Liposome composition, method for promoting cell proliferation, and method for preparing cultured cells

Abstract

To provide a liposome composition that has reduced toxicity to cells compared to conventional liposome compositions while having the function of delivering a drug to cells.SOLUTION: There is provided a liposome composition for cell culture, containing a liposome containing cholesterol and a lipid, the lipid containing an anionic lipid, the total ratio of cholesterol and anionic lipid among the cholesterol and lipid constituting the lipid bilayer being 40 mol% or more, and the zeta potential of the liposome being negative.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a liposome composition, a cell proliferation promoting method using the liposome composition, and a method for producing cultured cells.

Background Art

[0002] Liposomes can be used as carriers for drugs, genes and the like; carriers for cosmetics applied to the skin; carriers for insecticides for plants; carriers for enzymes, supplements and the like in foods; carriers for cosmetics applied to the skin, and the like. Since cells are generally negatively charged, cationic liposomes are used from the viewpoint of introducing target substances into cells. However, cationic carriers also have the drawback of inhibiting the normal function of cells by non-specifically adsorbing to various intracellular proteins. Under such circumstances, the development of liposomes with lower cytotoxicity is desired.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of Invention

Problems to be Solved by the Invention

[0005] The present invention aims to provide a liposome composition that has the function of delivering drugs to cells while reducing toxicity to cells compared to conventional liposome compositions. [Means for solving the problem]

[0006] Under these circumstances, after much trial and error, the inventors prepared liposomes (synthetic exosomes) with a lipid composition similar to that of natural exosomes secreted from cells, and investigated the uptake ability of these liposomes by cells. They found that these liposomes were well taken up by cells despite having a negative charge on their particle surface, and that they had low cytotoxicity. Based on this novel finding, the inventors researched the lipid composition of the liposomes, the types of drugs to be encapsulated, etc., and as a result, completed the present invention. Accordingly, as a typical embodiment, the present invention provides the following: Item 1. A liposome composition for cell culture comprising liposomes containing cholesterol and lipids, wherein the lipids include anionic lipids, the total proportion of cholesterol and anionic lipids among the cholesterol and lipids constituting the lipid bilayer is 40 mol% or more, and the zeta potential of the liposomes is negative.

[0007] Item 2. A liposome composition comprising liposomes containing cholesterol and lipids, wherein the lipids include anionic lipids, the total proportion of cholesterol and anionic lipids among the cholesterol and lipids constituting the lipid bilayer is 40 mol% or more, the zeta potential of the liposomes is negative, and the liposomes contain a hydrophilic agent and a hydrophobic agent.

[0008] Item 3. The liposome composition according to claim 2 for promoting cell proliferation.

[0009] Item 4. The liposome composition according to any one of claims 1 to 3, wherein the average particle size of the liposomes is 50 to 150 nm.

[0010] Item 5. The liposome composition according to any one of claims 2 to 4, wherein the hydrophilic agent is an antioxidant and the hydrophobic agent is a p53 inhibitor.

[0011] Item 6. A method for promoting cell proliferation, comprising the step of adding a liposome composition according to any one of claims 1 to 5 to cells.

[0012] Item 7. A step of culturing cells in the presence of the liposome composition according to any one of claims 1 to 5. Steps to collect the cells obtained from the culture as described above. A method for preparing cultured cells, including [the specified term]. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a liposome composition that has the function of delivering drugs to cells while reducing toxicity to cells compared to conventional liposome compositions. Therefore, the liposome composition of the present invention is effective for introducing target substances into various targets, particularly living cells. While the introduction of target substances into living cells is used in various applications, in a preferred embodiment, it can be used, for example, in cell therapy. Specifically, in cell therapy, which treats diseases using cells collected from the blood of the patient themselves (or a healthy donor), the collected cells are cultured, proliferated, and activated in vitro before being administered. However, prolonged culture causes cells to age, reducing therapeutic effects and proliferation rates. Therefore, there is a need for methods to suppress cellular senescence during in vitro expansion culture.

