Polyphenol-treated therapeutic lyophilized cells, preparation method therefor, and pharmaceutical composition comprising same

Abstract

The present invention relates to therapeutic lyophilized or frozen cells with surfaces treated with polyphenols, a preparation method therefor, and a pharmaceutical composition comprising same. The polyphenol-treated therapeutic frozen (lyophilized) cells (lyocells) of the present invention function as bio-reservoirs capable of controlling the release of various bioactive molecules including endogenous cellular substances and therapeutic drugs. In addition, the polyphenol-treated therapeutic frozen cells contain increased and / or concentrated intracellular bioactive substances, for example, growth factors, immunoregulatory factors, and the like, and thus can be utilized as a composition for the prevention or treatment of ischemic diseases through an angiogenesis promoting effect and as a composition for the prevention, alleviation, or treatment of inflammatory diseases through an enhanced immunomodulatory function. Furthermore, when the therapeutic cells (lyocells) are used as an appropriate drug delivery technology before the freezing (lyophilization) process, it is possible to expect the construction of a cell medicine system that can be simply and effectively utilized.

Description

Freeze-dried cells for polyphenol-treatment therapy, method for preparing the same, and pharmaceutical composition comprising the same

[0001] The present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the project number 00215511, and the research management specialized institution of the project is the Inter-Ministry Regenerative Medicine Technology Development Project Foundation (Foundation), the research project name is “Inter-Ministry Regenerative Medicine Technology Development Project”, the research project name is “Development of High-Functional Pulmonary Fibrosis Cell Therapy Based on Sustained-Release Drug Delivery System-Stem Cell Hybrid Technology”, the main institution is the Industry-Academic Cooperation Foundation of Sungkyunkwan University, and the research period is from 2023.04.01 to 2023.12.31.

[0002] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0125918, filed with the Korean Intellectual Property Office on September 20, 2023, the disclosure of which is incorporated herein by reference.

[0003] The present invention relates to polyphenol-treated lyophilized cells for therapeutic use, a method for preparing the same, and a pharmaceutical composition comprising the same. Specifically, the present invention relates to frozen or lyophilized polyphenol-treated cells for therapeutic use, a method for preparing the same, and a pharmaceutical composition comprising the same.

[0004]

[0005] Cell-based therapies, including mesenchymal stem cells (MSCs), fibroblasts, dendritic cells, and T cells, have been reported to have tremendous potential in the treatment of various intractable diseases, but existing treatment methods using these cells have low therapeutic efficacy as a limitation.

[0006] In particular, living-cell-based therapies face significant limitations in commercialization. Specifically, therapeutic cells, including mesenchymal stem cells, face challenges in standardizing their isolation, culture, and evaluation. Furthermore, they carry the risk of exerting effects in non-target areas. Furthermore, it may be difficult to expect appropriate therapeutic effects depending on the condition and / or severity of each patient. Furthermore, therapeutic cells exhibit low cell viability in harsh in vivo environments. Furthermore, storage, transportation, and utilization of these cells before their use as therapeutic agents pose significant challenges.

[0007] In general, lyophilization (freeze drying) is well known as an important technology for long-term storage, delivery, and use of biological products (antibodies, proteins, peptides, etc.) without compromising biological activity prior to clinical application (Arto Merivaara et al., 2021). However, when freeze-drying living cells, the cell structure is destroyed during the freeze-drying process, leading to cell death. Therefore, various freeze-drying protectants, such as dimethylsulfoxide (DMSO), glycerol, trehalose, and serum albumin, are used to prevent cell damage during freeze-drying and enable long-term storage of cells. However, to date, no study has demonstrated the therapeutic potential of freeze-dried therapeutic cells.

[0008] The present inventors sought to demonstrate the therapeutic potential of lyophilized therapeutic cells based on the hypothesis that maintaining high cell viability is only necessary for some treatments (e.g., chimeric antigen receptor (CAR)-T cells) and that apoptotic cells or dead cells can also induce certain therapeutic effects.

[0009]

[0010] The present inventors have diligently researched and developed therapeutic cells containing various bioactive substances, including endogenous substances and / or therapeutic drugs, for the effective treatment of various diseases. As a result, it was confirmed that mesenchymal stem cells treated with polyphenol and then freeze-dried exhibited cell death and loss of cell viability, but intracellular bioactive substances were stably preserved. Furthermore, the inventors have completed the present invention by elucidating that the polyphenol-treated freeze-dried mesenchymal stem cells have angiogenesis-promoting effects, enhanced immunomodulatory functions, and potential use as a drug delivery system.

[0011] Accordingly, it is an object of the present invention to provide freeze-dried cells for polyphenol-treatment therapy.

[0012] Another object of the present invention is to provide a method for producing freeze-dried cells for polyphenol-treatment treatment of the present invention.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating ischemic disease, comprising polyphenol-treated frozen (dried) cells.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating inflammatory diseases, comprising polyphenol-treated therapeutic freeze-dried cells.

[0015] Another object of the present invention is to provide a pharmaceutical composition for drug delivery comprising polyphenol-treated therapeutic freeze-dried cells.

[0016]

[0017] The present inventors have diligently researched and developed therapeutic cells containing various bioactive substances, including endogenous substances and therapeutic drugs, for the effective treatment of various diseases. As a result, we confirmed that mesenchymal stem cells treated with polyphenol and then freeze-dried exhibited cell death and loss of cell viability, but intracellular bioactive substances were stably preserved. We also elucidated that the polyphenol-treated freeze-dried mesenchymal stem cells have angiogenesis-promoting effects, enhanced immunomodulatory functions, and potential as a drug delivery system.

[0018] According to one aspect of the present invention, the present invention provides a polyphenol-treated frozen cell for treatment.

