Extracellular vesicles or extracellular vesicle contents

Abstract

The object is to provide extracellular vesicles or extracellular vesicle-containing materials derived from microalgae that have a characteristic composition and are more stable. [Solution] Extracellular vesicles or extracellular vesicle contents containing fatty acids and glycerolipids in a certain ratio are provided.

Description

[Technical Field]

[0001] The present invention relates to extracellular vesicles or extracellular vesicle contents derived from microalgae. [Background technology]

[0002] Many cell types secrete lipid bilayer membrane-containing vesicles. These vesicles are called extracellular vesicles (ECVs), and typically contain functional molecules such as proteins and nucleic acids within the lipid bilayer membrane. ECVs are involved in the delivery of functional molecules within the body, and in recent years, they have been shown to be involved in various biological phenomena and diseases.

[0003] For example, it has been disclosed that a population prepared by culturing under specific conditions stem cells that produce cell-derived vesicles can promote angiogenesis (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2019-501179 Summary of the Invention [Problem to be solved by the invention]

[0005] There are many unknowns about extracellular vesicles, and in particular, there are insufficient reports on the composition and function of extracellular vesicles derived from microalgae. Furthermore, the stability and absorbability of extracellular vesicles generally vary depending on their membrane structure, and their lipid distribution can change depending on the manufacturing process and the properties of the starting materials.

[0006] In particular, the lipids that make up the membrane of extracellular vesicles contribute greatly to the stability of the extracellular vesicles themselves.

[0007] Therefore, an object of the present invention is to provide extracellular vesicles or extracellular vesicle-containing materials derived from microalgae that have a characteristic composition and are more stable. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors conducted extensive research and found that extracellular vesicles or extracellular vesicle-containing materials containing a certain proportion of fatty acids having 16 to 18 carbon atoms and glyceroglycolipids are highly stable even when stored at low temperatures, leading to the completion of the present invention.

[0009] That is, the present invention provides the following extracellular vesicles or extracellular vesicle-containing materials.

[0010] [1] Extracellular vesicles or extracellular vesicle-containing materials derived from microalgae, which contain sulfoquinovosyl diacylglycerol (SQDG) and fatty acids, and the proportion of fatty acids with 16 to 18 carbon atoms among the fatty acids is 70 to 100%. [2] [1] Extracellular vesicles or extracellular vesicle contents, in which the ratio of SQDG to glyceroglycolipids is 80 to 100%. [3] [1] Extracellular vesicles or extracellular vesicle-containing substances, wherein the content of SQDG in the total amount of SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG and their stereoisomers is 80 to 100%. [4] An extracellular vesicle or extracellular vesicle-containing substance further containing diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), wherein the proportion of DGTS in the betaine lipid is 5 to 35% [1]. [5] The extracellular vesicles or extracellular vesicle-containing matter according to [1], wherein the content of DGTS in the total amount of DGTA, DGCC, MGTA, MGCC, DGTS, and their stereoisomers is 5 to 35%. [6] The extracellular vesicles or extracellular vesicle contents of [1], wherein the microalgae contain chloroplasts or thylakoids. [7] [6] The extracellular vesicles or extracellular vesicle-containing material according to [6], wherein the microalgae are microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella. [8] The extracellular vesicles or the material containing extracellular vesicles according to any one of [1] to [7], wherein the content of unsaturated fatty acids in the fatty acids having 16 to 18 carbon atoms is 5 to 20%.

[0011] Furthermore, the present invention can also provide the following aspects.

[0012] [7] An extracellular vesicle or extracellular vesicle-containing substance according to any one of [1] to [6], which contains stigmasterol as a sterol metabolite. [8] [7] Extracellular vesicles or extracellular vesicle contents in which the detected peak area of ​​stigmasterol is 35 to 60% of the total detected peak area of ​​sterol metabolites. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide microalgae-derived extracellular vesicles or extracellular vesicle-containing materials having a characteristic composition, and it is also possible to provide microalgae-derived extracellular vesicles or extracellular vesicle-containing materials that are highly stable when stored at low temperatures. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Extracellular vesicles or extracellular vesicle contents] The present invention relates to microalgae-derived extracellular vesicles or extracellular vesicle-containing materials, which contain sulfoquinovosyl diacylglycerol (SQDG) and fatty acids, wherein the fatty acids have 16 to 18 carbon atoms in an amount of 70 to 100%. Here, the extracellular vesicle-containing material may be a culture supernatant of microalgae itself, or may be a culture supernatant-derived material obtained by crude purification of the culture supernatant.

[0015] (Microalgae culture supernatant) In this specification, microalgae refers to organisms that perform oxygen-producing photosynthesis, excluding mosses, ferns, and spermatophytes, and have a cell size of 0.1 μm to 1000 μm in diameter.

[0016] Microalgae include, for example, prokaryotic organisms of the phylum cyanobacteria, as well as eukaryotic organisms of the phylum Glaucophyta, phylum Rhodophyta (red algae), phylum Chlorophyta, phylum Cryptophyta (cryptophyta), phylum Haptophyta (haptophyta), phylum Heterokontophyta, phylum Dinophyta (dinoflagellates), phylum Euglenida, and phylum Chlorarachniophyta.

[0017] Examples of microalgae include microalgae containing chloroplasts or thylakoids. From the viewpoint of significantly achieving the effects of the present invention, microalgae containing chloroplasts or thylakoids are preferred, and microalgae containing chloroplasts are more preferred.

[0018] Examples of microalgae containing chloroplasts include Prokaryotic Chlorophyceae, Glaucophyceae, Rhodophyceae, Prasinophyceae, Ulvaphyceae, Chlorophyceae, Treboxiophyceae, Charophyceae, Cryptophyceae, Chlorarachniophyceae, Euglenophyceae, Dinophyceae, Chrysophyceae, Raphidophyceae, Euglenoplastphyceae, Xanthophyceae, Phaeophyceae, Bacillariophyceae, Dictyophyceae, Pelagophyceae, and Haptophyceae. The above-listed microalgae may be used singly or in combination of two or more.

