Liquid composition for phospholipid membrane protection

Abstract

The present disclosure aims to provide a protective agent having excellent protective effect on phospholipid membranes. The following liquid composition for phospholipid membrane protection has excellent protective effect on phospholipid membranes, the liquid composition for phospholipid membrane protection comprising (A) a magnesium salt, and (B) an acetic acid buffer, a citric acid buffer, a carbonic acid buffer, and / or a succinic acid buffer, preferably further comprising at least any one of (C) a polyhydric alcohol, (D) a sugar and / or a sugar alcohol, (E) a polyethylene glycol, and (F) an amino acid.

Description

Technical Field

[0001] This disclosure relates to liquid compositions for phospholipid membrane protection. More specifically, this disclosure relates to liquid compositions comprising magnesium ions and specific buffer solutions for protecting various structures with phospholipid membranes, such as cells, extracellular vesicles, and liposomes, from various types of damage, including freezing damage, vibration damage, and damage caused by contact with hydrophobic materials. Background Technology

[0002] Phospholipids possess an amphiphilic structure, allowing them to arrange themselves continuously in space to form supramolecular structures. These supramolecular structures include Langmuir monolayers formed by phospholipids, phospholipid bilayers (layered structures) formed by the hydrophobic portions of the monolayers facing each other, and multilayered structures formed by the superposition of layered structures. Depending on the type of phospholipid, they sometimes form microcluster structures, hexagonal II-phase structures, and sometimes spherical vesicle structures formed by the spatial closure of phospholipid bilayers. Phospholipid membranes are major components of cell membranes and exhibit excellent biocompatibility. Therefore, they have been used in pharmaceuticals and medical devices.

[0003] In particular, the expansion into pharmaceuticals is being vigorously promoted, centered on DDS (Drug Delivery System), and many pharmaceuticals using phospholipid membranes as components have been put into practical use. For example, liposomal pharmaceuticals, which deliver drugs by encapsulating the active ingredient within the inner surface of vesicle structures, and LNPs (Lipid Nanoparticles) used in mRNA vaccines against SARS-CoV-2, also have phospholipid membranes formed on their particle surfaces. In recent years, exosomes, vesicles with phospholipid membranes released by cells, have attracted attention as materials for diagnostic or therapeutic applications. Furthermore, attempts are being actively made to use cells themselves, with phospholipid membranes as cell membranes, in cell medicine or regenerative therapies.

[0004] Cell medicine includes practical applications such as hematopoietic stem cell transplantation, CAR-T therapy, and cardiomyocyte transplantation, which have made significant contributions to society. Additionally, there are promising next-generation cell medicines such as MSCs, CAR-monocytes, and iPS cells. Cell medicine is a highly active field, currently implementing social applications and anticipating further possibilities. Furthermore, from an industry perspective, phospholipid membrane technologies closely related to exosomes or cell medicine are also attracting considerable attention. Exosomes or cell medicine are considered the next breakthrough in the pharmaceutical field. As seen with immunotherapy drugs like Opdivo and mRNA vaccines against SARS-CoV-2, these breakthroughs have dramatically expanded not only pharmaceuticals but also related markets such as research. Therefore, phospholipid membrane technologies related to them are of great industrial significance.

[0005] However, phospholipid membranes have the same composition as biological membranes, which makes them highly biocompatible and flexible, and they have the potential for a wide range of applications. On the other hand, because they have the same composition as biological membranes, it is difficult to ensure their stability.

[0006] For example, in liposome pharmaceuticals, the stability of the formulation is difficult to ensure due to the disintegration of the phospholipid membrane caused by contact. In exosomes, contact with hydrophobic materials such as plastics can cause exosome disintegration and leakage of the encapsulated active ingredients, leading to reduced therapeutic efficacy, which is a problem. In cell medicine, during cell cryopreservation, the cell membrane, i.e., the phospholipid membrane, disintegrates due to ice crystals forming inside and outside the cell, leading to cell death and loss of efficacy. In particular, the phospholipid membrane is extremely fragile when frozen, a major obstacle to the practical application of exosomes and cell medicine.

[0007] To date, various techniques have been utilized for the protection of phospholipid membranes. For example, in liposome pharmaceuticals, modifications to the phospholipid membrane surface by Peg or MPC polymers have been studied. Additionally, techniques for removing water by freeze-drying after adding protective agents, such as sugars, have been extensively investigated.

[0008] For LNPs, in addition to using Peg lipids as a protective agent, the use of protective polymers such as polysarcosine has also been studied as a new generation of protective agents (Non-Patent Literature 1). In the field of liquid biopsy, the addition of Peg or cyclodextrin encapsulated with Peg has been studied to protect blood (Patent Literature 1). Furthermore, the use of antioxidants to protect phospholipid membranes from oxidation has been reported. In the field of exosomes, various polymers have been synthesized to protect exosomes and are practically used as additives. Moreover, considering human administration, techniques for stabilizing exosomes using pharmacologically permissible materials have been studied (Non-Patent Literature 2).

[0009] In the field of cell cryopreservation, highly permeable membrane materials such as DMSO or ethylene glycol are used as cryoprotectants. In recent years, the addition of dextran or glucose has also been studied as cryoprotectants (Patent Document 2). Furthermore, the use of synthetic polymers such as trehalose and carboxylated polylysine, which are sugars, as cryoprotectants has been investigated. In particular, trehalose has been reported to have high membrane-protecting activity during cryopreservation, and its combination with other materials or delivery into cells has been studied (Patent Document 3).

[0010] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2021-153464 Patent Document 2: International Publication No. 2007 / 058308 Patent Document 3: Japanese Patent Application Publication No. 2022-79113 Non-patent literature Non-patent literature 1: Pharmaceutics, 2023, 15, 2068 Non-patent literature 2: Adv. Healthcare Mater. 2022, 11, 2100538 1-16 Summary of the Invention

[0011] The problem that the invention aims to solve Given the importance of phospholipid membrane structures in the pharmaceutical field, there is a need for protective agents with superior protective effects. Therefore, the purpose of this disclosure is to provide a protective agent with excellent protective effects against phospholipid membranes.

[0012] Methods for solving problems The inventors of this application focused on a recent research report using radiation light, which indicated that magnesium ions in aqueous solutions are concentrated on the surface of phospholipid membranes (Appl. Phys. Lett. 116, 133701 (2020)). Further research revealed that magnesium ions in aqueous solutions possess protective activity against phospholipid membranes, and that by combining specific buffers with phospholipid membrane-protective activity, excellent protective effects against phospholipid membranes can be achieved. This disclosure was completed through further iterative research based on these findings.

[0013] That is, this disclosure provides the invention of the manner disclosed below.

[0014] Item 1. A liquid composition for phospholipid membrane protection, comprising: (A) a magnesium salt; and (B) an acetate buffer, a citrate buffer, a carbonate buffer, and / or a succinate buffer.

[0015] Item 2. The liquid composition as described in Item 1, wherein the aforementioned component (A) is selected from the group consisting of magnesium chloride, magnesium sulfate and magnesium gluconate.

[0016] Item 3. The liquid composition as described in Item 1 or 2, further comprising (C) a polyol.

[0017] Item 4. The liquid composition as described in Item 3, wherein the aforementioned component (C) is glycerin.

[0018] Item 5. The liquid composition of any one of items 1 to 3, further comprising (D) sugars and / or sugar alcohols.

[0019] Item 6. The liquid composition as described in Item 5, wherein the aforementioned component (D) is mannitol.

[0020] Item 7. The liquid composition of any one of items 1 to 6, further comprising (E) polyethylene glycol.

[0021] Item 8. The liquid composition of any one of items 1 to 7, further comprising (C) glycerol, (D) mannitol, and (E) polyethylene glycol.

[0022] Item 9. The liquid composition as described in Item 7 or 8, wherein the average molecular weight of the aforementioned (E) component is 270 or more.

[0023] Item 10. The liquid composition as described in Item 7 or 8, wherein the aforementioned component (E) comprises a plurality of polyethylene glycols having an average molecular weight difference of 2000 or more.

[0024] Item 11. The liquid composition as described in any one of items 7 to 10, wherein the aforementioned component (B) is a mixture of acetate buffer and citrate buffer, and the content of the aforementioned component (E) is 15 mg / mL or more.

[0025] Item 12. The liquid composition of any one of items 1 to 11, wherein the aforementioned component (B) comprises: acetate buffer and citrate buffer; or, acetate buffer, citrate buffer or succinate buffer.

[0026] Item 13. The liquid composition of any one of items 1 to 12, further comprising (F) an amino acid class selected from the group consisting of amino acids, dipeptides and their salts.

[0027] Item 14. The liquid composition as described in Item 13, wherein the aforementioned component (F) is selected from the group consisting of arginine, arginine dipeptide, glutamic acid, proline and their salts.

[0028] Item 15. The liquid composition as described in any one of items 1 to 14, wherein the content of the aforementioned component (A) is 0.1 mg / mL or more.

[0029] Item 16. The liquid composition as described in any one of items 1 to 15, wherein the content of the aforementioned component (A) is 0.1 mM or more, calculated as magnesium ion concentration.

[0030] Item 17. The liquid composition as described in any one of items 1 to 16, wherein the content of the aforementioned component (B) is 5 mM or more in terms of buffer concentration.

[0031] Item 18. The liquid composition as described in any one of items 3 to 17, wherein the content of the aforementioned component (C) is 0.01 mg / mL or more.

[0032] Item 19. The liquid composition as described in any one of items 5 to 18, wherein the content of the aforementioned component (D) is 0.01 mg / mL or more.

[0033] Item 20. The liquid composition as described in any one of items 7 to 19, wherein the content of the aforementioned component (E) is 0.01 mg / mL or more.

[0034] Item 21. The liquid composition as described in any one of items 13 to 20, wherein the content of the aforementioned component (F) is 1 mg / mL or more.

[0035] Item 22. The liquid composition as described in any one of items 1 to 21, having an osmotic pressure of 200 to 1220 mOsm / kg.

[0036] Item 23. The liquid composition as described in any one of items 1 to 22, used for protection against freezing damage.

[0037] Item 24. The liquid composition as described in Item 23, wherein the aforementioned freezing damage is caused by repeated freezing and thawing.

[0038] Item 25. The liquid composition as described in any one of items 1 to 24, for protection against vibration damage.

[0039] Item 26. The liquid composition of any one of items 1 to 25, for protection against damage caused by contact with a hydrophobic material.

[0040] Item 27. The liquid composition as described in any one of items 1 to 26, used for protection against heat damage.

[0041] Item 28. The liquid composition of any one of items 1 to 27, for the preservation of cells, extracellular vesicles and / or liposomes.

[0042] Item 29. A pharmaceutical composition comprising cells, extracellular vesicles and / or liposomes, and a liquid composition as described in any one of items 1 to 28.

[0043] Item 30. The pharmaceutical composition as described in Item 29, which is contained in a container of a hydrophobic material.

[0044] Item 31. The pharmaceutical composition as described in Item 29 or 30, wherein it is in a frozen state.

[0045] It should be noted that although the mechanism by which the liquid composition of the present invention protects the phospholipid film is not clear, it can be speculated as follows.