[0019] The term "therapeutic cell" as used herein refers to various types of cells used for the purpose of treating diseases. The therapeutic cell may be a variety of cells, such as mesenchymal stem cells, dendritic cells, T-lymphocytes, natural killer cells, fibroblasts, myeloid cells, etc., and includes both primary cells and secondary cells.

[0020] The term "mesenchymal stem cells (MSCs)" used herein refers to a type of adult stem cell, which is a multipotent progenitor cell derived from various tissues including umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0021] The above mesenchymal stem cells (MSCs) have the ability to differentiate into various connective tissues, such as cartilage, bone tissue, ligaments, and bone marrow stroma. In addition to these structural support functions, they also have functions such as promoting angiogenesis, immunoregulating, and suppressing, thereby inhibiting inflammatory responses and making them suitable stem cells for tissue regeneration, making them highly useful as cell therapy. However, therapies based on MSCs face many limitations in their universal expansion and commercialization. In particular, living stem cell-based therapies face difficulties in producing and storing them on a clinically relevant scale.

[0022] In general, the use of freezing techniques such as freezing (drying) is essential for the long-term preservation of living cells isolated from various body tissues, including mesenchymal stem cells (MSCs). This freezing (drying) method is very useful not only for the long-term storage of living cells in case of emergency, but also for the differentiation of stem cells into various cells for subsequent tissue regeneration and transplantation for treatment.

[0023] Therefore, in one embodiment of the present invention, the frozen cells may be lyophilized.

[0024] The term "freezing" as used herein means rapidly lowering the temperature of a container to cool a sample in a liquid state, etc., thereby solidifying or crystallizing the sample into ice, or converting a sample in a coolable solution state into ice, and is typically used to make it easy to preserve cells or microorganisms having biological activity for a long period of time by placing them in an environment such as 0°C to -170°C, -10°C to -170°C, -20°C to -170°C, -50°C to -170°C, -70°C to -170°C, or -80°C to -170°C.

[0025] The freezing may include any suitable method commonly used in the art to which the present invention pertains, and on a small scale, for example, in a laboratory, it may be performed by placing the material in a freeze-drying flask and rotating the flask in a bath known as a shell freezer, for example, by cooling through a freezing machine, dry ice mixed with an alcohol such as ethanol or methanol, or liquid nitrogen, and it is also possible to use a commercially available freeze-drying apparatus.

[0026] The term "freeze drying" as used herein is also known as lyophilisation, lyophilization, or cryodesiccation, and refers to a drying method in which the temperature of a container is rapidly lowered to cool a sample in a liquid state, etc., and then the pressure inside the container is vacuumed to remove moisture in the solidified (or ice-crystallized) sample by sublimation.

[0027] The above freezing (drying) method is a method that minimizes damage to heat-sensitive materials and enables effective long-term preservation. It is widely used for the preservation of samples containing water, including biological samples, and has the advantages of maintaining the original state of the sample, preventing contamination, and being useful in storage, transportation, and economic efficiency. However, since ice particles are created during the freezing (drying) process, which can cause damage to the structure or function of the sample, a process of adding a "freeze (drying) protectant" is necessary to protect the sample from the freezing phenomenon and restore its function upon rehydration.

[0028] The above "freeze (drying) protectant" refers to a substance having a protective effect during freezing or lyophilization that is commonly used in the technical field to which the present invention belongs. The freeze (drying) protectant can be purchased and used as a commercially available one, and its type is not particularly limited. The freeze (drying) protectant that is commonly used in the technical field to which the present invention belongs can be manufactured by including sugar, protein, and water-soluble polymer substances, and for example, DMSO (dimethylsulfoxide), dextran, sucrose, glycerol, mannitol, sorbitol, fructose, trehalose, raffinose, serum albumin, propylene glycol, or polyacrylamide can be used in combination according to the purpose.

[0029] However, there is an advantage in that bacteria, viruses, serum, vaccines, etc. can be preserved for a long period of time at room temperature by freezing (drying) using the above-mentioned freeze (dry) protecting agent, but in the case of eukaryotic cells, even if the above-mentioned freeze (dry) protecting agent is used, it is difficult to stably preserve the cells without damaging the cell structure such as the cell membrane during the freezing or freeze-drying process, and there is a disadvantage in that the function of the physiologically active substances within the cells cannot be maintained.

[0030] The polyphenol-treated frozen cells of the present invention are characterized in that intracellular physiologically active substances are stably preserved during the freezing (drying) process.

[0031] Specifically, the polyphenol-treated frozen cells for treatment are characterized by having lost their biological activity (viability) during the freezing or freeze-drying process, that is, being dead cells, with damaged cell membranes but maintaining a cellular morphology identical / similar to that before freezing (drying), and also stably preserving intracellular physiologically active substances.

[0032] The term "polyphenol" as used herein refers to a type of aromatic alcohol compound found in plants, and generally the core structure of the polyphenol comprises one or more phenol units having one or more hydroxyl (- OH) groups bonded to a benzene ring (a cyclic structure composed of six carbon atoms and six hydrogen atoms), and a polyphenol is formed by the bonding of multiple such phenol units. There are various types and classes of polyphenols, but some of the most well-known examples include flavonoids (anthocyanins, flavanols, catechins, etc.), stilbenes (resveratrol), lignans, and tannins.

[0033] In one embodiment of the present invention, the polyphenol may be a water soluble polyphenol.

