WO2023008787A1 - Microalga-derived exosome mimetic and manufacturing method therefor - Google Patents

Microalga-derived exosome mimetic and manufacturing method therefor Download PDF

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WO2023008787A1
WO2023008787A1 PCT/KR2022/010227 KR2022010227W WO2023008787A1 WO 2023008787 A1 WO2023008787 A1 WO 2023008787A1 KR 2022010227 W KR2022010227 W KR 2022010227W WO 2023008787 A1 WO2023008787 A1 WO 2023008787A1
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exosome
microalgae
analogues
making
methods
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PCT/KR2022/010227
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French (fr)
Korean (ko)
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김진웅
백휘라
강정이
고유리
조중현
신경희
김준오
김영석
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성균관대학교산학협력단
주식회사 신세계인터내셔날
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Publication of WO2023008787A1 publication Critical patent/WO2023008787A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

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  • the present invention relates to a method for preparing a large amount of exosome analogues derived from microalgae through an extrusion processing method.
  • Microalgae are unicellular photosynthetic organisms, and their types include Euglena, Dunaliella, Chlorella, Spirulina, and the like. Since these microalgae produce a high rate of biomass based on their excellent photosynthetic ability, they have been intensively developed as next-generation biofuels. The type and size of the market is growing.
  • microalgae as a natural raw material to replace chemically synthesized raw materials in the health food and pharmaceutical industries.
  • microalgae are mostly raw materials in the form of powder.
  • viscosity may occur depending on the oil content, and even after drying, product development and commercialization are difficult due to problems such as low water dispersibility and low oxidation stability of the powder. Therefore, it is necessary to develop a new type of formulation that can fully utilize the useful substances of microalgae by supplementing these disadvantages.
  • Exosomes are microvesicles of about 50-150 nm secreted by almost all cells, and contain substances (transmembrane proteins, water-soluble proteins, DNA, mRNA, miRNA, etc.) contained in cells. Exosomes mediate cell-to-cell signal transduction, and exosomes themselves are sometimes used to diagnose diseases.
  • exosomes contain various cell-derived physiologically active substances and have many advantages, such as being able to pass through the blood-brain barrier, studies are being actively conducted to utilize exosomes themselves as drug delivery systems.
  • exosomes have been mostly researched and developed focusing on animal cells, including stem cells, and research on exosomes of plant cells, including algae, is very scarce.
  • exosomes are natural substances secreted by cells, many purification processes are required to obtain pure exosomes. Examples of processes for obtaining exosomes include ultracentrifuge, density gradient centrifuge, ultrafiltration, and precipitation.
  • One object of the present invention is to provide a method for preparing exosome analogs derived from microalgae in high yield through extrusion processing.
  • Another object of the present invention is to provide an exosome analogue derived from microalgae prepared by the above production method.
  • Another object of the present invention is to provide a skin antioxidant cosmetic composition comprising the microalgae-derived exosome analogue as an active ingredient.
  • a method for preparing exosome analogues according to an embodiment of the present invention includes the step of preparing nanovesicles by extruding a buffer solution containing microalgae.
  • the extrusion process is a first step of preparing a first mixed solution by passing the buffer solution through a first membrane having first pores, a second step of preparing the first mixed solution smaller than the first pores A second step of preparing a second mixed solution by passing it through a second membrane having pores, and a third step of passing the second mixed solution through a third membrane having third pores smaller than the second pores.
  • the first pores may be 5 to 12 ⁇ m
  • the second pores may be 1.0 to 2.0 ⁇ m
  • the third pores may be 100 to 200 nm.
  • each of the first step, the second step, and the third step may be repeatedly performed at least three times.
  • the microalgae are Euglena, Dunaliella, Chlorella, Spirulina, Colpodella, Chromera, Nanochloropsis, Haematococcus fluvialis, Thalasiocyra pseudonana and Chlamydomonas It may include any one or more selected from Reinhardy.
  • the buffer solution before the extrusion may further include a surfactant.
  • the surfactant may be a biolipid surfactant.
  • the biolipid surfactant may include a mannosylerythritol lipid (MEL) moiety and a polyethylene glycol (PEG) moiety combined with the mannosylerythritol lipid (MEL).
  • MEL mannosylerythritol lipid
  • PEG polyethylene glycol
  • the biolipid surfactant may have a structure represented by Chemical Formula 1 below.
  • n and m are each an integer of 1 to 13.
  • the concentration of the biolipid surfactant may be 50 to 1000 ⁇ M.
  • the concentration of the microalgae may be 1 x 10 5 to 1 x 10 6 cells/ml.
  • the microalgae-derived exosome analogue according to an embodiment of the present invention is prepared by the above method, has a size of 100 to 200 nm, and has a nanovesicle shape.
  • a polyethylene glycol (PEG) chain may be introduced into at least a portion of the surface of the nanovesicle.
  • another embodiment of the present invention includes a skin antioxidant cosmetic composition containing the microalgae-derived exosome analogue as an active ingredient.
  • the skin antioxidant cosmetic composition may contain the microalgae-derived exosome analogue in an amount of 0.01 to 10.0% by weight based on the total weight of the cosmetic composition.
  • the present invention has the advantage of being able to mass-produce exosome analogues derived from microalgae based on the extrusion processing method.
  • the present invention can increase the yield of vesicle production of exosome analogues derived from microalgae by selectively applying a biolipid surfactant that enhances extrusion processing efficiency.
  • FIG. 1 schematically shows a method for preparing an exosome analogue according to an embodiment of the present invention.
  • Figure 4 shows the fluorescence measurement value according to the ROS generation according to the concentration of the exosome analogue prepared in Example 2 of the present invention, and (b) shows the fluorescence image of the cell according to the concentration of the exosome analogue of the present invention.
  • FIG. 1 schematically shows a method for preparing an exosome analogue according to an embodiment of the present invention.
  • the method for preparing exosome analogues includes the step of preparing nanovesicles by extruding a buffer solution containing microalgae (S100).
  • the method for producing exosome analogues of the present invention is based on an extrusion process, which means a method for producing nano-sized vesicles by applying physical pressure to microalgae themselves By doing so, it can be used as an alternative process to conventional extraction of exosome analogues.
  • the microalgae when exosome analogues are extracted through extrusion processing, the microalgae may undergo degradation and recombination. In this process, the microalgae can be changed into small nanovesicles while passing through a membrane having pores of a certain size.
  • a surfactant capable of facilitating the deformation of the cell membrane of microalgae and reducing the surface activity can be used.
  • the buffer solution before the extrusion may further include a surfactant.
  • the surfactant may be a PEG-based surfactant such as a Tween-based surfactant or a Span-based surfactant, and is most preferably a biolipid surfactant.
  • the biolipid surfactant is a mannosylerythritol lipid (hereinafter referred to as MEL) moiety and polyethylene glycol (hereinafter referred to as polyethylene glycol) combined with the mannosylerythritol lipid (MEL). referred to as PEG) moiety.
  • MEL mannosylerythritol lipid
  • PEG polyethylene glycol
  • the biolipid surfactant may have a structure represented by Chemical Formula 1 below.
  • n and m are each an integer of 1 to 13.
  • the mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) may be bonded through an organic reaction. Specifically, the bonding may be achieved by a click reaction. For example, when maleimide introduced at the terminal of mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) containing a thiol group are reacted, the maleimide and the thiol group are reacted.
  • Mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) may be bonded by a thiol click reaction.
  • the mannosylerythritol lipid is a glycolipid-based material in which a fatty acid is bound to a sugar and has high surface activity, so it is one of representative biosurfactants, and the polyethylene glycol (PEG) is water-soluble (or hydrophilic). ) and non-toxic biocompatible polymers, widely used in biomedicine and other applications.
  • MEL mannosylerythritol lipid
  • PEG polyethylene glycol
  • the concentration of the biolipid surfactant is preferably 50 to 1000 ⁇ M.
  • the concentration of the biolipid surfactant increases, the yield of nanovesicles increases, and the size of nanovesicles produced may decrease. Therefore, the yield and size of nanovesicles can be appropriately controlled by adjusting the concentration of the biolipid surfactant.
  • the microalgae used in the present invention are Euglena, Dunaliella, Chlorella, Spirulina, Colpodella, Chromera, Nanochloropsis, Haematococcus fluvialis, Thalasiocyra pseudonana and Chlamydo It is preferable to include any one or more selected from Monas reinhati, but is not limited thereto, and a person skilled in the art may vary the type of microalgae according to the purpose.
  • the concentration of the microalgae is preferably 1 x 10 5 ⁇ 1 x 10 6 cells / ml.
  • the buffer solution refers to a solution in which the pH does not change significantly even when an acid or base is added, and may be a material that serves as a buffer to reduce the change in pH caused by metabolites of microalgae.
  • the buffer solution may be, for example, phosphate-buffered saline (PBS), secondary distilled water, or the like.
  • the extrusion process may be performed in 3 steps.
  • the extrusion process is a first step of preparing a first mixed solution by passing the buffer solution through a first membrane having first pores (S110), the first mixed solution smaller than the first pores
  • Step 3 (S130) may be included.
  • the first step, the second step, and the third step are preferably performed repeatedly at least three times, respectively, and through repetition, loss of microalgae remaining on the syringe wall, etc. can be reduced, and exosomes
  • the parent body can be evenly dispersed in a certain size.
  • the first, second and third membranes have a structure including pores and can selectively pass a material, so that they can have permeability capable of performing filtration, separation and delivery.