[0046] As mentioned above, studies using radiation have reported that magnesium ions in aqueous solutions are concentrated near phosphate-rich regions on the surface of phospholipid membranes. Furthermore, in gene delivery studies, magnesium ions, as divalent cations, are known to cause negatively charged nucleic acids to aggregate through electrostatic cross-linking. Based on these facts, it is believed that the concentration of magnesium ions on the phospholipid membrane surface, resulting in the strong bonding of adjacent phospholipid membranes, contributes to the overall stabilization and protection of the phospholipid membrane. Furthermore, it is believed that by combining substances that influence magnesium ions with other materials that protect the phospholipid membrane, a multi-faceted stabilizing effect can be achieved in multiple regions of the phospholipid membrane, thereby enhancing its protective effect. It should be noted that the present invention is not limited to this conjecture.

[0047] Invention Effects According to this disclosure, a protective agent with excellent protective effect on phospholipid films can be provided. Attached Figure Description

[0048] [Figure 1 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experimental Example 1 are shown.

[0049] [ Figure 2 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 2 are shown.

[0050] [ Figure 3 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 3 are shown.

[0051] [ Figure 4A The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 4 are shown.

[0052] [ Figure 4B The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 4 are shown.

[0053] [ Figure 5 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experimental Example 5 are shown.

[0054] [ Figure 6 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 6 are shown.

[0055] [ Figure 7 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 7 are shown.

[0056] [ Figure 8 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment Example 8 are shown.

[0057] [ Figure 9 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment 15 are shown.

[0058] [ Figure 10 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment 16 are shown.

[0059] [ Figure 11 The results of the evaluation of phospholipid membrane protection of fluorescent liposomes against cryodamage obtained in Experiment 16 are shown.

[0060] [ Figure 12 The results of the evaluation of phospholipid membrane protection of exosomes damaged by contact with hydrophobic materials are shown in Test Example 19.

[0061] [ Figure 13 The results of the evaluation of phospholipid membrane protection of exosomes damaged by contact with hydrophobic materials are shown in Test Example 19.

[0062] [ Figure 14 The results of the evaluation of phospholipid membrane protection of exosomes with cryodamage obtained in Experiment Example 20 are shown.

[0063] [ Figure 15 The results of the evaluation of phospholipid membrane protection of exosomes against vibration damage obtained in Experiment Example 21 are shown.

[0064] [ Figure 16A The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 22 are shown.

[0065] [ Figure 16B The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 22 are shown.

[0066] [ Figure 16C The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 22 are shown.

[0067] [ Figure 17 The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 23 are shown.

[0068] [ Figure 18A The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 24 are shown.

[0069] [ Figure 18B The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 24 are shown.

[0070] [ Figure 19 The results of the evaluation of phospholipid membrane protection for cells with cryogenic damage obtained in Experiment 31 are shown. Detailed Implementation

[0071] 1. Liquid composition for phospholipid film protection The liquid composition for phospholipid membrane protection disclosed herein is characterized by comprising (A) a magnesium salt (hereinafter also referred to as "(A) component"), and (B) an acetate buffer, citrate buffer, carbonate buffer, and / or succinate buffer (hereinafter also referred to as "(B) component" or "specified buffer"). In a preferred embodiment, the liquid composition for phospholipid membrane protection disclosed herein may further comprise at least one of (C) a polyol (hereinafter also referred to as "(C) component"), (D) a sugar and / or sugar alcohol (hereinafter also referred to as "(D) component"), (E) polyethylene glycol (hereinafter also referred to as "(E) component"), and (F) an amino acid selected from the group consisting of amino acids, dipeptides, and their salts (hereinafter also referred to as "(F) component"). The liquid composition for phospholipid membrane protection disclosed herein is described in detail below.

[0072] 1-1. (A) Magnesium salts The magnesium salt, as component (A), is an effective component of the liquid composition for phospholipid film protection disclosed herein. That is, component (A) alone also has the function of protecting the phospholipid film.

[0073] As for component (A), there are no particular limitations as long as it is water-soluble and pharmaceutically permissible; examples include magnesium chloride, magnesium sulfate, and magnesium gluconate. These magnesium salts can be used alone or in combination of two or more.

[0074] As a source of supply for component (A), anhydrous magnesium salts or hydrates of magnesium salts (e.g., magnesium chloride hexahydrate, magnesium sulfate heptahydrate) can be used.

[0075] The content of component (A) in the liquid composition for phospholipid film protection disclosed herein is not particularly limited and may be appropriately set according to the desired degree of phospholipid film protection effect.

[0076] As for the specific content of component (A), for example, 0.1 mg / mL or more can be mentioned. From the viewpoint of further improving the protective effect of phospholipid membrane, it is preferable to have a content of 0.5 mg / mL or more, 0.7 mg / mL or more, 0.9 mg / mL or more, 1 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, or 2.5 mg / mL or more. More preferably, it is preferable to have a content of 9 mg / mL or more, 10 mg / mL or more, 20 mg / mL or more, 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, or 100 mg / mL or more.

[0077] Regarding the range of the content of component (A) in the liquid composition for phospholipid membrane protection disclosed herein, there is no particular upper limit, which can be appropriately determined by those skilled in the art considering osmotic pressure, etc., for example, 300 mg / mL or less, 250 mg / mL or less, 210 mg / mL or less, 150 mg / mL or less, 110 mg / mL or less, 70 mg / mL or less, 55 mg / mL or less, 35 mg / mL or less, 25 mg / mL or less, or 15 mg / mL or less. From the viewpoint of further improving the phospholipid membrane protection effect brought about by the combination with the preferred buffer (specifically, acetate buffer, citrate buffer, succinate buffer), it is preferable to have a content of 8 mg / mL or less, 6 mg / mL or less, 4 mg / mL or less, 3 mg / mL or less, 2 mg / mL or less, 1.5 mg / mL or less, 1 mg / mL or less, 0.8 mg / mL or less, 0.6 mg / mL or less, or 0.4 mg / mL.

[0078] As for the specific range of the content of component (A) in the liquid composition for phospholipid film protection of this disclosure, the above-mentioned upper and lower limits can be arbitrarily combined, for example, 0.1~300mg / mL, 0.1~250mg / mL, 0.5~210mg / mL, 0.5~150mg / mL, 0.7~110mg / mL, 0.7~70mg / mL, 0.9~55mg / mL, 0.9~35mg / mL, 1~25mg / mL, 1~15mg / mL, 1~8mg / mL, 1.5~6mg / mL, 2~4mg / mL, 2.5~3mg / mL, etc.

[0079] Furthermore, the content of component (A) in the liquid composition for phospholipid film protection disclosed herein, in terms of magnesium ion concentration, can be, for example, 0.1 mM or more, 0.2 mM or more, 0.3 mM or more, or 0.4 mM or more. From the viewpoint of further improving the phospholipid film protection effect, it is preferable to have a content of 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 6 mM or more, or 8 mM or more. More preferably, it can have a content of 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 100 mM or more, 200 mM or more, 300 mM or more, 400 mM or more, 500 mM or more, 600 mM or more, 700 mM or more, 800 mM or more, or 900 mM or more.

[0080] The upper limit of the range of magnesium ion concentration in the liquid composition for phospholipid membrane protection disclosed herein can also be appropriately determined by those skilled in the art, taking into account factors such as osmotic pressure. Examples of upper limits include 1500 mM or less, 1300 mM or less, 1000 mM or less, 710 mM or less, 510 mM or less, 310 mM or less, 210 mM or less, 110 mM or less, 75 mM or less, 55 mM or less, or 35 mM or less. From the viewpoint of further improving the phospholipid membrane protection effect brought about by the combination with the preferred buffer (specifically, acetate buffer or citrate buffer), examples of upper limits include 22 mM or less, 15 mM or less, 12 mM or less, 7 mM or less, 5 mM or less, 3.5 mM or less, 2.5 mM or less, 1.5 mM or less, or 0.8 mM or less.

[0081] As for the specific range of magnesium ion concentration in the liquid composition for phospholipid film protection in this disclosure, the above-mentioned upper and lower limits can be arbitrarily combined. For example, 0.1~1500mM, 0.2~1300mM, 0.3~1000mM, 0.4~710mM, 1~510mM, 2~310mM, 2~210mM, 3~110mM, 3~75mM, 4~55mM, 435~mM, 6~22mM, 6~15mM, 8~12mM, etc.

[0082] 1-2. (B) Specified buffer solution The specified buffer solution, as component (B), also has the function of protecting the phospholipid membrane on its own, but it exerts an excellent phospholipid membrane protection effect when combined with component (A).

[0083] (B) Specifically, the components are acetate buffer, citrate buffer, carbonate buffer and / or succinate buffer (hereinafter also referred to as "specified buffers"). These buffers may be used alone or in combination of two or more.

[0084] In a preferred embodiment, component (B) may include acetate buffer, citrate buffer, and / or succinate buffer. In a more preferred embodiment, component (B) may include a combination of acetate buffer and citrate buffer. In this case, by incorporating component (E) described later (e.g., incorporating component (E) at a concentration of 15 mg / mL or higher), the phospholipid membrane protection effect can be further improved. In a further preferred embodiment, component (B) may include a combination of acetate buffer, citrate buffer, and succinate buffer.

[0085] Acetic acid buffer, citrate buffer, carbonate buffer, and succinate buffer are aqueous solutions of acetic acid, citric acid, carbonic acid, and succinate (hereinafter also referred to as "the specified acids") and their conjugate bases (hereinafter referred to as "the conjugate bases of the specified acids"), respectively.

[0086] In order to incorporate the desired component (B) into the phospholipid membrane protection liquid composition of this disclosure, at least one buffer selected from the specified buffer solutions may be incorporated, or at least one acid selected from the specified acids and at least one salt of the conjugate base selected from the specified acids may be dissolved in the presence of water, or at least one acid selected from the specified acids and / or at least one salt of the conjugate base selected from the specified acids may be dissolved in at least one buffer selected from the specified buffer solutions.

[0087] The content of component (B) in the liquid composition for phospholipid film protection disclosed herein is not particularly limited and may be appropriately set according to the desired degree of phospholipid film protection effect.

[0088] The content of the specific component (B), in terms of the total concentration of the buffer that constitutes the buffer solution, can be, for example, 5 mM or more. From the viewpoint of further improving the protective effect of the phospholipid membrane, it is preferable to have 8 mM or more, 10 mM or more, or 13 mM or more, and more preferably 16 mM or more, 18 mM or more, 20 mM or more, 25 mM or more, 30 mM or more, 35 mM or more, 40 mM or more, 45 mM or more, or 48 mM or more.

[0089] Regarding the range of the content of component (B) in the liquid composition for phospholipid film protection of this disclosure, there is no particular upper limit. Those skilled in the art can determine it appropriately by taking into account factors such as osmotic pressure. For example, based on the total concentration of the buffer, examples include 80 mM or less, 70 mM or less, 65 mM or less, 60 mM or less, 55 mM or less, 50 mM or less, 47 mM or less, 42 mM or less, 37 mM or less, 32 mM or less, 27 mM or less, 22 mM or less, or 17 mM or less.

[0090] Regarding the specific range of the content of component (B) in the liquid composition for phospholipid film protection of this disclosure, the upper and lower limits of the total concentration of the buffer can be arbitrarily combined, for example, 8~80mM, 10~70mM, 13~65mM, 16~65mM, 18~60mM, 20~60mM, etc.

[0091] 1-3. (C) Polyols The liquid composition for phospholipid film protection disclosed herein may contain a polyol as component (C) for the purpose of improving the phospholipid film protection effect.