[0019] Examples of microalgae containing thylakoids include microalgae belonging to the order Cyanobacteria. More specifically, examples include microalgae belonging to the orders Chlorococcales, Necrocapitales, Oscillatoriales, Prochlorococcales, Synechocystisales, and Plebydosphaerales. The above-listed microalgae may be used singly or in combination of two or more.

[0020] Examples of microalgae include microalgae that do not have a cell wall. From the viewpoint of significantly achieving the effects of the present invention, microalgae that do not have a cell wall are preferred. More specifically, examples include microalgae belonging to the genera Pavlova, Euglena, and Dunaliella. One type of the exemplified microalgae may be used alone, or two or more types may be used in combination.

[0021] Examples of microalgae include microalgae belonging to the genus Pavlova, Euglena (Japanese name: Midorimushi, protozoa / green algae), Spirulina (cyanophyceae), Chlorella (green algae), Dunaliella (green algae), Nannochloropsis (true eyespot algae), Licoricephala (green algae), and Botryococcus (green algae). The above-listed microalgae may be used singly or in combination of two or more.

[0022] Among microalgae, from the viewpoint of significantly achieving the effects of the present invention, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova, the genus Euglena, the genus Spirulina, and the genus Chlorella are preferred, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova, the genus Euglena, and the genus Spirulina are more preferred, microalgae belonging to at least one species selected from the group consisting of the genus Pavlova and the genus Euglena are even more preferred, and microalgae belonging to the genus Pavlova are particularly preferred.

[0023] Pavlova is included in the haptophyte division, and examples of microalgae of the genus Pavlova include P. calceolate, P. granifera, P. gyrans, P. lutheri, P. pinguis, and P. salina, with P. granifera and / or P. gyrans being preferred.

[0024] Examples of microalgae of the genus Euglena include E. gracilis, E. longa, E. caudata, E. oxyuris, E. tripteris, E. proxima, E. viridis, E. sociabilis, E. ehrenbergii, E. deses, E. pisciformis, E. spirogyra, E. acus, E. geniculata, E. intermedia, E. mutabilis, E. sanguinea, E. stellata, E. terricola, E. klebsi, E. rubra, and E. cyclopicola, with E. gracilis and / or E. longa being preferred.

[0025] Examples of microalgae of the genus Spirulina include the genus Altrospira, whose scientific name was changed from Spirulina, and include S. platensis (A. platensis) and S. maxima (A. maxima), with S. platensis being preferred.

[0026] Examples of microalgae of the genus Chlorella include C. vulgaris, C. saccharophila, C. Ellipsoidea, C. pyrenoidosa, C. sorokiniana, and C. lobophora, with C. vulgaris being preferred.

[0027] The microalgae exemplified above are widely distributed in seawater, freshwater such as ponds and marshes, and brackish water, and may be separated from these and used, or any microalgae that has already been isolated may be used.

[0028] The microalgae exemplified above include closely related species and mutant strains thereof, as long as they exhibit the effects of the present invention. Mutant strains also include those obtained by genetic methods such as gene recombination, transduction, and transformation.

[0029] As used herein, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained after culturing microalgae. For example, the term "microalgae culture supernatant" refers to the supernatant of a culture solution obtained by culturing microalgae using a known culture method and removing the microalgae using a known separation means such as centrifugation.

[0030] The culture conditions for preparing the culture supernatant of microalgae are not particularly limited as long as the effects of the present invention are achieved, and known methods can be used, including the following examples.

[0031] (Culture conditions) Microalgae can be cultured using a culture medium. A carbon source is added to the culture medium as a nutrient source, and examples of the carbon source include inorganic carbon sources (CO2, NaHCO3, Na2CO3, etc.) and organic carbon sources (glucose, etc.). The culture medium is not limited, but it is preferable to use an autotrophic medium that does not contain an organic carbon source such as glucose as a nutrient source. Examples include culture media supplemented with nutrient salts such as a nitrogen source, a phosphorus source, and minerals, such as Cramer-Myers medium and modified Cramer-Myers medium. In one embodiment, the medium may or may not contain phosphorus.

[0032] Depending on the type of microalgae, when seawater is used, it is possible to use a culture medium for marine microalgae or artificial seawater, or it is also possible to use commercially available products such as IMK medium.

[0033] The pH of the culture medium is not particularly limited as long as the effects of the present invention are achieved, and examples include 2 to 8, 3 to 8, 4 to 8, 5 to 8, 2 to 7.5, 3 to 7.5, 4 to 7.5, and 5 to 7.5.

[0034] The culture temperature is not particularly limited as long as the effects of the present invention are achieved, but examples include 15 to 40°C, 20 to 34°C, and 23 to 28°C.

[0035] The culture period is not particularly limited as long as the effects of the present invention are achieved, and examples include 4 to 30 days, 4 to 20 days, and 5 to 15 days.

[0036] Depending on the type of microalgae, the type of light source, the presence or absence of light and dark, aerobic conditions, anaerobic conditions, etc. can be appropriately selected.

[0037] In one embodiment, when culturing marine microalgae such as pavlova, known methods can be used as long as the effects of the present invention are achieved. For example, Seawater concentration: 50% seawater Medium concentration: IMK medium 2x concentration Culture solution volume: 800mL Light source: Side: Fluorescent lamp (100-150 μmol) Light / dark cycle: 12 hours light and 12 hours dark pH: pH approximately 7.4 at the start of each culture Aeration: Implementation Mechanical stirring: None Cultivation period: Approximately 10 days Culture temperature: 25℃~28℃ The cells can be cultured under conditions that satisfy at least one of the following:

[0038] (Collection of culture supernatant) After culturing microalgae under the culture conditions exemplified above, the culture supernatant is recovered using a known separation method such as centrifugation. However, the method is not particularly limited as long as it removes or substantially removes the microalgae and allows the culture supernatant to be appropriately recovered. In an exemplary embodiment, the centrifugation to obtain the culture supernatant may be carried out for 30 to 60 minutes at a centrifugal force of, for example, 1,000 to 20,000 × g, 1,500 to 20,000 × g, 1,500 to 15,000 × g, or 1,500 to 10,000 × g. The centrifugation may be carried out stepwise by varying the speed or time. For example, the culture supernatant may be separated by centrifugation at a low speed of 1,500 to 2,000 × g, followed by centrifugation at a high speed of 10,000 to 20,000 × g to further remove cells or cell-related debris and residues. The supernatant thus obtained is recovered and subjected to the next step.

