Composition for lyophilizing extracellular vesicles or artificial cell vesicles, method for producing lyophilized extracellular vesicles or artificial cell vesicles, and extracellular vesicles or artificial cell vesicles

Abstract

The present invention addresses the problem of providing a composition for lyophilizing extracellular vesicles or artificial cell vesicles, which makes it possible to prevent the extracellular vesicles or the artificial cell vesicles from being deteriorated in shape preserving properties or functions thereof or from being reduced in particle diameters when the extracellular vesicles or the artificial cell vesicles are recovered by hydration after lyophilization. The composition for lyophilizing extracellular vesicles or artificial cell vesicles is prepared, the composition being characterized by being intended to be added to extracellular vesicles or artificial cell vesicles upon use and also being characterized by containing the following hydrophobic substances: (a) phenylalanine or a methyl ester thereof; (b) a dipeptide which is composed of one or two kinds of hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine and contains at least one phenylalanine molecule as a constituent amino acid; and the like.

Description

Composition for freeze-drying extracellular vesicles or artificial cell vesicles, method for producing freeze-dried extracellular vesicles or artificial cell vesicles, and extracellular vesicles or artificial cell vesicles

[0001] The present invention relates to a composition for freeze-drying extracellular vesicles or artificial cell vesicles, a method for producing freeze-dried extracellular vesicles or artificial cell vesicles, extracellular vesicles or artificial cell vesicles, a pharmaceutical composition, and a cosmetic composition.

[0002] In recent years, extracellular vesicles such as exosomes and artificial cell vesicles have been attracting attention in the fields of cancer treatment, inflammatory disease treatment, regenerative therapy, and cosmetics. Freezing and freeze-drying techniques have been developed for the use of extracellular vesicles and artificial cell vesicles, but maintaining the membrane structure, which is important for intracellular introduction, is difficult, and membrane destruction due to freezing and thawing is an issue.

[0003] To date, cryopreservatives for exosomes containing methionine, mannitol, and trehalose have been disclosed (see Patent Documents 1 and 2), and a method for stabilizing extracellular vesicles by mixing a sample containing extracellular vesicles with a sugar and a chelating agent (see Patent Document 3).

[0004] Although clinical studies using frozen exosomes have progressed, satisfactory therapeutic effects have not yet been achieved. This is thought to be due to insufficient maintenance of the membrane structure.

[0005] JP 2021-523923 A JP 2021-526155 A JP 2023-153401 A

[0006] An object of the present invention is to provide a composition for freeze-drying extracellular vesicles or artificial cell vesicles that can suppress a decrease in shape retention, a decrease in particle size, or a decrease in function when the extracellular vesicles or artificial cell vesicles are freeze-dried and then rehydrated.

[0007] As a result of extensive research to solve the above problems, the inventors discovered that by adding specific amino acids or peptides and then freeze-drying, it is possible to prevent membrane collapse or functional deterioration even when extracellular vesicles or artificial cellular vesicles are restored after freeze-drying, and thus completed the present invention.

[0008] That is, the present invention is as follows. [1] A composition for freeze-drying extracellular vesicles or artificial cell vesicles, comprising any of the following hydrophobic substances: (a) phenylalanine or its methyl ester; (b) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; (c) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; or (d) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; characterized in that the composition is added to extracellular vesicles or artificial cell vesicles for use. [2] The composition for freeze-drying according to [1] above, wherein the hydrophobic substance is a combination of phenylalanine and leucine, or a dipeptide having phenylalanine as a constituent amino acid. [3] The composition for freeze-drying of extracellular vesicles or artificial cell vesicles according to [1] or [2] above, wherein the extracellular vesicles or artificial cell vesicles are exosomes. [4] The composition for freeze-drying according to any of [1] to [3] above, further comprising trehalose and / or ascorbic acid or derivatives thereof. [5] A method for producing freeze-dried extracellular vesicles or artificial cell vesicles, comprising the step of adding the composition for freeze-drying according to any of [1] to [4] above to extracellular vesicles or artificial cell vesicles. [6] A method for producing freeze-dried extracellular vesicles or artificial cell vesicles according to [5] above, characterized in that the composition for freeze-drying according to any one of [1] to [4] above is added to a solution containing the extracellular vesicles or artificial cell vesicles so that the concentration of the hydrophobic substance contained in the composition for freeze-drying is 0.3 to 100 mg / mL.[7] A method for producing the freeze-dried extracellular vesicles or artificial cell vesicles described in [5] or [6] above, comprising the following steps (a) and (b): adding the composition for freeze-drying described in any of [1] to [4] above to extracellular vesicles or artificial cell vesicles and freezing them at -196 to -20°C for 8 hours or more; and (b) drying the extracellular vesicles or artificial cell vesicles frozen in step (a) under vacuum conditions at -50 to 35°C for 10 to 72 hours. [8] Extracellular vesicles or artificial cell vesicles produced by the method described in any of [5] to [7] above and reconstituting the freeze-dried extracellular vesicles or artificial cell vesicles with a solvent. [9] A pharmaceutical composition comprising the reconstituted extracellular vesicles or artificial cell vesicles described in [8] above and a pharmaceutically acceptable additive.

[10] A cosmetic composition comprising the reconstituted extracellular vesicles or artificial cell vesicles described in [8] above.

[0009] According to the disclosure of the present specification, even if extracellular vesicles or artificial cell vesicles are freeze-dried, membrane collapse or functional deterioration can be prevented by restoration by hydration. Furthermore, extracellular vesicles or artificial cell vesicles produced according to the disclosure of the present specification can be easily stored, transported, or processed at room temperature.