[0034] The above water-soluble polyphenol refers to a polyphenol having two or more hydroxyl groups, and specific examples thereof include gallic acid, caffeic acid, chlorogenic acid (CGA), catechin, epicatechin gallate (EGCG), epicatechin, proanthocyanidins, luteolin, apigenin, baicalein, chrysoeriol, naringenin, hesperidin, eriodictyol, 2-methyl-1,4-naphthoquinone, flavinoquinones, tannic acid, gallotannins, ellagitannins, and derivatives thereof. It may be one or more selected from the group consisting of, but is not limited to, and if it is included in a water-soluble polyphenol, it can be applied to the present invention.

[0035] Additionally, in one embodiment of the present invention, the polyphenol may be a polyphenol derivative including a drug-polyphenol conjugate and a polymer-polyphenol conjugate, or a polyphenol-containing biomaterial.

[0036] More specifically, the polyphenol-treated therapeutic frozen cells are characterized by being treated with polyphenol before freezing / lyophilization, thereby losing the cell's biological activity (viability), i.e., being dead cells, with damaged cell membranes, but maintaining a cell morphology identical / similar to that before freezing (drying), and also stably preserving intracellular physiologically active substances.

[0037] In an embodiment of the present invention, various polyphenol groups were treated to mesenchymal stem cells (MSCs) before performing freeze (drying), and it was confirmed that the cell morphology and intracellular physiologically active substances (proteins, RNA, etc.) were stably preserved in all polyphenol-treated freeze (dried) cells.

[0038] In addition, in an embodiment of the present invention, various concentrations of polyphenol were treated to mesenchymal stem cells (MSCs) before performing freeze (drying), and it was confirmed that as the concentration of the treated polyphenol increased, the biological activity (viability) of the cells was lost and most of the cells were in a dead state. However, although the cells were in a dead state with a loss of biological activity, it was confirmed that the structural form of the cells was not damaged during the freeze (drying) process and that they had a surface that was completely maintained like a normal cell.

[0039] In addition, it was confirmed that various physiologically active substances and genetic materials, such as proteins and RNA, within the cells were stably preserved by having a surface that was completely maintained like a normal cell without damage to the structural form of the cells after freezing (drying), and it was also confirmed that secretion of physiologically active substances within the cells was continuously carried out.

[0040] The above “intact” means that after the freeze (dry) process, the structural morphology of the freeze (dried) cell remains identical or similar to the structural morphology of a normal cell.

[0041] The above “normal cells” are cells used as a control in the present invention, and refer to living cells that are not treated with polyphenol and are not freeze-dried.

[0042] Accordingly, the present invention provides a polyphenol-treated therapeutic frozen cell characterized in that the cell itself is lost in a state of biological activity (viability) by treating the cell with polyphenol, but has a surface in which the structural form is maintained intact without damage to the cell structure during the freezing (drying) process, thereby stably preserving the physiologically active substances within the cell.

[0043] Hereinafter, in the examples of the present invention, the freeze-dried mesenchymal stem cells whose cell surface is treated with polyphenol are named “lyocells”, and the normal cells used as a control, i.e., living cells that are not treated with polyphenol and frozen (dried), are named “norcells”.

[0044] Additionally, in an embodiment of the present invention, it was confirmed that the polyphenol-treated frozen cells (lyocells) for treatment showed a high distribution mainly in lung tissue when administered intravenously (IV injection), and a high distribution in the injection site when administered intramuscularly (IM injection).

[0045] That is, since the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention exhibit high persistence in tissues when administered in vivo, excellent therapeutic effects can be expected in the target subject (site) to be treated in the future.

[0046] In the present invention, any cell that can be used for the purpose of treating a disease can be applied as a therapeutic cell of the present invention.

[0047] In one embodiment of the present invention, the cells are preferably mesenchymal stem cells (MSCs), but are not particularly limited thereto, and may be at least one selected from the group consisting of mesenchymal stem cells (MSCs), macrophages, fibroblasts, and bone marrow cells.

[0048] According to research, mesenchymal stem cells (MSCs) contain various intracellular bioactive substances, and in particular, they are known to be able to sense changes in their microenvironment through direct or indirect interactions with host cells and secrete the bioactive substances contained within the cells. When the above-described MSCs are applied to the polyphenol-treated therapeutic cryocells of the present invention, the various bioactive substances contained within the MSCs exhibit beneficial therapeutic effects, and thus can be used clinically in pharmaceuticals or cosmetic compositions.

[0049] In one embodiment of the present invention, the polyphenol-treated therapeutic cryocells of the present invention may be primed with, but are not particularly limited to, (i) hypoxia; (ii) protein transport inhibitors; (iii) cytokines; (iv) protein synthesis inhibitors; (v) small molecule compounds; (vi) growth factors; or (iv) combinations thereof.

[0050] Specifically, in one embodiment of the present invention, the cells are not particularly limited thereto, but may be mesenchymal stem cells (MSCs), and may be mesenchymal stem cells in which various physiologically active substances exhibiting beneficial therapeutic effects within the cells are increased by the priming.

[0051] The term "priming" as used herein refers to a process of increasing / concentrating the content of intracellular bioactive substances by stimulating and activating cells, i.e., a process of enhancing beneficial intracellular bioactive substances and thereby enhancing the therapeutic effect of the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention.

[0052] The term "biomaterials" as used herein refers to specific substances that contain beneficial therapeutic effects that can affect the function of cells or the body, and specifically includes various factors such as inflammatory cytokines, cell growth factors, immunomodulatory factors, and extracellular vesicles (EVs). More specific examples of biomaterials include indoleamine 2,3-dioxygenase (IDO), Programmed Cell Death Ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), Fas ligand (FasL), TNF-stimulated gene 6 (TSG6), microRNA, messenger RNA (mRNA), extracellular vesicles (EV), and cyclooxygenase 2 (Cyclooxygenase 2). 2, COX2), interleukins (ILs), transforming growth factor (TGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor, collagen, fibronectin, antioxidant enzymes (superoxide dismutase (SOD), etc.), but are not particularly limited thereto.

