Lipid extracts and methods for producing the same, lipid compositions, and lipid membrane vesicles and methods for producing the same.

Abstract

This invention provides a method for extracting membrane-constituting lipids using reagents with lower biotoxicity, while maintaining their activity more stably, and for efficiently preparing lipid membrane vesicles. [Solution] (A) A method for producing a lipid extract, comprising the step of contacting a biological sample containing membrane-constituting lipids with an extraction solvent containing a dihydric alcohol to extract the membrane-constituting lipids.

Description

[Technical Field]

[0001] The present invention relates to lipid extracts and methods for producing the same, lipid compositions, and lipid membrane vesicles and methods for producing the same. [Background technology]

[0002] In the fields of biochemistry and biophysics, giant lipid membrane vesicles (GVs) are used as cell membrane models. Well-known methods for producing GVs include hydration and injection. However, these methods use organic solvents, making it difficult to utilize all components of the cell membrane, including functional molecules, and leaving behind biotoxic organic solvents, making them unsuitable for application to lipids derived from living organisms. Another method for producing GVs is the Giant Plasma Membrane Vesicle (GPMV) method, which involves chemically treating cultured cells to spontaneously change their shape, such as through budding. However, the GPMV method has a low GV yield and is difficult to stabilize in quality, making it unsuitable for mass production. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Colloids and Surfaces A 2018, 546, 74-82. [Overview of the project] [Problems that the invention aims to solve]

[0004] Furthermore, the present inventors have reported that they were able to obtain lipid membrane vesicles containing multiple synthetic lipids with high efficiency using an improved reverse-phase centrifugation method (Non-Patent Literature 1). However, the method in Non-Patent Literature 1 is characterized by the use of diethyl ether, an organic solvent that exhibits biotoxicity, and therefore cannot be applied to products used on the human body, such as pharmaceuticals and food products, and their raw materials.

[0005] This invention has been made in view of the current state of the prior art described above, and aims to provide a method for extracting membrane constituent lipids using reagents with lower biotoxicity, while maintaining their activity more stably, and for preparing lipid membrane vesicles with high efficiency. [Means for solving the problem]

[0006] The inventors have diligently conducted research to achieve the above-mentioned objectives. As a result, they have found that by contacting a biological sample containing membrane-constituting lipids with an extraction solvent containing a dihydric alcohol, membrane-constituting lipids can be extracted while maintaining their activity more stably, and furthermore, the obtained lipid extract is particularly suitable for the preparation of lipid membrane vesicles. The inventors have also found that by mixing the lipid extract obtained by this method with an oily medium containing vegetable oil and a first aqueous medium to form a W / O type emulsion, then removing the oil phase and further mixing it with a second aqueous medium, lipid membrane vesicles can be prepared with high efficiency while maintaining their activity more stably using reagents with lower biotoxicity. Based on these findings, the inventors have conducted further research and completed this lipid extraction invention. That is, the present invention encompasses the following configuration.

[0007] Item 1. (A) A method for producing a lipid extract, comprising the step of contacting a biological sample containing membrane-constituting lipids with an extraction solvent containing a dihydric alcohol to extract the membrane-constituting lipids. Item 2. The method according to Item 1, wherein the dihydric alcohol is a branched alcohol. Item 3. The method according to item 1 or 2, wherein the number of carbon atoms in the dihydric alcohol is 3 to 6. Item 4. The method according to any one of items 1 to 3, wherein the number of carbon atoms in the divalent polyhydric alcohol is 3 or 4. Item 5. The method according to any one of items 1 to 4, wherein the dihydric alcohol is 1,2-propanediol. Item 6. The method according to any one of items 1 to 5, wherein the biological sample is a cell lysate. Item 7. The method according to any one of items 1 to 6, wherein the lipid extract comprises glycolipids and / or membrane proteins. Item 8. The method according to any one of items 1 to 7, wherein step (A) is performed at a temperature of 10°C to 60°C. Section 9. A lipid extract obtained by the manufacturing method described in any one of sections 1 to 8. Item 10. Lipid extracts as described in Item 9, for use in the production of lipid membrane vesicles. Item 11. A lipid composition comprising a biological sample containing membrane-constituting lipids, an extraction solvent containing a dihydric alcohol, and an oily medium containing a vegetable oil. Section 12. (B) A step of forming a W / O type emulsion by mixing a lipid extract obtained by the manufacturing method described in any one of sections 1 to 8, an oily medium containing vegetable oil, and a first aqueous medium. (C) A step of removing the oil phase from the W / O type emulsion by centrifugal separation. (D) A step of mixing the result of step (C) with a second aqueous medium, A method for producing lipid membrane vesicles. Item 13. The method according to Item 12, wherein steps (B) to (D) are performed at a temperature of 10°C to 60°C. Item 14. The vegetable oil is at least one selected from the group consisting of soybean oil, corn oil, and olive oil, according to the method of Item 12 or 13. Item 15. The method according to any one of items 12 to 14, wherein the average particle size of the lipid membrane vesicles is 1 μm or more. Item 16. Vesicles with an average particle diameter of 1 μm or more and a coefficient of variation of particle diameter of 50% or less are defined as 1 × 10⁻⁶ 3 Lipid membrane vesicles containing more than 1 / mL. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a method for extracting membrane constituent lipids using reagents with lower biotoxicity while maintaining their activity more stably, thereby enabling the highly efficient preparation of lipid membrane vesicles. [Brief explanation of the drawing]

[0009] [Figure 1] It is a diagram showing an overview of Example 1. [Figure 2] It is a diagram showing an overview of Example 2. [Figure 3] It is a confocal laser microscopic image of lipid membrane vesicles obtained in Example 2. [Figure 4] It is the particle size distribution of lipid membrane vesicles obtained in Example 2. [Figure 5] It is a confocal laser microscopic image of lipid membrane vesicles labeled with FITC-WGA. [Figure 6] It is a confocal laser microscopic image of lipid membrane vesicles labeled with FITC-ConA. The arrow in the figure indicates FITC-ConA. [Figure 7] It is a confocal laser microscopic image of the lipid membrane obtained in Comparative Example 1.

Modes for Carrying Out the Invention

[0010] In this specification, "containing" is a concept that encompasses all of "comprise", "consist essentially of", and "consist of".

[0011] In this specification, the notation of the numerical range "A to B" means "A or more and B or less".

[0012] 1. Method for producing lipid extracts In one embodiment, the present invention provides a method for producing a lipid extract (membrane lipid extract), comprising the step of contacting a biological sample containing membrane-constituting lipids with an extraction solvent containing a dihydric alcohol to extract the membrane-constituting lipids. The lipid extract production method of the present invention having the above configuration allows for the extraction of membrane-constituting lipids using reagents with lower biotoxicity by using an extraction solvent containing a dihydric alcohol, and thus the lipids can be applied to products used on the human body, such as pharmaceuticals and foods, and their raw materials. Furthermore, the lipid extract production method of the present invention allows for the extraction of membrane-constituting lipids while maintaining their function and activity more stably by using an extraction solvent containing a dihydric alcohol, and furthermore, lipid membrane vesicles can be prepared with higher efficiency than with conventional extraction solvents using the lipids extracted in this way. This will be explained below.

[0013] 1-1. Process (A) In step (A), a biological sample containing membrane-constituting lipids is brought into contact with an extraction solvent containing a dihydric alcohol to extract the membrane-constituting lipids. In other words, step (A) is a step of extracting membrane-constituting lipids from a biological sample and leaching the extracted membrane-constituting lipids into the extraction solvent.