[0014] One of the main causes of cellular senescence is thought to be the accumulation of dysfunctional mitochondria (Mt) in cells. Mitophagy is responsible for the Mt quality control function that selectively removes dysfunctional Mt, but in senescent cells, a large amount of p53 protein highly expressed in the cytoplasm interacts with the Parkin protein required for mitophagy induction to form aggregates, thereby inhibiting the induction of mitophagy. Under such circumstances, the present inventors have advanced research on a method for suppressing cellular senescence by inducing mitophagy in order to obtain a cell population with a reduced proportion of senescent cells, and have developed a complex comprising a mitochondrial membrane potential lowering agent, a p53 inhibitor, and a carrier capable of co-delivering these agents (Patent Document 1). While having the function of delivering drugs to cells, the liposome composition of the present invention has lower toxicity to cells than conventional liposome compositions. Therefore, by using the liposome composition of the present invention as a carrier for a drug that induces mitophagy during cell culture to bring about suppression of cellular senescence, further long-term culture with suppressed cellular senescence becomes possible, and the number of cells for cell therapy can be significantly increased compared to conventional methods, which is extremely useful. [Brief Description of Drawings]

[0015] [Figure 1] Confocal laser micrographs of HCT116 cells administered with the liposomes containing Rhodamine B and FITC prepared in Examples 10, 11 and 12 are shown. [Figure 2] Confocal laser micrographs of mesenchymal stem cells administered with the liposomes containing Rhodamine B and FITC prepared in Examples 8 and 9 are shown. [Mode for Carrying Out the Invention]

[0016] Liposome composition In one embodiment, the present invention provides a liposome composition for cell culture, comprising liposomes containing cholesterol and lipids, wherein the lipids comprise an anionic lipid, and the zeta potential of the liposomes is negative.

[0017] The liposomes used in the present invention are characterized by containing anionic lipids as lipids. Examples of anionic lipids include diacylphosphatidylserine (diacylglycerophosphatidylserine), diacylphosphatidic acid, diacylphosphatidylglycerol, diacylphosphatidylinositol, monoacyllysophosphatidic acid, cardiolivin, and lysobismonocylphosphatidic acid. These anionic lipids can be used individually or in combination of two or more.

[0018] Furthermore, the liposome contains a lipid bilayer, and the proportion of anionic lipids among the cholesterol and lipids constituting the lipid bilayer is preferably 5 mol% or more, more preferably 10 mol% or more. There is no upper limit to the proportion of anionic lipids among the cholesterol and lipids constituting the lipid bilayer, but it is preferably 40 mol% or less, more preferably 30 mol% or less.

[0019] In the present invention, the proportion of cholesterol among the cholesterol and lipids constituting the lipid bilayer is preferably 15 mol% or more, and more preferably 20 mol% or more. The upper limit of the proportion of cholesterol among the cholesterol and lipids constituting the lipid bilayer is not limited, but is preferably 50 mol% or less, and more preferably 40 mol% or less.

[0020] In the present invention, the total proportion of cholesterol and anionic lipids among the cholesterol and lipids constituting the lipid bilayer is preferably 40 mol% or more, and more preferably 44 mol% or more. The upper limit of the total proportion of cholesterol and anionic lipids among the cholesterol and lipids constituting the lipid bilayer is not limited, but is preferably 75 mol% or less, and more preferably 60 mol% or less.

[0021] Furthermore, in the present invention, liposomes may contain neutral lipids and / or cationic lipids.

[0022] Examples of neutral lipids include diacylphosphatidylethanolamine, diacylphosphatidylcholine, lysophosphatidylcholine, sphingomyelin, sphingomyelin, ceramide, cerebroside, ganglioside, cholesterol, cholic acid, and diacylglycerol. Diacylphosphatidylethanolamine and diacylphosphatidylcholine are preferred, and dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC) are also preferred. These neutral lipids can be used individually or in combination of two or more.

[0023] In the present invention, when liposomes contain neutral lipids, the proportion of neutral lipids among the cholesterol and lipids constituting the lipid bilayer is preferably 25 mol% or more, and more preferably 40 mol% or more. The upper limit of the proportion of neutral lipids among the cholesterol and lipids constituting the lipid bilayer is not limited, but is preferably 65 mol% or less, and more preferably 50 mol% or less.

[0024] Examples of cationic lipids include N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium (DOTMA); didodecyldimethylammonium bromide (DDAB); 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP); 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP); dioleoyl-3-dimethylammoniumpropane (DODAP); dioctadecyl-dimethylammonium Examples include chloride (DODAC); 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (DMRIE); 2,3-dioleyloxy-N-[2-(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA); 3β-N-(N',N'-dimethyl-aminoethane-carbamoyl-cholesterol) (DC-Chol); and O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride. These cationic lipids can be used individually or in combination of two or more.