[0010] Figure 1 shows the results of scanning electron microscopy of exosomes derived from human mesenchymal stem cells lyophilized and restored using the additives in columns 3 and 19 in Example 2. Figure 2 shows the results of examining the number of exosomes lyophilized and restored using the additives in columns 3 and 19 in Example 3. Figure 3 shows the results of examining the proportion of CD63-positive and CD9-positive exosomes in exosomes lyophilized and restored using the additives in columns 3 and 19 in Example 4. Figure 4 shows the results of examining the cell proliferation rate when exosomes lyophilized and restored using the additives in columns 3 and 19 in Example 5 were added to human mesenchymal stem cells. Figure 5 shows the results of examining the proportion of ROS-positive cells when exosomes lyophilized and restored using the additives in columns 3 and 19 in Example 6 were added to THP-1 cells.

[0011] One aspect of the present invention is a hydrophobic substance selected from the group consisting of: (a) phenylalanine or a methyl ester thereof; (b) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; (c) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; or (d) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; The present invention relates to a composition for freeze-drying extracellular vesicles or artificial cell vesicles, characterized in that the composition comprises the above-mentioned compound, and is used by adding the composition to extracellular vesicles or artificial cell vesicles. Hereinafter, this composition will be referred to as the "composition for freeze-drying." Another aspect of the present invention is a method for producing freeze-dried extracellular vesicles or artificial cell vesicles, which comprises adding the above-mentioned composition for freeze-drying to extracellular vesicles or artificial cell vesicles. Hereinafter, this method will be referred to as the "method for producing freeze-dried extracellular vesicles or artificial cell vesicles." Another aspect of the present invention is extracellular vesicles or artificial cell vesicles obtained by reconstituting the above-mentioned freeze-dried cells with a solvent. Hereinafter, this method will be referred to as the "reconstituted extracellular vesicles or artificial cell vesicles." Another aspect of the present invention is a pharmaceutical composition or cosmetic composition comprising the above-mentioned reconstituted extracellular vesicles or artificial cell vesicles and a pharmaceutically acceptable additive. Hereinafter, this composition will be referred to as the "pharmaceutical composition" and the "cosmetic composition," respectively.Furthermore, one aspect of the present invention relates to the use of the following hydrophobic substances in the manufacture of a pharmaceutical composition for the prevention or treatment of a disease that is a target of regenerative medicine: (a) phenylalanine or a methyl ester thereof; (b) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; (c) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; or (d) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid. Furthermore, one aspect of the present invention is a method for preventing or treating a disease that is a target of regenerative medicine, which comprises administering the pharmaceutical composition to a patient in need thereof.

[0012] 1. Composition for Freeze-Drying (Hydrophobic Substance) The composition for freeze-drying contains a hydrophobic substance, such as: (a) phenylalanine or its methyl ester; (b) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; (c) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; or (d) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid.

[0013] A suitable example of (a) is phenylalanine.

[0014] As used herein, a dipeptide refers to a molecule in which two amino acids are linked by one peptide bond, a tripeptide refers to a molecule in which three amino acids are linked by two peptide bonds, and a tetrapeptide refers to a molecule in which four amino acids are linked by three peptide bonds.

[0015] The above-mentioned phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine are all hydrophobic amino acids, and each may independently be in either the D- or L-form. When two or more hydrophobic amino acids are combined, a mixture of D- and L-forms may be used, but it is preferable that all be in the L-form. Furthermore, the above-mentioned phenylalanine, leucine, glycine, valine, isoleucine, and tryptophan may each independently be a methyl ester. Examples of the methyl ester of phenylalanine include, but are not limited to, N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine 1-methyl ester (Neotame). The use of these hydrophobic amino acids makes it possible to form lattice-like fibers around extracellular vesicles or artificial cell vesicles when lyophilized, thereby making it possible to inhibit binding between extracellular vesicles or artificial cell vesicles.

[0016] When the dipeptide described in (b) above is composed of one type of hydrophobic amino acid, it is composed of two phenylalanines (phenylalanine-phenylalanine). When the dipeptide described in (b) above is composed of two types of hydrophobic amino acids, the combination of the two hydrophobic amino acids only needs to contain at least one phenylalanine as a constituent amino acid, and examples of the combination include phenylalanine and leucine; phenylalanine and glycine; phenylalanine and valine; phenylalanine and isoleucine; phenylalanine and tryptophan; and phenylalanine and alanine, with phenylalanine and leucine being preferred. The arrangement of the two hydrophobic amino acids is not particularly limited, and they may be arranged in either order from the N-terminus to the C-terminus.

[0017] When the tripeptide described in (c) above is composed of one type of hydrophobic amino acid, it is composed of three phenylalanines (phenylalanine-phenylalanine-phenylalanine).

[0018] In the tripeptide described in (c) above, when the tripeptide is composed of two hydrophobic amino acids, the combination of the two hydrophobic amino acids may contain at least one phenylalanine as a constituent amino acid, and examples thereof include combinations of phenylalanine and leucine; phenylalanine and glycine; phenylalanine and valine; phenylalanine and isoleucine; phenylalanine and tryptophan; and phenylalanine and alanine, with the combination of phenylalanine and leucine being preferred. The tripeptide described in (c) above may contain one hydrophobic amino acid of either of the two hydrophobic amino acids and two hydrophobic amino acids of the other, but preferably contains two phenylalanines. The arrangement of the three hydrophobic amino acids in the tripeptide described in (c) above is not particularly limited, and any hydrophobic amino acid may be arranged from the N-terminus to the C-terminus.