[0014] 1-1-1. Biologically Derived Samples Biologically derived samples contain membrane-constituting lipids. These membrane-constituting lipids are not particularly limited, as long as they are lipids capable of constituting biological membranes. Examples of membrane-constituting lipids include phospholipids, sphingolipids, glycolipids, etc., but are not limited to these; lipids contained in the biological membranes of the organism from which the sample originates can be broadly used. Membrane-constituting lipids are not limited to the above-mentioned complex lipids and may also include simple lipids such as neutral lipids and sterols.

[0015] Membrane-constituting lipids can be extracted individually or in combination of two or more types.

[0016] In addition to membrane-constituting lipids, biological samples may also contain lipids that, on their own, cannot constitute a lipid membrane or biological membrane.

[0017] Biological samples may contain membrane proteins in addition to membrane-constituting lipids. Membrane proteins are not particularly limited as long as they are proteins that can constitute biological membranes. Examples of membrane proteins include intrinsic membrane proteins and superficial membrane proteins.

[0018] The biological samples are not particularly limited as long as they contain membrane-constituting lipids of biological origin, and examples include animal, plant, bacterial, or viral samples.

[0019] While there are no particular limitations on the biological sample, cell lysates are preferred from the viewpoint of extracting membrane-constituting lipids. In this case, the extraction efficiency in step (A) can be improved by impairing the integrity of the biological membrane or cell membrane. However, the method is not limited to using cell lysates that have been pre-disrupted in step (A), and the disruption treatment may be performed on a biological sample containing cells or tissue in the presence of an extraction solvent.

[0020] Cell lysates are not particularly limited as long as they are biological materials containing cell fragments that have been disrupted in a way that facilitates the extraction of biological membranes. For example, cell lysates may be materials obtained by directly disrupting biological tissue containing cells (tissue lysates). Disruption methods are not particularly limited as long as they impair the integrity of the cell membranes, and examples include freeze-drying, freeze-thawing, hypotonic treatment, mechanical disruption, sonication, homogenization, and French press treatment. The amount of cell lysates used is not particularly limited, but in terms of cell number, it is 1 × 10⁻⁶. 5 A concentration of cells / mL or higher is preferable.

[0021] The cells may be animal cells, plant cells, or bacterial cells. Examples of animal cells include insect cells, bird cells, mammalian cells, and fish cells. Furthermore, examples of cells include cells without a nucleus, such as red blood cells and platelets.

[0022] Furthermore, examples of cell membranes include the cell wall and the outer membrane of bacteria, the membranes of extracellular vesicles (EVs) such as exosomes, and the membranes of organelles such as mitochondria, as well as the mantle (envelope) of enveloped viruses.

[0023] Examples of freeze-thaw treatments include freezing a biological sample dispersed in a buffer such as PBS(-) in a deep freezer at -80°C or in liquid nitrogen, and then allowing the biological sample to thaw at room temperature.

[0024] It is preferable to concentrate the biological sample after crushing, if necessary. Concentration methods include, for example, separating and removing unwanted components other than membrane-constituting lipids by separation treatment. Specific examples of separation and removal include methods that promote separation due to differences in specific gravity using centrifugation. The centrifugal force used in centrifugation should be sufficient to settle the membrane-constituting lipids, for example, around 1,000 × g to 15,000 × g. The centrifugation time should be sufficient to settle the membrane-constituting lipids, for example, around 5 minutes to 10 hours. The temperature for centrifugation is usually 4°C to 30°C and can be appropriately adjusted according to the various physical properties of the biological sample components, such as melting point and boiling point.

[0025] The biological sample may be in a liquid state, a dry state, or a solid state. From the viewpoint of preserving the function and activity of membrane-constituting lipids, it is preferable to appropriately adjust the amount of solvent contained in the sample so that the sample does not dry out. On the other hand, from the viewpoint of improving extraction efficiency, it is preferable to remove as much solvent as possible from the sample. For this reason, as the biological sample, a pellet-shaped group of cells or their lysates (cell lysates) from which as much solvent as possible has been removed is more preferable.

[0026] The solvents that may be included in biological samples may be polar or nonpolar solvents. From the viewpoint of improving extraction efficiency while maintaining the function and activity of membrane constituent lipids, it is preferable that biological samples contain a solvent such as water or a polar solvent other than water. Examples of solvents include buffers such as PBS(-) and culture media. The solvent may also include cell cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol, and polar solvents such as ethanol and DMF.

[0027] 1-1-2. Extraction solvent The extraction solvent contains a dihydric alcohol. In this specification, a dihydric alcohol means a compound having only two hydroxyl groups in a single molecule.

[0028] As a dihydric alcohol, branched alcohols are preferred from the viewpoint of the extraction efficiency of membrane constituent lipids and the efficiency of lipid membrane vesicle formation. The branched alcohol is not particularly limited as long as it is a dihydric alcohol having a branched structure. In the present invention, the branched alcohol may be, for example, a compound having a hydroxyl group as a substituent at the end of a branched alkyl chain, or a compound having a hydroxyl group as a substituent on a carbon atom inside a linear alkyl chain. Among these, as a branched alcohol, a compound having a hydroxyl group as a substituent on a carbon atom inside a linear alkyl chain (for example, 1,2-propanediol, 1,3-butanediol, etc.) is preferred.

[0029] The number of carbon atoms in the dihydric alcohol is not particularly limited, but from the viewpoint of the extraction efficiency of membrane constituent lipids and the formation efficiency of lipid membrane vesicles, an integer between 2 and 10 is preferred, an integer between 3 and 6 is more preferred, an integer between 3 and 5 is even more preferred, and 3 or 4 is particularly preferred.

[0030] Specific examples of dihydric alcohols include, but are not limited to, 1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 2,3-propanediol, 2-methyl-1,3-propanediol, 2-methyl-1,2-propanediol, and 2-methyl-2,3-butanediol. In particular, from the viewpoint of the extraction efficiency of membrane constituent lipids and the efficiency of lipid membrane vesicle formation, branched alcohols such as 1,2-propanediol and 1,3-butanediol are preferred, and 1,2-propanediol is more preferred.

[0031] Dihydric alcohols can be used individually or in combination of two or more types.

[0032] The content of dihydric alcohols is not particularly limited, but from the viewpoint of the extraction efficiency of membrane constituent lipids and the formation efficiency of lipid membrane vesicles, it is preferably 50% by mass or more, more preferably 70% to 100% by mass, even more preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, relative to the total mass of the extraction solvent.

[0033] The extraction solvent may be a mixed solvent containing a dihydric alcohol and a solvent other than a dihydric alcohol. Examples of solvents other than dihydric alcohols include water, buffer solutions such as PBS, etc. The extraction solvent may also contain cryoprotective agents for cells such as dimethyl sulfoxide (DMSO) and glycerol, and polar solvents such as ethanol and DMF. When the extraction solvent is a mixed solvent, the content of solvents other than dihydric alcohols is not particularly limited, but from the viewpoint of the extraction efficiency of membrane constituent lipids and the formation efficiency of lipid membrane vesicles, it is preferably 50% by mass or less, more preferably 30% by mass or less, even more preferably 10% by mass or less, and particularly preferably 5% by mass or less, relative to the total mass of the extraction solvent.