[0025] Examples of acyl groups contained in the above lipids include saturated or unsaturated acyl groups with 12 to 20 carbon atoms. More specifically, examples of acyl groups contained in the above lipids include dodecanoyl group (lauroyl group), tetradecanoyl group (myristoyl group), pentadecanoyl group, hexadecanoyl group (palmitoyl group), 9-hexadecenoyl group (palmitrenoyl group), heptadecanoyl group (margaroyl group), octadecanoyl group (stearoyl group), 9-octadecenoyl group (oleoyl group), and 11-octadecenoyl group (baxenoyl group). This includes groups such as 9,12-octadecadienoyl group (linoleoyl group), 9,12,15-octadecadrienoyl group (9,12,15-linolenoyl group), 6,9,12-octadecadrienoyl group (6,9,12-linolenoyl group), eicosaenoyl group (arachidinoyl group), 8,11-eicosadienoyl group, 5,8,11-eicosatrienoyl group, and 5,8,11-eicosatetraenoyl group (arachidonoyl group).

[0026] In this invention, the zeta potential of liposomes is negative, preferably -10 to -90 mV, and more preferably -30 to -70 mV. The zeta potential can be measured by electrophoresis (laser Doppler method) or the like.

[0027] Furthermore, in the present invention, the particle size (average particle size) of liposomes is not limited, but is preferably 40 to 200 nm, and more preferably 50 to 130 nm. The average particle size can be measured by dynamic light scattering, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), etc., and is typically measured by dynamic light scattering.

[0028] In a preferred embodiment of the present invention, liposomes may contain a drug. Examples of drugs include those that contribute to the suppression of cellular senescence, cell death, etc., drugs that contribute to the treatment of age-related diseases and lifestyle-related diseases, drugs that contribute to cancer treatment, and drugs that regulate the immune system. More specifically, examples include antioxidants, p53 inhibitors, epigenetic agents, anticancer drugs, microRNAs, etc.

[0029] Examples of antioxidants include porphyrin antioxidants, phthalocyanine antioxidants, ascorbic acid, glutathione, uric acid, melatonin, urobilinogen, tocopherol compounds, tocotrienol compounds, carotenoids, xanthophyll compounds, polyphenols, and flavonoid compounds, with porphyrin antioxidants being preferred.

[0030] Porphyrin antioxidants are represented by the following formula (I):

[0031] [ka]

[0032] (In the formula, M1 indicates a transition metal element or a base metal element. R1 is as follows:

[0033] [ka]

[0034] The formula indicates a group selected from the group consisting of the following, where R5, R6, R7, and R8 are each the same or different group, and each represents a group selected from the group consisting of hydrogen, alkyl groups, and alkoxy groups. R2, R3, and R4 are either the same or different groups, and are as follows:

[0035] [ka]

[0036] This formula represents a group selected from the group consisting of the following, where R9 is the same or different group, and each group is selected from the group consisting of hydrogen, alkyl groups, and alkoxy groups. It may also be a cationic metal porphyrin complex represented by .

[0037] The central metal M1 in formula (I) is not particularly limited as long as it is a transition metal element or a base metal element. Examples of transition metal elements include manganese, nickel, iron, copper, and cobalt atoms. Examples of base metal elements include alkali metals, alkaline earth metals, aluminum, or zinc atoms. In a preferred embodiment, the central metal M1 in formula (I) is a transition metal element, and more preferably a manganese atom.

[0038] In the cationic metal porphyrin complex represented by formula (I), the alkyl group may be a linear or branched alkyl group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 5 carbon atoms. The alkoxy group may also be an alkoxy group containing the aforementioned alkyl group.

[0039] In a preferred embodiment, R1 in the cationic metal porphyrin complex represented by formula (I) is

[0040] [ka]

[0041] In the formula, R5 and R6 are either the same or different groups, each selected from the group consisting of hydrogen, alkyl groups, and alkoxy groups, and preferably both R5 and R6 are methyl.

[0042] In another preferred embodiment, R2, R3, and R4 in the cationic metal porphyrin complex represented by formula (I) are together

[0043] [ka]

[0044] In the formula, R9 is the same or different group, each selected from the group consisting of hydrogen, alkyl groups, and alkoxy groups, and preferably all R9 are hydrogen.

[0045] In yet another preferred embodiment, the mitochondrial membrane potential lowering agent is given by formula (II)

[0046] [ka]

[0047] This is a cationic manganese porphyrin complex represented by [formula].