[0039] (extracellular vesicles) As used herein, extracellular vesicles refer to vesicles secreted by microalgae, typically having an average diameter of 10 nm to 1 μm. This average diameter is also referred to as the average particle size. Although not limited thereto, it is preferred that the extracellular vesicle-containing composition of the present invention does not contain microalgae cells themselves. Here, the term "diameter" or "particle size" used herein to specify the size of extracellular vesicles refers to the maximum dimension of the extracellular vesicles, since extracellular vesicles are not necessarily spherical. Diameter or particle size can be measured using microscopy techniques, particularly techniques for measuring nanoparticle size using a transmission electron microscope. Furthermore, measurement can also be performed using particle tracking analysis (PTA), which is based on the analysis of bright spots of particles undergoing Brownian motion. In the present invention, the diameter or particle size of extracellular vesicles is measured using a Viewsizer (HORIBA Scientific, ViewSizer 3000).

[0040] The average particle size of the extracellular vesicles of microalgae is not particularly limited as long as the effects of the present invention are achieved, but examples include 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, and 700 nm or less, 600 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, and the like. The average particle size of the extracellular vesicles of microalgae is, for example, 10 to 700 nm, 10 to 600 nm, 10 to 500 nm, 10 to 400 nm, 10 to 300 nm, 10 to 250 nm, 30 to 700 nm, 30 to 600 nm, 30 to 500 nm, 30 to 400 nm, 30 to 300 nm, 30 to 250 nm, 50 to 700 nm, 50 to 600 nm, 50 to Examples include 500 nm, 50 to 400 nm, 50 to 300 nm, 50 to 250 nm, 70 to 700 nm, 70 to 600 nm, 70 to 500 nm, 70 to 400 nm, 70 to 300 nm, 70 to 250 nm, 100 to 700 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm, 100 to 300 nm, and 100 to 250 nm.

[0041] The cumulative 10% value (D10) of particle diameter of pavlova-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 50 to 120 nm, 60 to 110 nm, and 65 to 100 nm. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 100 to 180 nm, 110 to 170 nm, and 115 to 160 nm. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 180 to 360 nm, 190 to 350 nm, and 200 to 340 nm. As used herein, the cumulative 10% value (D10) of particle diameter of extracellular vesicles refers to the diameter below which 10% of all extracellular vesicles are. The cumulative 50% value (D50) of particle diameter of extracellular vesicles refers to the diameter below which 50% of all extracellular vesicles are, and is synonymous with the median diameter. The cumulative 90% particle diameter (D90) of extracellular vesicles means the diameter below which 90% of all extracellular vesicles fall.

[0042] The cumulative 10% value (D10) of particle diameter of Spirulina-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 50 to 110 nm, 60 to 100 nm, 70 to 90 nm, etc. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 100 to 160 nm, 110 to 150 nm, 120 to 140 nm, etc. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 190 to 250 nm, 200 to 240 nm, 210 to 230 nm, etc.

[0043] The cumulative 10% value (D10) of particle diameter of chlorella-derived extracellular vesicles is not particularly limited as long as the effects of the present invention are achieved, and examples thereof include 90 to 150 nm, 100 to 140 nm, 110 to 130 nm, etc. Furthermore, the cumulative 50% value (D50) of the particle diameter is, for example, 180 to 240 nm, 190 to 230 nm, 200 to 220 nm, etc. Furthermore, the cumulative 90% value (D90) of the particle diameter is, for example, 320 to 380 nm, 330 to 370 nm, 340 to 360 nm, etc.

[0044] (Extracellular vesicle content) As used herein, the term "extracellular vesicle-containing material" refers to a concentrate or purified product containing extracellular vesicles obtained from the culture supernatant of microalgae and obtained by filtering the culture supernatant. In addition to the extracellular vesicles, the material may also contain secreted materials obtained by the culture.

[0045] The average particle number of extracellular vesicles of microalgae is, for example, 1.0 × 10 per ml if the purified product is in liquid form. 3 pcs or more, 1.0×10 4 pcs or more, 1.0×10 5 pcs or more, 1.0×10 6 pcs or more, 1.0×10 7 pcs or more, 1.0×10 8 pcs or more, 1.0×10 9 1.0×10 15 pcs or less, 1.0×10 14 pcs or less, 1.0×10 13 pcs or less, 1.0×10 12 pcs or less, 1.0×10 11 The average number of particles in the extracellular vesicles of microalgae is, for example, 1.0 × 10 4 ~1.0×10 15 pieces, 1.0×10 4 ~1.0×10 14 pieces, 1.0×10 4 ~1.0×10 13 pieces, 1.0×10 4 ~1.0×10 12 pieces, 1.0×10 4 ~1.0×10 11 pieces, 1.0×10 5 ~1.0×10 15 pieces, 1.0×10 5 ~1.0×10 14 pieces, 1.0×10 5 ~1.0×10 13 pieces, 1.0×10 5 ~1.0×10 12 pieces, 1.0×10 5 ~1.0×10 11 pieces, 1.0×10 6 ~1.0×10 15 pieces, 1.0×10 6 ~1.0×1014 pieces, 1.0×10 6 ~1.0×10 13 pieces, 1.0×10 6 ~1.0×10 12 pieces, 1.0×10 6 ~1.0×10 11 pieces, 1.0×10 7 ~1.0×10 15 pieces, 1.0×10 7 ~1.0×10 14 pieces, 1.0×10 7 ~1.0×10 13 pieces, 1.0×10 7 ~1.0×10 12 pieces, 1.0×10 7 ~1.0×10 11 pieces, 1.0×10 8 ~1.0×10 15 pieces, 1.0×10 8 ~1.0×10 14 pieces, 1.0×10 8 ~1.0×10 13 pieces, 1.0×10 8 ~1.0×10 12 pieces, 1.0×10 8 ~1.0×10 11 pieces, 1.0×10 9 ~1.0×10 15 pieces, 1.0×10 9 ~1.0×10 14 pieces, 1.0×10 9 ~1.0×10 13 pieces, 1.0×10 9 ~1.0×10 12 pieces, 1.0×10 9 ~1.0×10 11 The number of extracellular vesicles can be measured using a Viewsizer (HORIBA Scientific, ViewSizer3000) or other particle tracking analysis (PTA) based on the analysis of bright spots of particles undergoing Brownian motion.