[0019] When the tripeptide described in (c) above is composed of three hydrophobic amino acids, the combination of the three hydrophobic amino acids only needs to contain at least one phenylalanine as a constituent amino acid, and examples of the combinations include phenylalanine, leucine, and glycine; phenylalanine, leucine, and valine; phenylalanine, leucine, and isoleucine; phenylalanine, leucine, and tryptophan; phenylalanine, leucine, and alanine; phenylalanine, glycine, and valine; phenylalanine, glycine, and isoleucine; phenylalanine, glycine, and tryptophan; phenylalanine, glycine, and alanine; phenylalanine, valine, and isoleucine; phenylalanine, valine, and tryptophan; phenylalanine, valine, and alanine; phenylalanine, isoleucine, and tryptophan; phenylalanine, isoleucine, and alanine; phenylalanine, tryptophan, and alanine; and combinations containing phenylalanine and leucine are preferred. The arrangement of the three hydrophobic amino acids in the tripeptide described in (c) above is not particularly limited, and any hydrophobic amino acids may be arranged from the N-terminus to the C-terminus.

[0020] In the tetrapeptide described in (d) above, when it is composed of one type of hydrophobic amino acid, it is composed of four phenylalanines (phenylalanine-phenylalanine-phenylalanine-phenylalanine).

[0021] In the tetrapeptide described in (d) above, when the tetrapeptide is composed of two hydrophobic amino acids, the combination of the two hydrophobic amino acids may contain at least one phenylalanine as a constituent amino acid, and examples thereof include combinations of phenylalanine and leucine; phenylalanine and glycine; phenylalanine and valine; phenylalanine and isoleucine; phenylalanine and tryptophan; and phenylalanine and alanine, with the combination of phenylalanine and leucine being preferred. The tetrapeptide described in (d) above may contain one hydrophobic amino acid of either of the two hydrophobic amino acids and three hydrophobic amino acids of the other, but preferably contains three phenylalanines. The arrangement of the four hydrophobic amino acids in the tetrapeptide described in (d) above is not particularly limited, and any hydrophobic amino acids may be arranged from the N-terminus to the C-terminus.

[0022] In the tetrapeptide described in (d) above, when the tetrapeptide is composed of four hydrophobic amino acids, the combination of the four hydrophobic amino acids may contain at least one phenylalanine as a constituent amino acid, and the combinations include phenylalanine, leucine, glycine, and valine; phenylalanine, leucine, glycine, and isoleucine; phenylalanine, leucine, glycine, and tryptophan; phenylalanine, leucine, glycine, and alanine; phenylalanine, leucine, valine, and isoleucine; phenylalanine, leucine, valine, and tryptophan; phenylalanine, leucine, valine, and alanine; phenylalanine, leucine, Examples of suitable combinations include: phenylalanine, leucine, isoleucine, and tryptophan; phenylalanine, leucine, isoleucine, and alanine; phenylalanine, leucine, tryptophan, and alanine; phenylalanine, glycine, valine, and isoleucine; phenylalanine, glycine, valine, and tryptophan; phenylalanine, glycine, valine, and alanine; phenylalanine, glycine, isoleucine, and tryptophan; phenylalanine, glycine, isoleucine, and alanine; phenylalanine, glycine, tryptophan, and alanine; and a combination containing phenylalanine and leucine is preferred. The arrangement of the four hydrophobic amino acids in the tetrapeptide described in (d) above is not particularly limited, and any hydrophobic amino acids may be arranged from the N-terminus to the C-terminus.

[0023] Furthermore, the hydrophobic substance may be a combination of (a) phenylalanine or its methyl ester, (b) dipeptide, (c) tripeptide, or (d) tetrapeptide, or may be a combination of (a) to (d) phenylalanine or its methyl ester, dipeptide, tripeptide, or tetrapeptide with one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine. Specifically, (a) phenylalanine may be further combined with leucine.

[0024] The dipeptide, tripeptide or tetrapeptide may be straight-chain, cyclic or branched, but is preferably straight-chain.

[0025] The hydrophobic amino acids (monoamino acids) can be produced by known amino acid synthesis methods. The dipeptides, tripeptides, and tetrapeptides can be produced by known peptide synthesis methods such as solid-phase synthesis and liquid-phase synthesis. Examples of solid-phase synthesis methods include the fluorenylmethyloxycarbonyl method (Fmoc method) and the t-butyloxycarbonyl method (tBoc method). Alternatively, commercially available hydrophobic amino acids, dipeptides, tripeptides, and tetrapeptides may be used.

[0026] Furthermore, when phenylalanine is used in combination with another hydrophobic amino acid selected from the group consisting of leucine, glycine, valine, isoleucine, tryptophan, and alanine in the above (a), the weight ratio of phenylalanine to the other hydrophobic amino acid is not particularly limited, but examples thereof include 0.1 to 5 parts by weight, 0.2 to 3 parts by weight, 0.3 to 2 parts by weight, 0.5 to 1.5 parts by weight, or 0.8 to 2 parts by weight of the other hydrophobic amino acid per 1 part by weight of phenylalanine.

[0027] (Extracellular vesicles or artificial cell vesicles to be added) In the present composition for freeze-drying, extracellular vesicles refer to membrane vesicles composed of a lipid bilayer membrane derived from cells, and examples thereof include exosomes, microvesicles, and apoptotic vesicles.

[0028] The extracellular vesicles are released from most cell types and can be found in many body fluids. They encapsulate proteins and nucleic acids such as DNA, mRNA, or microRNA. The extracellular vesicles can be obtained by centrifuging or ultrafiltrating the culture supernatant of a cell population to remove cells and other contaminants and recover the supernatant. The centrifugation can be performed, for example, at 1,000 to 3,000 rpm for 2 to 10 minutes. The extracellular vesicles can then be further purified and recovered using a commercially available extracellular vesicle isolation kit or by known techniques. Alternatively, the extracellular vesicles can be purified or recovered by ultracentrifugation (e.g., 100,000 to 1,000,000 g for 30 to 12 hours).

