WO2021070778A1

WO2021070778A1 - Nanodisc

Info

Publication number: WO2021070778A1
Application number: PCT/JP2020/037729
Authority: WO
Inventors: 恵広柳澤; 忠夫辻; 井村　知弘; 敏彰平
Original assignee: 株式会社カネカ; 国立研究開発法人産業技術総合研究所
Priority date: 2019-10-10
Filing date: 2020-10-05
Publication date: 2021-04-15

Abstract

The purpose of the present invention is to provide: a nanodisc that can be produced easily, that makes it possible to suitably disperse a hydrophobic compound or the like in water, and that has excellent transparency and stability; and a cosmetic containing the nanodisc. A nanodisc according to the present invention includes a lipid bilayer and a lipopeptide biosurfactant and is characterized in that the lipid bilayer includes a lecithin and a lysolecithin and the ratio of the lysolecithin to the total of the lecithin and the lysolecithin is 1-20 mass%.

Description

Nanodisc

The present invention relates to nanodiscs which can be easily produced, which can disperse hydrophobic compounds and the like well in water and have excellent transparency and stability, and cosmetics containing the nanodiscs. ..

Many of the active ingredients contained in personal care compositions such as cosmetics and pharmaceutical compositions are hydrophobic and difficult to disperse in aqueous solvents. Organic solvents may be used to disperse hydrophobic compounds, but even ethanol and isopropanol may be reduced or avoided in personal care and pharmaceutical compositions applied to the human body. Tend to do. Therefore, especially in personal care compositions, surfactants are often used to disperse hydrophobic compounds in aqueous solvents.

However, it may not be possible to sufficiently disperse the hydrophobic compound simply by adding a surfactant. Therefore, various techniques for enhancing the dispersibility of hydrophobic compounds have been developed.

For example, it is known that when the concentration of a surfactant in an aqueous solution increases, micelles or vesicles in which the hydrophilic portions of the surfactant are aggregated facing outward are formed, and the hydrophobic portion inside thereof is hydrophobic. It is known that a hydrophobic compound can be dispersed by encapsulating the compound. Vesicles formed of phospholipids are called liposomes. In the invention described in Patent Document 1, a water-soluble polymer is further blended to form a liposome having a multi-membrane structure, and membrane formation efficiency and membrane stability are improved. Further, Patent Document 2 and Patent Document 3 disclose liposomes containing phospholipids and lysophospholipids.

However, the size of the liposome is relatively large, and the dispersion liquid of the liposome is usually cloudy. Therefore, in recent years, very fine particles called nanodisks having a lipid bilayer structure similar to that of liposomes have been developed.

The nanodisc has a structure in which the hydrophobic side of the disc composed of a lipid bilayer is coated with an amphipathic membrane scaffold protein (Non-Patent Documents 1, Patent Documents 4 and 5), and is a layer of nanodiscs. The thickness is several nm and the diameter is less than 10 nm, which is very fine. Patent Document 6 also discloses a composition containing a lipid and a surfactant, which is a polymer aggregate having a diameter of less than 100 nm. Therefore, the dispersion liquid of nanodisc is very transparent. In addition, since the lipid bilayer membrane of nanodiscs is similar to the lipid bilayer membrane of cells, it is considered that the membrane proteins contained in nanodiscs retain the structure and activity of the cell membranes as they are. It is expected that research on membrane proteins will progress dramatically. The present inventors have also developed a nanodisk characterized by containing a lipid bilayer and lipopeptide biosurfactant (Patent Document 7).

Japanese Unexamined Patent Publication No. 2008-94809 Japanese Unexamined Patent Publication No. 2008-127327 Japanese Unexamined Patent Publication No. 2007-269635 Special Table 2007-525490 Special Table 2016-504312 Special Table 2010-51103A International Publication No. 2018/181538 Pamphlet

As described above, fine nanodiscs containing a lipid bilayer have been developed, and it is considered that hydrophobic compounds and the like can be satisfactorily dispersed in an aqueous solvent by using nanodiscs.
However, although nanodiscs are expected to be applied to cosmetics having excellent transparency, the transparency may decrease over time and precipitation may occur in some cases.
Therefore, the present invention provides nanodiscs which can be easily produced, can disperse hydrophobic compounds and the like well in water, and have excellent transparency and stability, and cosmetics containing the nanodiscs. The purpose is.

The present inventors have conducted intensive studies to solve the above problems. As a result, they have found that by using lipopeptide biosurfactant as a surfactant and using a specific ratio of lecithin and lysolecithin as lipids, nanodiscs having excellent transparency and stability can be easily produced, and the present invention has been completed. did.
Hereinafter, the present invention will be shown.

[1] Contains lipid bilayer and lipopeptide biosurfactant
The lipid bilayer contains lecithin and lysolecithin,
A nanodisk characterized in that the ratio of the lysolecithin to the total of the lecithin and the lysolecithin is 1% by mass or more and 20% by mass or less.
[2] The nanodisc according to the above [1], wherein the lipopeptide biosurfactant is bound as a membrane scaffold protein to the side surface of the lipid bilayer membrane.
[3] The nanodisc according to the above [1] or [2], which has a thickness of 2 nm or more and 10 nm or less.
[4] The nanodisc according to any one of the above [1] to [3], which has a diameter of 6 nm or more and 40 nm or less.
[5] The nanodisc according to any one of the above [1] to [4], wherein the ratio is 10% by mass or less.
[6] The nanodisc according to any one of the above [1] to [5], wherein the lipopeptide biosurfactant is a surfactin represented by the following formula (I) or a salt thereof.