[0046] (Method for Obtaining Extracellular Vesicles or Extracellular Vesicle Contents) Methods for obtaining the extracellular vesicles or extracellular vesicle-containing materials of the present invention include preparation from the culture supernatant of algae.

[0047] In one embodiment, the extracellular vesicles or extracellular vesicle-containing material of the present invention are obtained by filtering a culture supernatant of microalgae.

[0048] The membrane pore size of the filter used for filtration is preferably 0.001 to 0.5 μm, more preferably 0.005 μm to 0.3 μm, and even more preferably 0.01 μm to 0.25 μm.

[0049] In another embodiment, the extracellular vesicles or extracellular vesicle-containing materials of the present invention are not subjected to heating. Although not limited thereto, the treatment is carried out at a temperature of 0 to 60°C, for example, preferably 10 to 50°C, more preferably 20 to 40°C.

[0050] The method for obtaining extracellular vesicles or extracellular vesicle contents of the present invention can also include a purification step. Specific embodiments of the purification step include purification by ultrafiltration (e.g., tangential flow filtration), ultracentrifugation, and affinity purification using antibodies. Of these, purification by ultrafiltration is preferred, although not limited thereto.

[0051] When purification by ultrafiltration is used, it may be performed using a hollow fiber membrane to filter from the inside to the outside of the fiber, using a spiral membrane including a filtration membrane and a support membrane, using a tubular membrane on a hollow cylinder, or using a flat membrane. Of these, but not limited to, it is preferable to perform filtration using a hollow fiber membrane to obtain a concentrated liquid containing extracellular vesicles on the inside of the hollow fiber membrane.

[0052] The molecular weight cutoff of a membrane is preferably 10 kDa to 1,000 kDa, 50 kDa to 1,000 kDa, or 100 kDa to 1,000 kDa, and more preferably 100 kDa to 500 kDa. Cutoff membranes of 10 kDa or more, 50 kDa or more, 100 kDa or more, or 150 kDa or more are preferred, and cutoff membranes of 2,000 kDa or less, 1,500 kDa or less, 1,000 kDa or less, 750 Dka or less, or 500 Dka or less are also preferred.

[0053] The extracellular vesicles or extracellular vesicle-containing materials of the present invention may be prepared by one or a combination of two or more of these preparation methods, for example, but not limited to, a combination of a purification step and a filter filtration process.

[0054] The extracellular vesicles or the material containing extracellular vesicles may be in the form of a purified liquid of the culture supernatant, or may be a solid or semi-solid obtained by drying the purified liquid.

[0055] The extracellular vesicles can optionally be further purified to provide a pure population of extracellular vesicles.

[0056] The extracellular vesicles may be in the form of a purified liquid of the culture supernatant itself, or may be a solid or semi-solid obtained by drying the purified liquid.

[0057] (Lipid analysis of extracellular vesicles or extracellular vesicle contents) The extracellular vesicles or extracellular vesicle contents of the present invention contain fatty acids and SQDG as lipids.

[0058] Here, the method for analyzing lipids conforms to the analytical method specifically described in the Examples.

[0059] In one embodiment, the extracellular vesicles or extracellular vesicle-containing materials of the present invention contain fatty acids. In a specific embodiment, the extracellular vesicles or extracellular vesicle-containing materials of the present invention contain fatty acids having 16 to 18 carbon atoms in a detection peak area ratio of 70% or more, preferably 70 to 100%, more preferably 80 to 100%, even more preferably 85 to 100%, and even more preferably 90 to 100% of the total fatty acids. Alternatively, the detection peak area ratio may be 75% or more, 77% or more, 80% or more, 83% or more, 85% or more, 88% or more, 90% or more, 100% or less, 99.9% or less, 99.8% or less, or 99.7% or less.

[0060] In another aspect of the present invention, the detection peak area of ​​fatty acids having 16 to 18 carbon atoms in extracellular vesicles or extracellular vesicle-containing materials is preferably 70% or more, preferably 70 to 100%, more preferably 80 to 100%, even more preferably 85 to 100%, and even more preferably 90 to 100% of the total detection peak area of ​​fatty acids having 16 or more carbon atoms detected in extracellular vesicles or extracellular vesicle-containing materials. Alternatively, it may be 75% or more, 77% or more, 80% or more, 83% or more, 85% or more, 88% or more, 90% or more, 100% or less, 99.9% or less, 99.8% or less, or 99.7% or less.

[0061] In another aspect of the present invention, the content of unsaturated fatty acids in the fatty acids having 16 to 18 carbon atoms in the extracellular vesicles or the material contained in the extracellular vesicles is preferably 5 to 20%, more preferably 7 to 18%, and even more preferably 10 to 15%.