[0029] The extracellular vesicles are preferably derived from mammalian cells such as human, dog, cat, monkey, cow, horse, mouse, rat, hamster, guinea pig, rabbit, goat, pig, or sheep, or from plant cells. In the case of mammalian cell-derived extracellular vesicles, examples include extracellular vesicles derived from stem cells, such as mesenchymal stem cells present in bone marrow, umbilical cord blood, dental pulp, placenta, adipose tissue, etc. In this specification, extracellular vesicles derived from human cells refer to extracellular vesicles isolated from a living body.

[0030] In the present composition for freeze-drying, the artificial cellular vesicles include (1) liposomes, which are vesicles containing phospholipids as a constituent component and containing a bilayer membrane of the phospholipids, and (2) vesicles containing a surfactant other than phospholipids as a constituent component and containing a bilayer membrane of the surfactant. The vesicles containing the bilayer membrane may be small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), which have one bilayer membrane, or multilamellar vesicles (MVLs), which have two or more bilayer membranes.

[0031] Examples of the phospholipid include phosphoethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE); and phosphocholines such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and diphthanoyl-sn-glycero-3-phosphocholine (DPhPC).

[0032] Examples of surfactants other than the phospholipids include phosphorus-free lipids and Gemini surfactants. Examples of the phosphorus-free lipids include N-(2,3-dioleoyloxy-1-propyl)trimethylammonium methylsulfate (DOTAP), N-[1-(2,3-oleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), and dioctadecylamido-glycylspermine (DOGS). The Gemini surfactants are dimeric surfactants having two types of surface-active units or trimeric surfactants having three types of surface-active units, each of which has a hydrophilic group and a hydrophobic group in a single molecule, and which are linked to each other via the hydrophilic group via a spacer group.

[0033] Examples of the hydrophobic group constituting the Gemini surfactant include a linear hydrocarbon chain having 8 to 24 carbon atoms, preferably 12 to 18 carbon atoms, and more preferably 12 or 16 carbon atoms, and a linear saturated hydrocarbon chain is preferred from the viewpoint of the stability of the Gemini surfactant. Furthermore, the linear hydrocarbon chains constituting the two or three types of hydrophobic groups may be the same or different, but are preferably the same.

[0034] (Other Components) The present composition for freeze-drying may contain other components in addition to the phenylalanine or its methyl ester, the dipeptide, the tripeptide, and the tetrapeptide, such as trehalose, ascorbic acid, mannitol, and / or polyethylene glycol or derivatives thereof. Examples of the ascorbic acid derivatives include L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 2-O-α-D-glucopyranosyl-L-ascorbic acid, and glyceryl ascorbate. Examples of the polyethylene glycol derivatives include polyethylene glycol monostearate. For example, the composition for freeze-drying of the present invention may be a combination of phenylalanine and ascorbyl 2-phosphate sesquimagnesium hydrate, a combination of phenylalanine and polyethylene glycol monoesterate, a combination of phenylalanine and trehalose, a combination of phenylalanine and mannitol, a combination of phenylalanine, leucine, and ascorbyl 2-phosphate sesquimagnesium hydrate, a combination of phenylalanine, leucine, and trehalose, a combination of phenylalanine, leucine, and polyethylene glycol monoesterate, a combination of phenylalanine, leucine, ascorbyl 2-phosphate sesquimagnesium hydrate, and trehalose, or a combination of phenylalanine, trehalose, ascorbyl 2-phosphate sesquimagnesium hydrate, and polyethylene glycol monoesterate.

[0035] The composition for freeze-drying of the present invention may also contain a dispersion medium, etc. Examples of the dispersant include water, a buffer solution, a water-soluble solvent, a liquid medium, an isotonic solution, etc., and these may be used alone or in combination of two or more.

[0036] Examples of the water include distilled water, ion-exchanged water, ultrafiltered water, and pure water. The water content is preferably 70% by mass or more, more preferably 80% by mass or more and 90% by mass or less, and even more preferably 90% by mass or more and 100% by mass or less, based on the total amount of the dispersion medium.

[0037] Examples of the buffer solution include phosphate buffer solution (PBS), trishydroxymethylaminomethane buffer solution (TRIS), hydroxyethylpiperazineethanesulfonic acid buffer solution (HEPES), borate buffer solution, acetate buffer solution, etc. The pH of the buffer solution is not particularly limited, but is preferably from pH 5 to pH 10, and more preferably from pH 6 to pH 9.

[0038] As the water-soluble solvent, alcohols such as methanol and ethanol can be mentioned from the viewpoint of the solubility of lipids.

[0039] Examples of the liquid medium include known media used for cell culture, such as DMEM (Dulbecco's Modified Eagle's Medium), DMEM:F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), EMEM (Eagle's minimal essential medium), and liquid media obtained by adding cell growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and nerve growth factor (NGF), as well as commercially available liquid media for cell growth.

[0040] 2. Method for Producing Freeze-Dried Extracellular Vesicles or Artificial Cell Vesicles (Step of Adding to Extracellular Vesicles or Artificial Cell Vesicles) In the method for producing freeze-dried extracellular vesicles or artificial cell vesicles, the step of adding the composition for freeze-drying to the extracellular vesicles or artificial cell vesicles is not particularly limited, but an example is a method in which the composition for freeze-drying is added to a predetermined solution containing the extracellular vesicles or artificial cell vesicles. Note that adding the composition for freeze-drying to the extracellular vesicles or artificial cell vesicles means that the composition for freeze-drying and the extracellular vesicles or artificial cell vesicles can be brought into contact with each other, and the extracellular vesicles or artificial cell vesicles may be added to the composition for freeze-drying.