[During the ceremony,
X indicates an amino acid residue selected from leucine, isoleucine and valine;
R ¹ represents a C _9-18 alkyl group]
[7] The nanodisc according to any one of the above [1] to [6], which further contains a hydrophobic compound.
[8] The nanodisc according to any one of the above [1] to [7], which further contains a hydrophilic compound.
[9] A cosmetic comprising the nanodisc according to any one of the above [1] to [8].

Conventionally, membrane scaffold protein has been used for manufacturing nanodiscs, but it took a lot of time and effort to design and manufacture membrane scaffold protein. On the other hand, the lipopeptide biosurfactant, which is the surfactant used in the present invention, is produced by a microorganism and can be produced relatively easily. Further, the nanodisc according to the present invention can be manufactured by a very simple method as compared with the conventional nanodisk which could not be manufactured without going through a complicated process. Further, by using the nanodisc according to the present invention, it is possible to satisfactorily disperse the hydrophobic compound in an aqueous solvent to obtain a visually transparent composition, and the transdermal properties of the hydrophobic compound are remarkably improved. There is a possibility of doing so. In addition, the nanodisk of the present invention, which has a lipid bilayer structure similar to that of cells, may significantly improve the transdermal properties of hydrophilic compounds existing in the vicinity thereof. In addition, the nanodisc of the present invention has excellent stability and can maintain the transparency of the preparation for a long period of time. Therefore, the present invention is industrially excellent as a technique that contributes to the development of personal care compositions and pharmaceutical compositions having excellent properties.

FIG. 1 is an external photograph of the lipid preparation according to the present invention after a stability test. FIG. 2 is an external photograph of a lipid preparation outside the scope of the present invention after a stability test.

The nanodisc according to the present invention contains a lipid bilayer and lipopeptide biosurfactant.

A general nanodisc is stabilized by a structure in which the hydrophobic part of the amphipathic membrane scaffold protein is bound to the side hydrophobic part of the lipid bilayer to cover it, and the hydrophilic part faces outward. There is. On the other hand, the nanodisc according to the present invention is stabilized by a structure in which the hydrophobic portion of the lipopeptide biosurfactant is bound to the side hydrophobic portion of the lipid bilayer to cover it and the hydrophilic portion faces outward. Conceivable. Further, it is also known that a cyclic peptide is pierced into a lipid bilayer as an ion channel, or a lipid having a bulky hydrophobic portion and a surface active compound having a bulky hydrophilic portion form a bilayer. Therefore, even in the nanodisc according to the present invention, it is possible that some lipopeptide biohydrophobics form a bilayer together with lipids.

The size of the nanodisk according to the present invention may be 2 nm or more and 10 nm or less, and the diameter may be 5 nm or more and 40 nm or less. The thickness is preferably 3 nm or more, more preferably 4 nm or more, preferably 8 nm or less, and more preferably 6 nm or less. The diameter is preferably 6 nm or more, more preferably 7 nm or more, preferably 20 nm or less, and more preferably 15 nm or less.

The phospholipid constituting the lipid bilayer is a compound having a hydrophilic phosphate base and a hydrophobic tail, and in the present invention, lecithin and lysolecithin are used in combination.

Lecithin is a general term for lipid products containing phospholipids, and is produced by separating and drying components that precipitate by adding water to raw materials containing phospholipids, such as soybean oil and egg yolk, or by extracting with ethanol or the like. Lecithin made from soybeans is called soybean lecithin, and lecithin made from egg yolk is called egg yolk lecithin. The components constituting lecithin are not particularly limited, and examples thereof include glycerophospholipids, sphingolipids, glycolipids, synthetic lipids, and sterols.

Glycerophospholipids are double-stranded phospholipids having a complex structure of two fatty acids, glycerin, phosphate and choline, such as dipalmitoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DLPC), and dipalmitoylphosphatidylcholine (DMPC). ), Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-stearoyl-2-myristoylphosphatidylcholine, dilinoleylphosphatidylcholine and the like.

Examples of the glycerophospholipid include a glycerophospholipid represented by the following formula (II).

[In the formula, R ² and R ³ independently represent a C _10-24 alkyl group or a C _10-24 alkenyl group]

In the above formula (II) _{, examples of the C 10-24} alkyl group include n-decyl, 8-methylnonade, n-undecyl, 9-methyldecyl, n-dodecyl, 10-methylundecyl, n-tridecyl, 11-. Examples thereof include methyldodecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecylic, n-icosyl, n-docosyl, n-tetracosyl and the like. Examples of the C _10-24 alkenyl group include decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, icosenyl, docosenyl, tetracosenyl and the like.