[0048] In the present invention, the p53 inhibitor is not particularly limited as long as it is a compound that can inhibit the function or action of p53, but examples include compounds represented by the following formulas (III), (IV), or (V):

[0049] [ka]

[0050] Here, the p53 inhibitor of formula (III) is pifislin-α hydrobromide (PFT-α, also known as 2-(2-imino-4,5,6,7-tetrahydrobenzothiazole-3-yl)-1-p-tolyleuthanone hydrobromide). The p53 inhibitor of formula (IV) is pifislin-μ (PFT-μ, also known as pifitrin-μ, 2-phenylethinesulfonamide). The p53 inhibitor of formula (V) is p-nitropifislin-α hydrobromide (p-nitro-PFT-α, also known as 1-(4-nitrophenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazole)ethanone hydrobromide). Among these p53 inhibitors, pifislin-α hydrobromide (structural formula: formula (III)) is preferred.

[0051] Examples of epigenetic agents include, but are not limited to, DNA demethylating agents such as azacitidine and decitabine, various small molecule inhibitors such as vorinostat, panobinostat, romidepsin, belinostat, and trichostatin A, EZH2 inhibitors such as dazberg, and BET inhibitors such as (+)-JQ1, as well as plasmid DNA expressing histone modifying enzymes.

[0052] While not particularly limited, examples of anticancer drugs include platinum-based drugs such as oxaliplatin, carboplatin, cisplatin, and nedaplatin; anticancer antibiotics such as actinomycin D, acralubicin, idarubicin, epirubicin, dinostatin stimulamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, peplomycin, mitomycin C, and mitoxantrone; and irinotecan, etoposide, eribulin, sobuzoxane, docetaxel, nogitecan, paclitaxel, and vinorel. Plant alkaloids such as vin, vincristine, vindesine, and vinblastine; enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine, cytarabine ocphosphonate, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, doxifluridine, nerarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, methotrexate and other antimetabolites; ifosfamide, cyclophosph Alkylating agents such as amide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ranimustine; molecular targeted drugs such as ibritumomab tiuxetan, imatinib, everolimus, erlotinib, gefitinib, gemtuzumab ozogamicin, sunitinib, cetuximab, sorafenib, dasatinib, tamibarotene, trastuzumab, tretinoin, panitumumab, bevacizumab, bortezomib, lapatinib, and rituximab; anastrozole, exemestane, and est. Examples include hormones such as ramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, phosfestrol, mitotane, methyltestosterone, medroxyprogesterone, mepitiostane, leuprorelin, and letrozole, as well as biological response modifiers such as interferon-α, interferon-β, interferon-γ, interleukin, ubenimex, dried BCG, and lentinan.

[0053] Furthermore, the drugs that may be encapsulated in liposomes in this invention can be classified into hydrophobic drugs, hydrophilic drugs, etc. In this invention, hydrophobicity means having a weak interaction with water and a weak affinity for water. Therefore, for example, the partition coefficient P(n-octanol / water), that is, the ratio of the concentration of the drug dissolved in n-octanol to the concentration of the drug dissolved in water, can serve as an indicator of the hydrophilicity or hydrophobicity of the drug. If the partition coefficient is greater than 1.0, it is classified as a hydrophobic drug, and if it is smaller, it is classified as a hydrophilic drug. Also, when the partition coefficient is expressed logarithmically (logP), hydrophobic (lipid-soluble) drugs are positive (+) and hydrophilic (water-soluble) drugs are negative (-), and the higher the lipid solubility or hydrophilicity, the larger the absolute value of logP.

[0054] In this embodiment, the drug contained in the liposome can be used alone or in combination of two or more, and it is preferable to use a combination of two or more (for example, two types, two to three types, two to four types, etc.). When using two or more drugs, it is preferable to include both a hydrophilic drug and a hydrophobic drug from the viewpoint of structural stability of the liposome and control of the amount of drug contained.

[0055] In the present invention, the liposome dispersion itself may be used as the liposome composition, but the liposome composition of the present invention may contain the above-mentioned drugs, pH adjusters, preservatives, stabilizers, antioxidants, etc.

[0056] Examples of pH adjusting agents include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, and alkali metal citrates such as sodium citrate.

[0057] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and other quaternary ammonium salts, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine.

[0058] Examples of stabilizers include sodium bisulfite.

[0059] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols.

[0060] The liposome composition of the present invention can be formed, for example, by the following procedure. 1. The lipids and cholesterol constituting the liposomes are first mixed in an organic solvent, and then the organic solvent is removed by distillation to obtain a dry thin film; 2. Add water to the obtained dried thin film and perform ultrasonic stirring to obtain a liposome dispersion.