[0092] Regarding the polyols used as component (C), there are no particular limitations as long as they are pharmaceutically permissible. Examples include diols such as propylene glycol, 1,3-butanediol, ethylene glycol, isopentyl glycol, diethylene glycol, and dipropylene glycol; and terols such as glycerin. It should be noted that the sugars and sugar alcohols in component (D) and the polyethylene glycol in component (E) are distinct from component (C). These polyols can be used individually or in combination of two or more.

[0093] Among these polyols, from the viewpoint of further improving the protective effect of phospholipid membranes, triols are preferred, and glycerols are more preferred.

[0094] The content of component (C) in the liquid composition for phospholipid film protection disclosed herein is not particularly limited and may be appropriately set according to the desired degree of phospholipid film protection effect.

[0095] As for the specific content of component (C), for example, 0.01 mg / mL or more can be mentioned. From the viewpoint of further improving the protective effect of phospholipid membrane, 0.1 mg / mL or more is preferred, 1 mg / mL or more is more preferred, 8 mg / mL or more or 10 mg / mL or more is even more preferred, 13 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, 25 mg / mL or more, 30 mg / mL or more, 35 mg / mL or more, 40 mg / mL or more, 45 mg / mL or more, 50 mg / mL or more, 55 mg / mL or more, or 60 mg / mL or more is even more preferred.

[0096] Regarding the range of the content of component (C) in the liquid composition for phospholipid membrane protection of this disclosure, there is no particular upper limit. Those skilled in the art can determine it appropriately considering factors such as osmotic pressure. For example, a content of 150 mg / mL or less is possible. From the viewpoint of further improving the phospholipid membrane protection effect, a content of 100 mg / mL or less or 80 mg / mL or less is preferred, 70 mg / mL or less is more preferred, and 60 mg / mL or less or 55 mg / mL or less is even more preferred. Furthermore, since the liquid composition for phospholipid membrane protection of this disclosure exhibits excellent phospholipid membrane protection effect, the upper limit of the content range of component (C) can also be lower, for example, it can be 40 mg / mL or less, 30 mg / mL or less, 20 mg / mL or less, or 18 mg / mL or less. For the phospholipid film protection liquid composition of this disclosure, from the viewpoint of improving the protective effect against heat damage, the upper limit of the range of the content of component (C) is preferably 40 mg / mL or less, more preferably 30 mg / mL or less, and even more preferably 20 mg / mL or less.

[0097] The specific range of the content of component (C) in the liquid composition for phospholipid film protection disclosed herein can be any combination of the above-mentioned upper and lower limits, for example, 0.01~150 mg / mL, 0.1~100 mg / mL, 1~80 mg / mL, 1~70 mg / mL, 8~60 mg / mL, 8~55 mg / mL, 8~40 mg / mL, 10~30 mg / mL, 10~20 mg / mL, etc.

[0098] 1-4. (D) Sugars and / or sugar alcohols The liquid composition for phospholipid film protection disclosed herein may contain sugars and / or sugar alcohols as component (D) for the purpose of improving the phospholipid film protection effect.

[0099] As for sugars, there are no particular limitations as long as they are pharmaceutically permissible. Examples include monosaccharides and disaccharides. Examples of monosaccharides include glucose, fructose, galactose, and mannose. Examples of disaccharides include sucrose, trehalose, maltose, and lactose. These sugars can be used alone or in combination of two or more.

[0100] As sugar alcohols, there are no particular limitations as long as they are pharmaceutically permissible. Examples include sorbitol, xylitol, isomaltose, erythritol, maltitol, mannitol, lactitol, palaginose, and reduced palaginose. These sugar alcohols can be used alone or in combination of two or more.

[0101] Among these (D) components, from the viewpoint of further improving the protective effect of phospholipid membranes, sugar alcohols are preferred, and mannitol is more preferred.

[0102] The content of component (D) in the liquid composition for phospholipid film protection disclosed herein is not particularly limited and may be appropriately set according to the desired degree of phospholipid film protection effect.

[0103] As for the specific content of component (D), for example, 0.01 mg / mL or more can be mentioned. From the viewpoint of further improving the protective effect of phospholipid membrane, 0.1 mg / mL or more is preferred, 0.8 mg / mL or more or 1 mg / mL or more is more preferred, 8 mg / mL or more or 10 mg / mL or more is even more preferred, 13 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, 25 mg / mL or more, 30 mg / mL or more, 35 mg / mL or more, 40 mg / mL or more, 45 mg / mL or more, 50 mg / mL or more, 55 mg / mL or more, or 60 mg / mL or more is even more preferred.

[0104] Regarding the range of the content of component (D) in the liquid composition for phospholipid film protection disclosed herein, there is no particular upper limit, which can be appropriately determined by those skilled in the art taking into account osmotic pressure, etc., for example, 100 mg / mL or less, 70 mg / mL or less, 60 mg / mL or less, 55 mg / mL or less, 50 mg / mL or less, 45 mg / mL or less, 40 mg / mL or less, 35 mg / mL or less, 30 mg / mL or less, or 20 mg / mL or less.

[0105] The specific range of the content of component (D) in the liquid composition for phospholipid film protection disclosed herein can be any combination of the above-mentioned upper and lower limits, for example, 0.01~100 mg / mL, 0.1~70 mg / mL, 0.8~60 mg / mL, 1~55 mg / mL, 8~50 mg / mL, 10~45 mg / mL, 13~40 mg / mL, 15~35 mg / mL, etc.

[0106] 1-5. (E) Polyethylene glycol The liquid composition for phospholipid film protection disclosed herein may include polyethylene glycol as component (E) for the purpose of improving the phospholipid film protection effect.

[0107] There is no particular limitation on the average molecular weight of polyethylene glycol. For example, it can be 270 or more. From the viewpoint of further improving the protective effect of phospholipid film, it is preferable to have a molecular weight of 350 or more, 400 or more, or 500 or more. More preferably, it can have a molecular weight of 550 or more, 600 or more, 800 or more, 1000 or more, 1500 or more, 2000 or more, or 2500 or more. Even more preferably, it can have a molecular weight of 3000 or more, or 3400 or more.

[0108] There is no particular upper limit to the range of average molecular weight of polyethylene glycol. Examples include below 30,000, below 25,000, below 20,000, below 15,000, below 10,000, below 8,000, below 6,000, below 4,000, below 2,000, below 1,000, below 900, below 700, below 500, or below 450.

[0109] The specific range of the average molecular weight of polyethylene glycol can be any combination of the upper and lower limits mentioned above. For example, 270~30000, 270~25000, 270~20000, 270~15000, 350~10000, 350~8000, 350~6000, and 350~4000 can be listed.

[0110] It should be noted that the method for determining the average molecular weight of polyethylene glycol (PEG) should follow the test method for PEG 200 (p1378) listed in the Pharmaceutical Industry Standard for Quasi-Pharmaceutical Raw Materials 2006 (Pharmaceutical Daily). Specifically, 42g of phthalic anhydride is added to a 1L light-proof, stoppered bottle containing 300mL of freshly distilled pyridine. The mixture is vigorously shaken to dissolve, and then left to stand for at least 16 hours. 25mL of this liquid is accurately measured and transferred to a pressure-resistant, stoppered bottle of approximately 200mL. Approximately 0.8g of PEG is accurately measured and added to the bottle. The bottle is sealed tightly and placed in a water bath heated to 98℃±2℃. After maintaining the temperature at 98℃±2℃ for 30 minutes, the bottle is removed from the water bath and left to air until it reaches room temperature. Next, 50mL of 0.5N sodium hydroxide solution is accurately added, and the liquid is titrated using the 0.5N sodium hydroxide solution. The indicator used here is 5 drops of phenolphthalein-pyridine solution (1→100). The titration endpoint is set when the liquid turns a pale pink color for 15 seconds. A blank test is performed using the same method.

[0111] Substitute the obtained value into the following formula to calculate the average molecular weight.

[0112] Average molecular weight = {amount of sample (g) × 4000} / (ab) Where, a: the amount of 0.5N sodium hydroxide solution consumed (mL) in the blank test. b: The amount of 0.5N sodium hydroxide solution consumed (mL) during the test of the sample. Specific examples of polyethylene glycol include PEG200 (average molecular weight 190-210), PEG300 (average molecular weight 285-315), PEG400 (average molecular weight 380-420), PEG1000 (average molecular weight 950-1050), PEG2000 (average molecular weight 1800-2200), PEG4000 (average molecular weight 2700-3400), PEG6000 (average molecular weight 7400-9000), and PEG20000 (average molecular weight 18000-25000).

[0113] These polyethylene glycols can be used alone or in combination of two or more.

[0114] In a preferred embodiment, as component (E), a combination of various polyethylene glycols with different average molecular weights can be used. Examples of the difference in average molecular weight of these various polyethylene glycols include 2000 or more, preferably 2100-5000 or 2100-4000, and more preferably 2200-3500.

[0115] The content of component (E) in the liquid composition for phospholipid film protection disclosed herein is not particularly limited and may be suitably set according to the desired degree of phospholipid film protection effect.

[0116] As for the specific content of component (E), for example, 0.01 mg / mL or more can be mentioned. From the viewpoint of further improving the protective effect of phospholipid membrane, 0.1 mg / mL or more is preferred, 0.8 mg / mL or more, 1 mg / mL or more, or 1.3 mg / mL or more can be mentioned more, and 1.5 mg / mL or more, 3 mg / mL or more, 5 mg / mL or more, 7 mg / mL or more, 10 mg / mL or more, or 15 mg / mL or more can be mentioned even more.

[0117] Regarding the range of the content of component (E) in the liquid composition for phospholipid film protection disclosed herein, there is no particular upper limit, which can be appropriately determined by those skilled in the art taking into account osmotic pressure, etc., for example, 200 mg / mL or less, 150 mg / mL or less, 100 mg / mL or less, 70 mg / mL or less, 50 mg / mL or less, 30 mg / mL or less, 20 mg / mL or less, 15 mg / mL or less, 10 mg / mL or less, 6 mg / mL or less, 4 mg / mL or less, or 2 mg / mL or less.

[0118] The specific range of the content of component (E) in the liquid composition for phospholipid film protection disclosed herein can be any combination of the above-mentioned upper and lower limits, for example, 0.01~200 mg / mL, 0.1~150 mg / mL, 0.8~100 mg / mL, 1~100 mg / mL, 1.3~70 mg / mL, 1.5~70 mg / mL, 3~50 mg / mL, 5~50 mg / mL, 7~50 mg / mL, etc.

[0119] 1-6. (F) Amino acids The liquid composition for phospholipid film protection disclosed herein may contain amino acids as component (F) for the purpose of improving the phospholipid film protection effect.

[0120] Amino acids are selected from groups consisting of amino acids, dipeptides, and their salts.

[0121] Examples of amino acids include asparagine, aspartic acid, alanine, arginine, cysteine, glutamine, glutamic acid, glycine, proline, serine, histidine, lysine, and threonine. As a dipeptide, it can be a compound composed of two of the above amino acids linked by a peptide bond. As a salt, there are no particular limitations as long as it is pharmaceutically permissible; examples include alkali metal salts such as potassium and sodium salts, alkaline earth metal salts such as calcium salts, ammonium salts, and triethanolamine salts.

[0122] These amino acids can be used alone or in combination of two or more.

[0123] Among these amino acids, preferred examples include arginine, histidine, glycine, aspartic acid, glutamic acid, proline, and threonine; dipeptides comprising at least one of them (preferably formed from two of them); and their salts. More preferred examples include arginine, glycine, glutamic acid, proline, dipeptides comprising at least one of them, and their salts. Even more preferred examples include arginine, arginine dipeptide (preferably arginine glutamate formed from arginine and glutamic acid), glutamic acid, proline, and their salts.