Sphingolipid has a structure in which phosphoric acid and a base are bound to ceramide in which a fatty acid is amide-bonded to sphingoid. That is, the sphingolipid is a double-chain phospholipid having a long-chain hydrocarbon group derived from sphingosine and a long-chain hydrocarbon group derived from a long-chain fatty acid. Examples of sphingolipids include sphingomyelin.

Examples of glycolipids include mannosyl erythritol lipid, sophorolipid, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride, galactosyl cerebroside, lactosyl cerebroside, and ganglioside.

Examples of sterols include animal-derived sterols such as cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols such as stigmasterol, cytosterol, campesterol, and brushcasterol (phytosterols); thymosterol. , Sterols derived from microorganisms such as ergosterol and the like.

Lysolecithin is a single-stranded phospholipid that has only one hydrophobic tail, whereas major lecithin such as glycerophospholipids and sphingolipids are double-stranded phospholipids that have two hydrophobic tails. is there. Lysolecithin is produced, for example, by treating glycerophospholipids and sphingolipids with phospholipase A2 to remove one of the hydrophobic tails. Examples of lysolecithin include lysophosphatidylcholine having a structure in which the fatty acid at the 2-position of phosphatidylcholine is removed by phospholipase A2, lysophosphatidic acid having a structure in which choline is removed from lysophosphatidylcholine, sphingosine monophosphate, sphingosine phosphorylcholine and the like. Can be mentioned.

Lecithin and lysolecithin generally contain a carbon-carbon double bond in the long-chain fatty acid moiety, but lecithin and lysolecithin used in the present invention may be saturated hydrogenated additives to which hydrogen is added or unsaturated. A mixture of mold and saturated mold may be used.

The lipid bilayer of the nanodisc according to the present invention is mainly composed of lecithin and lysolecithin. A general lipid bilayer is mainly composed of double-stranded phospholipids, which are planar because they have a well-balanced hydrophilic and hydrophobic parts and occupy an elongated rectangular parallelepiped space. It has the property of easily forming an infinite aggregate. In general nanodiscs, it is considered that a membrane scaffold protein, which is a polymer, forms a disc state by surrounding the lipid bilayer. The present inventors considered that in the nanodisc in the present invention, the role played by the membrane scaffold protein, which is a polymer, is played by the molecular aggregate formed by the lipopeptide biosurfactant. In this way, since the shape is maintained by the molecular aggregate instead of the polymer, stabilization is necessary for practical use, and it was considered that the addition of the third component is effective for that purpose. Here, since lysolecithin is a single chain, the hydrophobic part is small with respect to the hydrophilic part, the shape is different from lecithin, and it fills the intermolecular gap that causes the instability of nanodiscs. It was thought that nanodiscs could be stabilized by using an appropriate amount of lysolecithin together. Therefore, in the present invention, the ratio of lecithin to lysolecithin is adjusted to improve the stability of nanodiscs.

Specifically, in the lipid bilayer of the nanodisk according to the present invention, the ratio of lysolecithin to the total of lecithin and lysolecithin is 1% by mass or more and 20% by mass or less. By adjusting the ratio within this range, as shown in the results of the examples described later, the nanodisc stabilizing effect can be obtained by using lysolecithin in combination with lecithin. As the above ratio, 1.5% by mass or more is preferable, 2% by mass or more is more preferable, 3% by mass or more is further preferable, 15% by mass or less is preferable, and 10% by mass or less is more preferable.

The total amount of lecithin and lysolecithin can be appropriately adjusted within the range in which nanodiscs can be satisfactorily produced. For example, the total amount of lecithin and lysolecithin with respect to the solvent containing the nanodisk of the present invention can be 0.01% by mass or more and 10% by mass or less.

Lipopeptide biosurfactant has a hydrophobic group and a peptide containing a hydrophilic moiety, exhibits a surface-active effect, and is produced by a microorganism. Examples of the lipopeptide biosurfactant include surfactin, arslovactin, itulin, fendisin, serawettin, leikecin, and viscocin.

As the lipopeptide biosurfactant, surfactin represented by the above formula (I) or a salt thereof (hereinafter referred to as "surfactin (I)") is preferable. In formula (I), the roots of the carboxymethyl group and the carboxyethyl group represent the optically active sites.

X represents any one amino acid residue selected from leucine, isoleucine and valine. The amino acid residue as X may be L-form or D-form, but L-form is preferable.

R ¹ represents a C _9-18 alkyl group. Here, the "C _9-18 alkyl group" refers to a linear or branched monovalent saturated hydrocarbon group having 9 or more and 18 or less carbon atoms. For example, n-nonyl, 6-methyloctyl, 7-methyloctyl, n-decyl, 8-methylnonyl, n-undecyl, 9-methyldecyl, n-dodecyl, 10-methylundecyl, n-tridecyl, 11-methyldodecyl. , N-tetradecyl, n-pentadecylic, n-hexadecyl, n-heptadecyl, n-octadecyl and the like.

The above-mentioned surfactin (I) may be used alone or in combination of two or more. For example, the C _9-18 ^{alkyl group of R 1} may contain a plurality of different surfactins (I).

Surfactin (I) can be used by culturing a microorganism, for example, a strain belonging to Bacillus subtilis, and separating it from the culture solution according to a known method, and it can be used as a refined product or an unrefined product. For example, the culture solution can be used as it is. Further, those obtained by a chemical synthesis method can also be used in the same manner.