[0034] The amount of extraction solvent used (relative amount) is not particularly limited and can be appropriately adjusted according to the amount of membrane-constituting lipids present in the biological sample or the amount of membrane-constituting lipids required. The amount of extraction solvent used is usually 0.001 mL to 100 mL per 1 mg of membrane-constituting lipids.

[0035] The amount (absolute amount) of extraction solvent used is not particularly limited and can be appropriately adjusted according to the amount of membrane-constituting lipids present in the biological sample or the amount of membrane-constituting lipids required. According to the present invention, membrane-constituting lipids can be extracted on a wide range of scales, from μg to kg.

[0036] 1-1-3. Others The method of contacting the biological sample described above with the extraction solvent described above is not particularly limited. Contact may be carried out by adding the extraction solvent to the biological sample, or by adding the biological sample to the extraction solvent.

[0037] In step (A), it is preferable to mix the biological sample with the extraction solvent. In this case, the probability of contact between the membrane constituent lipids and the dihydric alcohol is improved, which makes it easier to improve the extraction efficiency of the membrane constituent lipids. The mixing method is not particularly limited, and a wide range of known mixing methods can be used, such as shaking, stirring, inversion mixing, sonication, homogenization, etc.

[0038] The temperature in step (A) is not particularly limited and can be adjusted as appropriate depending on the solubility, melting point, and boiling point of the biological sample and the extraction solvent. In particular, from the viewpoint of suppressing the denaturation of membrane constituent lipids, step (A) is preferably carried out at a temperature of 10°C to 60°C, and more preferably at a temperature of 20°C to 40°C.

[0039] Process (A) is not particularly limited and may be carried out in an atmospheric environment or in an inert gas atmosphere.

[0040] The extraction time is not particularly limited and can be adjusted as appropriate depending on the desired extraction volume. The extraction time can be, for example, the time from contact between the biological sample and the extraction solvent until the membrane-constituting lipids are leached from the biological sample into the extraction solvent, and is usually between 1 minute and 24 hours.

[0041] The extract obtained in step (A) can be obtained in a mixed state of the biological sample and the extraction solvent. Specifically, the extract obtained in step (A) may be a solution or dispersion in which the biological sample (particularly membrane-constituting lipids) is dissolved or dispersed in the extraction solvent (particularly a dihydric alcohol). Although not particularly limited, it is preferable to store the obtained extract in a frozen state after extraction until immediately before use.

[0042] The extract obtained in step (A) may contain insoluble solids. Examples of insoluble solids include cell or tissue debris. If the extract contains solids, the solids may be separated and removed from the extract by conventional removal methods, or they may be stored without being removed from the extract. Alternatively, when the extract is used in the lipid membrane vesicle production method described later, separation of lipid membrane vesicles and solids may be achieved by removing the solids before or after at least one of steps (B) to (D). Conventional solid-liquid separation methods such as sedimentation, filtration, and centrifugation can be used for removal. In this case, the extraction efficiency of membrane constituent lipids can be improved by applying step (A) again to the separated and removed solids as a biological sample.

[0043] The centrifugal force used in centrifugation should be sufficient to settle the membrane lipids, for example, around 1,000 × g to 15,000 × g. The centrifugation time should be sufficient to settle the membrane lipids, for example, around 5 minutes to 10 hours. The temperature for centrifugation is preferably 10°C to 60°C, and more preferably 20°C to 40°C, from the viewpoint of suppressing the denaturation of the membrane lipids.

[0044] 1-2. Lipid extract In one embodiment, the present invention provides a lipid extract (membrane-constituting lipid extract). The lipid extract of the present invention (which may also be simply referred to as "extract" in this specification) can be obtained by the method for producing the lipid extract described above. Therefore, the lipid extract obtained by the method for producing the lipid extract described above is also of biological origin.

[0045] The extract contains the biological sample used and some or all of the membrane-constituting lipids contained therein. Preferably, the extract contains at least two types of membrane-constituting lipids from the biological sample, and more preferably substantially all types of membrane-constituting lipids. Conventional extracts have problems such as difficulty in utilizing all components of highly functional membrane-constituting lipids and the residue of biotoxic organic solvents. On the other hand, according to the present invention, membrane-constituting lipids can be extracted from biological samples with high efficiency while maintaining their activity more stably, using reagents with lower biotoxicity.

[0046] In the extract, the content of membrane-constituting lipids is not particularly limited and can be appropriately adjusted according to the amount of membrane-constituting lipids present in the biological sample or the amount of membrane-constituting lipids required. The content of membrane-constituting lipids is usually 0.01 mg to 1000 mg per 1 mL of dihydric alcohol.

[0047] The extract may contain glycolipids. Furthermore, the extract may contain membrane proteins in addition to lipids. According to the present invention, functional molecules such as glycolipids and / or membrane proteins can be extracted while maintaining their function and activity more stably, and they can be effectively utilized.

[0048] Furthermore, the extract may contain the extraction solvent used and some or all of the dihydric alcohol contained therein. In the extract, it is preferable that the membrane constituent lipids are present together with the dihydric alcohol, that is, dissolved in the dihydric alcohol.

[0049] From the viewpoint of maintaining the function and activity of membrane-constituting lipids, it is preferable to appropriately adjust the amount of solvent contained in the extract so that the extract does not dry out. When the extract is applied to the method for producing lipid membrane vesicles and similar methods described later, it is preferable to concentrate it as much as possible from the viewpoint of lipid membrane vesicle formation efficiency.

[0050] The volume of the extract is not particularly limited and can be appropriately adjusted according to the amount of membrane-constituting lipids present in the biological sample or the amount of membrane-constituting lipids required. According to the present invention, membrane-constituting lipids can be extracted on a wide range of scales, from μg to kg.

[0051] The extract of the present invention is particularly suitable as a lipid extract for use in the production of lipid membrane vesicles. For reasons that are not entirely clear, when the extract of the present invention is applied to the method for producing lipid membrane vesicles and similar methods described later, lipid membrane vesicles can be prepared with higher efficiency compared to extracts obtained using conventional extraction solvents.

[0052] In addition to the uses described above, the extract of the present invention can be used in applications such as food, beverages, animal feed, cosmetics, and pharmaceuticals. Examples of food products include bread, confectionery, noodles, rice dishes, pasta, dressings, health foods, and foods for the sick or elderly. Foods for the sick or elderly include semi-solid and liquid foods such as dysphagia foods and foods for people with difficulty chewing. Examples of beverages include juices, dairy drinks, alcoholic beverages, and tea drinks. Examples of animal feed include pet feed, livestock feed, and fish and shellfish feed. Examples of cosmetics include moisturizers and beauty agents. Examples of cosmetic forms include lotions, creams, and emulsions. Examples of pharmaceuticals include anti-obesity agents, blood glucose level inhibitors, cancer cell proliferation inhibitors, and anti-inflammatory agents. Examples of pharmaceutical forms include tablets, powders, microparticle formulations, and capsules.