[0061] Examples of organic solvents that can be used include methanol, chloroform, ethanol, and acetone. pH adjusters, preservatives, stabilizers, antioxidants, etc., may be added to the above organic solvent, water, etc., or to the liposome dispersion. Furthermore, when using a hydrophobic agent among the aforementioned agents, for example, when obtaining a dry thin film in step 1, the lipids and cholesterol constituting the liposomes and the hydrophobic agent can be mixed in an organic solvent first, and then the organic solvent can be removed by distillation to incorporate the hydrophobic agent into the lipid bilayer. Furthermore, when using a hydrophilic agent among the aforementioned agents, for example, in step 2, an aqueous solution containing the hydrophilic agent can be added to the dry thin film instead of water, and ultrasonic stirring can be performed to incorporate the hydrophilic agent into the internal aqueous phase of the liposomes.

[0062] Methods for promoting cell proliferation In one embodiment, the present invention provides a method for promoting cell proliferation, comprising the step of adding the liposome composition to cells.

[0063] The target cells are not particularly limited, but examples include those derived from mammals, amphibians, birds, etc. Examples of mammals include humans, rats, mice, dogs, cats, monkeys, cattle, horses, sheep, goats, hamsters, pigs, etc., with humans being preferred. The cells may be primary cultured cells, established cell lines, etc. The cells may be isolated cells, aggregated in a planar form or as a cell mass, or tissue fragments, etc. Therefore, typically in the present invention, the liposome composition is added to cells in vitro. The amount of liposome composition to be added is not particularly limited, but for example, it can be set in the range of 1 to 100 μg / mL, preferably 10 to 50 μg / mL, in a cell culture medium. The temperature when adding the liposome composition to cells is not particularly limited, but for example, it can be set in the range of 15 to 30°C, preferably 20 to 25°C. In this embodiment, it is preferable that the liposome composition contains drugs that contribute to the suppression of cellular senescence, cell death, etc. The liposome composition of the present invention is useful because, despite having a negative zeta potential, it is easily taken up by cells, and the liposomes themselves have low cytotoxicity. By adding a liposome composition containing drugs that contribute to the suppression of cellular senescence and cell death to cells, cellular senescence and cell death are suppressed, and as a result, cell proliferation is promoted.

[0064] Method for preparing cultured cells In another embodiment, the present invention provides a step of culturing cells in the presence of the liposome composition described above. Steps to collect the cells obtained from the culture as described above. This invention provides a method for producing cultured cells, including [specific details omitted].

[0065] The target cells, the concentration of the liposome composition in the culture process, etc., are the same as described above. Liquid media are preferred as the culture medium. In addition to basic media such as DMEM medium, MEM medium, and RPMI medium, stem cell media such as PRIME-XV MSC Expansion® and Essential 8®, immune system cell media such as NK cell subculturing medium and αβ T cell subculturing medium, and cancer cell proliferation media can be used as liquid media. Additives such as serum, buffers, growth factors, differentiation factors, stabilizers, antioxidants, antibiotics, physiologically active molecules (amino acids, nucleic acids, vitamins, lipids, proteins, carbohydrates, insulin, transferrin, serine (sodium selenite), sodium pyruvate, glutamine, ethanolamine, linoleic acid, oleic acid, bovine serum albumin (BSA), etc.), cholesterol, lactoferrin, glucagon, cyclodextrin, and yeast extract may be added to the medium. The culture temperature is 37°C. The culture time is not particularly limited and can be set in the range of 12 to 360 hours, preferably 24 to 72 hours. The recovery step can be any method widely used in the field of cell culture to which the present invention belongs. Methods that can be used in the recovery step include washing, peeling, centrifugation, filtration, and adsorption.

[0066] In this embodiment as well, it is preferable that the liposome composition contains agents that contribute to the suppression of cellular senescence, cell death, etc., as described above. The liposome composition of the present invention is easily taken up by cells despite having a negative zeta potential, and the liposome itself has low cytotoxicity. Therefore, by adding a liposome composition containing agents that contribute to the suppression of cellular senescence, cell death, etc., to cells, cellular senescence and cell death are suppressed, making it useful to obtain a cell population with a high proportion of non-senescent cells.