[0041] The predetermined solution may be any solution capable of suspending the extracellular vesicles or artificial cellular vesicles, and examples thereof include isotonic solutions such as phosphate buffered saline (PBS), physiological saline, and simulated body fluids, and liquid media.

[0042] In the step of adding the present composition for freeze-drying to extracellular vesicles or artificial cell vesicles, the present composition for freeze-drying can be added to a predetermined solution containing extracellular vesicles or artificial cell vesicles so that the concentration of the hydrophobic substance contained in the composition for freeze-drying is 0.1 to 100 mg / mL. When a hydrophobic amino acid is included as the hydrophobic substance, the concentration can be adjusted appropriately depending on the solubility of the hydrophobic amino acid and the type of predetermined solution used, but can be set to 0.2 to 60 mg / mL, preferably 0.6 to 50 mg / mL, 1 to 48 mg / mL, 1.5 to 20 mg / mL, 3 to 15 mg / mL, 5 to 13 mg / mL, 7 to 12 mg / mL, or 20 to 45 mg / mL per hydrophobic amino acid. Specifically, when only phenylalanine or phenylalanine methyl ester is used as the hydrophobic substance, the concentration of phenylalanine or phenylalanine methyl ester can be 0.2 to 20 mg / mL, preferably 0.3 to 18 mg / mL, more preferably 1 to 15 mg / mL, 3 to 13 mg / mL, 5 to 12 mg / mL, or 7 to 11 mg / mL. Specifically, when phenylalanine and leucine are used in combination as the hydrophobic substances, the concentration of phenylalanine can be set to 0.2 to 60 mg / mL, preferably 0.3 to 50 mg / mL, more preferably 1 to 48 mg / mL, 1.5 to 20 mg / mL, 3 to 15 mg / mL, 5 to 13 mg / mL, or 7 to 12 mg / mL, and the concentration of leucine can be set to 0.2 to 60 mg / mL, preferably 0.3 to 50 mg / mL, more preferably 1 to 48 mg / mL, 1.5 to 20 mg / mL, 3 to 15 mg / mL, 5 to 13 mg / mL, or 7 to 12 mg / mL. Furthermore, when a dipeptide, tripeptide, or tetrapeptide is used as the hydrophobic amino acid, the concentration of the dipeptide, tripeptide, or tetrapeptide can be 0.1 to 20 mg / mL, 0.2 to 20 mg / mL, 0.3 to 20 mg / mL, 0.3 to 10 mg / mL, 0.3 to 3 mg / mL, 0.4 to 5 mg / mL, or 0.5 to 1 mg / mL.

[0043] In the method for producing the freeze-dried extracellular vesicles or artificial cell vesicles of the present invention, the freeze-drying method is not particularly limited, but preferably comprises the following steps (a) and (b): step (a) of adding the freeze-drying composition of the present invention to the extracellular vesicles or artificial cell vesicles and freezing them at a temperature of −196 to −20° C., preferably −196 to −80° C., for 8 hours or more, preferably 12 hours or more; and step (b) of drying the cells frozen in step (a) in a vacuum, for example, under conditions of −1 MPa or less, at −50 to 35° C. for 10 hours or more, preferably 12 to 72 hours.

[0044] The above step (a) can be carried out at normal pressure. Step (a) enables a predetermined solution containing extracellular vesicles or artificial extracellular vesicles to be rapidly solidified by freezing at a low temperature.

[0045] The drying in step (b) may be performed in one step or multiple steps. For example, when the drying is performed in two steps, the drying time can be adjusted appropriately depending on the type, shape, size, and degree of drying of the extracellular vesicles or artificial cell vesicles to be dried. Examples of the primary drying method include slow drying at -40°C and -1 MPa or less for 12 to 48 hours, and rapid drying at -20°C and -1 MPa or less for 4 to 24 hours. Examples of the secondary drying method include drying at -10°C and -1 MPa or less for 4 to 12 hours. Step (b) enables the solvent contained in the predetermined solution containing the extracellular vesicles or artificial cell vesicles solidified by freezing to be sublimated and removed.

[0046] After the step (b), a step (c) of further drying at normal pressure and room temperature for 2 hours or more may be carried out.

[0047] The freeze-dried extracellular vesicles or artificial cell vesicles produced by the method for producing freeze-dried extracellular vesicles or artificial cell vesicles of the present invention can be stored for long periods at room temperature or in a refrigerator. The storage period is not particularly limited, and examples thereof include 0.5 days to 50 years, 1 day to 10 years, 1 week to 5 years, and 1 month to 1 year.

[0048] 3. Restored Extracellular Vesicles or Artificial Cell Vesicles The restored extracellular vesicles or artificial cell vesicles can be obtained by restoring freeze-dried extracellular vesicles or artificial cell vesicles prepared by the method for preparing freeze-dried extracellular vesicles or artificial cell vesicles with a solvent, and examples of such solvents include distilled water, PBS, physiological saline, and simulated body fluid. The time required for restoring with the solvent can be adjusted appropriately depending on the subsequent use and storage conditions, but can be from 1 minute to 12 hours, and the temperature for restoring can be from 0 to 40°C, 4 to 37°C, or room temperature. The restored cells can be stored at a temperature appropriate for maintaining the function or morphology of the cells, with the solvent replaced as necessary.

[0049] 4. Pharmaceutical Composition The pharmaceutical composition of the present invention may contain reconstituted extracellular vesicles or artificial cell vesicles and pharmaceutically acceptable additives, such as saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers, surfactants, buffers, preservatives, tonicity agents, bulking agents, and lubricants.