[0053] In one embodiment of the present invention, when the amount of physiologically active substances in cells is increased by priming, and then polyphenol is treated and freeze-dried, the cells have a surface in which the shape (structure) of the cells is completely maintained during the freeze-drying process, thereby stably preserving the physiologically active substances increased by the priming within the cells.

[0054] Specifically, in one embodiment of the present invention, the polyphenol-treated freeze-dried cells (lyocells) can preserve the intracellular physiologically active substances increased by the priming at a level corresponding to that of normal cells (norcells).

[0055] Therefore, when using the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention, various physiologically active substances exhibiting increased beneficial therapeutic efficacy due to priming are stably preserved within the cells, suggesting the possibility of use as an effective therapeutic agent capable of exhibiting improved therapeutic effects.

[0056] Additionally, in another embodiment of the present invention, the cell (lyocell) may contain a therapeutic drug.

[0057] Specifically, the above cells (lyocells) are characterized by having a surface in which the shape (structure) of the cells is maintained intact without being damaged during the freeze (dry) process, and by additionally including a step of including a therapeutic drug into the cells before freeze (dry) the cells, the therapeutic drug is stably preserved within the cells and can be used as an effective therapeutic agent thereafter.

[0058] In one specific example of the present invention, the drug may be supported on nanoparticles.

[0059] The above "nanoparticle" refers to one of the drug delivery systems that can increase the stability of a therapeutic drug while efficiently delivering it to a target site to be treated. For example, the nanoparticle of the present invention includes, but is not limited to, one polymer nanoparticle selected from the group consisting of poly(alkyl cyanoacrylates) (PACA), poly(ε-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly-L-lysine (PLL), polyethylene glycol (PEG), chitosan (CS), gelatin, albumin, collagen, hyaluronic acid, and derivatives thereof.

[0060] Meanwhile, in one embodiment of the present invention, the cell (lyocell) is characterized by having long-term storage stability under low-temperature conditions.

[0061] Specifically, in one embodiment of the present invention, the cells (lyocells) are stable even when stored at -120°C to 10°C for 5 to 12 months or more. For example, the temperature conditions are -120°C to 10°C, -120°C to 0°C, -120°C to -20°C, -120°C to -40°C, -120°C to -60°C, -120°C to -80°C, -120°C to -100°C -100°C to 10°C, -100°C to 0°C, -100°C to -20°C, -100°C to -40°C, -100°C to -60°C, -100°C to -80°C, -80°C to 10°C, -80°C to 0°C, -80°C to -20°C, -80°C to -40°C, -80°C to -60°C, -60°C to 10°C, -60°C to 0°C, -60°C to -20°C, -60°C to -40°C, -40°C to It may be, but is not limited to, 10°C, -40°C to 0°C, -40°C to -20°C, -20°C to 10°C, -20°C to 0°C, or 0°C to 10°C.

[0062] The above “stability” means that the cell shape, cell size, level of intracellular physiologically active substances, and therapeutic effect by the cells remain the same as before storage under the conditions described above.

[0063]

[0064] According to another aspect of the present invention, a method for preparing polyphenol-treated frozen cells for treatment is provided, comprising the following steps:

[0065] (a) a step of treating cells with polyphenol; and

[0066] (b) A step of freezing or lyophilizing the cells.

[0067] In one embodiment of the present invention, the therapeutic cells may be, but are not limited to, mesenchymal stem cells, dendritic cells, T-lymphocytes, NK cells, fibroblasts, or bone marrow cells.

[0068] In one embodiment of the present invention, the therapeutic cell may include, but is not limited to, therapeutic cells derived from any mammal, such as a human, monkey, pig, sheep, cow, horse, dog, or cat, and specifically, may be a therapeutic cell derived from a human.

[0069] In one embodiment of the present invention, the polyphenol in step (a) may be a water-soluble polyphenol.

[0070] Specifically, in one embodiment of the present invention, the polyphenol is characterized in that it is one selected from the group consisting of gallic acid, caffeic acid, chlorogenic acid (CGA), catechin, epicatechin gallate (EGCG), epicatechin, proanthocyanidins, luteolin, apigenin, baicalein, chrysoeriol, naringenin, hesperidin, eriodictyol, 2-methyl-1,4-naphthoquinone, flavinoquinones, tannic acid, gallotannins, ellagitannins, and derivatives thereof.

[0071] Preferably, in one embodiment of the present invention, the polyphenol may be tannic acid (TA).

[0072] Additionally, in one embodiment of the present invention, the polyphenol may be a polyphenol derivative or a polyphenol-containing biomaterial including a drug-polyphenol conjugate and a polymer-polyphenol conjugate.

[0073] Polyphenols, including the above tannic acid (TA), have been reported to condense macromolecules and proteins in solution, and in particular, when cells are treated with an appropriate concentration of tannic acid (TA), the viability of the cells is lost and the cells die, but the structure of the killed cells is maintained intact, so that the intracellular physiologically active components can be preserved / concentrated.

[0074] In this regard, the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention are characterized by having a surface in which the structural form of the cells is completely maintained like that of normal cells even after freezing (drying), although they exhibit a dead cell state with loss of biological activity (viability).

[0075] In one specific example of the present invention, in the case of cells killed by a method other than polyphenol treatment, specifically, heat-inactivated (HI) cells, liquid nitrogen treated (LNT) cells, and spontaneously apoptotic (SA) cells, it was confirmed that the three types of cell groups killed by a method other than polyphenol treatment were similar to the therapeutic cells (lyocells) of the present invention in that they were apoptotic cells, but the cell structure was not completely maintained and intracellular physiologically active substances (e.g., proteins, mRNA, etc.) were not preserved.