[0053] 2. Method for producing lipid membrane vesicles In one embodiment, the present invention, (B) A step of forming a W / O type emulsion by mixing the lipid extract obtained by the above-described method for producing the lipid extract (especially step (A)), an oily medium containing vegetable oil, and a first aqueous medium. (C) A step of removing the oil phase from the W / O type emulsion by centrifugal separation, (D) A step of mixing the result of step (C) with a second aqueous medium. The present invention provides a method for producing lipid membrane vesicles, including the above configuration. Even when using lipid extracts extracted from biological samples, the present invention allows for the preparation of lipid membrane vesicles with high efficiency using reagents with lower biotoxicity. Furthermore, compared to conventional methods for producing lipid membrane vesicles, the present invention allows for the preparation of uniform and highly concentrated lipid membrane vesicles with higher efficiency. This will be explained below.

[0054] 2-1. Process (A) The method for producing lipid membrane vesicles of the present invention may optionally include step (A) as described in "1-1. Step (A)". In this case, it is preferable to perform step (A) before step (B).

[0055] 2-2. Process (B) In step (B), a W / O emulsion is formed by mixing (emulsifying) the lipid extract obtained by the above-described method for producing the lipid extract (particularly step (A)), an oily medium containing vegetable oil, and a first aqueous medium. That is, step (B) is a step in which a lipid film is formed on the surface of water droplets in the W / O emulsion.

[0056] 2-2-1. Lipid extract The lipid extract is not particularly limited as long as it is obtained by step (A), that is, as long as it contains membrane-constituting lipids of biological origin. Specific examples of lipid extracts include those described above in "1-2. Lipid Extracts," those described later in "3. Lipid Compositions," and similar lipid extracts. According to the present invention, functional lipids contained in biologically derived materials can be extracted while maintaining their activity more stably without requiring reagents that exhibit biological toxicity, and these can be utilized in the production of lipid membrane vesicles.

[0057] The lipid extract preferably contains the extraction solvent used in step (A) and the dihydric alcohol contained therein. In this case, the extraction solvent can be one of those described in "1-1-2. Extraction Solvent". In the extract, the membrane constituent lipids are preferably present together with the dihydric alcohol.

[0058] Lipid extracts can be chemically labeled for purposes such as tracking, quantitative analysis, and qualitative analysis of membrane-constituting lipids. Examples of labeling methods include fluorescent labeling and radioactive labeling. In the case of fluorescent labeling, membrane lipids and lipid membranes can be labeled by adding pre-labeled membrane-constituting lipids to the lipid extract, or by adding a lipid labeling reagent to the lipid extract. The content of labeled membrane-constituting lipids is usually 10% or less of the total mass of membrane-constituting lipids, and preferably 5% or less.

[0059] 2-2-2. Oil-based medium The oily medium includes vegetable oil. The vegetable oil is not particularly limited as long as it is an oil derived from the seeds or fruits of plants. Examples of vegetable oils include seed oils such as linseed oil, rapeseed oil, sesame oil, safflower oil, sunflower oil, palm kernel oil, soybean oil, corn oil, and cottonseed oil; and fruit oils such as olive oil, rice oil, palm oil, and coconut oil. Among these, soybean oil, olive oil, rapeseed oil, castor oil, sesame oil, corn oil, and maize oil are preferred as vegetable oils, and soybean oil, corn oil, and olive oil are more preferred.

[0060] Vegetable oils can be used individually or in combination of two or more types.

[0061] The vegetable oil content is not particularly limited, but from the viewpoint of lipid membrane vesicle formation efficiency and biotoxicity, it is preferably 50% by mass or more, more preferably 70% to 100% by mass, even more preferably 90% to 100% by mass, and particularly preferably 95% to 100% by mass, relative to the total mass of the oily medium.

[0062] The oily medium may be a mixed solvent containing vegetable oil and other fats and oils. Examples of fats and oils other than vegetable oil include animal oils such as marine animal oils and land animal oils. When the oily medium is a mixed solvent, the content of solvents other than vegetable oil is not particularly limited, but from the viewpoint of lipid membrane vesicle formation efficiency and biotoxicity, it is preferably 50% by mass or less, more preferably 30% by mass or less, even more preferably 10% by mass or less, and particularly preferably 5% by mass or less, relative to the total mass of the oily medium.

[0063] The amount of oily medium used is not particularly limited and can be adjusted as appropriate depending on the amount of membrane constituent lipids present or the required amount of lipid membrane vesicles. Typically, the amount of oily medium used is 0.001 mL to 100 mL per 1 mg of membrane constituent lipids.

[0064] 2-2-3. First aqueous medium The first aqueous medium is not particularly limited as long as it is a medium containing water. From the viewpoint of the stability of lipid membrane vesicles, an aqueous solution containing a solute is preferred as the aqueous medium, and an aqueous sugar solution is more preferred.

[0065] The aqueous sugar solution is not particularly limited as long as it is an aqueous solution containing dissolved sugar. Examples of sugars include monosaccharides such as erythritolose, erythrose, threose, ribulose, xylulose, ribose, arabinose, xylose, lyxose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine, mannose, fructose, allose, talose, growth, altrose, idose, psicose, sorbose, tagatose, and sedoheptulose; disaccharides such as lactose, sucrose, maltose, trehalose, turanose, and cellobiose; oligosaccharides such as raffinose, melegitose, maltotriose, acarbose, stachyose, fructooligosaccharides, galactooligosaccharides, and lactulose oligosaccharides; and polysaccharides such as starch, amylose, amylopectin, glycogen, cellulose, and pectin. In particular, from the viewpoint of the stability of lipid membrane vesicles, monosaccharides, disaccharides, and oligosaccharides are preferred as the sugars contained in the first aqueous medium, with monosaccharides and disaccharides being more preferred.

[0066] In particular, an aqueous solution of a relatively large molecular weight sugar, such as a disaccharide or oligosaccharide, is preferred as the first aqueous medium to be brought into contact with the lipid extract. In this case, since the internal aqueous phase of the lipid membrane vesicles is substantially occupied by the aqueous solution, by selecting an aqueous solution of a sugar with a smaller molecular weight than the internal aqueous phase (first aqueous medium) as the washing solution or external aqueous phase (second aqueous medium), it becomes easy to recover the lipid membrane vesicles by utilizing the difference in specific gravity.

[0067] The first aqueous medium is not particularly limited and may contain functional substances or physiologically active substances other than sugars (in this specification, these may be collectively referred to as "functional molecules"). In this case, the functional molecules can be encapsulated in the resulting lipid membrane vesicles, specifically in the inner aqueous phase of the lipid membrane vesicles. Functional molecules are not particularly limited as long as they are substances that exhibit function or activity in living organisms, and examples include the sugars, proteins, nucleic acids (e.g., DNA, RNA, etc.), dyes, and other drugs (pharmaceuticals) mentioned above. Functional molecules are preferably water-soluble, but their lipid solubility is not a barrier.

[0068] The proteins are not particularly limited as long as they are water-soluble proteins, and examples include enzymes, receptors, antibodies, antigens, vaccines, interferons, cytokines such as interleukins, chemokines, and transport proteins.

[0069] The nucleic acids are not particularly limited as long as they are water-soluble nucleic acids, and examples include DNA, RNA, DNA-RNA chimeric nucleic acids, and DNA / RNA hybrids.

[0070] The pigment is not particularly limited as long as it is water-soluble, and examples of fluorescent pigments include fluorescein isothiocyanate (FITC), calcein, rhodamine B (RhoB), and indocyanine green.