  • the first, second and third membranes may be membranes made of polycarbonate.
  • the material forming the first, second and third membranes is not particularly limited, but is preferably formed of a material that does not affect the microalgae extrusion process without cytotoxicity.
  • the first pores may be about 5 to 12 ⁇ m
  • the second pores may be about 1.0 to 2.0 ⁇ m
  • the third pores may be 100 to 200 nm.
  • the microalgae in the buffer solution have a smaller size due to the first pores of the first membrane through the first step, and then pass through the second step to have a second smaller size than the first pores. It has a smaller size due to the pore, and then, through the third step, nanovesicles (or exosome analogues) having a smaller size can be formed due to the third pore having a smaller size than the second pore.
  • microalgae-derived exosome analogue prepared according to the preparation method of the present invention may have a size of 100 to 200 nm and may have a nanovesicle shape. (See Figure 1)
  • a polyethylene glycol (PEG) chain may be introduced into at least a part of the surface of the nanovesicle, thereby reducing the cytotoxicity of the nanovesicle. reduced and increased hydrophilicity.
  • a skin antioxidant cosmetic composition containing the microalgae-derived exosome analogue as an active ingredient may be mentioned.
  • the microalgae-derived exosome analogue may be contained in an amount of 0.01 to 10.0% by weight based on the total weight of the cosmetic composition.
  • the cosmetic composition of the present invention contains microalgae-derived exosome analogues as an active ingredient and may have a skin antioxidant effect that reduces the amount of active oxygen produced by skin cells.
  • the present invention based on the extrusion processing method, it is difficult to commercialize due to problems such as low oxidation stability and low water dispersibility, and the process of separating and purifying only specific components is difficult and expensive. Since it can be manufactured in large quantities, it has the advantage of being able to expand the application of microalgae-derived active ingredients to the food, cosmetic, and medical markets.
  • the present invention can increase the yield of vesicle production of exosome analogues derived from microalgae by selectively applying a biolipid surfactant that enhances extrusion processing efficiency.
  • a biolipid surfactant containing mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) is prepared by attaching a maleimide group to the end of the MEL and PEG containing the maleimide group and a thiol group (hereinafter referred to as PEG-thiol) It was prepared by performing the step of reacting.
  • MEL mannosylerythritol lipid
  • PEG-thiol polyethylene glycol
  • the biolipid surfactant of the present invention a process of introducing a maleimide group at the terminal of MEL was first performed. After dissolving the purified MEL in anhydrous methylene chloride, pyridine and 4-nitrophenylchloroformate were added, and the solution was stirred at room temperature for about 1 hour to prepare a mixed solution did Subsequently, the reaction was terminated by adding a saturated aqueous solution of NH 4 Cl to the mixed solution, and the organic layer was extracted by separating the layers using a separatory funnel.
  • the extracted organic layer was dried over MgSO 4 and filtered, and the remaining solvent of the organic layer, ethylene chloride, was concentrated using a rotary evaporator, and the resulting residue was purified using column chromatography on silica gel to obtain a colorless oil Orthoformate compound 1 of was obtained.
  • N, N-diisopropylethylamine N, N-Diisopropylethylamine
  • N- (2-aminoethyl) maleimide trifluoroacetate salt N -(2-aminoethyl) maleimide trifluoro acetate salt
  • MEL-maleimide and PEG-thiol synthesized above were quantified to a molar ratio of 1:1 and then completely dissolved in chloroform. Then, after continuously shaking and reacting on a shaker at room temperature for more than 8 hours to achieve sufficient synthesis, chloroform as a solvent was completely evaporated and removed using a rotary evaporator to obtain a synthesized powder in powder form.
  • a biolipid surfactant hereinafter referred to as MEL-PEG
  • MEL-PEG biolipid surfactant
  • MEL-PEG prepared in Example 1 was added at 0 to 1000 ⁇ M and mixed by vortexing to prepare a microalgae mixed solution.
  • the density of the microalgae was quantified using a cell counting hemocytometer and used between 1 x 10 5 and 1 x 10 6 cells/ml.
  • the microalgae mixed solution was put in a gas tight syringe and then continuously applied to a polycarbonate membrane having a pore size of 5 ⁇ m through a mini-extruder three times. A first mixed solution was obtained through the primary extrusion process through which the mixture was passed through.
  • the first cell mixture solution was successively passed through a polycarbonate membrane having a pore size of 1 ⁇ m three times, and further passed through a polycarbonate membrane having a pore size of 200 nm three times in succession.
  • exosome analogues in the form of nanovesicles having a size of about 100 - 200 nm were obtained.
  • ⁇ M Surfactants Concentration ( ⁇ M) microalgae types sample 1 no additives 0 Euglena gracilis sample 2 MEL-PEG 250 Euglena gracilis sample 3 MEL-PEG 500 Euglena gracilis sample 4 MEL-PEG 1000 Euglena gracilis
  • nanovesicles generated through nanoparticle tracking analysis (Samples 1 - 4, of microalgae The density is shown in Table 2 below by quantifying the concentration of 200,000 cells/ml) and measuring the size.
  • Example 2 the morphology of the exosome analogue (sample 1) prepared in Example 2 was confirmed using a transmission electron microscope (TEM), and the results are shown in FIG. 2.
  • TEM transmission electron microscope
  • microalgae-derived exosome analogue has a size of 100 to 200 nm and exhibits a nanovesicle form.
  • beta-glucan which is the main component of Euglena
  • aniline blue staining was used.
  • the beta-glucan content was calculated by substituting the fluorescence values of Euglena and sample 1 of the present invention. shown in
  • exosome analogues (Samples 4 to 5) were prepared using Dunaliella and Chlorella in the same manner as Sample 3 in Example 2.
  • nanovesicles concentration of nanovesicles (Samples 3 - 5, the density of microalgae was 200,000 cells/ml) was quantified through nanoparticle tracking analysis (NTA), and is shown in Table 3 below.
  • HaCaT cells were dispensed into each well in a 96-well plate at a concentration of 2.5 x 10 4 and cultured for 24 hours, then the medium was removed, washed first with PBS, and 20 ⁇ M DCFDA (2',7'-dichlorofluorescindiacetate ) solution was dispensed by 100 ⁇ l and incubated at 36° C. for 30 minutes. Then, after removing the DCFDA solution and washing with PBS, 25 ⁇ l, 50 ⁇ l, 75 ⁇ l, and 100 ⁇ l of Sample 1 of the present invention were dispensed and cultured for 3 hours.
  • DCFDA 2',7'-dichlorofluorescindiacetate

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Abstract

Disclosed herein are a microalga-derived exosome mimetic and a manufacturing method therefor. The method for manufacturing an exosome mimetic of the present invention comprises a step of exclusion processing a buffer solution containing microalgae to prepare nanovesicles. The present invention has the advantage of being able to mass-produce exosome mimetics derived from microalgae on the basis of an extrusion processing method. In particular, the present invention can increase the yield of vesicle production of exosome mimetics derived from microalgae by selectively applying a biolipid surfactant that enhances extrusion processing efficiency.

Description

미세 조류 유래 엑소좀 유사체 및 이의 제조 방법Microalgae-derived exosome analog and method for preparing the same
본 발명은 압출 가공법을 통해 미세 조류 유래 엑소좀 유사체를 대량으로 제조하는 방법에 관한 것이다.The present invention relates to a method for preparing a large amount of exosome analogues derived from microalgae through an extrusion processing method.
미세 조류(microalgae)는 단세포 광합성 생물로서 그 종류로는 유글레나(Euglena), 두날리엘라(Dunaliella), 클로렐라(Chlorella), 스피룰리나 (Spirulina) 등이 있다. 이러한 미세 조류들은 우수한 광합성 능력을 바탕으로 고비율의 바이오매스를 생산하므로 차세대 바이오 연료로서 집중적으로 개발되었으며, 최근에는 미세 조류가 함유하고 있는 미네랄, 아미노산, 비타민, 지방산 등의 다양한 유용 물질에 집중하여 그 시장의 종류와 규모가 커지고 있는 추세이다.Microalgae are unicellular photosynthetic organisms, and their types include Euglena, Dunaliella, Chlorella, Spirulina, and the like. Since these microalgae produce a high rate of biomass based on their excellent photosynthetic ability, they have been intensively developed as next-generation biofuels. The type and size of the market is growing.
특히, 이러한 성분들은 생물체가 생산하는 천연 물질이기 때문에 건강식품 및 의약품 산업에서 화학적으로 합성되는 원료를 대체하여 미세 조류를 천연 원료로 사용하고자 하는 수요가 증가하고 있다.In particular, since these components are natural substances produced by living organisms, there is an increasing demand for using microalgae as a natural raw material to replace chemically synthesized raw materials in the health food and pharmaceutical industries.
통상적으로 미세 조류는 대부분 분말의 형태로 원료화되고 있다. 그러나, 미세 조류를 분말로 제조 시 오일 함량에 따라 점성이 생길 수 있으며, 건조된 후에도 분말의 낮은 수분산성, 낮은 산화안정성 등의 문제로 인해 제품 개발과 상용화에 난항을 겪고 있다. 따라서, 이러한 단점들을 보완하여 미세 조류의 유용 물질을 온전히 이용할 수 있는 새로운 형태의 제제 개발이 필요하다.Typically, microalgae are mostly raw materials in the form of powder. However, when microalgae are prepared as powder, viscosity may occur depending on the oil content, and even after drying, product development and commercialization are difficult due to problems such as low water dispersibility and low oxidation stability of the powder. Therefore, it is necessary to develop a new type of formulation that can fully utilize the useful substances of microalgae by supplementing these disadvantages.