[0124] The content of component (F) in the liquid composition for phospholipid film protection disclosed herein is not particularly limited and may be appropriately set according to the desired degree of phospholipid film protection effect.

[0125] The content of specific component (F) can be, for example, 1 mg / mL or more or 5 mg / mL or more. From the viewpoint of further improving the protective effect of phospholipid membrane, it is preferable to have 8 mg / mL or more or 10 mg / mL or more, and more preferably 18 mg / mL or more or 20 mg / mL or more.

[0126] Regarding the range of the content of component (F) in the liquid composition for phospholipid membrane protection disclosed herein, there is no particular upper limit, which can be appropriately determined by those skilled in the art taking into account osmotic pressure, etc., for example, 60 mg / mL or less, 55 mg / mL or less, 50 mg / mL or less, 45 mg / mL or less, 40 mg / mL or less, 35 mg / mL or less, 30 mg / mL or less, 25 mg / mL or less, 22 mg / mL or less, 20 mg / mL or less, 18 mg / mL or less, 16 mg / mL or less, 14 mg / mL or less, 12 mg / mL or less, or 10 mg / mL or less.

[0127] The specific range of the content of component (F) in the liquid composition for phospholipid film protection disclosed herein can be any combination of the above-mentioned upper and lower limits, for example, 1~60 mg / mL, 5~60 mg / mL, 8~55 mg / mL, 10~50 mg / mL, etc.

[0128] 1-7. Other ingredients The liquid composition disclosed herein may contain other components, or may not contain other components, in addition to components (A) and (B) above, or further, as needed, components (C), (D), (E) and / or (F).

[0129] Examples of other components, whether or not they are included, include isotonic agents and pH adjusters. When the liquid composition of this disclosure contains other components, one of these components may be used alone, or two or more may be used in combination. Examples of isotonic agents include sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, sodium carbonate, phosphoric acid, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, and potassium dihydrogen phosphate. One isotonic agent may be used alone, or two or more may be used in combination. Examples of pH adjusters include ammonia, hydrochloric acid, dilute hydrochloric acid, succinic acid, disodium succinate, acetic acid, glacial acetic acid, tartaric acid, disodium tartrate, ascorbic acid, sodium ascorbate, potassium hydroxide, sodium hydroxide, tromethamine hydrochloride, carbon dioxide, lactic acid, sodium lactate, sulfuric acid, and dilute sulfuric acid. One pH adjuster may be used alone, or two or more may be used in combination.

[0130] It should be noted that, among the preferred components included in the liquid composition of this disclosure that are not included as other components besides those mentioned above, are dimethyl sulfoxide (DMSO), polypeptides (such as poly-L-lysine (PLL)), proteins (such as ovalbumin, serum albumin, etc.), dextran, culture medium, etc.

[0131] 1-8. pH There is no particular limitation on the pH (25°C) of the liquid composition of this disclosure, which can be suitably determined by those skilled in the art. Due to its excellent protective effect on the phospholipid film, the liquid composition of this disclosure can exhibit excellent phospholipid film protection over a wide pH range. Specific pH values ​​include, for example, 4.5 to 10.5. From the viewpoint of further improving the phospholipid film protection effect, 5.0 to 10.0 is preferred, 5.0 to 9.0 is more preferred, 5.0 to 8.5 is even more preferred, and 5.0 to 8.3 is even more preferred. Furthermore, for the pH range of 5.0 to 8.3, the upper or lower limit can be set to any one of 5.5, 6.0, 6.5, 7.0, 7.5, 7.7, and 8.0.

[0132] 1-9. Osmotic pressure The liquid composition disclosed herein can be any of hypotonic, isotonic, or hypertonic. There is no particular limitation on the specific range of osmotic pressure values ​​(mOsm / kg) of the liquid composition disclosed herein; it can be any two points selected from 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 950, 1000, 1100, 1220, or 1220 mOsm / kg as the upper and lower limits. More specific ranges for the osmotic pressure values ​​(mOsm / kg) of the liquid compositions disclosed herein include, for example, 200 to 1220 mOsm / kg, preferably 400 to 840 mOsm / kg, and more preferably 500 to 800 mOsm / kg. It should be noted that the osmotic pressure is determined using the freezing point depression method. This osmotic pressure determination can be performed using, for example, an osmotic pressure analyzer such as the OSMOMAT3000.

[0133] 1-10. Uses The liquid composition disclosed herein is used for the protection of phospholipid membranes. Specifically, the liquid composition of this disclosure can be used to protect phospholipid membrane structures from potential damage to the phospholipid membrane, thereby improving their preservation. The types of damage are not particularly limited, but examples include freeze damage (especially damage caused by repeated freeze-thaw cycles of 1 to 2 to 5 times, preferably 3 to 5 times, more preferably 4 to 5 times), damage caused by contact with hydrophobic materials (polyolefins (polyethylene, polypropylene, etc.), polyvinyl chloride, polyethylene terephthalate, polycarbonate, polystyrene, etc.), vibration damage, and thermal damage.

[0134] Examples of phospholipid membrane structures include membrane particles containing phospholipids, such as cells (somatic cells, germ cells, stem cells, etc.), extracellular vesicles (exosomes, etc.), liposomes, and blood cells (red blood cells, white blood cells). Preferred examples of these phospholipid membrane structures include those used as active ingredients or carriers (specifically, carriers encapsulating active ingredients) in therapeutic or diagnostic pharmaceuticals.

[0135] 2. Pharmaceutical compositions The pharmaceutical composition disclosed herein includes a phospholipid membrane structure and the phospholipid membrane protection liquid composition described in “1. Liquid composition for phospholipid membrane protection” above.

[0136] Regarding phospholipid membrane structures, see “1-10. Applications” above.

[0137] The pharmaceutical composition disclosed herein is highly effective in protecting the phospholipid membrane from damage caused by contact with hydrophobic materials. Therefore, in a preferred embodiment, it can be contained in a container made of hydrophobic material. Hydrophobic materials are as described in "1-10. Uses" above. Specific examples of containers include tubes, syringes (pre-filled syringes), centrifuge tubes, cryovials, culture flasks, culture containers and other containers, bags (non-freezing bags, frozen bags), petri dishes, culture dishes, well plates, and other common containers. In addition to the common containers described above, hollow structures such as tubing can also be cited as specific examples of containers.

[0138] The pharmaceutical composition of this disclosure is highly effective in protecting the phospholipid membrane from freeze-thaw damage; therefore, in a preferred embodiment, it can be in a frozen state. In this case, it can also be in a frozen state where it is contained in a container made of the aforementioned hydrophobic material.

[0139] Example The following embodiments are shown to illustrate the invention in more detail. However, the various configurations and combinations thereof in each embodiment are merely examples, and appropriate additions, omissions, substitutions, and other changes to the configuration can be made without departing from the spirit of the invention. This disclosure is not limited to the embodiments, but only to the scope of the claims.

[0140] [A] Use reagents Sterilized water (manufactured by Nacalai Tesque) • D-PBS (Duborough phosphate-buffered saline: manufactured by Nacalai Tesque), hereinafter also abbreviated as PBS • Physiological saline solution (Note by Otsuka Naoya: manufactured by Otsuka Pharmaceutical Co., Ltd.) • TE buffer pH 7.5 × 20 (Promega) • Sephadex G100 (manufactured by Cytiva) • 0.1 mol / L phosphate buffer, pH 7.4 (Nacalai Tesque) • Anhydrous sodium carbonate (manufactured by FUJIFILM Wako Pure Chemical Corporation) • Sodium acetate (for molecular biology) (manufactured by Nacalai Tesque) • 1 mol / L acetic acid (manufactured by Nacalai Tesque) • Trisodium citrate dihydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) Citric acid monohydrate (manufactured by Nacalai Tesque) • Sodium succinate hexahydrate (manufactured by Nacalai Tesque) • Pyranine (made by Alfa Aesar) •DPX (p-xylene-bis-pyridinium bromide: manufactured by Invitrogen) • Biochemical use of EYPC (3-sn-phosphatidylcholine, derived from egg yolk: manufactured by FUJIFILM Wako Pure Chemical Corporation) MES (2-(Morpholino)ethanesulfonic acid: manufactured by NacalaiTesque) • Ethanol (for molecular biology use) (manufactured by Nacalai Tesque) Triton-X100 (manufactured by Tokyo Chemical Industry Co., Ltd.) • SYTOX blue dead cell staining (SYTOX: manufactured by ThermoFisher Scientific) Magnesium chloride hexahydrate (manufactured by Nacalai Tesque) Magnesium sulfate heptahydrate (manufactured by Nacalai Tesque) Magnesium gluconate (manufactured by Tokyo Chemical Industry Co., Ltd.) Arginine hydrochloride (manufactured by Nacalai Tesque) Arginine glutamate (manufactured by BLDpharm) Histidine hydrochloride monohydrate (manufactured by Nacalai Tesque) • Sodium aspartate (manufactured by Nacalai Tesque) Monosodium glutamate (manufactured by Nacalai Tesque) • Glycine (for molecular biology use) (manufactured by Nacalai Tesque) • Alanine (manufactured by Nacalai Tesque) • DMSO (for molecular biology) (Dimethyl sulfoxide: manufactured by Nacalai Tesque) • Ethylene glycol (biotechnology grade) (manufactured by Nacalai Tesque) • Propylene glycol (biotechnology grade) (manufactured by Nacalai Tesque) • Concentrated glycerol (for molecular biology use) (manufactured by Nacalai Tesque) • Glucose (produced by Nacalai Tesque) • Sugar (produced by Nacalai Tesque) Mannitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) Sorbitol (manufactured by FUJIFILM Wako Pure Chemical Corporation) Lactose monohydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation) Polyethylene glycol 300 has an average molecular weight of 285-315. Polyethylene glycol 400 has an average molecular weight of 380-420. Polyethylene glycol 600 has an average molecular weight of 570-630. Polyethylene glycol 4000 has an average molecular weight of 2700~3400. • Polyethylene glycol 20000, with an average molecular weight of 18000~25000 • Polyethylene glycol 40000, with an average molecular weight of 25000~40000 • Tris (Good's buffer) (2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol: manufactured by Nacalai Tesque) • Tween20 (Made by Tokyo Chemical Industry Co., Ltd.) Sorbitan monooleate (Span80) (manufactured by Nacalai Tesque) • Sodium carboxymethyl cellulose (manufactured by Nacalai Tesque) Milk exosomes (manufactured by COSMOBIO Co., Ltd.) ·LabAssay Phospholipid (manufactured by FUJIFILM Wako Shibayagi Corporation) • RPMI medium (manufactured by Nacalai Tesque) • FBS (Fetal Bovine Serum: manufactured by Gibco) • Penicillin-streptomycin mixed solution (stabilized) (manufactured by Nacalai Tesque) • Hemolytic reagent (VersaLyse: manufactured by Beckman-Coulter) • 0.40% trypan blue solution (Bio-rad) [B]Using equipment • Electronic balance (Shimadzu Corporation, TX223N) • Electronic balance (Sartorius, secura125D) pH meter (made by Horiba Manufacturing Co., Ltd., LAQUA_D200_2) • Ultrasonic irradiation device (BRANSONIC, CPX2800H-J) • Pharmaceutical refrigerator (manufactured by Panasonic, MPR-414FRS-PJ) • Biomedical refrigerator (manufactured by Panasonic, MDF-U538D-PJ) • Ultra-low temperature freezer (manufactured by PHcbi, MDFDU300H-PJ) • CO2 incubator (Panasonic, MCO-5AC-PJ) • Cleanroom workbench (Panasonic, MHE-S1301 A2PJ) • Fully automated cell counter (Bio-rad, TC-20) • Culture microscope (Olympus, CKX53) • Disposable cell counting chamber (manufactured by Digital Bio, C-Chip) Fluorescence microscope (KEYENCE, BZ-X710) • Fluorescence spectrophotometer (Hitachi High-Tech, F-7000) • Ultraviolet-visible spectrophotometer (Hitachi High-Tech, U-2900) • Refrigerated centrifuge (Tomy Corporation, MDX-310) • Miniature refrigerated centrifuge (Made by KUBOTA, 3740) • High-pressure autoclave (Tomy Corporation, LSX-700) • Laboratory heat sterilizer (Yamato, SK401) • Water bath (manufactured by AS ONE, TR-1α) • Osmometer (Gobatec, OSMOMAT3000) • High-speed oscillator (manufactured by AS ONE, ASCM-1) [C] Preparation of Evaluation Samples (Lipid Membrane Protection Liquid Composition of the Examples and Liquid Composition of the Comparative Examples) Evaluation samples with the compositions shown in the tables were prepared. For samples containing buffers, buffer solutions of various concentrations were prepared, the pH was appropriately adjusted using a pH adjuster, and then all other reagents, weighed using an electronic balance, were dissolved to prepare the evaluation sample. Additionally, in some comparative examples, water was used as the evaluation sample.