[0062] In this specification, fatty acid refers to an unsaturated or saturated monocarboxylic acid. The carbon chain of the fatty acid may contain an oxygen group (e.g., a ketone group). The fatty acid is preferably an unsaturated or saturated fatty acid having 10 to 32 carbon atoms.

[0063] In one embodiment, the extracellular vesicles or extracellular vesicle-containing compositions of the present invention contain SQDG. SQDG is a type of glyceroglycolipid. In a specific embodiment, the detection peak area of ​​SQDG relative to the total detection peak area of ​​glyceroglycolipids in the extracellular vesicles or extracellular vesicle-containing compositions of the present invention may be 70% or more, 73% or more, 75% or more, 78% or more, 80% or more, 82% or more, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, or 95% or less. This percentage is relative to the total amount of all glyceroglycolipids contained in the extracellular vesicles or extracellular vesicle-containing compositions.

[0064] As used herein, "glyceroglycolipid" refers to a compound in which a fatty acid diester of glycerin is glycosylated. The hydroxyl group to which the sugar chain is covalently bonded is a hydroxyl group that is not ester-bonded to a fatty acid. Examples of glyceroglycolipids include, but are not limited to, one or more selected from the group consisting of sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride. More specifically, sulfoquinovosyldiacylglycerol (SQDG), monoglycosyldiacylglycerol and / or monogalactosyldiacylglycerol (MGDG), diglycosyldiacylglycerol and / or digalactosyldiacylglycerol (DGDG), diglycosylmonoacylglycerol and / or digalactosylmonoacylglycerol (DGMG), glucuronosyldiacylglycerol (GADG), GADG hexose adducts (Hex The glycosylglycerol may be one or more selected from the group consisting of glycosylglycerol and / or galactosyldiacylglycerol (Gal-DAG), glycosylmonoacylglycerol and / or monogalactosylmonoacylglycerol (MGMG), gluconosylmonoacylglycerol and / or galactosylmonoacylglycerol (GAMG), monoglycosyldiacylglycerol and / or monogalactosyldiacylglycerol (MGDG), sulfoquinovosylmonoacylglycerol (SQMG), acylsulfoquinovosyldiacylglycerol (ASQDG), acylmonoglycosyldiacylglycerol and / or acylmonogalactosyldiacylglycerol (AMGDG), and acyldiglycosyldiacylglycerol and / or acyldigalactosyldiacylglycerol (ADGDG). That is, in this specification, these lipids may have either a glycosyl group or a galactosyl group, and both are represented by the same abbreviation. Furthermore, these lipids may have oxidized carbon chains of glycerol.

[0065] In another embodiment of the present invention, the detected peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 70 to 100%, more preferably 75 to 99%, and even more preferably 80 to 95% of the total detected peak area of ​​SQDG, MGDG, DGDG, GADG), HexGADG, MGMG, DGMG, GAMG, SQMG, ASQDG, SQDG[sn2_O], AMGDG, MGDG[sn2_O], DGDG[O], DGMG[sn2_O], MGMG[O], and ADGDG detected in the extracellular vesicles or extracellular vesicle-containing materials. It may also be 70% or more, 73% or more, 75% or more, 78% or more, 80% or more, 82% or more, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, or 95% or less.

[0066] In another embodiment of the present invention, the detected peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 70 to 100%, more preferably 75 to 99%, and even more preferably 80 to 95% of the total detected peak area of ​​SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG, and their stereoisomers. It may also be 70% or more, 73% or more, 75% or more, 78% or more, 80% or more, 82% or more, 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, or 95% or less. It is also possible for some components not to be included among the above-listed components.

[0067] In another embodiment of the present invention, the detected peak area of ​​SQDG in extracellular vesicles or extracellular vesicle-containing materials is preferably 70 to 100%, more preferably 75 to 99%, and even more preferably 80 to 95% of the total detected peak area of ​​SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, and ADGDG. Some components listed above may not be contained.

[0068] In one embodiment, the extracellular vesicles or extracellular vesicle-containing materials of the present invention contain sterol metabolites as lipids. Examples of sterol metabolites include, but are not limited to, stigmasterol, β-sitosterol, cholesterol sulfate, sitosterol sulfate, stigmasterol sulfate, hydroxycampestanol sulfate, hydroxystigmastanol sulfate, and brassicasterol sulfate. In a specific embodiment, the extracellular vesicles or extracellular vesicle-containing materials of the present invention contain stigmasterol as a sterol metabolite. The detection peak area of ​​stigmasterol in the extracellular vesicles or extracellular vesicle-containing materials of the present invention is preferably 35 to 60%, more preferably 37 to 60%, more preferably 40 to 55%, and even more preferably 43 to 53%, relative to the total detection peak area of ​​sterol metabolites. Alternatively, the detection peak area may be 35% or more, 40% or more, 43% or more, 100% or less, 80% or less, 60% or less, 55% or less, 53% or less, etc.

[0069] In another embodiment of the present invention, the detection peak area of ​​stigmasterol in extracellular vesicles or extracellular vesicle-containing materials is preferably 35 to 60%, more preferably 37 to 60%, more preferably 40 to 55%, and even more preferably 43 to 53%, of the total detection peak area of ​​stigmasterol, β-sitosterol, cholesterol sulfate, sitosterol sulfate, stigmasterol sulfate, hydroxycampestanol sulfate, hydroxystigmastanol sulfate, and brassicasterol sulfate detected in the extracellular vesicles or extracellular vesicle-containing materials. Alternatively, it may be 35% or more, 40% or more, 43% or more, 100% or less, 80% or less, 60% or less, 55% or less, 53% or less, etc.

[0070] In other embodiments, the extracellular vesicles or extracellular vesicle contents of the present invention may contain, as lipids, betaine lipids, acylhexoses, sphingolipids, neutral glycerolipids, and / or sphingoglycolipids in addition to glyceroglycolipids, fatty acids, and sterol metabolites.