한편, 최근 미세 조류에 대한 연구가 활발히 이루어지면서 미세 조류가 분비하는 엑소좀에 관한 관심도 증가하고 있다. 엑소좀(exosome)이란 거의 모든 세포가 분비하는 약 50-150nm 의 미세소포체(extracellular vesicle)로, 세포가 함유하는 물질(막관통단백질, 수용성단백질, DNA, mRNA, miRNA 등)을 포함하고 있다. 엑소좀은 세포와 세포간의 신호전달을 매개하기도 하며, 엑소좀 자체가 질병의 진단에 이용되기도 한다.Meanwhile, as research on microalgae has been actively conducted recently, interest in exosomes secreted by microalgae is also increasing. Exosomes are microvesicles of about 50-150 nm secreted by almost all cells, and contain substances (transmembrane proteins, water-soluble proteins, DNA, mRNA, miRNA, etc.) contained in cells. Exosomes mediate cell-to-cell signal transduction, and exosomes themselves are sometimes used to diagnose diseases.
무엇보다도, 엑소좀은 세포 유래의 다양한 생리활성물질을 함유하며 혈관-뇌장벽을 통과할 수 있는 등의 많은 이점이 있기 때문에, 엑소좀 자체를 약물 전달체로서 활용하고자 하는 연구가 활발히 진행되고 있다.Above all, because exosomes contain various cell-derived physiologically active substances and have many advantages, such as being able to pass through the blood-brain barrier, studies are being actively conducted to utilize exosomes themselves as drug delivery systems.
그동안 엑소좀은 대부분 줄기세포를 포함하는 동물세포에 집중되어 연구 및 개발되어 왔으며, 조류를 포함하는 식물세포의 엑소좀에 대한 연구는 매우 부족한 실정이다.So far, exosomes have been mostly researched and developed focusing on animal cells, including stem cells, and research on exosomes of plant cells, including algae, is very scarce.
또한, 엑소좀은 세포가 분비하는 천연 물질이기 때문에 순수한 엑소좀을 얻기 위해선 많은 정제 과정이 필요하다. 엑소좀을 얻기 위한 공정 예로는 초원심분리(ultracentrifuge), 밀도 원심 분리(density gradient centrifuge), 초미세여과법(ultrafilteration), 침전법(precipitation) 등이 보고되어 있다.In addition, since exosomes are natural substances secreted by cells, many purification processes are required to obtain pure exosomes. Examples of processes for obtaining exosomes include ultracentrifuge, density gradient centrifuge, ultrafiltration, and precipitation.
하지만, 종래의 방법들은 복잡한 공정과 높은 비용에 비해 얻을 수 있는 엑소좀의 수율이 현저히 낮아 상업화에 어려움을 겪고 있다. 뿐만 아니라, 이러한 정제 과정을 통해서 얻어진 미세 조류의 엑소좀은 기존 분말 형태의 원료보다 유용 물질의 함량이 현저히 떨어져 그 이용가치가 매우 낮은 문제점이 있다. 따라서, 미세 조류 유래의 엑소좀을 상용화할 수 있는 새로운 제조 공법의 개발이 절실히 필요하다.However, conventional methods have difficulties in commercialization due to the remarkably low yield of exosomes that can be obtained compared to complicated processes and high costs. In addition, the exosomes of microalgae obtained through such a purification process have a problem in that the content of useful substances is significantly lower than that of conventional powdered raw materials, and thus their usefulness is very low. Therefore, it is urgently needed to develop a new manufacturing method capable of commercializing exosomes derived from microalgae.
본 발명의 일 목적은 압출 가공법을 통해 미세 조류 유래 엑소좀 유사체를 높은 수율로 제조할 수 있는 엑소좀 유사체 제조 방법을 제공하는 것이다.One object of the present invention is to provide a method for preparing exosome analogs derived from microalgae in high yield through extrusion processing.
본 발명의 다른 목적은 상기 제조 방법에 의해 제조된 미세 조류 유래 엑소좀 유사체를 제공하는 것이다.Another object of the present invention is to provide an exosome analogue derived from microalgae prepared by the above production method.
본 발명의 또 다른 목적은 상기 미세 조류 유래 엑소좀 유사체를 유효 성분으로 포함하는 피부 항산화 화장료 조성물을 제공하는 것이다.Another object of the present invention is to provide a skin antioxidant cosmetic composition comprising the microalgae-derived exosome analogue as an active ingredient.
본 발명의 일 실시예에 따른 엑소좀 유사체 제조 방법은 미세 조류를 포함하는 버퍼 용액을 압출가공하여 나노베지클을 제조하는 단계를 포함한다.A method for preparing exosome analogues according to an embodiment of the present invention includes the step of preparing nanovesicles by extruding a buffer solution containing microalgae.
일 실시예에서, 상기 압출가공은, 상기 버퍼 용액을 제1 기공을 갖는 제1 멤브레인에 통과시켜 제1 혼합 용액을 제조하는 제1 단계, 상기 제1 혼합 용액을 상기 제1 기공보다 작은 제2 기공을 갖는 제2 멤브레인에 통과시켜 제2 혼합 용액을 제조하는 제2 단계, 및 상기 제2 혼합 용액을 상기 제2 기공보다 작은 제3 기공을 갖는 제3 멤브레인에 통과시키는 제3 단계를 포함할 수 있다.In one embodiment, the extrusion process is a first step of preparing a first mixed solution by passing the buffer solution through a first membrane having first pores, a second step of preparing the first mixed solution smaller than the first pores A second step of preparing a second mixed solution by passing it through a second membrane having pores, and a third step of passing the second mixed solution through a third membrane having third pores smaller than the second pores. can
일 실시예에서, 상기 제1 기공은 5 내지 12 ㎛ 이고, 상기 제2 기공은 1.0 내지 2.0 ㎛ 이고, 상기 제3 기공은 100 내지 200 nm 일 수 있다.In one embodiment, the first pores may be 5 to 12 μm, the second pores may be 1.0 to 2.0 μm, and the third pores may be 100 to 200 nm.
일 실시예에서, 상기 제1 단계, 제2 단계 및 제3 단계는 각각 적어도 3회 이상 반복적으로 수행할 수 있다.In one embodiment, each of the first step, the second step, and the third step may be repeatedly performed at least three times.
일 실시예에서, 상기 미세 조류는 유글레나, 두날리엘라, 클로렐라, 스피룰리나, 콜포델라류, 크로메라류, 나노클로롭시스류, 헤마토코쿠스 플루비아리스, 탈라시오시라 슈도나나 및 클라미도모나스 레인하티 중에서 선택된 어느 하나 이상을 포함할 수 있다.In one embodiment, the microalgae are Euglena, Dunaliella, Chlorella, Spirulina, Colpodella, Chromera, Nanochloropsis, Haematococcus fluvialis, Thalasiocyra pseudonana and Chlamydomonas It may include any one or more selected from Reinhardy.
일 실시예에서, 상기 압출가공 전의 상기 버퍼 용액은 계면활성제를 더 포함할 수 있다.In one embodiment, the buffer solution before the extrusion may further include a surfactant.
일 실시예에서, 상기 계면활성제는 바이오리피드 계면활성제일 수 있다.In one embodiment, the surfactant may be a biolipid surfactant.
일 실시예에서, 상기 바이오리피드 계면활성제는 만노실에리트리톨 지질(MEL) 모이에티 및 상기 만노실에리트리톨 지질(MEL)과 결합된 폴리에틸렌글리콜(PEG) 모이에티를 포함할 수 있다.In one embodiment, the biolipid surfactant may include a mannosylerythritol lipid (MEL) moiety and a polyethylene glycol (PEG) moiety combined with the mannosylerythritol lipid (MEL).
일 실시예에서, 상기 바이오리피드 계면활성제는 하기 화학식 1의 구조를 가질 수 있다.In one embodiment, the biolipid surfactant may have a structure represented by Chemical Formula 1 below.
<화학식 1><Formula 1>
Figure PCTKR2022010227-appb-img-000001
Figure PCTKR2022010227-appb-img-000001
상기 화학식 1에서, n 및 m은 각각 1 내지 13의 정수이다.In Formula 1, n and m are each an integer of 1 to 13.
일 실시예에서, 상기 바이오리피드 계면활성제의 농도는 50 내지 1000μM 일 수 있다.In one embodiment, the concentration of the biolipid surfactant may be 50 to 1000 μM.
일 실시예에서, 상기 미세 조류의 농도는 1 x 105 내지 1 x 106 cells/ml 일 수 있다.In one embodiment, the concentration of the microalgae may be 1 x 10 5 to 1 x 10 6 cells/ml.
한편, 본 발명의 일 실시예에 따른 미세 조류 유래 엑소좀 유사체는 상기 방법으로 제조되고, 100 내지 200 nm 의 크기를 가지며, 나노베지클 형태를 갖는다.On the other hand, the microalgae-derived exosome analogue according to an embodiment of the present invention is prepared by the above method, has a size of 100 to 200 nm, and has a nanovesicle shape.
일 실시예에서, 상기 나노베지클 표면의 적어도 일부에 폴리에틸렌글리콜(PEG) 사슬이 도입될 수 있다.In one embodiment, a polyethylene glycol (PEG) chain may be introduced into at least a portion of the surface of the nanovesicle.