A salt of surfactin (I) can also be used. The counter cation constituting the salt is not particularly limited, and examples thereof include alkali metal ions and ammonium ions.

The alkali metal ion that can be used for the salt of surfactin (I) is not particularly limited, but represents lithium ion, sodium ion, potassium ion, or the like. Further, the two alkali metal ions may be the same as each other or may be different from each other.

Examples of the substituent of the ammonium ion include alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl; aralkyl groups such as benzyl, methylbenzyl and phenylethyl; phenyl, toluyl, xsilyl and the like. Examples include organic groups such as the aryl group of. Examples of the ammonium ion include tetramethylammonium ion, tetraethylammonium ion, pyridinium ion and the like.

In the salt of surfactin (I), the two counter cations may be the same or different from each other. Also, one of the carboxy group -COOH or -COO ^- shall may be in the state of.

The amount of lipopeptide biosurfactant used may be appropriately adjusted within the range in which nanodisks are formed. For example, the amount of lipopeptide biosurfactant with respect to the total of lecithin and lysolecithin can be 0.5 mass times or more and 5 mass times or less. If the amount is 0.5 mass times or more, nanodisks are formed more reliably, and if the amount is 5 mass times or less, the amount of lipopeptide biosurfactant that is not involved in the formation of nanodisks is so excessive. Not economical. The amount is preferably 1 mass times or more, preferably 4 mass times or less, and more preferably 3 mass times or less.

The nanodisc according to the present invention may contain components other than lecithin, lysolecithin and lipopeptide biosurfactant. For example, an aqueous solvent is mainly used as a solvent for cosmetics and pharmaceutical compositions, but there is a problem that it is difficult to disperse a hydrophobic compound in the aqueous solvent. However, if the nanodisc according to the present invention is used, a hydrophobic compound can probably be taken in and dispersed in an aqueous solvent, so that a highly transparent composition can be obtained. In the present invention, the "aqueous solvent" refers to water and a mixed solvent of a water-miscible organic solvent and water. The water-miscible organic solvent refers to an organic solvent that can be miscible with water without limitation, and examples thereof include C _1-3 alcohol, preferably ethanol or isopropanol. The proportion of the water-miscible organic solvent in the mixed solvent is preferably 10% by mass or less, more preferably 5% by mass or less, still more preferably 2% by mass or less. The aqueous solvent may be a buffer solution, and its pH is not particularly limited, but is preferably 5.0 or more and 13.0 or less.

Hydrophobic compound refers to a compound that has a hydrophobic group, is sparingly soluble in water, and is soluble in oil. Here, poorly soluble means that 100 mL or more of water is required to dissolve 1 g of the hydrophobic compound at 20 ± 5 ° C. The hydrophobic compound used in the present invention is not particularly limited as long as it is an active ingredient to be blended in cosmetics, pharmaceutical compositions, etc., but for example, a whitening agent, an anti-aging agent, an antioxidant, a moisturizer, a hair restorer, and cell activation. Examples include agents, vitamins and amino acids. More specifically, fat-soluble vitamins such as tocopherol, coenzyme Q10, reduced coenzyme Q10, and derivatives thereof; oils such as squalane; steroidal anti-inflammatory agents such as hydrocortisone, prednisolone, dexamethasone, betamethasone; acetyl. Non-steroidal anti-inflammatory agents such as salicylic acid, ibuprofen, indomethacin, loxoprofen; antibiotics; antifungal agents; preservatives such as methylparaben; fragrances such as menthol; pigments such as alizanin. In addition, albutin and its derivatives; L-ascorbic acid and its derivatives; hydroquinone and its derivatives; glutathione and its derivatives; placenta extract; pantothenic acid and its derivatives; tranexamic acid and its derivatives; Derivatives; Plant extracts such as chamomile extract and citrus extract; Carotenoids such as beta-carotene; Astaxanthin and its derivatives; Flavonoids; Catechin and its derivatives; Vitamin A and its derivatives; α-lipoic acid and its derivatives; Glycyrrhizic acid And its derivatives; thiotaurine and its derivatives; urea and its derivatives; glycerin derivatives; salicylic acid and its derivatives; nicotinic acid and its derivatives; L-amino acids and its derivatives; adenosine and its derivatives; plant female hormone-like components such as isoflavone; Grabridine and its derivatives; magnolignan and its derivatives; minoxidil and its derivatives; finasteride and its derivatives; flavanones; carpronium chloride and its derivatives; ferulic acid and its derivatives; fullerene and its derivatives; lactoferrin; D-amino acids and its derivatives; Oligosaccharides and their derivatives; Nucleic acid and its derivatives; D-glucosamine and its derivatives; Sodium chondroitin sulfate and its derivatives; Dipropylene glycol and its derivatives; Ceramide and its derivatives; Sorbitol and its derivatives; Trehalose and its derivatives; Hyaluronic acid and its derivatives Derivatives thereof; Propropylene glycol fatty acid ester and its derivatives; Martitol and its derivatives; Raffinose and its derivatives; Arginic acid and its derivatives; Caffeine and its derivatives; Carboxyvinyl polymers and its derivatives; Xanthan gum and its derivatives; Polyvinylpyrrolidone and its derivatives; keratin degradation products and their derivatives; silk protein degradation products and their derivatives; γ-aminobutyric acid and its derivatives; allantin and its derivatives; γ-orizanol and its derivatives; L-carnitine and its derivatives; β -1,3-Glucane and its derivatives; biotin and its derivatives and the like.