[0071] In this specification, betaine lipids refer to lipids that have a diacylglycerol moiety as the basic skeleton and in which a methylated hydroxyamino acid and the diacylglycerol moiety are ester-bonded. Betaine lipids are unique lipids commonly found in lower organisms such as microalgae, and it is said that the lipids contained in the cell membranes of organisms have been replaced by phospholipids over the course of evolution.

[0072] Examples of betaine lipids include, but are not limited to, one or more selected from the group consisting of diacylglyceryl hydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA), diacylglyceryl hydroxymethylcholine (DGCC), diacylglyceryl 3-O-4-(N,N-trimethyl)homoserine (DGTS), monoacylglyceryl hydroxymethyl-N,N,N-trimethyl-β-alanine (MGTA), monoacylglyceryl-3-O-carboxyhydroxymethylcholine (MGCC), and monoacylglyceryl 3-O-4-(N,N-trimethyl)homoserine (MGTS).

[0073] In one embodiment, the extracellular vesicles or extracellular vesicle-containing compositions of the present invention may contain one or more of these betaine lipids. Other components may also be absent. Preferably, the extracellular vesicles or extracellular vesicle-containing compositions of the present invention contain one or more betaine lipids selected from the group consisting of DGTA, DGTS, DGCC, MGTA, and MGCC. More preferably, the extracellular vesicles or extracellular vesicle-containing compositions of the present invention contain DGTA or DGTS. Even more preferably, the extracellular vesicles or extracellular vesicle-containing compositions of the present invention contain DGTS. Alternatively, the extracellular vesicles or extracellular vesicle-containing compositions of the present invention contain all of DGTA, DGTS, DGCC, MGTA, and MGCC as betaine lipids.

[0074] In one embodiment of the present invention, the detection peak area of ​​DGTS in extracellular vesicles or extracellular vesicle contents is preferably 5 to 35%, more preferably 10 to 30%, and even more preferably 12 to 25%, of the total detection peak area of ​​betaine lipids detected in the extracellular vesicles or extracellular vesicle contents.

[0075] In another aspect of the present invention, the detection peak area of ​​DGTS in extracellular vesicles or extracellular vesicle-containing materials is preferably 5 to 35%, more preferably 10 to 30%, and even more preferably 12 to 25% of the total detection peak area of ​​DGTA, DGCC, MGTA, MGCC, DGTS, and their stereoisomers detected in the extracellular vesicles or extracellular vesicle-containing materials.

[0076] DGTS is a phosphate-free polar lipid that is known to contribute to maintaining membrane fluidity in Nannochloropsis oceanica at low temperatures (Hiroki Murakami et al., Betaine Lipid Is Crucial for Adapting to Low Temperature and Phosphate Deficiency in Nannochloropsis, Plant Physiology, May 2018, Vol. 177, pp. 181-193).

[0077] Examples of acylhexoses include diacylhexoses and / or monoacylhexoses.

[0078] Sphingolipids include, for example, ceramides and / or sulfonoceramides.

[0079] Neutral glycerolipids include, for example, triacylglycerols and / or diacylglycerols.

[0080] An example of glycosphingolipids is glucosylceramide.

[0081] The extracellular vesicles or extracellular vesicle-containing compositions of the present invention have improved stability at low temperatures. In this specification, "low temperature" refers to room temperature or below, for example, 20°C or below, 15°C to -100°C, 10°C to -80°C, 4°C to -60°C, 0°C to -30°C, or -5°C to -20°C. In a more specific embodiment, the rate of change in the average particle size of the extracellular vesicles or extracellular vesicle-containing compositions of the present invention after storage at -80°C for one week is 20% or less, for example, 18% or less. In another specific embodiment, the rate of change in the particle number of the extracellular vesicles or extracellular vesicle-containing compositions of the present invention after storage at -80°C for one week is 35% or less, for example, 30% or less, 25% or less, or 20% or less.

[0082] (Compositions comprising extracellular vesicles or extracellular vesicle contents) In one aspect of the present invention, a composition containing either the extracellular vesicles or the contents of the extracellular vesicles thus obtained can be prepared.

[0083] [Application] The composition containing the extracellular vesicles or extracellular vesicle-containing material of the present invention can be added to or mixed with pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet food. Alternatively, the extracellular vesicles or extracellular vesicle-containing material can be used as is as pharmaceuticals, quasi-drugs, cosmetics, foods, beverages, feed, or pet food.

[0084] When the composition containing the extracellular vesicles or extracellular vesicle-containing material of the present invention is added to or mixed with so-called topical preparations such as pharmaceuticals, quasi-drugs, and cosmetics, it can be used as pharmaceuticals, quasi-drugs, and cosmetics that explicitly or implicitly claim to improve skin elasticity, firmness, wrinkles, texture, moisture, dryness, roughness, etc. as efficacy, effects, or functions.

[0085] When the composition containing the extracellular vesicles or extracellular vesicle-containing material of the present invention is added to or mixed with food, beverage, feed, or pet food, it can be used as a food or beverage that explicitly or implicitly claims to have the functionality of improving skin elasticity, firmness, wrinkles, texture, moisture, dryness, roughness, etc., i.e., as a health food, functional food, food for patients, or food for specified health uses. Furthermore, even if the above functionality is not explicitly or implicitly claimed, it can be used as a so-called doctor's supplement recommended or suggested by doctors in internal medicine, orthopedic surgery, veterinary clinics, etc. in hospitals and / or clinics.

[0086] When the above functionality is not explicitly stated, it is also possible to use the composition of the present invention by indicating, for example, that it is suitable for those who seek skin elasticity, those who seek skin firmness, those who are concerned about wrinkles, those who are concerned about fine lines, those who want to smooth skin texture, those who seek skin moisture, those who are concerned about dry skin, those who are concerned about rough skin, those who are concerned about pores, those who are concerned about skin fluctuations, those who are concerned about pores, or similar indications.