[0067] Specific embodiments of the present invention will be described in detail below using examples, but the present invention is not limited to these examples. In the following examples, the liposome particle size (average particle size) was measured by dynamic light scattering. [Examples]

[0068] Example 1: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added, and then 37.3 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl, 42.1 μL of 10 mg / mL cholesterol (Chol) in CHCl, and 20.6 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0069] To the obtained dried thin film, 1000 μL of distilled water and 1 mg / mL of the solution were added, and vortexing was performed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0070] Example 2: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 15.2 μL of 10 mg / mL dioleoylphosphatidylethanolamine (DOPE) in CHCl3, 11.5 μL of 10 mg / mL dioleoylphosphatidylcholine (DOPC) in CHCl3, 15.8 μL of 10 mg / mL cholesterol (Chol) in CHCl3, 33.1 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl3, and 14.4 μL of 10 mg / mL sphingomyelin (SM) in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0071] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed at 45°C for 30 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0072] Example 3: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added, and then 350.0 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl, 324.0 μL of 10 mg / mL cholesterol (Chol) in CHCl, and 326.0 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0073] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0074] Example 4: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 20.1 μL of 10 mg / mL dioleoylphosphatidylethanolamine (DOPE) in CHCl3, 25.5 μL of 10 mg / mL dioleoylphosphatidylcholine (DOPC) in CHCl3, 17.9 μL of 10 mg / mL cholesterol (Chol) in CHCl3, 17.5 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl3, and 19.0 μL of 10 mg / mL sphingomyelin (SM) in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0075] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed at 45°C for 30 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0076] Example 5: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added, and then 354.8 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl, 326.8 μL of 10 mg / mL cholesterol (Chol) in CHCl, and 318.4 μL of 10 mg / mL lysophosphatidylglycerol (Lyso PG) in CHCl were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0077] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0078] Example 6: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added, and then 350.2 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl, 324.5 μL of 10 mg / mL cholesterol (Chol) in CHCl, and 325.3 μL of 10 mg / mL phosphatidylglycerol (DOPG) in CHCl were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0079] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0080] Example 7: Preparation of liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added, and then 351.4 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl, 325.1 μL of 10 mg / mL cholesterol (Chol) in CHCl, and 323.5 μL of 10 mg / mL phosphatidic acid (DOPA) in CHCl were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0081] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0082] Example 8: Preparation of Rhodamine B and FITC-containing liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 50.0 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl3, 24.0 μL of 10 mg / mL cholesterol (Chol) in CHCl3, 26.0 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl3, and 50 μL of 1 mg / mL FITC in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0083] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Rhodamine B in H2O were added, and the mixture was vortexed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0084] Example 9: Preparation of Rhodamine B and FITC-containing liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added, and then 20.1 μL of 10 mg / mL dioleoylphosphatidylethanolamine (DOPE) in CHCl3, 25.5 μL of 10 mg / mL dioleoylphosphatidylcholine (DOPC) in CHCl3, 10 mg / mL cholesterol (Chol) in CHCl3, 17.9 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl3, 17.5 μL of 10 mg / mL sphingomyelin (SM) in CHCl3:CH3OH=2:1, and 50 μL of 1 mg / mL FITC in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0085] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Rhodamine B in H2O were added, and the mixture was vortexed at 45°C for 30 minutes. Subsequently, sonic agitation was performed for 30 minutes to obtain the desired liposome dispersion.

[0086] Example 10: Preparation of Rhodamine B and FITC-containing liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 54.8 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl3, 26.8 μL of 10 mg / mL cholesterol (Chol) in CHCl3, 18.4 μL of 10 mg / mL lysophosphatidylglycerol (Lyso PG) in CHCl3, and 50 μL of 1 mg / mL FITC in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0087] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Rhodamine B in H2O were added, and the mixture was vortexed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0088] Example 11: Preparation of Rhodamine B and FITC-containing liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 50.2 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl3, 24.5 μL of 10 mg / mL cholesterol (Chol) in CHCl3, 25.3 μL of 10 mg / mL phosphatidylglycerol (DOPG) in CHCl3, and 50 μL of 1 mg / mL FITC in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0089] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Rhodamine B in H2O were added, and the mixture was vortexed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0090] Example 12: Preparation of Rhodamine B and FITC-containing liposome nanocarriers In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl3 (51.4 μL), 10 mg / mL cholesterol (Chol) in CHCl3 (25.1 μL), 10 mg / mL phosphatidic acid (DOPA) in CHCl3 (23.5 μL), and 1 mg / mL FITC in CHCl3:CH3OH=2:1 (50 μL) were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0091] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Rhodamine B in H2O were added, and the mixture was vortexed at 70°C for 10 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the desired liposome dispersion.

[0092] Example 13: Preparation of liposome nanocarriers containing Mn porphyrin and PFT-α In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 50.0 μL of 10 mg / mL distearoylphosphatidylcholine (DSPC) in CHCl3, 24.0 μL of 10 mg / mL cholesterol (Chol) in CHCl3, 26.0 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl3, and 50 μL of 1 mg / mL PFT-α in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0093] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Mn porphyrin in H2O were added, and the mixture was vortexed at 70°C for 10 minutes. Subsequently, sonic stirring was performed for 30 minutes to obtain the target Mn porphyrin and PFT-α-containing liposome dispersion. The particle size was 50.5 ± 11.8 nm.