[0076] In another specific embodiment of the present invention, it was confirmed that the polyphenol-treated frozen cells (lyocells) for treatment exhibited an improved therapeutic effect on ischemic disease by stably preserving the increased effective physiologically active substances within the cells through a step of priming the cells before polyphenol treatment. However, it was confirmed that the cell group killed by a method other than the polyphenol-treatment described above, for example, HI cells, exhibited a somewhat less effective therapeutic effect compared to the lyocells of the present invention.

[0077] Therefore, the characteristics of the therapeutic frozen cells (lyocells) of the present invention are exhibited by polyphenol treatment.

[0078] In one embodiment of the present invention, the treatment with the polyphenol can be performed at various concentrations.

[0079] Specifically, in one embodiment of the present invention, as the treatment concentration of the polyphenol increases, the number of cells that have lost biological activity, i.e., cells that have lost biological activity and died, increases.

[0080] Specifically, when treating tannic acid (TA) as the polyphenol of the present invention, the tannic acid may be treated at a concentration of 0.02 to 10%, and more specifically, the concentration of the tannic acid may be 0.02 to 10%, 0.02 to 8%, 0.02 to 6%, 0.02 to 4%, 0.02 to 2%, 0.02 to 1%, 0.02 to 0.5%, 0.02 to 0.1%, 0.1 to 10%, 0.1 to 8%, 0.1 to 6%, 0.1 to 4%, 0.1 to 2%, 0.1 to 1%, 0.1 to 0.5%, 0.5 to 10%, 0.5 to 8%, 0.5 to 6%, 0.5 to 4%, 0.5 to It may be 2%, 0.5 to 1%, 1 to 10%, 1 to 8%, 1 to 6%, 1 to 4%, 1 to 2%, 2 to 10%, 2 to 8%, 2 to 6%, 2 to 4%, 4 to 10%, 4 to 8%, 4 to 6%, 6 to 10%, 6 to 8%, or 8 to 10%, but is not limited thereto. Preferably, in the present invention, the tannic acid may be treated at a concentration of 1.0%.

[0081] In the present invention, in order to increase the stability of cells during the freeze (dry) process of step (b), a freeze (dry) protection agent may be added, and the freeze (dry) protection agent may include all of monosaccharides, disaccharides, and polysaccharides.

[0082] Specifically, in one embodiment of the present invention, the freezing (drying) may be performed by adding a sugar selected from the group consisting of dextrose, maltose, glucose, lactose, sucrose, trehalose, mannose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, fructose, melezitose, melibiose, sorbitol, and triose.

[0083] In manufacturing the above polyphenol-treated frozen cells (lyocells) for treatment, the step (b) of freezing or freeze-drying the cells can be performed according to a general freeze (dry) method known in the art.

[0084] Meanwhile, in order to obtain beneficial therapeutic effects according to various physiologically active substances within the cell, a process of strengthening beneficial physiologically active substances within the cell by priming the cell may be additionally included.

[0085] In general, when cells are exposed to a harsh environment and die, cellular priming may not be sufficient, which may result in a decrease in the content of beneficial bioactive substances within the cells. In this regard, since the therapeutic frozen cells (lyocells) of the present invention utilize dead cells whose biological activity (viability) has been lost by polyphenol treatment, an additional process for priming the cells may be required before cell death occurs due to the polyphenol treatment, for example, tannic acid (TA) treatment.

[0086] Therefore, in one embodiment of the present invention, the method for producing therapeutic frozen cells (lyocells) may further include a step of priming the cells before performing step (a) of treating the cells with polyphenol.

[0087] Specifically, in one embodiment of the present invention, a step of priming cells is further included prior to step (a) of treating the polyphenol, thereby stimulating and activating the cells, thereby increasing the content of various physiologically active substances within the cells and concentrating them.

[0088] More specifically, in one embodiment of the present invention, the therapeutic cell (lyocell) of the present invention can exhibit excellent therapeutic efficacy by increasing and / or concentrating the content of beneficial physiologically active substances within the cell through priming of the cell, thereby increasing and / or concentrating the content of beneficial physiologically active substances within the cell.

[0089] In one embodiment of the present invention, the priming may be accomplished by (i) hypoxia; (ii) a protein transport inhibitor; (iii) cytokines; (iv) a protein synthesis inhibitor; (v) a small molecule compound; (vi) a growth factor; or (iv) a combination thereof.

[0090] The term "hypoxia" as used herein refers to a condition in which the amount of oxygen supplied to cultured cells is significantly lower than the normal atmospheric oxygen concentration (the atmospheric oxygen concentration is approximately 20%) in relation to in vitro experiments, and is usually set to an oxygen concentration of between 1 and 5%, but may be set differently depending on the experimental requirements.

[0091] Specifically, in one embodiment of the present invention, the hypoxic condition is comprised of an oxygen concentration of 0.5 to 10%. More specifically, the hypoxic conditions are 0.5 to 10%, 0.5 to 9%, 0.5 to 8%, 0.5 to 7%, 0.5 to 6%, 0.5 to 5%, 0.5 to 4%, 0.5 to 3%, 0.5 to 2%, 0.5 to 1%, 1 to 10%, 1 to 9%, 1 to 8%, 1 to 7%, 1 to 6%, 1 to 5%, 1 to 4%, 1 to 3%, 1 to 2%, 2 to 10%, 2 to 9%, 2 to 8%, 2 to 7%, 2 to 6%, 2 to 5%, 2 to 4%, 2 to 3%, 3 to 10%, 3 to 9%, 3 to 8%, It can be comprised of 3 to 7%, 3 to 6%, 3 to 5%, 3 to 4%, 4 to 10%, 4 to 9%, 4 to 8%, 4 to 7%, 4 to 6%, 4 to 5%, 5 to 10%, 5 to 9%, 5 to 8%, 5 to 7%, 5 to 6%, 6 to 10%, 6 to 9%, 6 to 8%, 6 to 7%, 7 to 10%, 7 to 9%, 7 to 8%, 8 to 10%, 8 to 9%, or 9 to 10%, and preferably, the hypoxic condition of the present invention can be an oxygen concentration condition of 1 to 5%, and more preferably, the hypoxic condition of the present invention can be an oxygen concentration condition of 1%.