[0087] Health foods, functional foods, foods for patients, and foods for specified health uses can be used in various dosage forms, such as solid preparations (tablets, orally disintegrating tablets, granules, fine granules, powders, capsules, chewable tablets, candy, etc.), liquid preparations (syrups, suspensions), and liquid foods. Food preparations can be manufactured in the same manner as known pharmaceutical preparations, by mixing the active ingredient with a food-acceptable carrier, such as a suitable excipient, and then manufacturing the preparation using conventional means. The dosage form is not limited, but is preferably an orally disintegrating tablet, chewable tablet, candy, granules, powder, or liquid preparation, from the viewpoint of significantly achieving the effects of the present invention. [Example]

[0088] Next, the present invention will be specifically explained by way of examples, but the present invention is not limited to the following examples.

[0089] Example 1 Pavlova sp. was cultured in a commercially available algae medium by a conventional method, and the culture supernatant was obtained by centrifugation at 3000×g at room temperature for 60 minutes.

[0090] 3000 mL of the resulting culture supernatant was centrifuged, and 3000 mL of the resulting supernatant was filtered through a 0.22 μm filter. The supernatant was then concentrated to a volume of 150 mL or less using a 300 kDa hollow fiber membrane filter (MiniKros sampler S04-E300-05-N: Repligen) by tangential flow. The concentrate was then washed with 10,000 mL of PBS, and 100 mL was finally recovered with PBS. Each concentrated sample was then sterilized using a 0.20 μm filter to obtain the extracellular vesicle-containing product.

[0091] Example 2 Pavlova sp. was cultured in a medium similar to a commercially available algae medium but without phosphorus components, using a standard method, and the culture supernatant was obtained by centrifugation at 3000 × g and room temperature for 60 minutes.

[0092] 3000 mL of the culture supernatant thus obtained was filtered through a 0.22 μm filter and then concentrated to a volume of 150 mL or less using a 300 kDa hollow fiber membrane filter (MiniKros sampler S04-E300-05-N: Repligen) by tangential flow. The concentrate was then washed with 10,000 mL of PBS, and a final volume of 100 mL was recovered with PBS. Each concentrated sample was then sterilized using a 0.20 μm filter to obtain extracellular vesicle-containing material.

[0093] (Comparative Example 1) Pavlova sp. was cultured in a commercially available algae medium using standard methods. The culture was heated at 90°C for 4 minutes and then centrifuged at 3000 x g at room temperature for 60 minutes to obtain the culture supernatant.

[0094] 3000 mL of the culture supernatant thus obtained was filtered through a 0.22 μm filter and then concentrated to a volume of 150 mL or less using a 300 kDa hollow fiber membrane filter (MiniKros sampler S04-E300-05-N: Repligen) by tangential flow. The concentrate was then washed with 10,000 mL of PBS, and a final volume of 100 mL was recovered with PBS. Each concentrated sample was then sterilized using a 0.20 μm filter to obtain extracellular vesicle-containing material.

[0095] The extracellular vesicle-containing materials of Example 1 and Comparative Example 1 were appropriately diluted with PBS(-), and the particle number, average particle size, and mode particle size were measured using a Viewsizer (ViewSizer3000, manufactured by HORIBA Scientific).

[0096] Extracellular vesicles of Example 1: 3.253 × 10 11 Particles / mL, average particle size 168.22nm, mode particle size 131.19nm Extracellular vesicles of Example 2: 1.68 × 10 11 Particles / mL, average particle size 172.76nm, mode particle size 130.03nm Extracellular vesicles of Comparative Example 1: 3.221 × 10 11 Particles / mL, average particle size 180.76nm, mode particle size 129.53nm

[0097] (Lipidomics measurement)

[0098] <Extraction operation> The extracellular vesicle contents of Examples 1 and 2 and Comparative Example 1 were each subjected to lipid analysis. 225 μL of extraction solution (CHCl3:MeOH = 1:2) containing the internal standard was added as the extraction solution. This extraction solution was prepared by mixing 4 μL of Equi SPLASH, 500 μL of CHCl3, and 996 μL of MeOH. The added extraction solution was then sonicated for 5 seconds.

[0099] Furthermore, 225 μL of the extraction solution (CHCl3:MeOH = 1:2) was added again. The preparation method of the extraction solution this time was carried out by mixing 500 μL of CHCl3 and 1000 μL of MeOH. Then, ultrasonic treatment was performed again for 30 seconds.

[0100] <Centrifugation> Centrifugation was carried out at 750 rpm for 20 minutes at 20 °C. By this operation, the solid components in the sample were separated. Next, 30 μL of water was added for homogenization of the sample. Centrifugation was carried out again at 750 rpm for 20 minutes at 20 °C. Further clarification of the sample was performed in this step.

[0101] Next, centrifugation was carried out at 1670 G for 10 minutes at 20 °C. By this centrifugation, fine solid components were completely removed.

[0102] <Sample Collection> Finally, 150 μL of the supernatant was collected twice from a total of 480 μL of the solution. Thus, the samples for analysis were obtained.

[0103] <LC-MS Analysis> The collected samples were injected into LC vials. The injection volume was 3 μL in negative ion mode and 1.5 μL in positive ion mode. The instruments used were UPLC (Waters) and TripleTOF 6600 (SCIEX). Ionization method: ESI Measurement mode: Negative-ion mode measurement, Positive-ion mode Ionization conditions: [Negative-ion mode] (Source / Gas) Ion Source: DuoSplay Ion Source Ion Source Gas1: 50 Ion Source Gas2: 50 Curtain Gas: 30 Temperature: 300 Ion Splay Voltage Floating:-4500 (Compound) Declustering Potential:-80 Collision Energy:-5 [Positive-ion mode] (Source / Gas) Ion Source:DuoSplay Ion Source Ion Source Gas 1:40 Ion Source Gas 2:80 Curtain Gas:30 Temperature: 250 Ion Splay Voltage Floating:5500 (Compound) Declustering Potential:80 Collision Energy: 5 Mobile phase: A: MeOH: ACN: Water (=1:1:3) *B: Isopropanol* (*Contains 5 mM ammonium acetate / 10 nM EDTA.) Analysis conditions: Gradient analysis and DDA were used. ”B_0% to 64%(0-6.5min) → 64% to 76.5%(6.5-13.5min) → 76.5% to 98%(13.5-18min) → 98% to 98%(19-20min)” Flow rate: 0.3 mL / min Column temperature: 45℃ MS range: 140-1700 MS / MS range: 70-1700 MS2 cleavage method: CID

[0104] <Analysis> For the analysis, qualitative and quantitative analyses were performed using In-house lipid screening (manufactured by Kazusa Genomic Technologies Co., Ltd.).