[0094] Example 14: Preparation of liposome nanocarriers containing Mn porphyrin and PFT-α In a 20 mL round-bottom flask, 1000 μL of chloroform and 500 μL of methanol were added. Then, 20.1 μL of 10 mg / mL dioleoylphosphatidylethanolamine (DOPE) in CHCl3, 25.5 μL of 10 mg / mL dioleoylphosphatidylcholine (DOPC) in CHCl3, 10 mg / mL cholesterol (Chol) in CHCl3, 17.9 μL of 10 mg / mL dioleoylphosphatidylserine (DOPS) in CHCl3, 17.5 μL of 10 mg / mL sphingomyelin (SM) in CHCl3:CH3OH=2:1, and 50 μL of 1 mg / mL PFT-α in CHCl3:CH3OH=2:1 were dissolved. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0095] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Mn porphyrin in H2O were added, and vortexing was performed at 45°C for 30 minutes. Subsequently, ultrasonic stirring was performed for 30 minutes to obtain the target Mn porphyrin and PFT-α-containing liposome dispersion. The particle size was 50.5 ± 11.8 nm.

[0096] Comparative Example 1: Preparation of Cationic Liposome Nanocarriers In a 20 mL round-bottom flask, 500 μL of chloroform and 500 μL of methanol were added. Then, 50 μL of 10 mg / mL dioleoylphosphatidylethanolamine (DOPE) in CHCl3:CH3OH = 2:1 and 50 μL of 10 mg / mL N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium (DOTMA) in CHCl3:CH3OH = 2:1 were added and dissolved in 2 mL of chloroform. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0097] 1000 μL of distilled water was added to the obtained dried thin film, and vortexing was performed for 20 minutes while cooling with ice water. Subsequently, ultrasonic stirring was performed for 20 minutes to obtain the desired liposome dispersion.

[0098] Comparative Example 2: Preparation of Mn porphyrin and PFT-α-containing cationic liposome nanocarriers In a 20 mL round-bottom flask, 500 μL of chloroform and 500 μL of methanol were added. Then, 50 μL of 10 mg / mL dioleoylphosphatidylethanolamine (DOPE) in CHCl3:CH3OH = 2:1, 50 μL of 10 mg / mL N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium (DOTMA) in CHCl3:CH3OH = 2:1, and 50 μL of 1 mg / mL LPFT-α in CHCl3:CH3OH = 2:1 were added and dissolved in 2 mL of chloroform. The solvent was removed using a rotary evaporator to prepare a dry thin film.

[0099] To the obtained dried thin film, 950 μL of distilled water and 50 μL of 1 mg / mL Mn porphyrin in H2O were added, and the mixture was vortexed for 20 minutes while cooling with ice water. Subsequently, ultrasonic stirring was performed for 20 minutes to obtain the target Mn porphyrin and PFT-α-containing liposome dispersion.

[0100] [Table 1]

[0101] [Table 2]

[0102] [Table 3]

[0103] Example 15: Evaluation of intracellular uptake of liposomes and co-delivery of encapsulated drugs Liposomes containing Rhodamine B and FITC, prepared in Examples 10, 11, and 12, were mixed with HCT116 cells (in DEEM medium at a lipid concentration of 50 μg / mL, 5.0 × 10⁶ cells). 4 The cells were administered to cells seeded at a concentration of cells / mL and observed 24 hours later using a confocal laser microscope. The results are shown in Figure 1. As shown in Figure 1, both types of fluorescent dyes were taken up into the cells in each liposome, and the encapsulated drug could be co-delivered into the cells.

[0104] Example 16: Evaluation of liposome stability The liposomes prepared in Examples 1-7 were left standing in a dispersion state at a lipid concentration of 1 mg / mL, 4°C, and the average particle size was measured before storage, and after one week, two weeks, and three weeks. The average particle size was measured by dynamic light scattering. The results are shown in Table 4. As shown in Table 4, no change in the average particle size was observed for any of the liposomes, indicating that their morphology was very stable.