[0050] The dosage of the pharmaceutical composition of the present invention, when in a suspension state, is 5 x 10 as the number of the restored extracellular vesicles or artificial extracellular vesicles contained therein. ７ ~1 x 10 １２ pieces / time, preferably 1 x 10 8 ~1 x 10 １１ The above dose may be administered as a single dose multiple times, or the above dose may be divided into multiple doses and administered.

[0051] The method of administration of the pharmaceutical composition of the present invention is not particularly limited as long as it can provide the desired preventive or therapeutic effect on a disease, and examples thereof include intravenous injection, subcutaneous injection, and intramuscular injection.

[0052] The pharmaceutical composition can be used to prevent or treat diseases that are targets of regenerative medicine. Examples of the above diseases include vascular disorders such as cerebrovascular disease, peripheral vascular disease, coronary artery disease, diabetic vasculopathy, and lymphatic vascular disease; skin ulcers such as bedsores, arteriosclerosis obliterans, severe limb ischemia, venous insufficiency, diabetic skin ulcers, collagen diseases, and vasculitis; immune diseases such as graft-versus-host disease, graft rejection, autoimmune diseases, chronic inflammatory diseases, inflammatory pain, and neuropathic pain; eye diseases such as bullous keratopathy; spinal cord injury, traumatic cartilage defect, endometriosis, uterine fibroids; and cancers such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer.

[0053] 5. Cosmetic Composition The cosmetic composition of the present invention may contain restored extracellular vesicles or artificial cell vesicles and additives acceptable for use in cosmetics. Examples of additives that may be incorporated into the cosmetic include moisturizers, whitening agents, soaps, anti-inflammatory agents, cooling agents, UV absorbers, UV scattering agents, vitamins, plant extracts, skin astringents, cell activators, vasodilators, blood circulation promoters, skin function enhancers, disinfectants (antibacterial agents), pH adjusters, thickeners, antioxidants, chelating agents, surfactants, emulsifiers, colorants (dyes, pigments), fragrances, and preservatives. Specific examples of the cosmetic include facial cleansers, lotions, creams, emulsions, beauty serums, and packs.

[0054] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0055] [Example 1] Extraction and freeze-drying of exosomes (Extraction and freeze-drying of exosomes) For the extraction of exosomes, ExoQuick-TC ＴＭwas used. 10 mL of human mesenchymal stem cell culture supernatant was collected, 2 mL of ExoQuick reagent was added, and the mixture was thoroughly stirred and allowed to stand at 4°C for 1 day. Centrifuged at 1500g for 30 minutes to obtain exosome precipitates. The obtained exosomes and the additives listed in the comparative products 1 to 6 and the practical products 1 to 23 columns in Table 1 below were dispersed in 2 mL of phosphate buffer solution (PBS), filled into glass vials, and pre-frozen for 12 to 84 hours in liquid nitrogen or a deep freezer (-80°C). The pre-frozen glass vials were evacuated using a vacuum freeze dryer (VFD-03: AS ONE Corporation) and dried in the following order to prepare freeze-dried products, which were used in the subsequent examples. -20°C, -1 MPa, 24 hours -10°C, -1 MPa, 4 hours

[0056] [Example 2] Scanning electron microscope images of freeze-dried exosomes. Freeze-dried exosomes prepared by the method described in Example 1 using the additives listed in columns Comparative Product 3 and Example Product 19 in Table 1 below were evaluated using a scanning electron microscope (SEM). The samples were removed while maintaining their freeze-dried state, platinum was deposited, and the samples were observed using a high-resolution scanning electron microscope (FE-SEM) JSM-7500F (JEOL Ltd.). The results are shown in Figure 1.

[0057] As shown in Figure 1, when freeze-dried with the addition of trehalose (comparison product 3), the exosomes formed layers and did not maintain their shape. On the other hand, when freeze-dried with the addition of phenylalanine, leucine, and ascorbic acid ester (product 19), lattice-like fibers formed around the exosomes, with the exosomes located in the gaps, and the shape of the exosomes themselves was maintained.

[0058] [Example 3] Average Particle Size of Exosomes The particle size of exosomes lyophilized and reconstituted using the additives in columns 1-3 for Comparative Products and columns 1-3 for Example Products according to the method of Example 1 was evaluated using dynamic light scattering (DLS). Lyophilized exosomes prepared without lyophilization or using the additives in columns 3 and 19 for Example Product according to the method of Example 1 were diluted 50-fold with PBS or PBS containing 10% mannitol and reconstituted. 1 mL of each solution was loaded into a square cell for particle size measurement, and the average particle size of the exosomes was measured using a Zetasizer Pro. The results are shown in Table 1. In Table 1, the unit of average particle size is nm, and the amount added is the amount (mg) of additive per 2 mL of phosphate buffer solution before lyophilization.

[0059]

[0060] As is clear from Table 1, when exosomes were freeze-dried using the additives in Examples 1 to 23, the average particle size of the restored exosomes remained at least 50% of the average particle size of the non-freeze-dried control exosomes. On the other hand, when exosomes were freeze-dried using the additives in Comparative Examples 1 to 3, i.e., trehalose added in conventional freeze-drying methods, the average particle size was less than 1 / 300, and when exosomes were freeze-dried using the additives in Comparative Examples 4 to 6, i.e., polyethylene glycol added in conventional freeze-drying methods, the average particle size was less than 1 / 26, and the shape could not be maintained and the exosomes collapsed.