한편, 본 발명의 다른 실시 형태로 상기 미세 조류 유래 엑소좀 유사체를 유효 성분으로 함유하는 피부 항산화 화장료 조성물을 들 수 있다.Meanwhile, another embodiment of the present invention includes a skin antioxidant cosmetic composition containing the microalgae-derived exosome analogue as an active ingredient.
일 실시예에서, 상기 피부 항산화 화장료 조성물은 상기 미세 조류 유래 엑소좀 유사체를 화장료 조성물 총 중량에 대하여 0.01 내지 10.0 중량%로 함유할 수 있다.In one embodiment, the skin antioxidant cosmetic composition may contain the microalgae-derived exosome analogue in an amount of 0.01 to 10.0% by weight based on the total weight of the cosmetic composition.
본 발명은 압출 가공법을 기반으로 하여 미세 조류 유래 엑소좀 유사체를 대량으로 제조할 수 있는 장점이 있다. 특히, 본 발명은 압출 가공 효율을 증진시키는 바이오리피드 계면활성제를 선택적으로 적용함으로써 미세 조류 유래 엑소좀 유사체의 베지클 생성 수율을 증가시킬 수 있다.The present invention has the advantage of being able to mass-produce exosome analogues derived from microalgae based on the extrusion processing method. In particular, the present invention can increase the yield of vesicle production of exosome analogues derived from microalgae by selectively applying a biolipid surfactant that enhances extrusion processing efficiency.
따라서, 본 발명에 따르면, 낮은 산화안정성, 낮은 수분산성 등의 문제로 인해 제품화가 어렵고, 특정 성분만 분리 및 정제하는 공정이 까다롭고 고비용이어서 상업화에 어려웠던 미세 조류로부터 엑소좀 유사체를 대량으로 제조할 수 있어, 미세 조류 유래 효능 성분들을 식품, 화장품, 의료 시장까지 확대 적용 가능한 장점이 있다.Therefore, according to the present invention, it is difficult to commercialize due to problems such as low oxidation stability and low water dispersibility, and the process of separating and purifying only specific components is difficult and expensive. Therefore, there is an advantage in that the active ingredients derived from microalgae can be expanded to the food, cosmetic, and medical markets.
도 1은 본 발명의 일 실시예에 따른 엑소좀 유사체 제조 방법을 개략적으로 나타낸 것이다.1 schematically shows a method for preparing an exosome analogue according to an embodiment of the present invention.
도 2의 (a)는 Euglena gracilis, (b) 본 발명의 실시예 2를 통해 제조된 엑소좀 유사체의 TEM 이미지, (c)는 엑소좀 유사체의 크기 분포를 나타낸다.2 (a) shows a TEM image of Euglena gracilis, (b) an exosome analogue prepared in Example 2 of the present invention, and (c) shows the size distribution of the exosome analogue.
도 3의 (a)는 베타글루칸의 기준 곡선(standard curve), (b)는 유글레나 및 본 발명의 실시예 2를 통해 제조된 엑소좀 유사체의 베타글루칸 함량을 나타낸다. 3 (a) shows a standard curve of beta-glucan, and (b) shows the beta-glucan content of Euglena and exosome analogues prepared in Example 2 of the present invention.
도 4의 (a)는 본 발명의 실시예 2를 통해 제조된 엑소좀 유사체의 농도별 ROS 발생에 따른 형광측정값, (b)는 엑소좀 유사체의 농도에 따른 세포의 형광이미지를 나타낸다.Figure 4 (a) shows the fluorescence measurement value according to the ROS generation according to the concentration of the exosome analogue prepared in Example 2 of the present invention, and (b) shows the fluorescence image of the cell according to the concentration of the exosome analogue of the present invention.
이하, 첨부한 도면을 참조하여 본 발명의 실시예에 대해 상세히 설명한다. 본 발명은 다양한 변경을 가할 수 있고 여러 가지 형태를 가질 수 있는 바, 특정 실시예들을 도면에 예시하고 본문에 상세하게 설명하고자 한다. 그러나 이는 본 발명을 특정한 개시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 각 도면을 설명하면서 유사한 참조부호를 유사한 구성요소에 대해 사용하였다. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the present invention may have various changes and various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. Like reference numerals have been used for like elements throughout the description of each figure.
본 출원에서 사용한 용어는 단지 특정한 실시 예를 설명하기 위해 사용된 것으로서 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 출원에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 단계, 동작, 구성요소, 부분품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 단계, 동작, 구성요소, 부분품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Terms used in this application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as "comprise" or "have" are intended to designate that there is a feature, step, operation, component, part, or combination thereof described in the specification, but one or more other features or steps However, it should be understood that it does not preclude the possibility of existence or addition of operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't
도 1은 본 발명의 일 실시예에 따른 엑소좀 유사체 제조 방법을 개략적으로 나타낸 것이다.1 schematically shows a method for preparing an exosome analogue according to an embodiment of the present invention.
도 1을 참조하면, 본 발명의 일 실시예에 따른 엑소좀 유사체 제조 방법은 미세 조류를 포함하는 버퍼 용액을 압출가공하여 나노베지클을 제조하는 단계(S100)를 포함한다.Referring to FIG. 1 , the method for preparing exosome analogues according to an embodiment of the present invention includes the step of preparing nanovesicles by extruding a buffer solution containing microalgae (S100).
도 1에 나타나듯이, 본 발명의 엑소좀 유사체 제조 방법은 압출가공 방법을 기반으로 한다, 상기 압출 가공은 미세 조류 자체에 물리적인 압력을 가하여 나노 크기의 베지클(vesicle)을 제조하는 방법을 의미하는 것으로, 종래의 엑소좀 유사체 추출의 대체 공정으로 사용될 수 있다.As shown in Figure 1, the method for producing exosome analogues of the present invention is based on an extrusion process, which means a method for producing nano-sized vesicles by applying physical pressure to microalgae themselves By doing so, it can be used as an alternative process to conventional extraction of exosome analogues.
구체적으로 압출 가공을 통해 엑소좀 유사체를 추출하는 경우 상기 미세 조류는 분해와 재조합이 발생할 수 있다. 이러한 과정에서 상기 미세 조류는 일정 크기의 기공을 갖는 멤브레인을 통과하면서 작은 나노베지클(nano vesicle) 형태로 변화될 수 있다.Specifically, when exosome analogues are extracted through extrusion processing, the microalgae may undergo degradation and recombination. In this process, the microalgae can be changed into small nanovesicles while passing through a membrane having pores of a certain size.
한편, 본 발명은 이러한 과정에서 나노베지클의 생산 효율을 높이기 위해 미세 조류의 세포막의 변형을 용이하게 하고 계면활성력을 저하시킬 수 있는 계면활성제를 이용할 수 있다.On the other hand, in the present invention, in order to increase the production efficiency of nanovesicles in this process, a surfactant capable of facilitating the deformation of the cell membrane of microalgae and reducing the surface activity can be used.
즉, 상기 S100 단계에서, 상기 압출가공 전의 상기 버퍼 용액은 계면활성제를 더 포함할 수 있다. 여기서, 상기 계면활성제는 Tween 계열, Span 계열 등의 PEG 기반의 계면활성제일 수 있고, 바이오리피드 계면활성제인 것이 가장 바람직하다.That is, in the step S100, the buffer solution before the extrusion may further include a surfactant. Here, the surfactant may be a PEG-based surfactant such as a Tween-based surfactant or a Span-based surfactant, and is most preferably a biolipid surfactant.
일 실시예에서, 상기 바이오리피드 계면활성제는 만노실에리트리톨 지질(Mannosylerithritol lipid, 이하에서 MEL이라 함) 모이에티 및 상기 만노실에리트리톨 지질(MEL)과 결합된 폴리에틸렌글리콜(Polyethylene glycol, 이하에서 PEG이라 함) 모이에티를 포함할 수 있다.In one embodiment, the biolipid surfactant is a mannosylerythritol lipid (hereinafter referred to as MEL) moiety and polyethylene glycol (hereinafter referred to as polyethylene glycol) combined with the mannosylerythritol lipid (MEL). referred to as PEG) moiety.
일 실시예에서, 상기 바이오리피드 계면활성제는 하기 화학식 1의 구조를 가질 수 있다.In one embodiment, the biolipid surfactant may have a structure represented by Chemical Formula 1 below.
<화학식 1><Formula 1>
Figure PCTKR2022010227-appb-img-000002
Figure PCTKR2022010227-appb-img-000002
상기 화학식 1에서, n 및 m은 각각 1 내지 13의 정수이다.In Formula 1, n and m are each an integer of 1 to 13.
상기 화학식 1의 구조를 참조하면, 본 발명의 바이오리피드 계면활성제는 상기 만노실에리트리톨 지질(MEL)과 폴리에틸렌글리콜(PEG)의 결합은 유기 반응에 의해 이루어 질 수 있다. 구체적으로, 상기 결합은 클릭 반응(Click reaction)에 의해 이루어질 수 있다. 예를 들어, 만노실에리트리톨 지질(MEL)의 말단에 도입된 말레이미드(maleimide)와 티올기(thiol group)를 포함하는 폴리에틸렌글리콜(PEG)을 반응시키는 경우, 상기 말레이미드(maleimide와 상기 티올(thiol)의 클릭 반응에 의해 만노실에리트리톨 지질(MEL)과 폴리에틸렌글리콜(PEG)이 결합될 수 있다.Referring to the structure of Chemical Formula 1, in the biolipid surfactant of the present invention, the mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) may be bonded through an organic reaction. Specifically, the bonding may be achieved by a click reaction. For example, when maleimide introduced at the terminal of mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) containing a thiol group are reacted, the maleimide and the thiol group are reacted. Mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) may be bonded by a thiol click reaction.