[0092] The term "protein transport inhibitor" as used herein refers to a substance that can cause protein accumulation within a cell by blocking the process of protein transport within the cell.

[0093] Specifically, in one embodiment of the present invention, the protein transport inhibitor is characterized by being brefeldin A (BFA), monensin, or a combination thereof.

[0094] In one specific example of the present invention, when mesenchymal stem cells (MSCs) were primed under hypoxic conditions and a protein transport inhibitor and then treated with polyphenol to produce polyphenol-treated therapeutic frozen cells (lyocells), it was confirmed that the priming increased / concentrated intracellular bioactive substances, and thus the therapeutic frozen cells (lyocells) were dead cells that had lost their viability, but had a level of bioactive substances similar to that of normal cells (norcells). In addition, it was confirmed that continuous release of intracellular bioactive substances occurred and that the amount of released bioactive substances continuously increased, showing a different pattern from normal cells.

[0095] More specifically, the content of intracellular angiogenic factors was increased, and it was confirmed that it can be effectively used in the treatment of ischemic diseases through a high angiogenic effect in a mouse model.

[0096] Therefore, in the present invention, before performing step (a) of treating the cells to be manufactured with polyphenol, a step of priming the cells, for example, a step of treating them with hypoxic conditions and a protein transport inhibitor, can be further added, thereby increasing and / or concentrating the content of effective physiologically active substances, specifically, growth factors, within the cells, and thus an excellent therapeutic effect can be expected.

[0097] The term "cytokines" as used herein refers to low-molecular-weight signaling proteins mainly secreted by immune cells, which are the main communication mechanisms used in the immune system, and include both inflammatory cytokines that cause various inflammatory symptoms in the body and / or anti-inflammatory cytokines that have the function of suppressing inflammatory symptoms. Examples of the cytokines may include chemokines (CCLs), tumor necrosis factors (TNFs), interferons (IFNs), interleukins (ILs), colony-stimulating factors (CSFs), growth factors, monocyte chemoattractant proteins, etc.

[0098] Specifically, in one embodiment of the present invention, the cytokine is characterized in that it is at least one selected from the group consisting of IL-1β, IL-4, IL-10, IL-17, GM-CSF, TGF-β, TNF-α, and IFN-γ.

[0099] According to research, the most notable characteristic of mesenchymal stem cells (MSCs), one of the therapeutic cells applicable to the present invention, is its ability to control inflammation, which can be achieved by soluble factors or direct cell-to-cell contact. Specifically, mesenchymal stem cells (MSCs) are known to regulate the immune system through various mechanisms, such as suppressing inflammation by secreting anti-inflammatory factors such as prostaglandin E2 (PGE2), interleukin-10 (IL-10), HLA-G, nitric oxide, and indoleamine 2,3-dioxygenase (IDO) when activated by inflammatory cytokines expressed at the site of inflammation, such as interferon-γ (IFN-γ) and tumor necrosis factor-alpha (TNF-α), or inducing the production of regulatory T cells (Treg) or the death of immune cells involved in apoptosis.

[0100] In one specific example of the present invention, when mesenchymal stem cells (MSCs) were primed with cytokines (e.g., IFN-γ) and then treated with polyphenol to produce polyphenol-treated therapeutic frozen cells (lyocells), it was confirmed that the content of beneficial physiologically active substances within the cells, such as immunomodulatory factors such as PD-L1 and IDO (indoleamine 2,3-dioxygenase), increased. In addition, it was confirmed that compared to normal cells (norcells), the cells exhibited an increased capacity to retain immunomodulatory factors within the cells and exhibited an extended release pattern thereof.

[0101] More specifically, it was confirmed that the cells can exhibit excellent T cell immunosuppressive effects by continuously releasing immunomodulatory factors, and that they can be effectively used to treat inflammatory diseases such as colitis through the extended release pattern of immunomodulatory factors.

[0102] Therefore, in the present invention, before performing step (a) of treating the cells to be manufactured with polyphenol, a step of priming the cells, for example, a step of treating cytokines, can be further added, thereby increasing and / or concentrating the content of effective physiologically active substances, specifically, immunomodulatory factors, within the cells, and thus an excellent therapeutic effect can be expected.

[0103] The term "small molecule compound" as used herein refers to pharmacological and chemical agents such as rapamycin, tetrandrine, kynurenic acid, curcumin, valproic acid, sphingosine-1-phosphate, and lipopolysaccharide (LPS).

[0104] The term "growth factor" as used herein refers to an important protein or peptide that regulates the growth, division, and survival of cells. For example, the growth factor may be one or more selected from the group consisting of Epidermal Growth Factor (EGF), Fibroblast Growth Factors (FGF), Insulin-like Growth Factors (IGF), Nerve Growth Factor (NGF), Platelet-Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), and the like, but is not limited thereto.

[0105] In another embodiment of the present invention, the method for producing therapeutic frozen cells (lyocells) may further include a step of including a therapeutic drug in the cells before performing step (a) of treating the cells with polyphenol.