[0141] [D] Preparation of culture medium For the culture medium used to culture or prepare cells, add 50 mL of FBS and the recommended amount of penicillin-streptomycin mixture to 500 mL of RPMI medium to obtain RPMI medium containing 10% serum.

[0142] [E] Preparation of fluorescently encapsulated liposomes EYPC liposomes, encapsulated with the fluorescent substance Pyranine and the matting agent DPX, were prepared as fluorescent encapsulated liposomes (hereinafter simply referred to as "liposomes") for the evaluation of phospholipid membrane protection. Specifically, 10 mg of EYPC was weighed into a 10 mL round-bottom flask, dissolved in an appropriate amount of ethanol, and then formed into a thin film using hot air. 1.0 mL of a Pyranine solution (Pyranine: 35 mM, DPX: 50 mM, MES: 25 mM, pH 7.4) was added to the film, and then dispersed using an ultrasonic irradiation device. The outer aqueous layer was then replaced using PBS or physiological saline and a Sephadex G100 column to obtain a dispersion of the fluorescent encapsulated liposomes.

[0143] [F] Preparation of exosome samples Prior to the experiment, the exosome solution, which was used as a reagent, was diluted 5-fold with PBS. Then, it was quickly mixed with the constructed evaluation samples at a 1:1 (vol / vol) ratio for each evaluation.

[0144] Preparation of [G] cells Cells were prepared from spleens of euthanized Balb / c or C57BL / 6 mice. Specifically, 5.0 mL of RPMI medium containing 10% serum was added to the spleens removed from the samples, and the spleens were treated with a 100 μm Falcon cell filter to prepare a cell suspension. Then, after washing with PBS, hemolysis was performed using a hemolytic agent. Following further multiple PBS-based washes, to suppress the effects of hemolysis, the cells were cultured in RPMI medium containing 10% serum for 2–8 hours. Then, after passing through a 100 μm Falcon cell filter again, multiple PBS-based washes and cell counts were performed to obtain a spleen cell dispersion. Cell counting was performed using a disposable cell counting chamber (C-Chip) manufactured by Digital Bio.

[0145] Preparation of [H] cell samples The spleen cell dispersion obtained in [G] above was prepared into a 1×10⁻⁶ solution. 6 Cells were aliquoted into individual microcentrifuge tubes and centrifuged at 250g to recover the cells. The supernatant was discarded, and cell particles were obtained. 1.0 mL of separately prepared evaluation sample was added to the cell particles to disperse them, thus obtaining the cell sample.

[0146] [I] Preparation of blood samples (blood cell samples) Blood samples (blood cell samples) were prepared from specimens of euthanized Balb / c or C57BL / 6 mice transferred to other animals. Specifically, the specimens of euthanized Balb / c or C57BL / 6 mice were dissected, and the blood remaining in the heart was recovered using a syringe. The blood (whole blood) was then immediately anticoagulated by mixing it with 3.2% wt% citric acid solution at a ratio of 9:1 (vol / vol). This yielded the blood samples (blood cell samples).

[0147] [J] Evaluation [J-1] Evaluation of phospholipid membrane protection: 1- Evaluation of the effect of protecting fluorescently encapsulated liposomes from freeze-thaw damage. The degree of phospholipid membrane protection was evaluated based on the destruction rate of fluorescently encapsulated liposomes. Specifically, 40 μL of a dispersion of fluorescently encapsulated liposomes and 40 μL of an evaluation sample were mixed to prepare a sample for cryodamage evaluation. The sample was placed in an ultra-low temperature freezer at -80°C for thorough freezing, and then thawed using room temperature water. This freeze-thaw cycle was repeated five times to obtain a thawed sample. 2970 μL of PBS and 30 μL of the thawed sample were added to a disposable cuvette for fluorescence measurement, and the fluorescence intensity was measured to determine the destruction rate of the fluorescently encapsulated liposomes. Fluorescent substances within the liposomes leak out along with the destruction of the phospholipid membrane, reducing the effect of the matting agent, thus increasing the fluorescence intensity. Therefore, higher fluorescence intensity indicates a greater degree of destruction. The destruction rate of the fluorescently encapsulated liposomes was calculated as a relative value (%) of 30 μL of Triton-X100 diluted 10-fold and stored as 100 (representing a completely destroyed state). Specifically, it is calculated using the following equation (1). Here, Lx is the fluorescence intensity of the sample after freezing and thawing, and L0 is the fluorescence intensity of the sample prepared by mixing PBS or physiological saline into the dispersion of fluorescently encapsulated liposomes (without freezing process). 100 The fluorescence intensity of samples (without freezing) prepared by adding 30 μL of Triton-X100 diluted 10-fold to a dispersion of fluorescently encapsulated liposomes was measured. The concentration of the fluorescently encapsulated liposomes was determined using LabAssay Phospholipid as a phospholipid concentration and adjusted with PBS or physiological saline to approximately 0.2 mg / mL. Fluorescence intensity was measured using a fluorescence spectrophotometer (excitation: 416 nm, measurement: 512 nm).

[0148] [Mathematical Expression 1] [J-2] Determination of osmotic pressure Collect 50 μL of the sample for osmotic pressure measurement into a dedicated microcentrifuge tube (Eppendorf tube), place it in the device (OSMOMAT3000 osmotic pressure analyzer), and perform the measurement.

[0149] [J-3] Determination of Phospholipid Concentration Phospholipid concentration was determined using LabAssay Phospholipid. After colorimetric reactions were performed with quantitative sample solutions containing phospholipids, the absorbance at a wavelength of 600 nm was measured using a UV spectrophotometer to determine the phospholipid concentration.

[0150] [J-4] Evaluation of phospholipid membrane protection 2 - Protecting exosomes from damage caused by contact with hydrophobic materials Evaluation of effects 100 μL of the prepared exosome sample solution was pipetted into a polypropylene microcentrifuge tube and incubated at room temperature for 10 minutes. The exosome sample solution was then transferred to a new microcentrifuge tube and incubated for 10 minutes in the same manner. This transfer process was repeated 0–8 times. The concentration of residual phospholipids was then evaluated using 50 μL of the exosome sample solution. Alternatively, the concentration of residual phospholipids was evaluated after transferring the exosome sample solution to a single microcentrifuge tube and incubating at room temperature for 0–6 hours.

[0151] [J-5] Evaluation of phospholipid membrane protection 3 - Evaluation of the effect of protecting exosomes from cryodamage The prepared exosome sample solution was placed into polypropylene microcentrifuge tubes and then frozen at -80°C. It was then thawed using room temperature water, constituting one freezing cycle. After 0–5 freezing cycles, the exosome sample solution was passed through a 0.22 μm PVDF rotary filter from a Merck Millipore Ultrafree centrifugal filter unit to remove any material agglomerated during freezing. The concentration of residual phospholipids in the filtrate was then evaluated.

[0152] [J-6] Evaluation of phospholipid membrane protection 4 - Evaluation of the effect of protecting exosomes from vibration damage The prepared exosome sample solution was placed in polypropylene microcentrifuge tubes and then subjected to vibration at 1,000 rpm for 0–2 hours using a high-speed shaker. The exosome sample solution was then passed through a 0.22 μm PVDF rotary filter from a Merck Millipore Ultrafree centrifugal filter unit to remove any material that had agglomerated due to freezing. The concentration of residual phospholipids in the filtrate was then evaluated.

[0153] [J-7] cell culture Cell culture was carried out using a 5% CO2 incubator set to 37°C (MCO20AIC).

[0154] [J-8] Evaluation of phospholipid membrane protection 5 - Evaluation of the effect of protecting cells from cryodamage 150 μL of cell samples were aliquoted into 1.5 mL CryoFreeze tubes (CryoTube) manufactured by AS ONE, serving as unfrozen (hereinafter referred to as pre-freeze-thaw or pre-FT) cell samples, cell samples for freeze-thaw (hereinafter referred to as post-freeze-thaw or post-FT) operations, and cell samples for storage at -80°C for a specified time after freezing. It should be noted that the freeze-thaw process for cell samples is similar to that for fluorescently encapsulated liposomes: the cell samples are placed in an ultra-low temperature freezer, allowed to freeze completely, and then thawed using room temperature water. After freezing and storing the cell samples for the specified time, they are stored in an ultra-low temperature freezer for the specified period. Cell viability is determined for each cell sample that has undergone these operations.

[0155] [J-9] Determination of cell viability For cell viability determination, the trypan blue exclusion method was used. Specifically, for cell samples at each stage, 0.4% trypan blue solution was mixed with the cell sample at a 1:1 (vol / vol) ratio, loaded onto a counting slide, and immediately counted using a fully automated cell counter. In the case of DMSO, the counting slide was photographed using a fluorescence microscope, and cell viability was determined by visual counting. At least five photographs were used for each sample.

[0156] [J-10] Evaluation of phospholipid membrane protection 6 - Evaluation of its effect on protecting leukocytes from thermal damage The prepared blood samples were placed into polypropylene microcentrifuge tubes and then sealed with vinyl bags. These tubes were then placed in a laboratory heat sterilizer set to 40°C and allowed to stand for one week. After standing, the blood samples were centrifuged at 6,000 rpm using a micro-refrigerated centrifuge, and the supernatant was collected. The collected supernatant was mixed 1:1 (vol / vol) with a SYTOX solution diluted 100-fold with TE buffer to obtain the nucleic acid free evaluation sample. 20 μL of the nucleic acid free evaluation sample was added to 3.0 mL of TE buffer, and the results were measured using a fluorescence spectrophotometer (excitation: 440 nm, measurement: 480 nm).