상기 만노실에리트리톨 지질(MEL)은 당에 지방산이 결합된 당지질계 물질로, 높은 계면활성력을 가지므로 대표적인 바이오 계면활성제(biosurfactants) 중 하나이며, 상기 폴리에틸렌글리콜(PEG)은 수용성(또는 친수성)이면서 독성이 없는 생체 적합성 고분자로, 생체 의학 및 기타 응용 분야에서 널리 사용되는 고분자이다. The mannosylerythritol lipid (MEL) is a glycolipid-based material in which a fatty acid is bound to a sugar and has high surface activity, so it is one of representative biosurfactants, and the polyethylene glycol (PEG) is water-soluble (or hydrophilic). ) and non-toxic biocompatible polymers, widely used in biomedicine and other applications.
따라서, 만노실에리트리톨 지질(MEL) 및 이에 결합된 폴리에틸렌글리콜(PEG)을 포함하는 바이오리피드 계면활성제를 본 발명의 엑소좀 유사체 제조 방법에 활용하는 경우, 만노실에리트리톨 지질(MEL)의 고유적인 계면활성력은 그대로 보유하고 있어 미세 조류의 압출가공이 용이하며, 폴리에틸렌글리콜(PEG) 사슬(chain)의 낮은 독성 및 높은 친수성으로 인해 버퍼 용액에 용해성이 높아, 압출가공 전의 버퍼 용액에 직접 용해시켜 미세 조류의 압출 가공에 효과적으로 적용할 수 있다.Therefore, when a biolipid surfactant containing mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) bound thereto is used in the method for producing exosome analogues of the present invention, the unique properties of mannosylerythritol lipid (MEL) It is easy to extrude microalgae as it retains its natural surface activity, and it is highly soluble in buffer solution due to low toxicity and high hydrophilicity of polyethylene glycol (PEG) chain, so it is directly dissolved in buffer solution before extrusion process. It can be effectively applied to the extrusion processing of microalgae.
또한, 상기 바이오리피드 계면활성제의 농도는 50 내지 1000 μM 인 것이 바람직하다. 상기 농도 범위 내에서, 바이오리피드 계면활성제의 농도가 증가할수록 나노베지클의 수율이 증가하고, 제조되는 나노베지클의 크기는 감소할 수 있다. 따라서, 바이오리피드 계면활성제의 농도를 조절하여 나노베지클의 수율 및 크기를 적절히 조절할 수 있다.In addition, the concentration of the biolipid surfactant is preferably 50 to 1000 μM. Within the above concentration range, as the concentration of the biolipid surfactant increases, the yield of nanovesicles increases, and the size of nanovesicles produced may decrease. Therefore, the yield and size of nanovesicles can be appropriately controlled by adjusting the concentration of the biolipid surfactant.
한편, 본 발명에서 사용되는 미세 조류는 유글레나, 두날리엘라, 클로렐라, 스피룰리나, 콜포델라류, 크로메라류, 나노클로롭시스류, 헤마토코쿠스 플루비아리스, 탈라시오시라 슈도나나 및 클라미도모나스 레인하티 중에서 선택된 어느 하나 이상을 포함하는 것이 바람직하나, 이에 제한되는 것은 아니며, 당업자가 목적에 따라 미세 조류의 종류를 달리하여 수행할 수 있다. 또한, 상기 미세 조류의 농도는 1 x 105 ~ 1 x 106 cells/ml 인 것이 바람직한데, 이보다 미세조류의 수가 많으면 포어의 막힘 현상 때문에 압출가공이 어렵고, 그 수가 적으면 효능 물질인 베타글루칸의 함량 및 최종 나노베지클의 수율이 낮아지기 때문이다.On the other hand, the microalgae used in the present invention are Euglena, Dunaliella, Chlorella, Spirulina, Colpodella, Chromera, Nanochloropsis, Haematococcus fluvialis, Thalasiocyra pseudonana and Chlamydo It is preferable to include any one or more selected from Monas reinhati, but is not limited thereto, and a person skilled in the art may vary the type of microalgae according to the purpose. In addition, the concentration of the microalgae is preferably 1 x 10 5 ~ 1 x 10 6 cells / ml. If the number of microalgae is greater than this, extrusion processing is difficult due to the clogging of pores, and if the number is small, beta-glucan, an effective substance This is because the content of and the yield of final nanovesicles are lowered.
상기 버퍼 용액은 산이나 염기를 가해도 pH가 크게 변하지 않는 용액을 의미하며, 미세 조류의 대사산물로 인해 발생되는 pH의 변화를 줄이기 위한 완충 작용의 역할을 하는 물질일 수 있다. 일 실시예에서, 상기 버퍼 용액은 예를 들어, 인산완충생리식염수(Phosphate-buffered saline, PBS), 2차 증류수 등일 수 있다.The buffer solution refers to a solution in which the pH does not change significantly even when an acid or base is added, and may be a material that serves as a buffer to reduce the change in pH caused by metabolites of microalgae. In one embodiment, the buffer solution may be, for example, phosphate-buffered saline (PBS), secondary distilled water, or the like.
또한, 상기 S100 단계에서, 상기 압출 가공은 3 step으로 이루어질 수 있다. 일 실시예에서, 상기 압출가공은 상기 버퍼 용액을 제1 기공을 갖는 제1 멤브레인에 통과시켜 제1 혼합 용액을 제조하는 제1 단계(S110), 상기 제1 혼합 용액을 상기 제1 기공보다 작은 제2 기공을 갖는 제2 멤브레인에 통과시켜 제2 혼합 용액을 제조하는 제2 단계(S120), 및 상기 제2 혼합 용액을 상기 제2 기공보다 작은 제3 기공을 갖는 제3 멤브레인에 통과시키는 제3 단계(S130)를 포함할 수 있다.In addition, in the step S100, the extrusion process may be performed in 3 steps. In one embodiment, the extrusion process is a first step of preparing a first mixed solution by passing the buffer solution through a first membrane having first pores (S110), the first mixed solution smaller than the first pores A second step (S120) of preparing a second mixed solution by passing it through a second membrane having second pores, and passing the second mixed solution through a third membrane having third pores smaller than the second pores. Step 3 (S130) may be included.
일 실시예에서, 상기 제1 단계, 제2 단계 및 제3 단계는 각각 적어도 3회 이상 반복적으로 수행하는 것이 바람직하며, 반복 수행을 통해 시린지 벽면 등에 남아있는 미세조류의 손실을 줄일 수 있으며 엑소좀 모사체를 일정한 크기로 고르게 분산시킬 수 있다.In one embodiment, the first step, the second step, and the third step are preferably performed repeatedly at least three times, respectively, and through repetition, loss of microalgae remaining on the syringe wall, etc. can be reduced, and exosomes The parent body can be evenly dispersed in a certain size.
한편, 상기 제1, 제2 및 제3 멤브레인은 기공을 포함하는 구조로 물질을 선택적 통과시킬 수 있어 여과, 분리 및 전달 등을 수행할 수 있는 투과성을 가질 수 있다. 일례로, 상기 제1, 제2 및 제3 멤브레인은 폴리카보네이트(polycarbonate)로 형성된 멤브레인일 수 있다. 본 발명에서는 제1, 제2 및 제3 멤브레인을 형성하는 물질은 특별히 한정하지는 않으나, 세포 독성이 없으면서 미세 조류 압출 공정에서 영향을 주지 않는 물질로 형성되는 것이 바람직하다.On the other hand, the first, second and third membranes have a structure including pores and can selectively pass a material, so that they can have permeability capable of performing filtration, separation and delivery. For example, the first, second and third membranes may be membranes made of polycarbonate. In the present invention, the material forming the first, second and third membranes is not particularly limited, but is preferably formed of a material that does not affect the microalgae extrusion process without cytotoxicity.
일 실시예에서, 상기 제1 기공은 약 5 내지 12 ㎛일 수 있고, 상기 제2 기공은 약 1.0 내지 2.0 ㎛일 수 있고, 상기 제3 기공은 100 내지 200 nm일 수 있다. 이처럼, 제1, 제2 및 제3 기공의 크기를 달리함으로써 나노 크기의 엑소좀 유사체를 용이하게 형성할 수 있다. 구체적으로, 상기 버퍼 용액의 미세 조류는 제1 단계를 거치면서 제1 멤브레인의 제1 기공으로 인해 보다 작은 크기를 갖게 되고, 이어서 제2 단계를 거치면서 상기 제1 기공보다 작은 크기를 갖는 제2 기공으로 인해 보다 더 작은 크기를 갖게 되고, 이어서 제3 단계를 거치면서 제2 기공보다 작은 크기를 갖는 제3 기공으로 인해 보다 더 작은 크기를 갖는 나노베지클(또는 엑소좀 유사체)을 형성할 수 있다.In one embodiment, the first pores may be about 5 to 12 μm, the second pores may be about 1.0 to 2.0 μm, and the third pores may be 100 to 200 nm. As such, by varying the sizes of the first, second and third pores, nano-sized exosome analogues can be easily formed. Specifically, the microalgae in the buffer solution have a smaller size due to the first pores of the first membrane through the first step, and then pass through the second step to have a second smaller size than the first pores. It has a smaller size due to the pore, and then, through the third step, nanovesicles (or exosome analogues) having a smaller size can be formed due to the third pore having a smaller size than the second pore. there is.