[0071] The drugs are not particularly limited as long as they are water-soluble, for example, antitumor agents, antihypertensive agents, antihypotensive agents, antipsychotics, analgesics, antidepressants, antimanic agents, anxiolytics, sedatives, hypnotics, antiepileptic agents, opioid agonists, asthma treatments, anesthetics, antiarrhythmics, arthritis treatments, antispasmodics, ACE inhibitors, decongestants, antibiotics, antianginic agents, diuretics, antiparkinson's disease agents, bronchodilators, antidiuretics, diuretics, antihyperlipidemia agents, immunosuppressants, immunomodulators, antiemetics, antiinfective agents, Examples include those that can act as antineoplastic agents, antifungal agents, antiviral agents, antidiabetic agents, antiallergic agents, antipyretics, antigout agents, antihistamines, antipruritics, bone regulators, cardiovascular agents, cholesterol lowering agents, antimalarial agents, antitussives, expectorants, mucolytics, nasal congestion medications, dopamine agonists, gastrointestinal medications, muscle relaxants, neuromuscular blocking agents, parasympathetic agonists, prostaglandins, stimulants, appetite suppressants, thyroid agents or antithyroid agents, hormones, anti-migraine agents, anti-obesity agents, anti-inflammatory agents, etc.

[0072] Functional molecules can be used individually or in combination of two or more.

[0073] If the first aqueous medium contains functional molecules (e.g., sugars, proteins, nucleic acids, dyes, or other agents), the amount is not particularly limited and can be appropriately adjusted depending on the amount to be encapsulated in the inner aqueous phase of the lipid membrane vesicle. The amount of functional molecules is usually 1 mM to 10 M.

[0074] The first aqueous medium is not particularly limited and may contain salts used in cell culture, such as sodium chloride, potassium chloride, lithium chloride, DPBS, EBSS, HBSS, HEPES, and PBS. If the first aqueous medium contains salts, the content is not particularly limited and is usually between 1 mM and 10 M.

[0075] The osmotic pressure of the first aqueous medium (internal aqueous phase) is not particularly limited and can be appropriately adjusted according to the stability of the lipid membrane vesicles and the osmotic pressure of the second aqueous medium (external aqueous phase).

[0076] The amount of the first aqueous medium used is not particularly limited and can be adjusted as appropriate depending on the amount of membrane constituent lipids present or the required amount of lipid membrane vesicles. The amount of the first aqueous medium used is usually 0.001 mL to 100 mL per 1 mg of membrane constituent lipids.

[0077] The amount of the first aqueous medium used is not particularly limited and can be adjusted as appropriate according to the amount of oily medium used. The amount of aqueous medium used is preferably 1% to 10,000% by volume, and more preferably 10% to 1,000% by volume, relative to 100% by volume of the amount of oily medium used.

[0078] 2-2-4. Others The method for forming a W / O emulsion is not particularly limited. From the viewpoint of the efficiency of W / O emulsion formation, it is preferable to obtain a mixture of lipid extract, oily medium, and aqueous medium by, for example, sequentially adding the oily medium and aqueous medium to the lipid extract. The mixing (emulsification) method is not particularly limited, and a wide range of known mixing (emulsification) methods can be employed, such as shaking, stirring, inversion mixing, sonication, and homogenization.

[0079] The temperature in step (B) is not particularly limited and can be adjusted as appropriate depending on the composition of the lipid extract. In particular, from the viewpoint of suppressing the denaturation of membrane constituent lipids, step (B) is preferably carried out at a temperature of 10°C to 60°C, and more preferably at a temperature of 20°C to 40°C.

[0080] The pressure in process (B) is not particularly limited and is usually at atmospheric pressure.

[0081] Process (B) is not particularly limited and may be carried out in an atmospheric environment or in an inert gas atmosphere.

[0082] The mixing (emulsification) time can be the time from the mixing of the lipid extract, oily medium, and aqueous medium until lipid membrane vesicles are formed, and is usually between 1 second and 24 hours.

[0083] The W / O emulsion is obtained in step (B) as water droplets in oil coated with a lipid film. Specifically, the W / O emulsion is obtained as a lipid film present on the oil-water interface in which a first aqueous medium is dispersed in an oily medium.

[0084] The W / O emulsion preferably contains the extraction solvent used in step (A) and the dihydric alcohol contained therein. In this case, the extraction solvent can be one of those described above in "1-1-2. Extraction Solvent". In the W / O emulsion, the membrane constituent lipids are preferably present together with the dihydric alcohol.

[0085] The W / O emulsion obtained in step (B) may contain insoluble solids derived from the extract. If the W / O emulsion contains solids, these can be separated and removed from the W / O emulsion by conventional removal methods. In this case, in step (C), the settled solids, or the solids suspended on the surface or interface of the oil or aqueous phase, can be easily removed.

[0086] 2-3. Process (C) In step (C), the oil phase is removed from the W / O emulsion obtained in step (B) by centrifugation. Removal is sufficient if it is only a portion of the oily medium used in step (B), but it is preferable to remove substantially all of the oily medium used in step (B).

[0087] The centrifugal force used in centrifugation should be sufficient to allow the lipid membrane vesicles to settle, for example, around 1,000 × g to 15,000 × g. The centrifugation time should be sufficient to allow the lipid membrane vesicles to settle, for example, around 5 minutes to 10 hours. The centrifugation temperature should be preferably between 10°C and 60°C, and more preferably between 20°C and 40°C, from the viewpoint of suppressing the denaturation of membrane constituent lipids.

[0088] The conditions for process (C) (e.g., temperature, pressure, atmosphere, etc.) are not particularly limited and the conditions described above in "2-2-4. Others" can be adopted. In particular, from the viewpoint of suppressing the denaturation of membrane constituent lipids, processes (B) and (C) are preferably carried out at a temperature of 10°C to 60°C, and more preferably at a temperature of 20°C to 40°C.

[0089] The result of step (C) is obtained as a lipid membrane vesicle precursor, which is a gel-like vesicle aggregate in the lower layer (aqueous phase) or an emulsion at the upper-lower interface (aqueous-oil phase interface). The lipid membrane vesicle precursor can be recovered by removing the oil phase from the W / O emulsion obtained in step (B). More specifically, it can be recovered, for example, by aspirating the upper layer (oil phase) or lower layer (aqueous phase) after centrifugation.

[0090] Thus, the lipid membrane vesicle precursor can be recovered in the form of a dispersion in a first aqueous medium (e.g., an aqueous solution). The lipid membrane vesicle precursor preferably contains a lipid membrane, and more preferably contains a lipid membrane that encapsulates the first aqueous medium.

[0091] The lipid membrane vesicle precursor preferably contains the extraction solvent used in step (A) and the dihydric alcohol contained therein. In this case, the extraction solvent can be one of those described in "1-1-2. Extraction Solvent". In the lipid membrane vesicle precursor, the membrane constituent lipids are preferably present together with the dihydric alcohol.

[0092] Furthermore, if the W / O emulsion contains solid matter such as cell residue, it is preferable to remove the precipitated solid matter by centrifugation.

[0093] Step (C) is preferably repeated multiple times along with any necessary washing steps. In this case, the recovery efficiency of lipid membrane vesicles can be improved. Washing can be performed, for example, by redispersing the lipid membrane vesicle precursors that have settled after centrifugation into a washing solution.

[0094] 2-4. Process (D) In step (D), the result of step (C) is mixed with a second aqueous medium. In this invention, by dispersing the lipid membrane vesicle precursor obtained in step (C) in the second aqueous medium, the lipid membrane self-assembles, and stable lipid membrane vesicles can be produced.