The nanodisc according to the present invention may contain a membrane protein. Membrane proteins are proteins that are bound to biological membranes, and are mainly biological membranes other than covalent bonds such as hydrophobic interactions and electrostatic interactions with endogenous membrane proteins, of which at least a part of them is present in the biological membrane. It is classified as a superficial membrane protein that is bound to. Conventionally, it has been considered extremely difficult to study the function of a membrane protein because the higher-order structure changes and the original function is lost when a membrane protein existing in a biological membrane such as a cell membrane is isolated. However, the nanodisc according to the present invention has a lipid bilayer similar to that of a biological membrane, and it is considered that the membrane protein maintains the structure in the biological membrane in the lipid bilayer. Research on functions will progress dramatically, and it is possible that the difficult industrial use of membrane proteins will be possible.

Further, the nanodisk according to the present invention may contain a hydrophilic compound. The hydrophilic compound is not particularly limited as long as it is a compound having an affinity for water molecules. Many hydrophilic compounds are water-soluble, and water-soluble hydrophilic compounds can be dissolved in aqueous solvents, but they may be adsorbed on the hydrophilic part of nanodiscs to further stabilize them. is there. Further, by adsorbing an active ingredient such as a metal oxide, which is hydrophilic but insoluble in water, on the hydrophilic portion of the nanodisk, it may be possible to disperse the active ingredient in an aqueous solvent. In some cases, a plurality of nanodisks are laminated with each other to form a multilayer structure. In this case, the hydrophilic compound may be present between the lipid bilayer layers. In the present disclosure, the hydrophilic compound refers to a compound in which the amount of water for dissolving 1 g at 20 ± 5 ° C. is less than 30 mL.

Examples of hydrophilic compounds include ultraviolet scattering agents such as titanium oxide particles and zinc oxide particles; inorganic pigments such as red iron oxide, titanium oxide and iron oxide; water-soluble vitamins such as ascorbic acid and its derivatives; locust bean gum and guar gum. Examples thereof include thickeners such as derivatives, carrageenan, pectin, xanthan gum, gellan gum, and alginic acid.

The nanodisc according to the present invention may contain a surfactant other than lipopeptide biosurfactant. Such a surfactant is not particularly limited, such as a nonionic surfactant, an anionic surfactant, a cationic surfactant, and an amphoteric surfactant.

The nonionic surfactant is not particularly limited, but for example, polyoxyethylene (hereinafter, also referred to as POE) -polyoxypropylene (hereinafter, also referred to as POP) block copolymer; POE-POP block copolymer addition of ethylene diamine such as poloxamine. Things: POE sorbitan fatty acid esters such as monolauric acid POE (20) sorbitan, monooleic acid POE (20) sorbitan, POE sorbitan monostearate, POE sorbitan tristearate; POE (5) hardened castor oil, POE (10) ) Hardened castor oil, POE (20) Hardened castor oil, POE (40) Hardened castor oil, POE (50) Hardened castor oil, POE (60) Hardened castor oil, POE (100) Hardened castor oil, etc. Classes; POE (3) castor oil, POE (10) castor oil, POE (35) castor oil and other POE castor oils; POE (9) POE alkyl ethers such as lauryl ether; POE (20) POP (4) POE / POP alkyl ethers such as cetyl ether; POE alkylphenyl ethers such as POE (10) nonionic phenyl ether; polyethylene glycol monostearate such as polyoxyl 40 stearate and the like can be mentioned. The numbers in parentheses indicate the average number of moles of POP or POE added.

The anionic surfactant is not particularly limited, and examples thereof include alkylbenzene sulfonate, alkyl sulfate, polyoxyethylene alkyl sulfate, aliphatic α-sulfomethyl ester, and α-olefin sulfonic acid.

The cationic surfactant is not particularly limited, and examples thereof include benzalkonium chloride and benzethonium chloride.

The amphoteric tenside is not particularly limited, and examples thereof include glycine type such as alkyldiaminoethylglycine, betaine type acetate such as lauryldimethylaminoacetic acid betaine, and imidazoline type amphoteric tenside.

General nanodiscs are manufactured by a complicated method in which a membrane scaffold protein is added to a phospholipid solution containing sodium cholic acid as a surfactant, and then the surfactant is removed by dialysis or the like. On the other hand, the nanodisc according to the present invention can be produced by an extremely simple method of mixing at least lecithin, lysolecithin and lipopeptide biosurfactant in an aqueous solvent.

The total concentration of lecithin and lysolecithin in the reaction solution may be appropriately adjusted, but can be, for example, 0.001% by mass or more and 40% by mass or less, preferably 0.1% by mass or more and 30% by mass or less. .. The concentration of lipopeptide biosurfactant in the reaction solution can also be 0.001 part by mass or more and 50% by mass or less, preferably 0.1% by mass or more and 45% by mass or less.