본 발명의 제조 방법에 따라 제조된 미세 조류 유래 엑소좀 유사체는 100 내지 200 nm 의 크기를 가질 수 있고, 나노베지클 형태를 가질 수 있다. (도 1 참조)The microalgae-derived exosome analogue prepared according to the preparation method of the present invention may have a size of 100 to 200 nm and may have a nanovesicle shape. (See Figure 1)
또한, 본 발명의 미세 조류 유래 엑소좀 유사체는 도 1에 나타난 것처럼, 상기 나노베지클 표면의 적어도 일부에 폴리에틸렌글리콜(PEG) 사슬이 도입될 수 있으며, 이로 인해 제조된 나노베지클의 세포 독성이 감소되고 친수성이 증가될 수 있다.In addition, as shown in FIG. 1, in the exosome analogue derived from microalgae of the present invention, a polyethylene glycol (PEG) chain may be introduced into at least a part of the surface of the nanovesicle, thereby reducing the cytotoxicity of the nanovesicle. reduced and increased hydrophilicity.
한편, 본 발명의 다른 실시 형태로, 상기 미세 조류 유래 엑소좀 유사체를 유효 성분으로 함유하는 피부 항산화 화장료 조성물을 들 수 있다.On the other hand, as another embodiment of the present invention, a skin antioxidant cosmetic composition containing the microalgae-derived exosome analogue as an active ingredient may be mentioned.
일 실시예에서, 상기 미세 조류 유래 엑소좀 유사체는 화장료 조성물 총 중량에 대하여 0.01 내지 10.0 중량%로 함유될 수 있다. 본 발명의 화장료 조성물은 미세 조류 유래 엑소좀 유사체를 유효 성분으로 함유하여 피부 세포의 활성산소 생성량을 감소시키는 피부 항산화 효능을 가질 수 있다.In one embodiment, the microalgae-derived exosome analogue may be contained in an amount of 0.01 to 10.0% by weight based on the total weight of the cosmetic composition. The cosmetic composition of the present invention contains microalgae-derived exosome analogues as an active ingredient and may have a skin antioxidant effect that reduces the amount of active oxygen produced by skin cells.
본 발명에 따르면, 압출 가공법을 기반으로 하여 낮은 산화안정성, 낮은 수분산성 등의 문제로 인해 제품화가 어렵고, 특정 성분만 분리 및 정제하는 공정이 까다롭고 고비용이어서 상업화에 어려웠던 미세 조류로부터 엑소좀 유사체를 대량으로 제조할 수 있어, 미세 조류 유래 효능 성분들을 식품, 화장품, 의료 시장까지 확대 적용 가능한 장점이 있다.According to the present invention, based on the extrusion processing method, it is difficult to commercialize due to problems such as low oxidation stability and low water dispersibility, and the process of separating and purifying only specific components is difficult and expensive. Since it can be manufactured in large quantities, it has the advantage of being able to expand the application of microalgae-derived active ingredients to the food, cosmetic, and medical markets.
특히, 본 발명은 압출 가공 효율을 증진시키는 바이오리피드 계면활성제를 선택적으로 적용함으로써 미세 조류 유래 엑소좀 유사체의 베지클 생성 수율을 증가시킬 수 있다.In particular, the present invention can increase the yield of vesicle production of exosome analogues derived from microalgae by selectively applying a biolipid surfactant that enhances extrusion processing efficiency.
이하 본 발명의 이해를 돕기 위해, 구체적인 실시예에 대해 상술한다. 다만, 하기 실시예는 본 발명의 일부 실시형태에 불과한 것으로서, 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, in order to aid understanding of the present invention, specific embodiments will be described in detail. However, the following examples are merely some embodiments of the present invention, and the scope of the present invention is not limited to the following examples.
<실시예 1: 바이오리피드 계면활성제(MEL-PEG)의 합성><Example 1: Synthesis of biolipid surfactant (MEL-PEG)>
만노실에리트리톨 지질(MEL) 및 폴리에틸렌글리콜(PEG)을 포함하는 바이오리피드 계면활성제는 MEL 말단에 말레이미드기를 붙이는 단계 및 상기 말레이미드기와 티올기를 포함하는 PEG(이하에서, PEG-thiol 이라 함)를 반응시키는 단계를 수행함으로써 제조하였다.A biolipid surfactant containing mannosylerythritol lipid (MEL) and polyethylene glycol (PEG) is prepared by attaching a maleimide group to the end of the MEL and PEG containing the maleimide group and a thiol group (hereinafter referred to as PEG-thiol) It was prepared by performing the step of reacting.
1) 말레이미드가 도입된 MEL(MEL-maleimide)의 합성1) Synthesis of MEL (MEL-maleimide) with maleimide introduced
본 발명의 바이오리피드 계면활성제를 제조하기 위해 먼저 MEL의 말단에 말레이미드기를 도입하는 과정을 수행하였다. 정제된 MEL을 무수의 메틸렌 클로라이드(anhydrous methylene chloride)에 녹인 후, 피리딘(pyridine) 및 4-니트로페닐클로로포름에이트(4-nitrophenylchloroformate)를 첨가하여 용액을 실온에서 약 1시간 동안 교반하여 혼합 용액을 제조하였다. 이어서, 상기 혼합 용액에 NH4Cl 포화 수용액을 첨가하여 반응을 종료하고, 분액깔대기를 이용하여 층을 분리시켜 유기층을 추출하였다.In order to prepare the biolipid surfactant of the present invention, a process of introducing a maleimide group at the terminal of MEL was first performed. After dissolving the purified MEL in anhydrous methylene chloride, pyridine and 4-nitrophenylchloroformate were added, and the solution was stirred at room temperature for about 1 hour to prepare a mixed solution did Subsequently, the reaction was terminated by adding a saturated aqueous solution of NH 4 Cl to the mixed solution, and the organic layer was extracted by separating the layers using a separatory funnel.
추출한 유기층을 MgSO4로 건조시켜 여과하고, 유기층의 잔여 용매인 에틸렌 클로라이드를 회전증발농축기 (rotary evaporator)를 이용하여 농축시킨 후, 생성된 잔류물을 실리카겔 상에서 컬럼 크로마토그래피를 이용하여 정제하여 무색 오일의 오소포메이트 화합물 1(orthoformate compound 1)을 수득하였다. 수득된 오소포메이트 화합물 1을 아세토니트릴(acetonitrile)에 녹인 후, N,N-디이소프로필에틸아민 (N,N-Diisopropylethylamine) 및 N-(2-아미노에틸)말레이미드 트리플루오르아세테이트 염(N-(2-aminoethyl)maleimide trifluoro acetate salt)을 넣고 실온에서 12 시간 교반하여 반응을 수행하였다. 반응이 끝난 후, 용매를 회전증발농축기로 제거하고, 잔여물을 메틸렌클로라이드(methylene chloride)에 용해한 후 회전증발농축기로 재농축하였고, 이를 실리카겔 상에서 컬럼 크로마토그래피로 정제하여 무색 오일의 바이오리피드 계면활성제(이하에서, MEL-maleimide 이라 함)를 수득하였다.The extracted organic layer was dried over MgSO 4 and filtered, and the remaining solvent of the organic layer, ethylene chloride, was concentrated using a rotary evaporator, and the resulting residue was purified using column chromatography on silica gel to obtain a colorless oil Orthoformate compound 1 of was obtained. After dissolving the obtained orthoformate compound 1 in acetonitrile (acetonitrile), N, N-diisopropylethylamine (N, N-Diisopropylethylamine) and N- (2-aminoethyl) maleimide trifluoroacetate salt (N -(2-aminoethyl) maleimide trifluoro acetate salt) was added and the reaction was performed by stirring at room temperature for 12 hours. After the reaction was completed, the solvent was removed with a rotary evaporator, and the residue was dissolved in methylene chloride and re-concentrated with a rotary evaporator, which was then purified by column chromatography on silica gel to obtain a colorless oily biolipid surfactant. (hereinafter referred to as MEL-maleimide) was obtained.
2) 말레이미드(maleimide)와 PEG-티올기의 반응2) Reaction between maleimide and PEG-thiol group
바이오리피드 계면활성제를 합성하기 위해 다양한 분자량의 PEG-thiol(Mw ~500, ~1000, ~2000)를 준비하였다. 상기에서 합성된 MEL-maleimide와 PEG-thiol을 몰 비율이 1:1이 되도록 정량한 뒤 클로로포름(Choloroform)에 완전히 용해시켰다. 이어서, 충분한 합성이 이루어지도록 상온에서 8시간 이상 진탕기(Shaker) 위에서 계속 흔들어주며 반응시킨 후, 회전증발농축기(Rotary Evaporator)를 이용하여 용매인 클로로포름을 완전히 증발시켜 제거함으로써, 파우더 형태의 합성된 바이오리피드 계면활성제(이하에서, MEL-PEG 이라 함)를 수득하였다. 수득된 MEL-PEG는 버퍼용액에 직접 용해시켜 원하는 농도로 제조하여 사용하였다.To synthesize biolipid surfactants, various molecular weights of PEG-thiol (Mw ~500, ~1000, ~2000) were prepared. MEL-maleimide and PEG-thiol synthesized above were quantified to a molar ratio of 1:1 and then completely dissolved in chloroform. Then, after continuously shaking and reacting on a shaker at room temperature for more than 8 hours to achieve sufficient synthesis, chloroform as a solvent was completely evaporated and removed using a rotary evaporator to obtain a synthesized powder in powder form. A biolipid surfactant (hereinafter referred to as MEL-PEG) was obtained. The obtained MEL-PEG was directly dissolved in a buffer solution to prepare a desired concentration and used.