[0095] 2-4-1. Second aqueous medium The second aqueous medium is not particularly limited as long as it is a medium containing water. From the viewpoint of the stability of lipid membrane vesicles, an aqueous solution containing some solute is preferred as the aqueous medium, and a sugar aqueous solution is more preferred. As the second aqueous medium, one of the above-mentioned "2-2-3. First aqueous medium" can be used.

[0096] When the first aqueous medium is a sugar aqueous solution, the second aqueous medium is preferably a sugar aqueous solution, and more preferably an aqueous solution of a relatively small molecular weight sugar such as a monosaccharide or disaccharide. In particular, by selecting an aqueous solution of a sugar with a smaller molecular weight than the inner aqueous phase (first aqueous medium) as the outer aqueous phase (second aqueous medium), it becomes easier to recover lipid membrane vesicles by utilizing the difference in specific gravity.

[0097] The osmotic pressure of the second aqueous medium (outer aqueous phase) is not particularly limited and can be appropriately adjusted according to the stability of the lipid membrane vesicles and the osmotic pressure of the first aqueous medium (inner aqueous phase). It is preferable that the osmotic pressure of the second aqueous medium is substantially equal to (especially isotonic) the osmotic pressure of the first aqueous medium.

[0098] In the second aqueous medium (outer aqueous phase), the sugar content is not particularly limited and can be appropriately adjusted according to the specific gravity of the inner aqueous phase of the lipid membrane vesicles. The sugar content is usually 1 mM to 10 M per 1 mg of membrane constituent lipids.

[0099] 2-4-2. Others The mixing method is not particularly limited, and a wide range of known mixing (emulsification) methods can be employed, such as shaking, stirring, inversion mixing, ultrasonic treatment, and homogenization.

[0100] The conditions for process (D) (e.g., temperature, pressure, atmosphere, etc.) are not particularly limited and the conditions described above in "2-2-4. Others" can be adopted. In particular, from the viewpoint of suppressing the denaturation of membrane constituent lipids, processes (B) to (D) are preferably carried out at a temperature of 10°C to 60°C, and more preferably at a temperature of 20°C to 40°C.

[0101] 2-5. Lipid membrane vesicles In one embodiment, the present invention provides lipid membrane vesicles. The lipid membrane vesicles of the present invention (which may also be simply referred to as "vesicles" in this specification) can be obtained by the method for producing lipid membrane vesicles described above.

[0102] Lipid membrane vesicles are not particularly limited as long as they include a lipid membrane composed of membrane-constituting lipids contained in the extract described above. Lipid membrane vesicles may be monolayer vesicles (liposomes) or multilayer vesicles (liposomes). Lipid membrane vesicles are preferably lipid bilayer vesicles, and monolayer lipid bilayer vesicles are more preferable.

[0103] Lipid membrane vesicles preferably contain glycolipids within the lipid membrane. Furthermore, lipid membrane vesicles preferably contain membrane proteins within the lipid membrane. According to the present invention, functional molecules such as glycolipids and / or membrane proteins can be impaired in lipid membrane vesicles without affecting their function and activity. Examples of sugars that may be contained in lipid membrane vesicles include α-mannose, α-galactose, N-acetyl-D-glucosamine, and sialic acid. The presence of glycolipids can be confirmed, for example, by adding fluorescently labeled lectins (e.g., concanavalin A, wheat malt agglutinin).

[0104] The lipid membrane constituting the lipid membrane vesicles is not particularly limited, but it is preferable that it corresponds to the composition of the membrane constituent lipids contained in the biological sample used.

[0105] Typically, after the production of lipid membrane vesicles, the internal aqueous phase of the lipid membrane vesicles is mainly a first aqueous medium, and the external aqueous phase of the lipid membrane vesicles is mainly a second aqueous medium. The external aqueous phase (especially the second aqueous medium) may permeate the membrane and infiltrate the internal aqueous phase, and the internal aqueous phase (first aqueous medium) may permeate the membrane and leach into the external aqueous phase.

[0106] The resulting lipid membrane vesicles (dispersion) can be stored under refrigerated or frozen conditions, for example, in a dispersed state in an aqueous solution or in a dry state, although this is not particularly limited.

[0107] The lipid membrane vesicles preferably contain the extraction solvent used in step (A) and the dihydric alcohol contained therein. In this case, the extraction solvent can be one of those described in "1-1-2. Extraction Solvent". In the lipid membrane vesicles, the membrane constituent lipids are preferably present together with the dihydric alcohol.

[0108] The average particle size of lipid membrane vesicles is not particularly limited, but is preferably 1 μm or larger, more preferably 3 μm or larger, and even more preferably 5 μm or larger. However, the lipid membrane vesicles can be adjusted to submicron or nano-sized dimensions by micronization treatment as needed. Examples of micronization treatments include sonication, homogenization, filtration, and extrusion using an extruder. When the size of the lipid membrane vesicles is nano-sized, the average particle size of the lipid membrane vesicles can be determined manually or automatically by subjecting images acquired with a confocal laser microscope or the like to image analysis. Alternatively, when the size of the lipid membrane vesicles is nano-sized, the average particle size can be determined by, for example, dynamic light scattering.

[0109] The coefficient of variation of the particle size of lipid membrane vesicles is not particularly limited, but is preferably 50% or less, more preferably 40% or less, and even more preferably 30% or less. The lower the coefficient of variation of the particle size of lipid membrane vesicles, the better; there is no particular lower limit, but it can be, for example, 1% or more.

[0110] The concentration of lipid membrane vesicles is not particularly limited, but is 1 × 10⁻⁶.3 Preferably, 1 × 10¹ / mL or more. 5 Preferably 1 × 10¹ / mL or more. 6 More preferably 1 × 10¹ / mL or more. 8 A concentration of vesicles / mL or higher is particularly preferred. While higher concentrations of lipid membrane vesicles are generally better, and no specific upper limit is set, for example, 1 × 10⁻⁶ 10 The concentration can be reduced to 1 / mL or less. The concentration of lipid membrane vesicles can be determined by image analysis. One method for determining the vesicle concentration is to place a silicone rubber sheet with a thickness d and a hole of a predetermined size on a glass slide as a spacer during microscopic observation of lipid membrane vesicles, fill the hole with a lipid membrane vesicle dispersion adjusted to a concentration that prevents the settled lipid membrane vesicles from overlapping, seal it with a coverslip, and calculate the formula: (vesicle concentration) = (number of lipid membrane vesicles in the field of view / volume of a rectangular parallelepiped with a height d and the actual area of ​​the field of view as the base).

[0111] In one embodiment, the lipid membrane vesicles of the present invention have an average particle diameter of 1 μm or more, and are 1 × 10⁻¹⁶ 3 The solution contains more than 100% of the lipid membrane vesicles per mL, and the coefficient of variation of the particle size of the lipid membrane vesicles is 50% or less. Conventional techniques for synthesizing giant lipid membrane vesicles (GVs) have made it difficult to synthesize GVs in large quantities. According to the present invention, it is possible to obtain novel lipid membrane vesicles and their dispersions that not only enable the synthesis of large quantities of GVs, but are also uniform and highly concentrated.