[0105] <Stability evaluation> The extracellular vesicle contents of the examples and comparative examples were filled into 20 ml light-shielding glass vials and stored at -80°C. The particle diameter and particle size were measured at the time point after one week had passed. The measurement conditions are as follows. Equipment used: Viewsizer https: / / www.horiba.com / jpn / scientific / products / detail / action / show / Product / viewsizer3000-1862 / Measurement temperature: 22°C Methods: Exosomes Videos count: 10 times

[0106] Average particle diameter fluctuation rate = |(d_mean_after one week / d_mean_immediately after preparation) × 100| Most frequent particle diameter fluctuation rate = |(d_mode_after one week / d_mode_immediately after preparation) × 100| Particle number fluctuation rate = |(n_after one week / n_immediately after preparation) × 100|[[ID=z7]] Note that || represents the absolute value.

[0107] The results of the lipid analysis of the extracellular vesicle contents of Examples 1 and 2 and Comparative Example 1 are shown in Tables 1 to 4, and the results of the stability tests of the examples and comparative examples are shown in Table 5.

[0108] Table 1 shows the results of the peak area ratios of fatty acids obtained by the method described in the <LC-MS analysis> section. The peak area ratio was calculated as the relative ratio of each fatty acid in the detected fatty acid species by normalizing each detected peak area with the peak area of the internal standard. Among the standard substances contained in Equi SPLASH, 18:1(d7)MAG was used as the internal standard.

[0109] Table 2 shows the results of the peak area ratios of saturated and unsaturated fatty acids in fatty acids with 16 to 18 carbon atoms.

[0110] Table 3 shows the results of the peak area ratios of glyceroglycolipids obtained by the method described in the <LC-MS analysis> section. The peak area ratio was calculated as the relative ratio of each glyceroglycolipid in the detected glyceroglycolipid species by normalizing each detected peak area with the peak area of the internal standard. Among the standard substances included in Equi SPLASH, 15:0~18:1(d7) DAG was used as the internal standard.

[0111] Table 4 shows the results of the peak area ratios of sterol metabolites obtained by the method described in the <LC-MS analysis> section. The peak area ratio was calculated as the relative ratio of each sterol metabolite in the detected sterol metabolite species by normalizing each detected peak area with the peak area of the internal standard. Among the standard substances included in Equi SPLASH, 18:1(d7) Chol Ester was used as the internal standard.

[0112] Table 5 shows the results of the peak area ratios of betaine lipids obtained by the method described in the <LC-MS analysis> section. The peak area ratio was calculated as the relative ratio of each betaine lipid in the detected betaine lipid species by normalizing the detected peak area of each betaine lipid with the peak area of the internal standard. Among the standard substances included in Equi SPLASH, 15:0~18:1(d7) PC was used as the internal standard.

[0113]

Table 1

[0114]

Table 2

[0115]

Table 3

[0116] [Table 4]

[0117] [Table 5]

[0118] The extracellular vesicle contents of the Examples contained a large amount of fatty acids with 16 to 18 carbon atoms, and a large amount of SQDG among glyceroglycolipids.Furthermore, the contents contained a larger amount of DGTS among betaine lipids than those of the Comparative Examples.

[0119] The average particle size change rate and particle number change rate after storage for one week were measured for the extracellular vesicle-containing products obtained in Examples 1 and 2 and Comparative Example 1 using the method described in the section <Stability Evaluation> above. The results are shown in Tables 6 and 7.

[0120] [Table 6]

[0121] [Table 7]

[0122] The extracellular vesicle-containing material of the example was found to have little fluctuation in particle number and particle size even after storage at low temperature, and to be more stable.

Claims

1. An extracellular vesicle or extracellular vesicle-containing substance derived from microalgae, which contains sulfoquinovosyl diacylglycerol (SQDG) and fatty acids, wherein the fatty acids contain 70 to 100% fatty acids having 16 to 18 carbon atoms.

2. The extracellular vesicles or extracellular vesicle-containing material according to claim 1, wherein the proportion of SQDG in glyceroglycolipids is 80 to 100%.

3. The extracellular vesicles or extracellular vesicle-containing composition according to claim 1, wherein the content of SQDG in the total amount of SQDG, SQMG, MGMG, MGDG, GADG, HexGADG, GAMG, DGDG, ASQDG, AMGDG, ADGDG and their stereoisomers is 80 to 100%.

4. The extracellular vesicles or extracellular vesicle-containing material according to claim 1, further comprising diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), wherein the proportion of DGTS in the betaine lipid is 5 to 35%.

5. The extracellular vesicles or extracellular vesicle-containing composition according to claim 1, wherein the content of DGTS is 5 to 35% of the total amount of DGTA, DGCC, MGTA, MGCC, DGTS, and their stereoisomers.

6. The extracellular vesicles or extracellular vesicle contents of claim 1, wherein the microalgae contain chloroplasts or thylakoids.

7. The extracellular vesicles or extracellular vesicle-containing material according to claim 6, wherein the microalgae are microalgae belonging to the genus Pavlova, Euglena, Spirulina, or Chlorella.

8. The extracellular vesicles or extracellular vesicle-containing material according to any one of claims 1 to 7, wherein the content of unsaturated fatty acids in the fatty acids having 16 to 18 carbon atoms is 5 to 20%.

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