[0157] [J-11] Evaluation of phospholipid membrane protection 7 - Evaluation of its effect on protecting erythrocytes from thermal damage The prepared blood samples were placed into polypropylene microcentrifuge tubes and then sealed with vinyl bags. These tubes were then placed in a laboratory heat sterilizer set to 40°C and allowed to stand for 3 days. After standing, the blood samples were centrifuged at 6,000 rpm using a micro-refrigerated centrifuge, and the supernatant was collected as the sample for free hemoglobin evaluation. 30 μL of the free hemoglobin evaluation sample was added to 3.0 mL of D-PBS, and the absorbance at 571 nm was measured using a UV-Vis spectrophotometer.

[0158] [Experimental Example 1] In this experimental example, to evaluate the phospholipid membrane protection in the experimental examples described later, the damage to the phospholipid membrane caused by the storage conditions of fluorescently encapsulated liposomes was confirmed. The procedure was performed based on the method described in "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescently Encapsulated Liposomes from Freeze-Dry Damage". The evaluation samples used and the measured fluorescence intensities are shown in Table 1 below. Figure 1 middle.

[0159] [Table 1] The degree of liposome damage (damage to the phospholipid membrane) can be evaluated by comparison with Reference Example 1a. With the addition of Triton as a surfactant, the fluorescence intensity increased dramatically from 25.5 to 899, indicating liposome damage (Comparative Example 1). While substantially no damage was observed after storage in water at 4°C for 24 hours (Reference Example 1b), damage was confirmed at 90°C for 10 minutes (Comparative Example 2). Furthermore, even after a single freeze-thaw cycle, liposome damage occurred, and the degree increased with repeated freeze-thaw cycles.

[0160] [Experimental Example 2] In this experimental example, the effect of magnesium salts on protecting phospholipid membranes from freeze-thaw damage was evaluated according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Thaw Damage". The evaluation samples used and the obtained membrane damage rates are shown in Tables 2A and 2B below. Figure 2 (The results of Reference Example 1b of Experiment 1 are also shown.)

[0161] [Table 2A] [Table 2B] In PBS, liposomes were destroyed by cryo-damage (Comparative Example 4). Furthermore, even with the addition of other components (Peg20,000, CMC, Tween20, or Span80), no protective effect was substantially confirmed (Comparative Example 6 series to Comparative Example 8 series) or only slightly confirmed (Comparative Example 5 series). However, with the addition of magnesium sulfate, when compared with the addition of the other components at the same concentration, a superior protective effect was found (Refer to Example 2 series).

[0162] [Experimental Example 3] In this experimental example, the protective effect of various magnesium salts (magnesium sulfate, magnesium chloride, magnesium gluconate) on phospholipid membranes against cryodamage was evaluated according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the effect of protecting fluorescently encapsulated liposomes from cryodamage". The evaluation samples used are shown in Table 3 below, and the membrane damage rate is shown in... Figure 3 middle.

[0163] [Table 3] like Figure 3 As shown, the protective effect of phospholipid membranes was confirmed for any magnesium salt (see Examples 2 to 4). In particular, when the magnesium salt concentration was 10 mg / mL or higher, i.e., on the order of 10 mM or higher as magnesium ion concentration, a particularly significant protective effect was confirmed (see Examples 2c to 2e, Examples 3c to 3f, Examples 4c to 4e).

[0164] [Experimental Example 4] In this experimental example, following the guidelines of "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage," the effectiveness of various base solutions (phosphate buffer, physiological saline, water, aqueous phosphate solution, acetate buffer, citrate buffer, carbonate buffer) combined with magnesium salts in protecting phospholipid membranes from freeze-dry damage was evaluated. The evaluation samples used are shown in Tables 4A and 4B below, and the membrane damage rate is shown in... Figure 4A and Figure 4B middle.

[0165] [Table 4A] [Table 4B] like Figure 4AAs shown, when magnesium salt (as component (A)) is combined with phosphate buffer, physiological saline, water or phosphate aqueous solution, no increase in the protective effect of phospholipid membrane due to the combination is confirmed across the entire concentration range of magnesium salt (see series of examples 3, 5, 6 and 7).

[0166] On the other hand, such as Figure 4B As shown, the protective effect on the phospholipid membrane was confirmed when the acetate buffer, citrate buffer, or carbonate buffer, which is component (B), was used alone (Reference Examples 8 to 10). Moreover, when the magnesium salt, which is component (A), is combined with the acetate buffer, citrate buffer, or carbonate buffer, which is component (B), especially in the low concentration range of component (A), the protective effect on the phospholipid membrane was confirmed to be enhanced by the combination (Examples 1a, 1b, 2a, 2b, 3a, 3b). This enhancement effect was particularly large when combined with the acetate buffer or citrate buffer, which is component (B) (Examples 1a, 1b, 2a, 2b).

[0167] [Experimental Example 5] In this experimental example, the effectiveness of buffers at various concentrations in protecting phospholipid membranes from cryodamage was evaluated according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage". The evaluation samples used are shown in Table 5 below, and the membrane damage rate is shown in... Figure 5 middle.

[0168] [Table 5] like Figure 5 As shown, compared to the protective effect of magnesium as component (A) alone on the phospholipid membrane (reference example 3d), the combination with acetate buffer or citrate buffer as component (B) resulted in a superior protective effect on the phospholipid membrane across the entire concentration range (Example 1 series, Example 2 series). When considered together with the results of Experimental Example 6 described later, the enhanced protective effect of the phospholipid membrane resulting from the combination with component (B) is a unique effect resulting from the combination with component (A).

[0169] [Experimental Example 6] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage", the effect of protecting the phospholipid membrane from freeze-dry damage when component (A) of Experimental Example 6 was replaced with other components was evaluated. The evaluation samples used are shown in Table 6 below, and the membrane damage rate is shown in... Figure 6 middle.

[0170] [Table 6] like Figure 6 As shown, the protective effect on the phospholipid membrane was confirmed when arginine and sorbitol were used alone (see Examples 11a and 12a), which is the same as that of magnesium salt as component (A) (Example 3 and 12a). Figure 3 (See Reference Examples 2e, 3e, and 4e). However, regarding the acetate buffer or citrate buffer as component (B), unlike when combined with component (A), the protective effect of the phospholipid membrane cannot be improved when combined with arginine or sorbitol (Reference Examples 11b-11f, 12b-12f, and 13b-13f).

[0171] [Experimental Example 7] In this experimental example, the protective effect of magnesium salts against freezing damage to phospholipid membranes at various pH values ​​was evaluated according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the effect of protecting fluorescently encapsulated liposomes from freezing damage". The evaluation samples used are shown in Table 7 below, and the membrane damage rate is shown in... Figure 7 middle. Figure 7 The bar charts, from left to right, show the results for pH 5.0, pH 6.0, pH 6.5, pH 7.4, pH 8.0, pH 9.0, and pH 10.0.

[0172] [Table 7] like Figure 7 As shown, magnesium salts were confirmed to exert a protective effect on phospholipid films over a wide pH range of 5–10. In particular, they were confirmed to maintain excellent and stable phospholipid film protection within the pH range of 5–8. Thus, the effect of magnesium salts is so excellent that they exhibit a high level of phospholipid film protection even in a pH range including alkaline conditions.

[0173] [Experimental Example 8] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) containing components (A) and (B) and additional components (component (C) as a polyol, component (D) as a sugar or sugar alcohol, component (E) as a polyethylene glycol, or component (F) as an amino acid or its derivative) on protecting the phospholipid membrane from freeze-dry damage was evaluated. Furthermore, in order to evaluate the degree of improvement in the phospholipid membrane protection effect due to the additional addition, a phospholipid protection enhancement index was derived based on the following formula. The evaluation sample used (liquid composition for phospholipid membrane protection, pH 7.4), membrane damage rate, and phospholipid protection enhancement index are shown in Tables 8A to 8C below.

[0174] [Mathematical Expression 2] [Table 8A] [Table 8B] [Table 8C] Compared with the liquid compositions for phospholipid film protection containing components (A) and (B) (Examples 1k, 1h), an improved protective effect on the phospholipid film was confirmed in the liquid compositions for phospholipid film protection that also contain components (C), (D), (E), or (F) (Examples 4-17). This improved effect was particularly significant in the cases where glycerol was contained as component (C) (Example 4 series), mannitol was contained as component (D) (Example 6 series), component (E) was contained (Example 10-13 series), and arginine or its derivatives were contained as component (F) (Examples 14, 15).

[0175] [Experimental Example 9] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) containing components (A) and (B) and various concentrations of additional components (component (C) as a polyol, component (D) as a sugar or sugar alcohol, and component (E) as a polyethylene glycol) on protecting the phospholipid membrane from freeze-dry damage was evaluated. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Tables 9A to 9E below.

[0176] [Table 9A] [Table 9B] [Table 9C] [Table 9D] [Table 9E] As shown in Tables 9A-9E, the additional components combined with components (A) and (B) (component (C) as a polyol, component (D) as a sugar or sugar alcohol, and component (E) as polyethylene glycol) showed improved phospholipid membrane protection across the entire concentration range (Example 4 series, Example 6 series, Example 11-13 series, Example 16 series). It should be noted that, as shown in Table 9A, when glycerol was used as component (C) (Examples 4b, 4h, 4i), the improvement in phospholipid membrane protection was higher than when ethylene glycol was used as component (C) (Examples 16a, 16b, 16c).

[0177] [Experimental Example 10] In this experimental example, the effect of the combination of component (B) and additional components ((C), (D), and (E)) on protecting the phospholipid membrane from freeze-thaw damage was evaluated according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the effect of protecting fluorescently encapsulated liposomes from freeze-thaw damage". The evaluation sample (pH 7.4) and membrane damage rate used are shown in Table 10 below.

[0178] [Table 10] As shown in Table 10, the protective effect of phospholipid membranes can be obtained by simply combining component (B) and additional components ((C), (D), and (E)) (see Reference Examples 17, 18, and 19). However, by further combining it with component (A), a more superior protective effect of phospholipid membranes is obtained (Example 6, 11, and 12).

[0179] [Experimental Example 11] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) containing components (A), (B), and (C) and various concentrations of component (E) on protecting the phospholipid membrane from freeze-damage was evaluated. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Tables 11A to 11C below.

[0180] [Table 11A] [Table 11B] [Table 11C] As shown in Tables 11A-11C, component (E), which is combined with components (A), (B), and (C), shows an improved protective effect on phospholipid membranes across the entire concentration range (Example 21 series, Example 22 series).

[0181] [Experimental Example 12] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) containing components (A), (B), (C), and (E), and various (D) components on protecting the phospholipid membrane from freeze-damage was evaluated. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Table 12 below.

[0182] [Table 12] As shown in Table 12, component (D), when combined with components (A), (B), (C), and (E), all showed an improved protective effect on the phospholipid membrane (Example 23 series, Example 24 series, Example 25, and Example 28 series). It should be noted that when mannitol was used as component (D) (Example 23 series), the improvement in the protective effect on the phospholipid membrane was significantly greater compared to when other sugars were used as component (D) (Example 24 series, Example 25, and Example 28 series c).

[0183] [Experimental Example 13] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) containing components (A), (B), and (E), and various concentrations of components (C) and (D) on protecting the phospholipid membrane from freeze-damage was evaluated. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) used and the membrane damage rate are shown in Table 13 below.

[0184] [Table 13] As shown in Table 13, components (C) and (D), which are combined with components (A), (B) and (E), all showed an improvement in the protective effect of phospholipid membranes across the entire concentration range.

[0185] [Experimental Example 14] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) obtained by adding component (E) with different molecular weights to components (A) and (B) was evaluated to protect the phospholipid membrane from freeze-dry damage. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Table 14 below, and the membrane damage rate is also shown in... Figure 8 middle.