<실시예 2: 엑소좀 유사체의 제조><Example 2: Preparation of exosome analogues>
미세 조류를 버퍼 용액인 2차 증류수에 분산시킨 뒤, 실시예 1에서 제조된 MEL-PEG 를 0 ~ 1000 μM 로 첨가하고 볼텍싱(voltexing)을 통해 혼합하여 미세 조류 혼합 용액을 제조하였다. 이 때, 미세 조류의 밀도는 세포 카운팅 헤모사이토미터(cell counting hemocytometer)를 이용해서 정량하여 1 x 105 ~ 1 x 106 cells/ml 사이로 사용하였다. 이후, 상기 미세 조류 혼합 용액을 가스 타이트 시린지(gas tight syringe)에 담은 뒤 미니-익스트루더(mini-extruder)를 통해 5μm 의 기공(pore) 크기를 갖는 폴리카보네이트(polycarbonate) 멤브레인에 3차례 연속적으로 통과시키는 1차 압출 가공을 통해 제1 혼합 용액을 얻었다. 이어서, 상기 제1 세포 혼합 용액은 다시 1μm 의 기공 크기를 갖는 폴리카보네이트(polycarbonate) 멤브레인에 3차례 연속적으로 통과시키고, 추가적으로 200 nm 의 기공 크기를 갖는 폴리카보네이트(polycarbonate) 멤브레인에 3차례 연속적으로 통과시켜, 최종적으로 약 100 - 200 nm 사이즈를 갖는 나노베지클 형태의 엑소좀 유사체(샘플 1 - 4)를 수득하였다.After dispersing the microalgae in double distilled water as a buffer solution, MEL-PEG prepared in Example 1 was added at 0 to 1000 μM and mixed by vortexing to prepare a microalgae mixed solution. At this time, the density of the microalgae was quantified using a cell counting hemocytometer and used between 1 x 10 5 and 1 x 10 6 cells/ml. Thereafter, the microalgae mixed solution was put in a gas tight syringe and then continuously applied to a polycarbonate membrane having a pore size of 5 μm through a mini-extruder three times. A first mixed solution was obtained through the primary extrusion process through which the mixture was passed through. Subsequently, the first cell mixture solution was successively passed through a polycarbonate membrane having a pore size of 1 μm three times, and further passed through a polycarbonate membrane having a pore size of 200 nm three times in succession. Finally, exosome analogues in the form of nanovesicles having a size of about 100 - 200 nm (Samples 1 - 4) were obtained.
샘플 1 - 4의 구체적인 제조 조건은 하기 표 1에 나타냈다.The specific manufacturing conditions of Samples 1 - 4 are shown in Table 1 below.
계면활성제Surfactants 농도 (μM)Concentration (μM) 미세조류 종류microalgae types
샘플 1sample 1 무첨가no additives 00 Euglena gracilisEuglena gracilis
샘플 2sample 2 MEL-PEGMEL-PEG 250250 Euglena gracilisEuglena gracilis
샘플 3sample 3 MEL-PEGMEL-PEG 500500 Euglena gracilisEuglena gracilis
샘플 4sample 4 MEL-PEGMEL-PEG 10001000 Euglena gracilisEuglena gracilis
<실험예 1: 계면활성제 농도에 따른 엑소좀 유사체 분석><Experimental Example 1: Analysis of exosome analogues according to surfactant concentration>
미세 조류의 압출 가공 과정에서 MEL-PEG 처리량에 따른 나노베지클의 수율을 확인하기 위해서, 나노 입자 추적 분석기(NTA, nanoparticle tracking analysis)를 통해 생성된 나노베지클(샘플 1 - 4, 미세 조류의 밀도는 200,000 cells/ml)의 농도를 정량하고 사이즈를 측정하여, 하기 표 2에 나타냈다.In order to confirm the yield of nanovesicles according to the MEL-PEG throughput in the process of extrusion processing of microalgae, nanovesicles generated through nanoparticle tracking analysis (NTA) (Samples 1 - 4, of microalgae The density is shown in Table 2 below by quantifying the concentration of 200,000 cells/ml) and measuring the size.
입자 농도 (particles/ml)Particle concentration (particles/ml) 사이즈 (nm)size (nm)
샘플 1sample 1 2.04×109 2.04×10 9 152.2152.2
샘플 2sample 2 7.78×109 7.78×10 9 140.3140.3
샘플 3sample 3 1.37×1010 1.37×10 10 138.6138.6
샘플 4sample 4 2.34×1010 2.34×10 10 132.5132.5
그 결과, 표 2에 나타난 것처럼, 계면활성제인 MEL-PEG의 농도가 증가할수록 나노베지클의 농도가 크게 증가하는 것을 확인하였고, 사이즈는 약 150 nm에서 130 nm 정도로 점차 줄어드는 것을 확인하였다.As a result, as shown in Table 2, it was confirmed that the concentration of nanovesicles increased significantly as the concentration of MEL-PEG, a surfactant, increased, and the size gradually decreased from about 150 nm to about 130 nm.
한편, 투과전자현미경(TEM)을 이용하여 실시예 2를 통해 제조된 엑소좀 유사체(샘플 1)의 모폴로지를 확인하고, 그 결과를 도 2에 나타냈다.Meanwhile, the morphology of the exosome analogue (sample 1) prepared in Example 2 was confirmed using a transmission electron microscope (TEM), and the results are shown in FIG. 2.
도 2를 보면, 미세 조류 유래 엑소좀 유사체는 100 내지 200 nm 의 크기를 갖고 나노베지클 형태를 나타내는 것을 확인할 수 있다.Referring to Figure 2, it can be seen that the microalgae-derived exosome analogue has a size of 100 to 200 nm and exhibits a nanovesicle form.
<실험예 2: 미세 조류 유래 엑소좀 유사체의 주성분 분석><Experimental Example 2: Main component analysis of microalgae-derived exosome analogues>
미세 조류 유래 엑소좀 유사체에서 유글레나의 주성분인 베타글루칸 함량을 확인하기 위해, 아닐린블루(Aniline blue) 염색법을 이용하였다.In order to confirm the content of beta-glucan, which is the main component of Euglena, in microalgae-derived exosome analogues, aniline blue staining was used.
구체적으로, 순수 베타글루칸을 농도 별로 아닐린블루 시약과 반응시켜 얻은 형광 값으로 기준 곡선(standard curve)을 그린 뒤, 유글레나 및 본 발명의 샘플 1의 형광값을 대입하여 베타글루칸 함량을 계산하여 도 3에 나타냈다.Specifically, after drawing a standard curve with the fluorescence values obtained by reacting pure beta-glucan with the aniline blue reagent for each concentration, the beta-glucan content was calculated by substituting the fluorescence values of Euglena and sample 1 of the present invention. shown in
그 결과, 도 3에 나타난 것처럼, 5.0 x 105 의 농도의 유글레나 및 유글레나 엑소좀 유사체에는 각각 약 494.63 ㎍/ml, 23.15 ㎍/ml 의 베타글루칸이 포함되어 있는 것을 확인하였다.As a result, as shown in FIG. 3, it was confirmed that about 494.63 μg/ml and 23.15 μg/ml of beta-glucan were contained in Euglena and Euglena exosome analogues at a concentration of 5.0×10 5 , respectively.
<실시예 3: 다양한 미세 조류를 이용한 엑소좀 유사체 제조><Example 3: Preparation of exosome analogues using various microalgae>
여러 가지 미세 조류에 대한 확대 적용성을 확인하기 위해, 두날리엘라 및 클로렐라를 이용하여 실시예 2의 샘플 3과 동일한 방법으로 엑소좀 유사체(샘플 4 - 5)를 제조하였다. In order to confirm the broad applicability to various microalgae, exosome analogues (Samples 4 to 5) were prepared using Dunaliella and Chlorella in the same manner as Sample 3 in Example 2.
이후, 나노 입자 추적 분석기(NTA, nanoparticle tracking analysis)를 통해 생성된 나노베지클(샘플 3 - 5, 미세 조류의 밀도는 200,000 cells/ml)의 농도를 정량하여, 하기 표 3에 나타냈다.Thereafter, the concentration of nanovesicles (Samples 3 - 5, the density of microalgae was 200,000 cells/ml) was quantified through nanoparticle tracking analysis (NTA), and is shown in Table 3 below.
미세조류 종류microalgae types 입자 농도 (particles/ml)Particle concentration (particles/ml)
샘플 3sample 3 Euglena gracilisEuglena gracilis 2.34×1010 2.34×10 10
샘플 4sample 4 DunaliellaDunaliella 2.05×1010 2.05×10 10
샘플 5sample 5 ChlorellaChlorella 1.98×1010 1.98×10 10
표 3을 참조하면, 샘플 3 내지 5의 농도는 유사한 수준으로 관찰되었으며, 이로부터 본 발명이 다양한 미세 조류에 적용 가능함을 확인할 수 있다.Referring to Table 3, the concentrations of samples 3 to 5 were observed at similar levels, from which it can be confirmed that the present invention is applicable to various microalgae.