When a hydrophobic compound and / or a hydrophilic compound is added, the amounts of the hydrophobic compound and the hydrophilic compound are preferably 0.1% by mass or more and 50% by mass or less, respectively, based on the total amount of lecithin and lysolecithin, respectively. It is more preferably 5.5% by mass or more and 20% by mass or less, and even more preferably 1% by mass or more and 10% by mass or less.

The reaction conditions of lecithin, lysolecithin and lipopeptide biosurfactant may be appropriately adjusted within a range in which the formation of nanodisks progresses satisfactorily. For example, the reaction temperature may be room temperature. For example, when a phospholipid such as DMPC is used as the lipid, the temperature can be set to 20 ° C. or higher and 30 ° C. or lower, which is near the general phase transition temperature. The reaction time can be 2 minutes or more and 50 hours or less. In addition, as compared with the production method via liposomes described later, nanodiscs may not be formed simply by mixing each component, but even in such a case, the reaction temperature is relatively high, for example, 40 ° C. or higher. , Nanodiscs may be formed by lowering the temperature to 90 ° C. or lower. The temperature is preferably 85 ° C. or lower, more preferably 80 ° C. or lower.

Further, the nanodisk according to the present invention can also be produced by first preparing liposomes from lecithin and lysolecithin, and then adding lipopeptide biosurfactant. That is, after dissolving lecithin and lysolecithin in a lower alcohol solvent such as methanol, ethanol and isopropanol or an organic solvent such as chloroform, the organic solvent is distilled off to form a lipid film, and an aqueous solvent is added and stirred to form liposomes. .. The concentration of the liposome dispersion liquid at this time may be appropriately adjusted, and for example, it can be 0.01% by mass or more and 40% by mass or less with respect to the organic solvent, and 0.05% by mass or more and 35% by mass or less. The following is preferable. Next, by adding lipopeptide biosurfactant, nanodiscs can be obtained. The concentration of the lipopeptide biosurfactant in the reaction solution may be appropriately adjusted, and may be, for example, 0.01% by mass or more and 40% by mass or less, 0.05% by mass or more, based on the amount of the liposome solution. It is preferably 35% by mass or less. In the case of producing conventional nanodisks, it is necessary to use and remove a surfactant such as cholic acid, but in the case of the present invention, no surfactant other than lipopeptide biosurfactant is particularly required. Further, when the temperature at which the lipopeptide biosurfactant is added is relatively high, for example, 40 ° C. or higher and 80 ° C. or lower, the formation of nanodisks may be facilitated.

Ingredients other than lecithin, lysolecithin and lipopeptide biosurfactant may be added in a timely manner. For example, the hydrophilic compound may be added to the aqueous solvent in a timely manner. Hydrophobic compounds, membrane proteins and water-insoluble hydrophilic compounds are preferably allowed to coexist with at least lecithin and lysolecithin in aqueous solvents. The amounts of the hydrophobic compound and the hydrophilic compound are preferably 0.1% by mass or more and 100% by mass or less, more preferably 0.5% by mass or more and 50% by mass or less, based on the total of lecithin and lysolecithin, respectively. More preferably, it is by mass% or more and 20% by mass or less. Since the biomolecules lecithin and lysolecithin exhibit a surface-active action, they assist the dispersion of these compounds in an aqueous solvent. Further, in the case of a membrane protein, it is also possible to bind the membrane protein to the nanodisk by cell-free synthesis of the membrane protein in the presence of the nanodisk according to the present invention.

The nanodisks may be freeze-dried and then the freeze-dried nanodisks may be dissolved again in an aqueous solvent. The nanodisc concentration of the redissolved solution can be adjusted arbitrarily.

Since the nanodisc according to the present invention can take in a hydrophobic compound therein and disperse it well in an aqueous solvent and is extremely fine, the dispersion in the aqueous solvent is visually sufficient. It looks transparent and is considered to have high permeability to skin tissues. In the cosmetics industry in particular, in general, a state in which the dispersoid cannot be visually confirmed and the dispersion liquid becomes visually transparent may be referred to as "solubilization". Therefore, it can be said that the nanodisc according to the present invention is particularly effective as a component of a personal care composition such as a cosmetic or a pharmaceutical composition containing a hydrophobic compound as an active ingredient.

This application claims the benefit of priority based on Japanese Patent Application No. 2019-186835 filed on October 10, 2019. The entire contents of the specification of Japanese Patent Application No. 2019-186835 filed on October 10, 2019 are incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can be adapted to the gist of the above and the following. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

Example 1: Production of Nanodiscs Containing Coenzyme Q10 According to the composition shown in Table 1, only soy lecithin (“Basis LP-20” manufactured by Nisshin Oillio Group) or soy lecithin and hydrogenated lysolecithin (“SLP-LPC70H”” A solution was prepared by stirring and mixing a combination with Tsuji Oil Co., Ltd.), coenzyme Q10 (manufactured by Kaneka Co., Ltd.) and an appropriate amount of chloroform with a vortex mixer. During the draft, chloroform was distilled off by spraying nitrogen gas on the obtained solution, and the solution was dried. A part of the phosphate buffer solution and paraben as a preservative were added to the obtained dried product, the mixture was heated to 60 to 70 ° C., and the mixture was stirred and mixed with a vortex mixer to obtain a solution. Sodium surfactin and the remaining amount of phosphate buffer were added to the obtained solution, the mixture was heated to 60 to 70 ° C., and stirred and mixed with a vortex mixer to obtain a solution.
The hydrogenated lysolecithin product used contains about 70% by mass of lysophosphatidylcholine as lysolecithin.