[0186] [Table 14] As shown in Table 14 and Figure 8 As shown, compared with the case of using a single component as component (E) (Example 12 series), the case of using a combination of components with different molecular weights (Example 27 series) confirmed an improvement in the protective effect of the phospholipid membrane.

[0187] [Experimental Example 15] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage", the effect of the evaluation samples (liquid compositions for phospholipid membrane protection) obtained by adding component (E) with different molecular weights to components (A), (B), and (C) was evaluated to protect the phospholipid membrane from freeze-dry damage. The evaluation samples used (liquid compositions for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Table 15 below, and the membrane damage rate is also shown in... Figure 9 middle.

[0188] [Table 15] As shown in Table 15 and Figure 9 As shown, compared with the case of using a single component as component (E) (Example 28 series), the protection effect of the phospholipid membrane was improved when components with different molecular weights were used in combination (Example 29 series). A comparison with the results of Experimental Example 14 revealed that the improvement in the protection effect of the phospholipid membrane resulting from combining components with different molecular weights as component (E) was even greater when component (C) was present.

[0189] [Experimental Example 16] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of the evaluation sample (liquid composition for phospholipid membrane protection) obtained by adding component (E) with different molecular weights to components (A), (B) (acetic acid buffer, or a combination of acetic acid buffer and citric acid buffer), (C) and (D) was evaluated to protect the phospholipid membrane from freeze-damage. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Tables 16A to 16D below. Figure 10 middle.

[0190] [Table 16A] [Table 16B] [Table 16C] [Table 16D] As shown in Tables 16A to 16D and Figure 10 As shown, when components with different molecular weights are used in combination as component (E) (Example 29 series), the improvement in the protective effect of the phospholipid membrane is confirmed, similar to the results of Experimental Example 15. Furthermore, compared to the case where only acetate buffer is used as component (B) (Example 34 series), the improvement in the protective effect of the phospholipid membrane resulting from combining acetate buffer and citric acid buffer as component (B) (Example 36 series, Example 38 series) is even greater.

[0191] [Experimental Example 17] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Dry Damage", the effect of protecting the phospholipid membrane from freeze-dry damage was evaluated by increasing the concentration of components other than component (B) in the evaluation sample (liquid composition for phospholipid membrane protection, Example 39 series, pH 7.4) obtained by adding components (A), (B), (C), (D), and (E). The membrane damage rate is shown in the figure. Figure 11 middle.

[0192] [Table 17] like Figure 11 As shown, with the increase of concentration (N), a tendency was confirmed that the protective effect of the phospholipid membrane improved.

[0193] [Experimental Example 18] In this experimental example, the osmotic pressure of various liquid compositions for phospholipid membrane protection (all compositions for which the protective effect of phospholipid membranes has been confirmed) was measured according to "[J-2] Determination of Osmotic Pressure". The results are shown in Tables 18A to 18D below.

[0194] [Table 18A] [Table 18B] [Table 18C] [Table 18D] As described above, liquid compositions for phospholipid membrane protection can be prepared over a wide range of osmotic pressures.

[0195] [Experimental Example 19] In this experimental example, the effect of the phospholipid membrane protection liquid composition (Example 40) on protecting exosomes from damage caused by contact with hydrophobic materials was evaluated according to "[J-4] Evaluation of Phospholipid Membrane Protection 2 - Evaluation of the effect of protecting exosomes from damage caused by contact with hydrophobic materials". The composition of the phospholipid membrane protection liquid composition is as follows. The results are shown below. Figure 12 and Figure 13 middle.

[0196] [Table 19] like Figure 12As shown in (A), exosomes in PBS are damaged with increasing number of transfers from the tube, resulting in a decrease in phospholipid concentration, i.e., the residual amount of exosomes. On the other hand, as... Figure 12 As shown in (B), the exosomes in the liquid composition for phospholipid membrane protection (Example 40) are provided with excellent protection even after 8 transfers, showing the same phospholipid concentration, i.e., the amount of exosomes remaining, as in the initial case (0 transfers).

[0197] like Figure 13 As shown in (A), exosomes in PBS are damaged with the passage of time following transfer (0 times), resulting in a decrease in phospholipid concentration, i.e., the residual amount of exosomes. On the other hand, as... Figure 13 As shown in (B), the exosomes in the liquid composition for phospholipid membrane protection (Example 40) are well protected even after standing for 6 hours, showing the same phospholipid concentration, i.e., the amount of exosomes remaining, as in the initial case (standing time 0 hours).

[0198] As described above, it was confirmed that the liquid composition for phospholipid membrane protection (Example 40) stabilizes the phospholipid membrane of exosomes and protects them from damage caused by contact with hydrophobic materials (polypropylene constituting the microcentrifuge tube).

[0199] [Experimental Example 20] In this experimental example, the effect of the liquid composition for phospholipid membrane protection (Example 40) on protecting exosomes from cryodamage was evaluated according to "[J-5] Evaluation of Phospholipid Membrane Protection 3 - Evaluation of the Effect of Protecting Exosomes from Freeze-Damage". The results are shown below. Figure 14 middle.

[0200] like Figure 14 As shown in (A), exosomes in PBS aggregate and become damaged with increasing freezing cycles, leading to a decrease in the phospholipid concentration in the filtrate, i.e., the amount of residual exosomes. The absorbance after five freezing cycles decreased from 0.043 before freezing to 0.04, a value close to the blank (0.039). On the other hand, as... Figure 14 As shown in (B), the exosomes in the liquid composition for phospholipid membrane protection (Example 40) exhibit excellent protective effects even with increasing freezing cycles, maintaining an absorbance of 0.057 even at the 5th freezing. Due to... Figure 14 The comparison in (A) confirmed that the liquid composition for phospholipid membrane protection (Example 40) stabilizes the phospholipid membrane of exosomes and protects them from freezing damage.

[0201] [Experimental Example 21] In this experimental example, the protection of exosomes from vibration damage achieved by the liquid composition for phospholipid membrane protection (Example 40) was tested according to "[J-6] Evaluation of Phospholipid Membrane Protection 4 - Evaluation of the effect of protecting exosomes from vibration damage". The results are shown below. Figure 15 middle.

[0202] like Figure 15 As shown in (A), exosomes in PBS aggregate and are damaged with increasing vibration duration, leading to a decrease in the phospholipid concentration in the filtrate, i.e., the amount of residual exosomes. After 2 hours of vibration stimulation, the absorbance decreased from 0.043 before stimulation to 0.04, a value close to the blank absorbance of 0.039. On the other hand, as... Figure 15 As shown in (B), the exosomes in the liquid composition for phospholipid membrane protection (Example 40) remained at 0.057 even after 2 hours of vibration stimulation. This is due to... Figure 15 The comparison in (A) confirmed that the liquid composition for phospholipid membrane protection (Example 40) stabilizes the phospholipid membrane of exosomes and protects them from vibration damage.

[0203] [Experimental Example 22] In this experimental example, according to "[J-8] Evaluation of Phospholipid Membrane Protection 5 - Evaluation of the Effect of Protecting Cells from Freezing Damage", the glycerol concentration of the liquid composition for phospholipid membrane protection [Example 41 (G16) and Example 42 (Citrate G16), their double-concentrated solutions (Example 41-2 (G16×2) and Example 42-2 (Citrate G16×2)) and Example 41 (G16) with variations in glycerol concentration (Example 41-3 (Gx)) was evaluated. b The protection of the cellular phospholipid membrane from cryodamage was examined. The results are presented in... Figures 16A-16C middle.

[0204] [Table 20] like Figure 16A As shown, cells in PBS were damaged by freeze-thaw (FT), with cell viability decreasing from approximately 84% to 12%, resulting in significant cell death. Furthermore, when using 10% DMSO (water), a commonly used cell preservation solution, the change in cell viability due to FT ranged from 3% to 0%. Moreover, when using the gold standard cell preservation solution—a liquid containing 10% DMSO in PBS (10% DMSO (PBS))—the change in cell viability due to FT ranged from 77% to 46%.

[0205] On the other hand, such as Figure 16BAs shown, for cells using the phospholipid membrane protection liquid composition (Example 41 (G16)), the change in cell viability caused by FT ranged from 86% to 51%, demonstrating a higher viability than general or gold standard cell preservation solutions. Furthermore, for the case using the phospholipid membrane protection liquid composition (Example 42 (Citrate G16)), the change in cell viability caused by FT ranged from 92% to 58%, also demonstrating a higher viability than general or universal cell preservation solutions.

[0206] In addition, such as Figure 16B As shown, when using a phospholipid membrane protection liquid composition (Example 41-2 (G16×2)) obtained by concentrating the phospholipid membrane protection liquid composition (Example 41 (G16)) by 2 times, the cell survival rate after FT increased to 67%, which was higher than that before concentration. When using a phospholipid membrane protection liquid composition (Example 42-2 (Citrate G16×2)) obtained by concentrating the phospholipid membrane protection liquid composition (Example 42 (Citrate G16)) by 2 times, the cell survival rate after FT was also higher than that before concentration, at 68%. On the other hand, when using a liquid (PBS×2) prepared by concentrating PBS by 2 times to achieve the same osmotic pressure as the phospholipid membrane protection liquid composition (Example 41-2 (G16×2)), the cell survival rate after FT was 14%, which was the same as that of PBS before concentration (12%).

[0207] In addition, such as Figure 16C As shown, when using the phospholipid membrane protection liquid compositions of Examples 41-3 (G20), 41-3 (G30), 41-3 (G40), 41-3 (G50), or 41-3 (G60) obtained by changing the glycerol concentration to 20, 30, 40, 50, or 60 mg / mL respectively to increase the osmotic pressure of the phospholipid membrane protection liquid composition (Example 41 (G16)), the cell viability generated by FT remained at a high level corresponding to the glycerol concentration.

[0208] The above confirms that various liquid compositions for protecting phospholipid membranes are highly effective in protecting cell phospholipid membranes from cryo-damage and are useful for cell cryopreservation.

[0209] [Experimental Example 23] In this experimental example, the protection of the cell's phospholipid membrane from cryodamage achieved by the liquid composition for phospholipid membrane protection [Example 43 (G16) and Example 44 (G16-aa)] was examined according to "[J-8] Evaluation of Phospholipid Membrane Protection 5 - Evaluation of the effect of protecting cells from cryodamage" (Example 43 (G16) and Example 44 (G16-aa)). The results are presented below. Figure 17middle.

[0210] [Table 21] like Figure 17 As shown, regarding cell viability after FT, the phospholipid membrane protection liquid composition without arginine (Example 43 (G16)) showed a 51% viability, while the phospholipid membrane protection liquid composition with arginine (Example 44 (G16-Arg)) showed a 63% viability, demonstrating a higher survival rate. Furthermore, the phospholipid membrane protection liquid composition with added amino acids (Example 44 (G16-ArgGlu, GluNa, Pro, Thr, Gly, His)) also showed a higher cell viability than Example 43 (G16) without added amino acids.

[0211] [Experimental Example 24] In this experimental example, the protection of the cell's phospholipid membrane from cryodamage achieved by the liquid composition for phospholipid membrane protection [Example 45 (G16ArgGlu) and Example 46 (G40ArgGlu)] was examined according to "[J-8] Evaluation of Phospholipid Membrane Protection 5 - Evaluation of the effect of protecting cells from cryodamage" (J-8). The results are presented below. Figure 18A and Figure 18B middle.