<실시예 4: 미세 조류 유래 엑소좀 유사체의 항산화 효능 평가><Example 4: Evaluation of antioxidant efficacy of microalgae-derived exosome analogues>
실시예 2에 따라 제조된 유글레나 엑소좀 유사체의 항산화 효능을 확인하기 위해, 피부 각질 세포(HaCaT cell)에 본 발명의 실시예에 따른 유글레나 엑소좀 유사체(샘플 1)를 농도별로 처리한 뒤 생성되는 활성 산소를 측정하였다.In order to confirm the antioxidant efficacy of the Euglena exosome analog prepared according to Example 2, skin keratinocytes (HaCaT cells) were treated with the Euglena exosome analog (sample 1) according to the embodiment of the present invention by concentration, and then generated Active oxygen was measured.
구체적으로, HaCaT 세포를 96 웰 플레이트에 2.5 x 104 농도로 각 웰에 분주하여 24시간 동안 배양한 후, 배지를 제거하고 PBS 로 1차 워싱한 뒤 20 μM DCFDA(2',7'-dichlorofluorescindiacetate) 용액을 100㎕ 씩 분주하여 30 분간 36℃ 에서 배양하였다. 이후, DCFDA 용액을 제거하고 PBS 로 워싱한 후, 본 발명의 샘플 1을 25㎕, 50㎕, 75㎕, 100㎕ 씩 분주하여 3시간 동안 배양하였다. 마지막으로, 산화 스트레스를 유발하기 위해 TBHP(tert-Butyl hydroperoxide) 용액을 최종 농도가 50μM 가 되도록 처리한 뒤 3시간 후 형광 플레이트 리더(fluorescence plate reader)로 Ex/Em = 485/535 nm에서 형광 값을 측정하고, 그 결과를 도 4에 나타냈다.Specifically, HaCaT cells were dispensed into each well in a 96-well plate at a concentration of 2.5 x 10 4 and cultured for 24 hours, then the medium was removed, washed first with PBS, and 20 μM DCFDA (2',7'-dichlorofluorescindiacetate ) solution was dispensed by 100 μl and incubated at 36° C. for 30 minutes. Then, after removing the DCFDA solution and washing with PBS, 25 μl, 50 μl, 75 μl, and 100 μl of Sample 1 of the present invention were dispensed and cultured for 3 hours. Finally, in order to induce oxidative stress, TBHP (tert-Butyl hydroperoxide) solution was treated to a final concentration of 50 μM, and after 3 hours, fluorescence values at Ex/Em = 485/535 nm with a fluorescence plate reader was measured, and the results are shown in FIG. 4 .
도 4를 보면, 본 발명의 샘플 1의 처리양이 증가할수록 활성산소 생성량이 감소하는 것을 확인할 수 있다. 이를 통해, 본 발명의 실시예에 따른 미세 조류 유래 엑소좀 유사체의 피부 항산화 효능을 확인하였다.Referring to FIG. 4 , it can be seen that the amount of active oxygen produced decreases as the treatment amount of Sample 1 of the present invention increases. Through this, the skin antioxidant efficacy of the microalgae-derived exosome analogues according to an embodiment of the present invention was confirmed.
상기에서는 본 발명의 바람직한 실시예를 참조하여 설명하였지만, 해당 기술 분야의 숙련된 당업자는 하기의 특허 청구 범위에 기재된 본 발명의 사상 및 영역으로부터 벗어나지 않는 범위 내에서 본 발명을 다양하게 수정 및 변경시킬 수 있음을 이해할 수 있을 것이다.Although the above has been described with reference to preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention described in the claims below. You will understand that you can.

Claims (15)

  1. 미세 조류를 포함하는 버퍼 용액을 압출가공하여 나노베지클을 제조하는 단계;를 포함하는,Preparing nanovesicles by extruding a buffer solution containing microalgae;
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  2. 제1항에 있어서,According to claim 1,
    상기 압출가공은,The extrusion process,
    상기 버퍼 용액을 제1 기공을 갖는 제1 멤브레인에 통과시켜 제1 혼합 용액을 제조하는 제1 단계;A first step of preparing a first mixed solution by passing the buffer solution through a first membrane having first pores;
    상기 제1 혼합 용액을 상기 제1 기공보다 작은 제2 기공을 갖는 제2 멤브레인에 통과시켜 제2 혼합 용액을 제조하는 제2 단계; 및a second step of preparing a second mixed solution by passing the first mixed solution through a second membrane having second pores smaller than the first pores; and
    상기 제2 혼합 용액을 상기 제2 기공보다 작은 제3 기공을 갖는 제3 멤브레인에 통과시키는 제3 단계;를 포함하는 것을 특징으로 하는,A third step of passing the second mixed solution through a third membrane having a third pore smaller than the second pore; characterized in that it comprises,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  3. 제2항에 있어서,According to claim 2,
    상기 제1 기공은 5 내지 12 ㎛ 이고,The first pores are 5 to 12 μm,
    상기 제2 기공은 1.0 내지 2.0 ㎛ 이고,The second pores are 1.0 to 2.0 μm,
    상기 제3 기공은 100 내지 200 nm 인 것을 특징으로 하는,Characterized in that the third pore is 100 to 200 nm,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  4. 제2항에 있어서,According to claim 2,
    상기 제1 단계, 제2 단계 및 제3 단계는 각각 적어도 3회 이상 반복적으로 수행함을 특징으로 하는,Characterized in that the first step, the second step and the third step are repeatedly performed at least three times or more,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  5. 제1항에 있어서,According to claim 1,
    상기 미세 조류는 유글레나, 두날리엘라, 클로렐라, 스피룰리나, 콜포델라류, 크로메라류, 나노클로롭시스류, 헤마토코쿠스 플루비아리스, 탈라시오시라 슈도나나 및 클라미도모나스 레인하티 중에서 선택된 어느 하나 이상을 포함함을 특징으로 하는,The microalgae are any selected from Euglena, Dunaliella, Chlorella, Spirulina, Colpodella, Chromera, Nanochloropsis, Hematococcus fluvialis, Thalassiocyra pseudonana, and Chlamydomonas Reynhati Characterized in that it contains one or more,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  6. 제1항에 있어서,According to claim 1,
    상기 압출가공 전의 상기 버퍼 용액은 계면활성제를 더 포함하는 것을 특징으로 하는,Characterized in that the buffer solution before the extrusion further comprises a surfactant,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  7. 제6항에 있어서,According to claim 6,
    상기 계면활성제는 바이오리피드 계면활성제임을 특징으로 하는,Characterized in that the surfactant is a biolipid surfactant,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  8. 제7항에 있어서,According to claim 7,
    상기 바이오리피드 계면활성제는 만노실에리트리톨 지질(MEL) 모이에티 및 상기 만노실에리트리톨 지질(MEL)과 결합된 폴리에틸렌글리콜(PEG) 모이에티를 포함함을 특징으로 하는,Characterized in that the biolipid surfactant comprises a mannosylerythritol lipid (MEL) moiety and a polyethylene glycol (PEG) moiety combined with the mannosylerythritol lipid (MEL).
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  9. 제8항에 있어서,According to claim 8,
    상기 바이오리피드 계면활성제는 하기 화학식 1의 구조를 갖는 것인, The biolipid surfactant has a structure represented by Formula 1 below,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
    <화학식 1><Formula 1>
    Figure PCTKR2022010227-appb-img-000003
    Figure PCTKR2022010227-appb-img-000003
    상기 화학식 1에서, n 및 m은 각각 1 내지 13의 정수이다.In Formula 1, n and m are each an integer of 1 to 13.
  10. 제7항에 있어서,According to claim 7,
    상기 바이오리피드 계면활성제의 농도는 50 내지 1000 μM 인 것을 특징으로 하는,Characterized in that the concentration of the biolipid surfactant is 50 to 1000 μM,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  11. 제1항에 있어서,According to claim 1,
    상기 미세 조류의 농도는 1 x 105 ~ 1 x 106 cells/ml 인 것을 특징으로 하는,Characterized in that the concentration of the microalgae is 1 x 10 5 ~ 1 x 10 6 cells / ml,
    엑소좀 유사체 제조 방법.Methods for making exosome analogues.
  12. 제1항 내지 제11항 중 어느 한 항에 따른 방법으로 제조되고,It is prepared by the method according to any one of claims 1 to 11,
    100 내지 200 nm 의 크기를 가지며, 나노베지클 형태를 갖는,It has a size of 100 to 200 nm and has a nanovesicle shape,
    미세 조류 유래 엑소좀 유사체.Microalgae-derived exosome analogues.
  13. 제12항에 있어서,According to claim 12,
    상기 나노베지클 표면의 적어도 일부에 폴리에틸렌글리콜(PEG) 사슬이 도입된 것을 특징으로 하는,Characterized in that a polyethylene glycol (PEG) chain is introduced to at least a part of the surface of the nanovesicle,
    미세 조류 유래 엑소좀 유사체.Microalgae-derived exosome analogues.
  14. 제12항에 따른 미세 조류 유래 엑소좀 유사체를 유효 성분으로 함유하는 피부 항산화 화장료 조성물.A skin antioxidant cosmetic composition containing the microalgae-derived exosome analogue according to claim 12 as an active ingredient.
  15. 제14항에 있어서,According to claim 14,
    상기 미세 조류 유래 엑소좀 유사체를 화장료 조성물 총 중량에 대하여 0.01 내지 10 중량%로 함유하는 피부 항산화 화장료 조성물.A skin antioxidant cosmetic composition containing the microalgae-derived exosome analogue in an amount of 0.01 to 10% by weight based on the total weight of the cosmetic composition.
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