[0061] [Example 4] Number of Exosomes Exosome quantification was performed using the EXOCET Exosome Quantitation Kit (SYSTEM BIOSCIENCES) using a colorimetric quantification kit that uses AChE activity as an indicator. Lyophilized exosomes prepared without lyophilization or using the additives listed in Comparative Product 3 and Example Product 19 in the method of Example 1 were reconstituted with PBS, thoroughly mixed, and incubated at 37°C for 5 minutes to release exosome proteins. After centrifugation at 1500g for 5 minutes, the supernatant was collected and placed on ice. An EXOCET reaction solution was prepared immediately before the reaction, mixed with the sample on a 96-well plate, and the colorimetric reaction was carried out at room temperature for 20 minutes. A standard curve was prepared using an EXOCET standard solution. Absorbance at 405 nm was measured using a plate reader. The number of exosomes n was calculated from the absorbance A using the following formula (I) (standard curve): Formula (I) A = 0.0012n + 0.0176 (R^2 = 0.9976) The results for the number of exosomes are shown in Figure 2.

[0062] As can be seen from Figure 2, when the additives in column 19 of Example Product, i.e., phenylalanine, leucine, and ascorbic acid ester, were used, the number of exosomes was maintained at approximately 75% compared to when no lyophilization was performed.

[0063] [Example 5] Percentage of CD63- and CD9-positive exosomes ExoStep ＴＭ The expression of exosome markers CD63 and CD9 was evaluated by flow cytometry using an exosome flow cytometry analysis kit (Cosmo Bio). Lyophilized exosomes prepared without lyophilization or using the additives listed in columns Comparison Product 3 and Example Product 19 in Example 1 were reconstituted by adding 100 μL of phosphate buffer. 50 μL of CD63 antibody-modified capture beads was then added, mixed, and allowed to stand overnight at room temperature. The mixture was centrifuged at 2500 g for 5 minutes to obtain a precipitate. PE-labeled CD90 antibody was added and allowed to react for 60 minutes at 4°C. After washing with assay buffer, CD90-positive exosomes were detected by flow cytometry (SA3800: Sony Corporation). The percentages of CD63-positive and CD9-positive exosomes are shown in Figure 3.

[0064] As shown in Figure 3, when the additives in column 19 of Example Product, i.e., phenylalanine, leucine, and ascorbic acid ester, were used, the number of CD63- and CD9-positive exosomes was maintained at approximately 72% compared to when no lyophilization was performed.

[0065] [Example 6] Cell proliferation rate with the addition of exosomes. Freeze-dried exosomes prepared without lyophilization or using the additives in the comparative product 3 and the example product 19 columns by the method of Example 1 were suspended in phosphate buffered saline (PBS) and added to human mesenchymal stem cells (PT-5006: LONZA). The cells were incubated at 5% CO ２ The cells were cultured for 24 hours in an incubator at 37°C and 2000 human adipose tissue-derived stem cells were seeded in a 96-well plate using an MTT cell proliferation assay kit (Cosmo Bio). 2000 human adipose tissue-derived stem cells were seeded per well, suspended in 10 μL of MTT standard reagent, and incubated at 37°C in CO ２ After 4 hours, 100 μL of formazan solubilization solution was added to each well and mixed thoroughly. ２ The cells were then placed in an incubator and the absorbance at 540 nm was measured using a microplate reader. The results of the cell proliferation rate are shown in Figure 4.

[0066] As can be seen from FIG. 4, when the additives in column 19 of Example Product, namely phenylalanine, leucine and ascorbic acid ester, were used, the cell proliferation rate was maintained at approximately 70% compared to the case without freeze-drying.

[0067] Example 7: ROS-Positive Cell Rate by Exosome Addition THP-1 cells differentiated into macrophages with lipopolysaccharide (LPS) were used to evaluate the rate of ROS-positive cells induced by exosomes. For the evaluation, the ROS Detection Cell-Based Assay Kit (DHE) (Cayman Chemical) was used. Lyophilized exosomes prepared without lyophilization or using the additives listed in Comparative Product 3 and Example Product 19 in the method of Example 1 were suspended in phosphate buffered saline (PBS), added to THP-1 cells, and exposed for 4 hours. Then, the cells were treated with 100 ng / ml LPS solution and DHE solution (10 μL / Petri dish). The rate of ROS-positive cells was quantified using a flow cytometer at PE wavelength. The results for the rate of ROS-positive cells are shown in Figure 5.

[0068] As can be seen from FIG. 5, when the additives in column 19 of Example Product, namely phenylalanine, leucine, and ascorbic acid ester, were used, the proportion of ROS-positive cells was maintained at approximately the same level as in the case without freeze-drying.

[0069] [Example 8] Preparation and freeze-drying of liposomes In the above Examples 1 to 7, exosomes were freeze-dried as extracellular vesicles. Next, liposomes, which are vesicles containing phospholipids as a constituent component and containing a bilayer membrane of the phospholipids, were freeze-dried as artificial extracellular vesicles.

[0070] Liposomes containing the above-mentioned DPPC as a phospholipid were prepared by the organic solvent injection method according to the following procedure. (1) 5.3 ml of PBS (pH 7.4 at 25°C) was placed in a beaker and stirred at 25°C. (2) 1990 μL of ethanol, 38.7 mg of cholesterol, and 11.1 mg of DPPC (a lipid amphiphilic molecule) were mixed in a centrifuge tube, and the mixture was heated to 75°C, above the phase transition temperature of the lipid, to completely dissolve the cholesterol and the lipid. (3) The entire mixture was taken with a syringe and rapidly mixed into the PBS. (4) The beaker was shielded from light and stirred at 25°C for 1 hour. (5) The obtained liposome solution was placed in an LF-STB liposophst stabilizer manufactured by Avestin, and sized by passing the solution through filters with pore sizes of 1000 nm, 800 nm, 400 nm, 200 nm, 100 nm, and 50 nm in this order while applying pressure.