[0106] The polyphenol-treated therapeutic frozen cells (lyocells) of the present invention are characterized by stably preserving intracellular physiologically active substances. When a therapeutic drug exhibiting a specific therapeutic effect is further included in the cells, the therapeutic drug is stably preserved in the cells, so that excellent therapeutic efficacy can be expected in the future.

[0107] The above therapeutic drug may include all glucocorticoid drugs, and as a specific example, may be at least one selected from the group consisting of dexamethasone, fluticasone, betamethasone, budesonide, and mometasone furoate, but is not particularly limited thereto.

[0108] The drug may have a specific particle size, specifically, about 300 to 1,000 nm. More specifically, the drug may have a size of about 300 to 1,000 nm, 500 to 1,000 nm, 700 to 1,000 nm, 800 to 1,000 nm, 900 to 1,000 nm, 300 to 900 nm, 500 to 900 nm, 700 to 900 nm, 800 to 900 nm, 300 to 800 nm, 500 to 800 nm, 700 to 800 nm, 300 to 700 nm, 500 to 700 nm, or 300 to 500 nm, and preferably, about 700 nm.

[0109] In another specific embodiment of the present invention, the drug may be supported on nanoparticles (NPs).

[0110] It is widely known that therapeutic drugs use polymeric nanoparticles (NPs), such as PLGA-NPs, as drug delivery systems to achieve sustained and targeted drug release. These polymeric nanoparticles are characterized by enhancing the stability of therapeutic drugs while enabling efficient delivery to the target site of treatment. Recent studies have demonstrated that small nanoparticles (less than 100 nm) exhibit higher penetration efficiency than larger nanoparticles, and several studies have confirmed that surface coating of nanoparticles can increase penetration into the target site. For example, various substances such as surfactants, lipids, ligands, chitosan, and albumin are being used to modify the surface of nanoparticles.

[0111] As a specific example, the polymer nanoparticle applicable to the present invention may be at least one selected from the group consisting of poly(alkyl cyanoacrylates), poly(ε-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), Poly-L-lysine (PLL), polyethylene glycol (PEG), chitosan (CS), gelatin, albumin, collagen, hyaluronic acid, and derivatives thereof, but is not particularly limited thereto.

[0112] However, nanoparticles (NPs) often exhibit variable biodistribution and short residence times in the body due to rapid phagocytosis and nonspecific uptake by other cells, and therefore, it is necessary to improve the stability and in vivo persistence of therapeutic drugs to use them more effectively.

[0113] The polyphenol-treated therapeutic frozen cells (lyocells) of the present invention are characterized by not only stably preserving intracellular physiologically active substances, but also exhibiting excellent in vivo persistence when administered in vivo. Therefore, when a therapeutic drug loaded onto the nanoparticles is applied to the present invention, not only can the in vivo stability of the nanoparticles be increased, but also excellent therapeutic effects are expected through high persistence at the target site after in vivo administration of the therapeutic drug loaded onto the nanoparticles.

[0114]

[0115] According to another aspect of the present invention, a pharmaceutical composition for the prevention and treatment of ischemic disease is provided, comprising polyphenol-treated frozen cells (lyocells).

[0116] The term "ischemic disease" as used herein refers to a disease that occurs due to partial blood insufficiency caused by various causes, such as blood leakage due to blood vessel damage, narrowing or constriction of arteries, thrombosis or embolism. In particular, in order for each part of the body to function normally, an appropriate supply of oxygen and nutrients through blood vessels must be made. However, when the blood supply to the heart and other tissues is blocked for various reasons, preventing the supply of oxygen and various nutrients contained in the blood, ischemic disease occurs, and thus, it can be said to be a representative disease that requires angiogenesis. Examples of ischemic diseases include, but are not limited to, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina pectoris, stroke, ischemic disease of the lower extremities, ischemic disease of the extremities, peripheral vascular disease, cerebrovascular dementia, cerebral failure, vascular insufficiency, and coronary artery insufficiency, and all ischemic diseases that require angiogenesis may be included in the scope of the present invention.

[0117] The above "angiogenesis" refers to the process of new blood vessel formation, i.e., the generation of new blood vessels into cells, tissues or organs, and includes both cases where new blood vessels are created (vasculogenesis) and cases where new blood vessels extend from existing blood vessels (angiogenesis).

[0118] That is, the pharmaceutical composition according to the present invention is expected to have the effect of functionally improving ischemic-related tissue damage and injury by providing blood vessels that supply nutrients, growth factors, and oxygen necessary for metabolism of hypoxic or ischemic tissues and organs due to the blood circulation disorder.

[0119] In one specific example of the present invention, when mesenchymal stem cells (MSCs) are primed under hypoxic conditions and / or with a protein transport inhibitor, and then the cell surface is treated with polyphenol to produce polyphenol-treated therapeutic frozen cells (lyocells), it was confirmed that the cells exhibit an enhanced angiogenic effect by containing increased physiologically active substances, specifically, increased angiogenic factors (VEGF), within the cells.

[0120] More specifically, when the conditioned medium in which the above-mentioned therapeutic cells (lyocells) were cultured (specifically, including primed and increased and / or concentrated angiogenic factor (VEGF)) were treated to HUVEC cells, it was confirmed that cell viability, cell mobility, and vascular tube formation were improved, and it was confirmed that this result was similar to that of EGM-2 (Endothelial growth medium 2).

[0121] In addition, more specifically, it was confirmed that the manipulated therapeutic cells (lyocells) have a therapeutic effect on ischemic disease by regulating the expression of angiogenesis-related genes (e.g., vegfa, fgf2, and pdgf genes) and inflammation-related genes (e.g., tnfa gene) in mice in which ischemic symptoms were induced.