Test Example 1: Measurement of Nanodisc Particle Size According to the composition shown in Table 2, soy lecithin (“Basis LP-20” manufactured by Nisshin Oillio Group) alone, or soy lecithin and hydrogenated chloroform (“SLP-LPC70H” Tsuji) A solution was prepared by stirring and mixing the combination with (manufactured by Oil & Refinery Co., Ltd.) and an appropriate amount of chloroform with a vortex mixer. During the draft, chloroform was distilled off by spraying nitrogen gas on the obtained solution, and the solution was dried. Sodium surfactin and a phosphate buffer were added to the obtained dried product, heated to 60 to 70 ° C., and stirred and mixed with a vortex mixer to obtain a solution.
The average diameter of the particles contained in each of the obtained solutions was measured using a dynamic light scattering photometer (“DLS-7000” manufactured by Otsuka Electronics Co., Ltd.). The results are shown in Table 2.

As shown in the results shown in Table 2, the particle size increases as the proportion of lysolecithin increases, but if the proportion of lysolecithin / (lysolecithin + lecithin) is 25% by mass or less, the appearance of the mixed solution is transparent and the average particle size. Was less than 100 nm, and nanodisks were formed. There are also jumbo liposomes with a size of about 100 μm, but the particle size of general liposomes is said to be 0.1 to 0.2 μm, and the mixed solution was somewhat opaque, so the ratio of lysolecithin / (lysolecithin + lecithin). When the content was 30% by mass or more, the main component was considered to be liposomes.

Test Example 2: Measurement of particle size of nanodisc According to the composition shown in Table 3, hydrogenated lecithin (“SLP-PC70HS” manufactured by Tsuji Oil Co., Ltd.) only, or hydrogenated lecithin and hydrogenated chloroform (“SLP-LPC70H” Tsuji Oil Co., Ltd.) A solution was prepared by stirring and mixing the combination with (manufactured by the company) and an appropriate amount of chloroform with a vortex mixer. During the draft, chloroform was distilled off by spraying nitrogen gas on the obtained solution, and the solution was dried. Sodium surfactin and a phosphate buffer solution were added to the obtained dried product, and the mixture was allowed to stand at room temperature for 10 minutes and then stirred and mixed with a vortex mixer for 3 minutes to obtain an opaque solution. The opaque solution was allowed to stand at 30 ° C. overnight, and the appearance was visually confirmed. Further, after visually confirming the appearance of each liquid obtained by heating at 70 ° C., the average diameter of the particles contained therein was measured using a dynamic light scattering photometer (“DLS-7000” manufactured by Otsuka Electronics Co., Ltd.). Was measured. The results are shown in Table 3.

As shown in the results shown in Table 3, it was found that the solubility decreased when hydrogenated lecithin was used, but a transparent solution could be obtained by heating to 70 ° C. The reason may be that hydrogenated lecithin strengthens the packing of molecules. Moreover, since the average particle size of the particles was less than 100 nm, it was confirmed that nanodiscs were formed. Only when hydrogenated lysolecithin was not added, turbidity occurred after heating at 70 ° C. for 1 day. Therefore, it was found that the addition of lysolecithin stabilizes the nanodiscs.

Test Example 3: Method of obtaining nanodiscs directly from powder According to the composition shown in Table 3, hydrogenated lecithin (“SLP-PC70HS” manufactured by Tsuji Oil Co., Ltd.) and hydrogenated lecithin (“SLP-LPC70H” manufactured by Tsuji Oil Co., Ltd.) The phosphate buffer solution was directly added to the powder of sodium surfactin, and the mixture was stirred with a vortex for 5 minutes, and then stirred at 180 rpm and 25 ° C. for 20 hours using a shaker (“TAITEC BioShaker BR-23FP”). did. The mixture was stirred and mixed with a vortex mixer for 3 minutes to obtain an opaque liquid. The temperature of this opaque solution was raised in a constant temperature bath from 25 ° C. to 70 ° C. in 5 ° C. increments, and changes in the solution were observed. As a result, it was found that a transparent solution was obtained by heating to 70 ° C., and nanodiscs could be obtained without using a solvent.