[0212] [Table 22] like Figure 18A and Figure 18B As shown, in terms of cell survival after FT, the phospholipid membrane protection liquid compositions with added arginine glutamate (Examples 45 (G16ArgGlu 10, 20, 30, 40) and Example 46 (G40ArgGlu 0)) all showed greater survival rates compared to the phospholipid membrane protection liquid compositions without added arginine glutamate (Example 45 (G16ArgGlu 0) and Example 46 (G40ArgGlu 0)).

[0213] [Experimental Example 25] In this experimental example, the protection of leukocyte phospholipid membranes from heat damage achieved by the liquid compositions for phospholipid membrane protection (Examples 47a-47f, Examples 48a-48c, Examples 49a-49c) was examined according to "[J-10] Evaluation of Phospholipid Membrane Protection 6 - Evaluation of the Effect of Protecting Leukocytes from Thermal Damage". For comparison, the protection of leukocyte phospholipid membranes after preservation in PBS or Triton X-100 aqueous solution was also examined. The results are shown in Tables 23A and 23B.

[0214] [Table 23A] [Table 23B] As shown in Comparative Example 12, when whole blood samples were stored in PBS at 40°C for one week, a significant amount of nucleic acid leaked out, confirming leukocyte disintegration due to heat damage. In a concentrated solution obtained by doubling the salt concentration of PBS (Comparative Example 13) and a liquid containing a surfactant (Comparative Example 14), even more nucleic acid leaked out after storage at 40°C for one week, confirming a higher degree of leukocyte disintegration due to heat damage. On the other hand, when whole blood samples were stored at 40°C for one week in the phospholipid membrane protection liquid compositions of Examples 47a-47f, 48a-48c, and 49a-49c, nucleic acid leakage and leukocyte disintegration were significantly suppressed. In other words, a significant phospholipid membrane protection effect was confirmed against heat stimulation.

[0215] In addition, in these embodiments, when citric acid buffer is included (Examples 48a-48c, Examples 49a-49c), a tendency to increase the phospholipid film protection effect was confirmed. In the case of Examples 49a-49c, where acetate buffer and citric acid buffer are combined as component (B), a tendency to further increase the phospholipid film protection effect was confirmed. Especially when the glycerol concentration is below 40 mg / mL (preferably below 30 mg / mL) (Examples 47a-47d, Examples 48a-48c, Examples 49a-49c), the amount of nucleic acid leakage can be suppressed to a level close to that of whole blood samples stored in PBS at 40°C for 1 week (without heat stimulation) (Reference Example 20), and an exceptionally significant phospholipid membrane protective effect is confirmed. In Examples 49a and 49b, where the acetate buffer and citrate buffer are combined as component (B) and the glycerol concentration is below 30 mg / mL, an extremely excellent protective effect is confirmed to suppress nucleic acid leakage to a degree greater than that of Reference Example 20 without heat stimulation.

[0216] [Experimental Example 26] In this experimental example, the protection of the phospholipid membrane of erythrocytes from heat damage achieved by the liquid composition for phospholipid membrane protection (Examples 47a, 48c, and 49c) was examined according to "[J-11] Evaluation of Phospholipid Membrane Protection 7 - Evaluation of the effect of protecting erythrocytes from heat damage". For comparison, the protection of the phospholipid membrane of erythrocytes stored in PBS or Triton X-100 aqueous solution was also examined. The results are shown in Tables 24A and 24B.

[0217] [Table 24A] [Table 24B] As shown in Comparative Example 12, when whole blood samples were stored in PBS at 40°C for 3 days, a significant amount of hemoglobin leaked out, confirming that red blood cells disintegrated due to heat damage. In a liquid containing a surfactant (Comparative Example 14), even more hemoglobin leaked out after storage at 40°C for 3 days, confirming a higher degree of red blood cell disintegration due to heat damage. On the other hand, when whole blood samples were stored at 40°C for 3 days in the phospholipid membrane protection liquid compositions of Examples 47a, 48c, and 49c, hemoglobin leakage was suppressed, and red blood cell disintegration was inhibited. That is, a phospholipid membrane protective effect was confirmed against heat stimulation.

[0218] In addition, in these embodiments, when a citrate buffer is included (Examples 48c and 49c), the amount of hemoglobin leakage can be suppressed to a level close to that of Reference Example 20 without heat stimulation, confirming a significant phospholipid membrane protective effect.

[0219] [Experimental Example 27] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of an evaluation sample (liquid composition for phospholipid membrane protection) containing component (A), component (B) (acetic acid buffer, or a combination of acetic acid buffer and citric acid buffer), component (C), component (D), and component (E) with different molecular weights (E) was evaluated to protect the phospholipid membrane from freeze-damage. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) used and the membrane damage rate are shown in Table 25 below.

[0220] [Table 25] As shown in Table 25, compared with the case where only acetate buffer is used as component (B) (Example 50), the protection effect of phospholipid membrane is improved to a greater extent when acetate buffer and citric acid buffer are combined as component (B) (Examples 38f, 49c, and 51 series).

[0221] [Experimental Example 28] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of an evaluation sample (liquid composition for phospholipid membrane protection) containing component (A), component (B) (a combination of acetate buffer and citrate buffer, or a combination of acetate buffer, citrate buffer and succinate buffer), component (C), component (D), and component (E) with different molecular weights (E) was evaluated to protect the phospholipid membrane from freeze-damage. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) used and the membrane damage rate are shown in Table 26 below.

[0222] [Table 26] As shown in Table 26, compared with the case where the acid buffer and citric acid buffer are combined as component (B) (Example 49c), the protection effect of the phospholipid membrane is improved to a higher degree when the acid buffer, citric acid buffer and succinic acid buffer are combined as component (B) (Example 52 series).

[0223] [Experimental Example 29] In this experimental example, according to "[J-1] Evaluation of Phospholipid Membrane Protection 1 - Evaluation of the Effect of Protecting Fluorescent Encapsulated Liposomes from Freeze-Damage", the effect of an evaluation sample (liquid composition for phospholipid membrane protection) containing component (A), component (B) (a combination of acetate buffer and citrate buffer), component (C), component (D), and component (E) with different molecular weights (E) was evaluated to protect the phospholipid membrane from freeze-damage. The evaluation sample (liquid composition for phospholipid membrane protection, pH 7.4) and the membrane damage rate are shown in Table 27 below.

[0224] [Table 27] [Experimental Example 30] In this experimental example, the osmotic pressure of various phospholipid membrane protection liquid compositions was measured according to "[J-2] Determination of Osmotic Pressure". The results are shown below.

[0225] [Table 28] As described above, liquid compositions for phospholipid membrane protection can be prepared over a wide range of osmotic pressures.

[0226] [Experimental Example 31] In this experimental example, the protection of the cell's phospholipid membrane from cryodamage achieved by Examples 49b, 49c, 54c, 54d, 54e, and 54f (see Table 28) was examined according to "[J-8] Evaluation of Phospholipid Membrane Protection 5 - Evaluation of the Effect of Protecting Cells from Freeze-Damage". The results are presented in... Figure 19 middle.

[0227] like Figure 19 As shown, in terms of cell survival rate after FT, Examples 49b, 49c, 54c, 54d, 54e, and 54f all exhibited superior survival rates. Their improvement in survival rate was more significant than... Figure 16B , 16C [Examples of Test Example 22: Examples 41 (G16) and 42 (Citrate G16), their double-concentrated solutions (Examples 41-2 (G16×2) and 42-2 (Citrate G16×2)), and variations in glycerol concentration of Example 41 (G16) (Examples 41-3 (Gx)).] b ))]、 Figure 17 [Examples 43 (G16) and 44 (G16-aa) of Experiment 23], and Figure 18A , 18B [Examples 45 (G16ArgGlu) and 46 (G40ArgGlu) of Experimental Example 24] are superior. That is, cell viability also reflects that: by including a combination of acetate buffer and citrate buffer as component (B) in the liquid composition for phospholipid membrane protection, the phospholipid membrane protection effect is improved, and by including a combination of acetate buffer, citrate buffer and succinate buffer, the phospholipid membrane protection effect is further improved.

Claims

1. A liquid composition for phospholipid membrane protection, comprising: (A) a magnesium salt; and (B) an acetate buffer, a citrate buffer, a carbonate buffer, and / or a succinate buffer.

2. The liquid composition of claim 1, wherein, The component (A) is selected from the group consisting of magnesium chloride, magnesium sulfate and magnesium gluconate.

3. The liquid composition of claim 1, further comprising (C) a polyol.

4. The liquid composition of claim 3, wherein, The component (C) is glycerin.

5. The liquid composition of claim 1, further comprising (D) sugars and / or sugar alcohols.

6. The liquid composition of claim 5, wherein, The component (D) is mannitol.

7. The liquid composition of claim 1, further comprising (E) polyethylene glycol.

8. The liquid composition of claim 1, further comprising (C) glycerol, (D) mannitol, and (E) polyethylene glycol.

9. The liquid composition of claim 7 or 8, wherein, The average molecular weight of component (E) is 270 or higher.

10. The liquid composition of claim 7 or 8, wherein, The (E) component comprises a variety of different polyethylene glycols with an average molecular weight difference of more than 2,000.

11. The liquid composition of claim 7 or 8, wherein, The component (B) is a mixture of acetate buffer and citrate buffer, and the content of the component (E) is 15 mg / mL or more.

12. The liquid composition of claim 1, wherein, The (B) component comprises: acetate buffer and citrate buffer; or, acetate buffer, citrate buffer or succinate buffer.

13. The liquid composition of claim 1, further comprising (F) an amino acid selected from the group consisting of amino acids, dipeptides and their salts.

14. The liquid composition of claim 13, wherein, The component (F) is selected from the group consisting of arginine, arginine dipeptide, glutamic acid, proline and their salts.

15. The liquid composition of claim 1, wherein, The content of component (A) is 0.1 mg / mL or higher.

16. The liquid composition of claim 1, wherein, The content of component (A) is 0.1 mM or more, calculated as magnesium ion concentration.

17. The liquid composition of claim 1, wherein, The content of component (B) is 5 mM or more, as measured by buffer concentration.

18. The liquid composition of claim 3 or 8, wherein, The content of component (C) is 0.01 mg / mL or higher.

19. The liquid composition of claim 5 or 8, wherein, The content of component (D) is 0.01 mg / mL or higher.

20. The liquid composition of claim 7 or 8, wherein, The content of component (E) is 0.01 mg / mL or higher.

21. The liquid composition of claim 13, wherein, The content of component (F) is 1 mg / mL or more.

22. The liquid composition of claim 1, wherein the osmotic pressure is 200~1220 mOsm / kg.

23. The liquid composition of claim 1, used for protection against freezing damage.

24. The liquid composition of claim 23, wherein, The freezing damage is caused by repeated freezing and thawing.

25. The liquid composition of claim 1, used for protection against vibration damage.

26. The liquid composition of claim 1, for protection against damage caused by contact with hydrophobic materials.

27. The liquid composition of claim 1, used for protection against heat damage.

28. The liquid composition of claim 1, for the preservation of cells, extracellular vesicles and / or liposomes.

29. A pharmaceutical composition comprising cells, extracellular vesicles and / or liposomes, and the liquid composition of claim 1.

30. The pharmaceutical composition of claim 29, wherein it is contained in a container of a hydrophobic material.

31. The pharmaceutical composition of claim 29, wherein it is in a frozen state.

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