[0071] The obtained liquid composition containing liposomes and the additives listed in the comparative products 7 to 10 and the working products 24 to 47 columns in Table 2 below were dispersed in 2 mL of phosphate buffer, filled into glass vials, and pre-frozen for 12 to 84 hours in liquid nitrogen or a deep freezer (-80°C). The pre-frozen glass vials were evacuated in a vacuum freeze dryer (VFD-03: AS ONE Corporation), and dried in the following order to prepare freeze-dried liposomes, which were used in the following examples: -20°C, -1 MPa, 24 hours -10°C, -1 MPa, 4 hours

[0072]

[0073] [Example 9] Average particle size of liposomes Lyophilized liposomes prepared without lyophilization or using the additives listed in columns 7 to 10 for comparison and 24 to 47 for examples prepared by the method of Example 8 were reconstituted by adding PBS. The particle size of the reconstituted liposomes was then evaluated using dynamic light scattering (DLS).

[0074] Freeze-dried liposomes prepared without lyophilization or using the additives listed in columns 7 to 10 for comparison and 24 to 47 for examples using the method of Example 8 were diluted 50-fold with PBS or PBS containing 10% mannitol to reconstitute, and 1 mL of each was filled into a square cell for particle size measurement, followed by measurement of the average particle size of the liposomes using a Zetasizer Pro. The results are shown in Table 2. In Table 2, the unit of average particle size is nm. In Table 2, the amount of each additive is the amount (mg) of additive per 2 mL of phosphate buffer solution before lyophilization.

[0075] As is clear from Table 2, when liposomes were freeze-dried using the additives in Examples 24 to 47, the average particle size of the reconstituted liposomes was 100 to 300 nm, and the liposome shape was maintained. On the other hand, when liposomes were freeze-dried using the additives in Comparative Examples 7 to 10, i.e., trehalose and mannitol added in conventional freeze-drying methods, the average particle size was 900 nm or more, and the liposomes had aggregated and become larger.

[0076] [Example 10] Freeze-drying of exosomes and liposomes In Examples 1 to 7 above, exosomes were freeze-dried, but if exosomes alone are not sufficient for physiological activity, other substances such as liposomes or low-molecular-weight compounds may be added. Therefore, we investigated the freeze-drying of exosomes and liposomes as a combination of vesicles with different particle sizes and compositions.

[0077] The exosomes extracted in Example 1 and the liposomes prepared in Example 8 were dispersed in 2 mL of phosphate buffer, filled into glass vials, and pre-frozen for 12 to 84 hours in liquid nitrogen or a deep freezer (-80°C). The pre-frozen glass vials were evacuated in a vacuum freeze dryer (VFD-03: AS ONE Corporation), and dried in the following order to prepare freeze-dried products: -20°C, -1 MPa, 24 hours; -10°C, -1 MPa, 4 hours.

[0078] Next, PBS was added to the freeze-dried product prepared above to reconstitute it. The particle size of the reconstituted liposomes was then evaluated using dynamic light scattering (DLS). The results are shown in Table 3. In Table 3, the unit of average particle size is nm, and the amount of each additive is the amount (mg) of additive per 2 mL of phosphate buffer solution before freeze-drying.

[0079]

[0080] The results in Table 3 confirm that even when a combination of exosomes and liposomes was freeze-dried using a hydrophobic substance containing phenylalanine, the shape was largely maintained when restored.

Claims

1. A composition for freeze-drying extracellular vesicles or artificial cell vesicles, comprising any of the following hydrophobic substances: (a) phenylalanine or its methyl ester; (b) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; (c) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; or (d) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, and containing at least one phenylalanine as a constituent amino acid; characterized in that the composition is added to extracellular vesicles or artificial cell vesicles for use.

2. The composition for freeze-drying according to claim 1, wherein the hydrophobic substance is a combination of phenylalanine and leucine, or a dipeptide having phenylalanine as a constituent amino acid.

3. A composition for freeze-drying extracellular vesicles or artificial cell vesicles according to claim 1 or 2, characterized in that the extracellular vesicles or artificial cell vesicles are exosomes.

4. A composition for freeze-drying according to any one of claims 1 to 3, further comprising trehalose and / or ascorbic acid or derivatives thereof.

5. A method for producing freeze-dried extracellular vesicles or artificial cellular vesicles, comprising the step of adding the composition for freeze-drying described in any one of claims 1 to 4 to extracellular vesicles or artificial cellular vesicles.

6. A method for producing freeze-dried extracellular vesicles or artificial cell vesicles according to claim 5, characterized in that the composition for freeze-drying according to any one of claims 1 to 4 is added to a solution containing the extracellular vesicles or artificial cell vesicles so that the concentration of the hydrophobic substance contained in the composition for freeze-drying is 0.3 to 100 mg / mL.

7. A method for producing freeze-dried extracellular vesicles or artificial cell vesicles according to claim 5 or 6, comprising the steps of: (a) adding the composition for freeze-drying according to any one of claims 1 to 4 to extracellular vesicles or artificial cell vesicles and freezing them at -196 to -20°C for 8 hours or more; and (b) drying the extracellular vesicles or artificial cell vesicles frozen in step (a) under vacuum conditions at -50 to 35°C for 10 to 72 hours.

8. Extracellular vesicles or artificial cell vesicles produced by the method according to any one of claims 5 to 7, which are freeze-dried and reconstituted with a solvent.

9. A pharmaceutical composition comprising the restored extracellular vesicles or artificial extracellular vesicles of claim 8 and a pharmaceutically acceptable excipient.

10. A cosmetic composition comprising the restored extracellular vesicles or artificial extracellular vesicles according to claim 8.

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