for collecting a skin surface substance according to any one of claims 1 to 4, wherein the skin surface substance contains a hydrophilic component and a hydrophobic component.

6. A method for collecting a skin surface material according to any one of claims 1 to 5, wherein the attachment step is performed by performing a step selected from (I-1) and (I-2) below: (I-1) A step of electrostatically spraying a composition containing one or more volatile substances selected from water, alcohol and ketones and a polymer having fiber-forming ability onto the skin. (I-2) A step of applying a deposit of fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less, obtained by electrospinning or melt-blown, to the skin.

7. The method for collecting a skin surface substance according to any one of claims 1 to 6, wherein the skin surface substance comprises one or more selected from proteins, nucleic acids, and lipids.

8. The method for collecting skin surface material according to claim 7, wherein the protein comprises one or more selected from immune response proteins, natural moisturizing factor-producing enzymes, ceramide metabolic enzymes, stratum corneum exfoliating proteases, oxidoreductases, desmosome constituent proteins and other plasma-derived proteins, the nucleic acid comprises DNA and / or RNA, and the lipid comprises lipids derived from sebaceous glands and / or lipids derived from keratinocytes.

9. The method for collecting skin surface material according to claim 6, wherein the volatile substance comprises one or more selected from ethanol, isopropyl alcohol, butyl alcohol, and water, preferably comprising one or more selected from ethanol and butyl alcohol, and more preferably comprising ethanol.

10. The method for collecting skin surface material according to claim 6, wherein the polymer is soluble in the volatile substance.

11. The method for collecting skin surface material according to any one of claims 1 to 10, wherein the skin to which the deposit is applied is one or more locations selected from the face, neck, hands, feet, back, shoulders, abdomen, chest, legs, and arms.

12. The method for collecting surface skin material according to any one of claims 1 to 11, wherein the average fiber diameter of the fibers contained in the sediment is 0.05 μm or more and 5 μm or less, preferably 0.1 μm or more and 3 μm or less.

13. The areal weight of the deposit is 0.4 g / m ２ or more and 40 g / m ２ or less, preferably 0.5 g / m ２ or more and 30 g / m ２ or less, more preferably 0.8 g / m ２ or more and 10 g / m ２ or less, still more preferably 1 g / m ２ or more and 10 g / m ２ or less. The method for collecting skin surface substances according to any one of claims 1 to 12.

14. The method for collecting a skin surface substance according to any one of claims 1 to 13, wherein the fiber mainly comprises a synthetic resin.

15. The method for collecting skin surface material according to claim 14, wherein the synthetic resin is a water-soluble or water-insoluble thermoplastic resin, the water-soluble thermoplastic resin preferably comprises one or more selected from substantially non-crosslinked partially saponified polyvinyl alcohol, water-soluble nylon, polyvinylpyrrolidone and polyethylene oxide, and the water-insoluble thermoplastic resin preferably comprises one or more selected from polyvinyl acetal, polyester, acrylic resin, polystyrene resin, polyvinyl butyral resin, polyurethane resin, polyamide resin, polyimide resin, polyamide, polyamideimide resin, olefin resin, fully saponified polyvinyl alcohol and crosslinked partially saponified polyvinyl alcohol.

16. The method for collecting skin surface material according to any one of claims 1 to 15, wherein the fiber comprises a thermoplastic resin having a glass transition temperature Tg of 45°C or more and 150°C or less, preferably a thermoplastic resin having a glass transition temperature Tg of 50°C or more and 120°C or less, and more preferably a thermoplastic resin having a glass transition temperature Tg of 50°C or more and 100°C or less.

17. A method for collecting skin surface material according to any one of claims 1 to 16, wherein the deposit is applied to the skin without using an adhesive.

18. A method for collecting surface material from the skin according to any one of claims 1 to 17, wherein the thickness of the deposit is 1 μm or more and 300 μm or less, preferably 1 μm or more and 100 μm or less, more preferably 1 μm or more and 20 μm or less, and particularly preferably 3 μm or more and 20 μm or less.