[0112] The lipid membrane vesicles of the present invention can efficiently coat a surface with membrane-constituting lipids by bringing them into contact with the surface of a solid material. In this case, the functions of the membrane-constituting lipids contained in the sample can be transferred to the solid material. Specifically, for example, if lipid membrane vesicles are produced using a lipid extract derived from blood cells, bringing these vesicles into contact with the surface of a solid material can impart to the solid material properties such as biocompatibility, blood retention, and stealth derived from the cell membrane of blood cells. Examples of solid materials include silica nanoparticles.

[0113] As described above, the lipid membrane vesicles of the present invention can optionally contain any membrane-constituting lipids in the lipid membrane and / or encapsulate any functional molecules in the internal aqueous phase. For this reason, the lipid membrane vesicles of the present invention can be particularly suitable as drug carriers for drug delivery. In this case, it is preferable to set the average particle size of the lipid membrane vesicles to 10 nm to 500 nm, and more preferably to 20 nm to 200 nm, by the above-described micronization treatment.

[0114] In addition to the above-mentioned uses, the lipid membrane vesicles of the present invention can be used in applications such as food, beverages, animal feed, cosmetics, and pharmaceuticals.

[0115] 3. Lipid composition In one embodiment, the present invention provides a lipid composition (membrane-constituting lipid composition). The lipid composition of the present invention (which may also be simply referred to as "composition" in this specification) comprises a biological sample containing membrane-constituting lipids, an extraction solvent containing a dihydric alcohol, and an oily medium containing vegetable oil. That is, the composition of the present invention is a composition obtained by contacting a lipid extract obtained by the above-described method for producing lipid extracts (particularly step (A)) with an oily medium containing vegetable oil.

[0116] The biological samples and membrane constituent lipids included in the composition can be those described above in "1-1-1. Biological Samples".

[0117] The extraction solvent and dihydric alcohol included in the composition can be those described above in "1-1-2. Extraction Solvent".

[0118] As for the vegetable oils and oily media that may be included in the composition, those described above in "2-2-2. Oily Media" can be used.

[0119] The composition is not particularly limited, and is usually, per 1 mg of membrane lipids, The content of the extraction solvent (especially dihydric alcohols) is 0.001 mL to 100 mL. The content of the oily medium (especially vegetable oil) is 0.001 mL to 100 mL. In particular, the composition of the mixture is such that, from the viewpoint of lipid membrane vesicle formation efficiency, 1 mg of membrane constituent lipids corresponds to: The content of the extraction solvent (especially dihydric alcohols) is 0.01 mL to 10 mL. The content of the oily medium (especially vegetable oil) is preferably 0.01 mL to 10 mL.

[0120] The ratio of the content of the extraction solvent to the oily medium in the composition (extraction solvent:oily medium) is not particularly limited, and is usually 10:1 to 1:10 by volume. In particular, from the viewpoint of the efficiency of lipid membrane vesicle formation, a ratio of 5:1 to 1:5 by volume is preferred.

[0121] The composition of the present invention may further contain an aqueous medium. Examples of aqueous media that may be included in the composition include those described in "2-2-3. First Aqueous Medium". That is, the composition of the present invention may also be the composition (mixture) used in step (B) of the method for producing lipid membrane vesicles of the present invention described above.

[0122] If the composition further contains an aqueous medium, the ratio of the oily medium to the aqueous medium in the composition (oily medium:aqueous medium) is not particularly limited, and is usually 10:1 to 1:10 by volume. In particular, from the viewpoint of lipid membrane vesicle formation efficiency, the ratio of the oily medium to the aqueous medium in the composition (oily medium:aqueous medium) is preferably 5:1 to 1:5 by volume.

[0123] The composition of the present invention is particularly suitable as a lipid composition for use in the production of lipid membrane vesicles. For reasons that are not fully understood, when the composition of the present invention is applied to the method for producing lipid membrane vesicles and similar methods described later, lipid membrane vesicles can be prepared with higher efficiency compared to compositions containing conventional extraction solvents. In addition to the production of lipid membrane vesicles, the lipid extract of the present invention can be used in applications such as food, beverages, animal feed, cosmetics, and pharmaceuticals. [Examples]

[0124] Hereinafter, reference examples and examples are shown to clarify the features of the present invention more clearly, but the present invention is not limited to the following examples.

[0125] In order to simplify the description of the specification, the following abbreviations may be used in the reference examples, examples, and tables in the examples. Abbreviations: As the meaning, PG: 1,2-propanediol (propylene glycol), GOL: glycerin, FITC: fluorescein isothiocyanate, Con A: concanavalin A, WGA: wheat germ agglutinin.

[0126] [Example 1: Extraction of membrane constituent lipids] Sf9 cells were cultured in a liquid medium of Sf-900 III SFM (1x, Gibco). Sf9 cells were washed and collected by a normal method and used in subsequent experiments. PBS(-) was used as the cell washing solution.

[0127] Sf9 cells (about 1.63×10 6 cells / mL) recovered from the culture solution were suspended in 1 mL of PBS(-) in a 1.5 mL tube, and then the frozen and stored one at a deep freezer (-80 °C) was thawed at room temperature, and the Sf9 cells were sedimented into pellets by centrifuging at FAST, 800 rpm, and 5 min using a tabletop micro high-speed centrifuge (CT-15E, HITACHI). Then, 1000 μL of 1,2-propanediol (Wako Pure Chemical Industries, Ltd.) was added as an extraction solvent and stirred using a vortex mixer. The obtained lipid extract was frozen and stored in a deep freezer (-80 °C, Panasonic).

[0128] [Example 2: Preparation of lipid vesicles] After thawing the lipid extract of Sf9 cells obtained in Example 1 at room temperature, 100 μL of the extract taken from the bottom was weighed into a 1.5 mL tube. 400 μL of soybean oil (Nacalai Tesque) was added and the mixture was stirred using a vortex mixer for about 5 minutes. Then, 500 μL of 300 mM trehalose (Wako Pure Chemical Industries) aqueous solution was added as the first aqueous medium, which mainly forms the inner aqueous phase of the lipid membrane vesicles, and the mixture was emulsified by stirring again using a vortex mixer for about 5 minutes. The solution was then centrifuged in a benchtop micro-high-speed centrifuge under the conditions FAST, 15000 rpm, 1 min, and 25°C. After centrifugation, the solution in the 1.5 mL tube separated into an upper layer (oil phase) and a lower layer (aqueous phase). Taking care to avoid aspirating the turbidity at the interface as much as possible, the soybean oil constituting the upper layer (oil phase) was removed by pipetting. Subsequently, approximately 500 μL of the supernatant of the remaining aqueous phase was transferred to another 1.5 mL tube to remove the cell debris that had settled at the bottom. Then, the solution was centrifuged in a benchtop micro-high-speed centrifuge at FAST, 15000 rpm, 1 min, and 25°C, and the upper layer (oil phase) formed by phase separation was removed in the same manner. This process was repeated 2-3 times to remove as much soybean oil as possible from the solution. After that, 500 μL of 300 mM glucose (Wako Pure Chemical Industries) solution was added as a second aqueous medium, mainly to form the outer aqueous phase of the lipid membrane vesicles, and the mixture was mixed by inversion. The obtained lipid membrane vesicles were then centrifuged in a benchtop micro-high-speed centrifuge at FAST, 1000 rpm, 10 min, and 25°C to settle at the bottom of a 1.5 mL tube and collected. Furthermore, when the obtained lipid membrane vesicles were to be subjected to confocal laser scanning microscopy observation as described later, the lipid membrane was fluorescently labeled by adding 10 μL of chloroform solution of Rhodamine-DOPE (0.01 mg / mL) to the lipid extract, and the inner aqueous phase was fluorescently labeled by adding calcein (final concentration 0.5%) to an aqueous trehalose solution.