Test Example 4: Formation of Nanodiscs Using Different Hydrogenated Lecithin According to the composition shown in Table 4, hydrogenated lecithin (“SLP-PC92H” manufactured by Tsuji Oil Co., Ltd.) and hydrogenated lecithin (“SLP-LPC70H” manufactured by Tsuji Oil Co., Ltd.) ) Was used to prepare nanodiscs. Specifically, hydrogenated lecithin, hydrogenated lysolecithin, and an appropriate amount of chloroform were stirred and mixed with a vortex mixer to prepare a solution. During the draft, chloroform was distilled off by spraying nitrogen gas on the obtained solution, and the solution was dried. Sodium surfactin and a phosphate buffer solution were added to the obtained dried product, and the mixture was allowed to stand at room temperature for 10 minutes and then stirred and mixed with a vortex mixer for 3 minutes to obtain an opaque solution. After heating this opaque liquid to 60 ° C., it was allowed to stand for one day, and the appearance was visually confirmed. The average diameter of the particles contained in each of the obtained solutions was measured using a dynamic light scattering photometer (“DLS-7000” manufactured by Otsuka Electronics Co., Ltd.). The results are shown in Table 4.

As shown in the results shown in Table 4, it was found that a transparent solution can be obtained by heating to 60 ° C. even if the type of hydrogenated lecithin is changed. Since the average particle size was less than 100 nm, it was clarified that nanodiscs were formed.

Test Example 5: Formation of Nanodiscs Using Different Soy Lecithin According to the composition shown in Table 5, soy lecithin (“SLP-White” manufactured by Tsuji Oil Co., Ltd.) and hydrogenated lecithin (“SLP-LPC70H” manufactured by Tsuji Oil Co., Ltd.) Nanodiscs were prepared with the combination of. Specifically, soybean lecithin, hydrogenated lysolecithin, and an appropriate amount of chloroform were stirred and mixed with a vortex mixer to prepare a solution. During the draft, chloroform was distilled off by spraying nitrogen gas on the obtained solution, and the solution was dried. Sodium surfactin and a phosphate buffer were added to the obtained dried product, allowed to stand at room temperature for 10 minutes, and then stirred and mixed with a vortex mixer for 3 minutes.
The average diameter of the particles contained in each of the obtained mixed solutions was measured using a dynamic light scattering photometer (“DLS-7000” manufactured by Otsuka Electronics Co., Ltd.). The results are shown in Table 5.

As shown in the results shown in Table 5, it was found that a transparent nanodisc solution can be obtained when the ratio of hydrogenated lysolecithin / (hydrogenated lysolecithin + lecithin) is 25% by mass or less.

Test Example 6: Stability test The mixture (10 mL) prepared in Example 1 was placed in a 50 mL falcon tube and placed in a constant temperature and humidity chamber (manufactured by Yamato) at 50 ° C, 25 ° C or 5 ° C for 30 days (1M). Alternatively, it was stored for 60 days (2M), or it was stored for 30 days or 60 days even under the condition that the cycle of raising and lowering the temperature in the range of −5 ° C. to 45 ° C. was repeated 3 times in 2 days. The appearance was observed after 30 days or 60 days, and the stability was evaluated according to the following criteria. The results are shown in Table 6. In addition, a photograph of the appearance of a mixed solution containing 0.95% by mass of soybean lecithin and 0.05% by mass of hydrogenated lysolecithin (lysolecithin / (lysolecithin + lecithin) = 3.5%) after 60 days is shown in FIG. 1, soybean lecithin 0. FIG. 2 shows a photograph of the appearance of a mixed solution containing .60% by mass and 0.40% by mass of hydrogenated lysolecithin (lysolecithin / (lysolecithin + lecithin) = 28.0%) after 60 days.
〇: Transparent △: Slightly reduced transparency is observed, but overall transparent ×: Clear turbidity and precipitation are observed.

As shown in the results shown in Table 6, if the ratio of lysolecithin / (lysolecithin + lecithin) is 20% by mass or less, the transparency of the nanodisc solution is maintained for 30 days even at a relatively high temperature of 50 ° C., and the ratio is 20% by mass. If it is% or less, the transparency is maintained even after 60 days except when it is held at 50 ° C. or 25 ° C., and if it is 10% by mass or less, the transparency is maintained after 60 days even if it is held at 50 ° C. Therefore, the stability of the nanodisk according to the present invention was demonstrated.

Claims

Contains lipid bilayer and lipopeptide biosurfactant
The lipid bilayer contains lecithin and lysolecithin,
A nanodisk characterized in that the ratio of the lysolecithin to the total of the lecithin and the lysolecithin is 1% by mass or more and 20% by mass or less.
The nanodisc according to claim 1, wherein the lipopeptide biosurfactant is bound as a membrane scaffold protein to the side surface of the lipid bilayer membrane.
The nanodisc according to claim 1 or 2, which has a thickness of 2 nm or more and 10 nm or less.
The nanodisc according to any one of claims 1 to 3, which has a diameter of 6 nm or more and 40 nm or less.
The nanodisc according to any one of claims 1 to 4, wherein the ratio is 10% by mass or less.
The nanodisc according to any one of claims 1 to 5, wherein the lipopeptide biosurfactant is a surfactin represented by the following formula (I) or a salt thereof.

[During the ceremony,
X indicates an amino acid residue selected from leucine, isoleucine and valine;
R 1 represents a C 9-18 alkyl group]
The nanodisc according to any one of claims 1 to 6, further comprising a hydrophobic compound.
The nanodisc according to any one of claims 1 to 7, further comprising a hydrophilic compound.
A cosmetic comprising the nanodisc according to any one of claims 1 to 8.