[0105] [Table 4]

[0106] Example 17: Evaluation of liposome cytotoxicity The empty liposomes prepared in Examples 3 and 4 were placed in 5.0 × 10¹⁶ XSFM medium containing aged mesenchymal stem cells (Prime XV MSCs) with a lipid concentration in the range of 0–50 μg / mL. 4 The liposome composition was administered to seeded cells at a concentration of cells / mL, and the number of viable cells was measured 48 hours later using the Cell Counting Kit-8 (CCK-8). The results are shown in Table 5. As shown in Table 5, while conventional liposome compositions composed of cationic and neutral lipids showed cytotoxicity when the dose exceeded 15 μg / mL, the number of viable cells did not change even when the concentration of the liposome composition of the present invention was increased to 50 μg / mL, indicating a significant reduction in toxicity to cells.

[0107] [Table 5]

[0108] Example 18: Evaluation of cell proliferation rate Liposomes containing Mn porphyrin and PFT-α prepared in Examples 13 and 14 were placed in aged mesenchymal stem cells (Prime XV MSC expansion XSFM medium) at lipid concentrations ranging from 0 to 50 μg / mL in a range of 5.0 × 10⁶ 4 The liposome was administered to seeded cells at a concentration of cells / mL, and the number of viable cells was measured 48 hours later using the Cell Counting Kit-8 (CCK-8). The results are shown in Table 6. As shown in Table 6, with conventional liposome compositions consisting of cationic and neutral lipids, cell viability began to decrease when the dose exceeded 15 μg / mL, whereas with the liposome composition of the present invention, cell viability increased in proportion to the added concentration, and recovery from cell proliferation arrest was possible while reducing toxicity to cells.

[0109] [Table 6]

[0110] Example 19: Evaluation of intracellular uptake of liposomes and co-delivery of encapsulated drugs Liposomes containing Rhodamine B and FITC prepared in Examples 8 and 9 were mixed in mesenchymal stem cell (Prime XV MSC) expansion XSFM medium at a lipid concentration of 50 μg / mL in 5.0 × 10⁶ cells. 4 The cells were administered to cells seeded at a concentration of cells / mL and observed 24 hours later using a confocal laser microscope. The results are shown in Figure 2. As shown in Figure 2, both types of fluorescent dyes were taken up into the cells in each liposome, and the encapsulated drug could be co-delivered into the cells.

[0111] Example 20: Evaluation of the cellular senescence inhibitory effect of the liposome composition Liposomes containing Mn porphyrin and PFT-α prepared in Example 13 were placed in aged mesenchymal stem cells (Prime XV MSC expansion XSFM medium) at a lipid concentration range of 0-80 μg / mL in a 1.5 × 10⁶ 5 The solution was administered to cells seeded at a concentration of cells / mL, and after 72 hours, changes in β-galactosylase activity were measured using a fluorescence microscope and flow cytometer with the SPiDER-βGal Cellular Senescence Detection Kit. The results are shown in Table 7. As shown in Table 7, senescent mesenchymal stem cells showed high β-galactosylase activity, while cells treated with the liposome composition of the present invention showed decreased β-galactosylase activity, indicating that recovery from cellular senescence was possible while reducing toxicity to cells.

[0112] [Table 7]

Claims

1. A liposome composition for cell culture comprising liposomes containing cholesterol and lipids, wherein the lipids include anionic lipids, the total proportion of cholesterol and anionic lipids among the cholesterol and lipids constituting the lipid bilayer is 40 mol% or more, the zeta potential of the liposomes is negative, the liposomes contain a hydrophilic agent and a hydrophobic agent, the hydrophobic agent is contained only inside the lipid bilayer of the liposomes, and the hydrophilic agent is contained only in the internal aqueous phase of the liposomes.

2. A liposome composition comprising liposomes containing cholesterol and lipids, wherein the lipids include anionic lipids, the total proportion of cholesterol and anionic lipids among the cholesterol and lipids constituting the lipid bilayer is 40 mol% or more, the zeta potential of the liposomes is negative, the liposomes contain a hydrophilic agent and a hydrophobic agent, the hydrophobic agent is contained only inside the lipid bilayer of the liposomes, and the hydrophilic agent is contained only in the internal aqueous phase of the liposomes.

3. A liposome composition according to claim 2 for promoting cell proliferation.

4. The liposome composition according to any one of claims 1 to 3, wherein the average particle size of the liposomes is 50 to 150 nm.

5. A liposome composition according to any one of claims 1 to 3, wherein the hydrophilic agent is an antioxidant and the hydrophobic agent is a p53 inhibitor.

6. A method for promoting cell proliferation, comprising the step of adding a liposome composition according to any one of claims 1 to 3 to cells.

7. A step of culturing cells in the presence of the liposome composition according to any one of claims 1 to 3, Steps to collect the cells obtained from the culture as described above. A method for preparing cultured cells, including [the specified term].

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