[0129] (Confocal laser scanning microscope observation) For observation of lipid membrane vesicles, an inverted microscope (IX-71, Olympus) equipped with a confocal unit (CSU-X1, Yokogawa Electric), a laser and its control unit (ALC5000, Andor Technology), an EM-CCD camera (iXon, Andor Technology), and a 60x oil immersion lens (60x, numerical aperture 1.45, Olympus) was used. The microscope slide containing lipid membrane vesicles was fixed on the stage, the field of view was manually positioned, and automatic imaging of bright-field, red fluorescence (ex 562nm, em 580-660nm), green fluorescence (ex 488nm, em 500-550nm), and blue fluorescence (ex 405nm, em 420-470nm) was performed using a measurement program designed on the control software (iQ2, Andor Technology). Images of lipid membrane vesicles obtained in Example 2 are shown in Figure 3.

[0130] In Figure 3, the upper left image shows the internal aqueous phase labeled with calcein (green), the upper center image shows the lipid membrane labeled with rhodaminine, and the upper right image is a composite of these. The results shown in Figure 3 confirm the formation of fluorescently labeled lipid membrane vesicles and the presence of calcein in the internal aqueous phase of these vesicles.

[0131] (Particle concentration measurement) The concentration of lipid membrane vesicles obtained in Example 2 was determined by analyzing images acquired with a confocal laser scanning microscope using ImageJ software. Specifically, a 0.1 mm thick silicone rubber sheet with a 6 mm diameter hole was placed on a glass slide as a spacer, the lipid membrane vesicle dispersion was placed in the hole, and the slide was sealed with a coverslip. The image was then observed at 60x and 250x magnification using a confocal microscope. Images were acquired in the field of view where vesicles were observed across the entire surface, and the number density (concentration) of lipid membrane vesicles was calculated by assuming that the lipid membrane vesicles contained in the field of view exist in a rectangular parallelepiped with a height of 0.1 mm and the image area (length × width) as the base. The number of lipid membrane vesicles contained in the field of view was analyzed manually or automatically using ImageJ software. As a result, the vesicle concentration was 1.66 × 10⁻¹⁶. 8 The count was cells / mL.

[0132] (Particle size measurement) The average particle size and coefficient of variation of the lipid membrane vesicles obtained in Example 2 were determined by analyzing images acquired with a confocal laser scanning microscope using ImageJ software. More specifically, the particle size of 100 lipid membrane vesicles that did not overlap the field of view was analyzed using ImageJ software. As a result, the average particle size was 6.78 μm, the standard deviation was 1.78 μm, and the coefficient of variation (CV) was 28%. The particle size distribution is shown in Figure 4.

[0133] (Lectin reaction experiment) To the lipid membrane vesicle dispersion obtained in Example 2, a fluorescently labeled lectin, either FITC-WGA (concentration 2 mg / mL) solution or FITC-Con A (concentration 2 mg / mL) solution, was added, and the mixture was mixed four times by pipetting. Here, a 300 mM mannitol (Wako Pure Chemical Industries) aqueous solution was used as the outer aqueous phase of the lipid membrane vesicles. In addition, the lipid membrane was fluorescently labeled by adding 10 μL of a chloroform solution of Rhodamine-DOPE (0.01 mg / mL) to the lipid extract.

[0134] Lipid membrane vesicles labeled with FITC-WGA and FITC-ConA are shown in Figures 5 and 6, respectively. In Figures 5 and 6, co-localization of FITC fluorescence and Rhodamine fluorescence was confirmed. Considering that some of the lipid membrane vesicles were ruptured, it is thought that the lipid membrane vesicles obtained in Example 2 contain glycolipids. Furthermore, since the FITC-labeled region is unevenly distributed in some of the lipid membrane vesicles, it is thought that membrane domains are formed within the lipid membrane.

[0135] [Comparative Example 1: Glycerin] The membrane constituent lipids were extracted and lipid membrane vesicles were prepared in the same manner as in Examples 1 and 2, except that 1,2-propanediol was replaced with glycerin. The results of the observation of the obtained lipid membrane are shown in Figure 7.

[0136] When glycerin was used as the extraction solvent, a sufficient amount of lipid membrane vesicles could not be obtained, and the particle size of the small amount of lipid membrane obtained was non-uniform (Figure 7). Furthermore, when glycerin was used as the extraction solvent, the intensity of the calcein-derived fluorescence detected in the aqueous phase of the lipid membrane was significantly lower compared to when 1,2-propanediol was used as the extraction solvent (Figure 3). Therefore, it is thought that when glycerin was used as the extraction solvent, the stability of the lipid membrane decreased, and complete lipid membrane vesicles could not be formed. For this reason, the average particle size and coefficient of variation of particle size of the lipid membrane obtained in Comparative Example 1 could not be measured.

[0137] Thus, according to the present invention, membrane constituent lipids can be extracted using reagents with lower biotoxicity while maintaining their activity more stably, and lipid membrane vesicles can be prepared with high efficiency.

Claims

1. (A) A method for producing a lipid extract, comprising the step of contacting a biological sample containing membrane-constituting lipids with an extraction solvent containing a dihydric alcohol to extract the membrane-constituting lipids.

2. The method according to claim 1, wherein the dihydric alcohol is a branched alcohol.

3. The method according to claim 1 or 2, wherein the number of carbon atoms in the dihydric alcohol is 3 to 6.

4. The method according to claim 1 or 2, wherein the number of carbon atoms in the dihydric alcohol is 3 or 4.

5. The method according to claim 1 or 2, wherein the dihydric alcohol is 1,2-propanediol.

6. The method according to claim 1 or 2, wherein the biological sample is a cell lysate.

7. The method according to claim 1 or 2, wherein the lipid extract comprises glycolipids and / or membrane proteins.

8. The method according to claim 1 or 2, wherein step (A) is carried out at a temperature of 10°C to 60°C.

9. A lipid extract obtained by the manufacturing method described in claim 1 or 2.

10. A lipid extract according to claim 9, for use in the production of lipid membrane vesicles.

11. A lipid composition comprising a biological sample containing membrane-constituting lipids, an extraction solvent containing a dihydric alcohol, and an oily medium containing vegetable oil.

12. (B) A step of forming a W / O type emulsion by mixing a lipid extract obtained by the manufacturing method described in claim 1 with an oily medium containing vegetable oil and a first aqueous medium. (C) A step of removing the oil phase from the W / O type emulsion by centrifugal separation, and (D) A step of mixing the result of step (C) with a second aqueous medium, A method for producing lipid membrane vesicles.

13. The method according to claim 12, wherein steps (B) to (D) are performed at a temperature of 10°C to 60°C.

14. The method according to claim 12 or 13, wherein the vegetable oil is at least one selected from the group consisting of soybean oil, corn oil, and olive oil.

15. The method according to claim 12 or 13, wherein the average particle size of the lipid membrane vesicles is 1 μm or more.

16. Vesicles with an average particle diameter of 1 μm or more and a coefficient of variation of particle diameter of 50% or less are divided into 1 × 10⁻¹⁶ ３ Lipid membrane vesicles containing more than 1 / mL.

Images