19. A method for collecting skin surface material according to any one of claims 1 to 18, which is non-invasive to the skin.

20. A method for collecting a skin surface substance according to any one of claims 1 to 19, wherein the time for which the deposit is retained on the skin is 2 hours or more and 12 hours or less, preferably 3 hours or more and 12 hours or less, more preferably 4 hours or more and 12 hours or less.

21. A method for collecting surface material according to any one of claims 1 to 20, comprising the step of separating surface material from the recovered sediment.

22. A method for inspecting skin surface material obtained by the sampling method described in any one of claims 1 to 21, comprising the step of separating the skin surface material from the recovered sediment.

23. The method for inspecting skin surface material according to claim 22, wherein the skin surface material is separated from the deposit by immersing the deposit in an inspection solution and extracting the skin surface material.

24. A method for inspecting a skin surface according to claim 22 or 23, wherein the constituent fibers of the deposit contain a thermoplastic resin, and the method comprises heating the deposit at a temperature between the glass transition temperature of the thermoplastic resin and 160°C, preferably between 60°C and 100°C.

25. A method for inspecting a skin surface according to any one of claims 22 to 24, wherein the constituent fibers of the deposit contain a thermoplastic resin, the thermoplastic resin contains polyvinyl butyral, and the temperature at which the deposit is heated is 70°C or more and 100°C or less, preferably 80°C or more and 100°C or less.

26. A method for inspecting a skin surface according to any one of claims 22 to 25, wherein the constituent fibers of the deposit contain a thermoplastic resin, the thermoplastic resin contains polypropylene, and the temperature at which the deposit is heated is 60°C or more and 100°C or less, preferably 70°C or more and 100°C or less.

27. The method for inspecting a surface material according to any one of claims 22 to 26, wherein the time for heating the deposit is 5 seconds or more and 120 minutes or less.

28. A method for inspecting a skin surface according to any one of claims 22 to 27, comprising inspecting the amount and / or type of compounds contained in the skin surface.

29. The method for examining a skin surface according to claim 28, wherein the examination of the amount and / or type of the compound comprises one or more selected from genome analysis, epigenetics, transcriptome analysis and proteome analysis.

30. A method for examining a skin surface according to claim 28 or 29, wherein the examination of the amount and / or type of the compound comprises protein analysis.

31. A method for monitoring a person's health status, comprising using a skin sample obtained by the sampling method described in any one of claims 1 to 21, or using the examination method described in any one of claims 22 to 30, wherein a skin sample is continuously collected from a subject's skin at predetermined intervals, the collected sample is examined, and the analysis results are accumulated.

32. The method for monitoring health status according to claim 31, wherein the subject of monitoring is a human being.

33. A fibrous deposit used to collect skin surface material, which is an endogenous substance present on the stratum corneum of the skin, and which contains fibers with an average fiber diameter of 0.01 μm or more and 7 μm or less.

34. The deposit according to claim 33, wherein the deposit is a single layer sheet, is laminated on a substrate to form a laminated sheet, or is provided onto the skin from an electrostatic spray device.

35. The deposit according to claim 33, wherein the fibers are obtained by electrospinning or meltblowing.

36. A kit comprising a sediment according to any one of claims 33 to 35 and an inspection liquid for immersing the sediment.

37. A test kit comprising a deposit according to any one of claims 33 to 35 and a liquid agent, wherein the test kit is used to apply the deposit to the skin, hold it for 0.5 hours or more to allow skin surface substances, which are internally derived substances present on the stratum corneum of the skin, to adhere to the deposit, then remove the deposit from the skin and collect the removed deposit, wherein the liquid agent is applied to the skin before or while the skin surface substances are attached to the deposit, and the liquid agent is a liquid agent containing one or more selected from water, polyols, and oils that are liquid at 20°C.

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