[0122] Therefore, in the present invention, the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention exhibit an excellent angiogenesis promoting effect by having intracellularly increased and / or concentrated growth factors, and thus can be easily used for the prevention or treatment of ischemic diseases.

[0123]

[0124] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease is provided, comprising lyocells treated with freeze-dried polyphenols.

[0125] The term "inflammatory disease" as used herein refers to a disease that occurs when the body's immune system overreacts due to the interaction of environmental and genetic factors, and is characterized by chronic inflammation resulting from an excessive immune response, resulting in pain, damage, and dysfunction.

[0126] In one specific example of the present invention, when mesenchymal stem cells (MSCs) were primed with cytokines (e.g., IFN-γ) and then treated with polyphenol to produce polyphenol-treated therapeutic frozen cells (lyocells), it was confirmed that the primed cells contained increased physiologically active substances, specifically, increased IDO (indoleamine 2,3-Dioxygenase) and PD-L1 (Programmed Death Ligand 1), thereby having an enhanced immune regulatory function.

[0127] More specifically, when the therapeutic cells (lyocells) containing the manipulated (specifically, primed, increased and / or enriched immunomodulatory factors (e.g., IDO and PD-L1) were treated in an acute colitis model (mouse) induced by dextran sodium sulfate (DSS), it was confirmed that the body weight and colon length of the mice were improved, indicating a therapeutic effect on colitis.

[0128] Therefore, in the present invention, the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention can not only stably preserve intracellular physiologically active substances, such as immunoregulatory factors such as IDO and PD-L1, but can also be effectively used for the prevention, improvement, or treatment of inflammatory diseases such as colitis through the increased and / or concentrated immunoregulatory function.

[0129] In one embodiment of the present invention, the inflammatory disease may be an inflammatory bowel disease selected from the group consisting of ulcerative colitis, Crohn's disease, Behcet's enteritis, infectious enteritis, radiation enteritis, ischemic bowel disease, irritable bowel syndrome, etc., and is not particularly limited thereto, and may include any inflammatory disease caused by an excessive immune response in the body.

[0130] In a specific embodiment of the present invention, the inflammatory disease may be a disease related to the lung. Examples of specific lung diseases include interstitial lung disease, progressive fibrosing interstitial lung disease, idiopathic interstitial pneumonia, nonspecific interstitial pneumonia, pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, alveolitis, pneumonia, emphysema, bronchitis, chronic obstructive pulmonary disease (COPD), combined pulmonary fibrosis and emphysema (CPFE), asthma, and respiratory infectious diseases (e.g., coronavirus disease 2019 (COVID-19)), and the lung disease may be an inflammatory lung disease caused by an inflammatory response.

[0131] The term “prevention” as used herein means any action that inhibits or delays the growth, spread or recurrence of a disease, disorder or symptom by administration of the composition of the present invention.

[0132] The term "treatment" as used herein means suppressing, alleviating, or eliminating the development of a disease, condition, or symptom by administration of the composition of the present invention. It also means any action by which a disease, condition, or symptom is improved or beneficially altered.

[0133] The term “administration” as used herein means providing a composition of the present invention to a subject by any suitable means.

[0134] The term "subject" as used herein refers to an animal whose disease, illness, or symptom can be improved by administering the composition of the present invention, and may be a vertebrate, preferably a mammal, and may include, but is not limited to, all animals such as humans, monkeys, dogs, cats, goats, pigs, rats, guinea pigs, hamsters, chimpanzees, or gorillas. More preferably, the subject of the present invention may be a human.

[0135] The term "therapeutically effective amount" as used herein means an amount sufficient to provide a therapeutic or prophylactic effect to a subject to whom the composition is to be administered, and includes a "prophylactically effective amount." This may be determined based on factors including the type and severity of the subject's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0136] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a suitable organic or inorganic carrier material that does not detrimentally react with the cells, compositions or components of the present invention, and may include, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, PBS (phosphate buffered saline) or medium, etc.

[0137] The pharmaceutical composition of the present invention may further comprise, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0138] The composition of the present invention, as a cell therapeutic agent, can be administered orally or parenterally. Parenteral administration can be achieved by intraperitoneal administration, intrarectal administration, subcutaneous administration, intravenous administration, intramuscular administration, intrauterine epidural administration, intrathecal administration, intracerebroventricular administration, intravascular injection, or intrathoracic injection, and can be administered directly to the site of the disease.

[0139] The above solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc. These solid preparations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.

[0140] The above-mentioned parenteral administration preparations may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0141] The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. The general dosage of mesenchymal stem cells, which are the active ingredient of the pharmaceutical composition of the present invention, is 10 per day for adults. 2 -10 10 It may be, but is not limited to, a cell.

[0142] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0143]

[0144] In addition, according to another aspect of the present invention, a pharmaceutical composition for drug delivery is provided, comprising polyphenol-treated therapeutic frozen cells (lyocells) carrying a drug.

[0145] The polyphenol-treated therapeutic frozen cells (lyocells) of the present invention are characterized by existing in a dead cell state with loss of cellular viability, but stably preserving intracellular physiologically active substances. That is, the therapeutic frozen (dried) cells of the present invention are advantageous in that they are easy to transport, store, and preserve because they exist in a dead state, and can stably preserve intracellular substances.

[0146] Therefore, a pharmaceutical composition for drug delivery can be provided by loading a therapeutic drug into the polyphenol-treated therapeutic frozen cell (lyocell) of the present invention, which suggests that the therapeutic cell (lyocell) of the present invention can be used as an effective drug delivery system.

[0147] The above "Drug Delivery System (DDS)" refers to a system that controls the release and absorption of a drug or delivers a drug to a specific part of the body, thereby reducing side effects of the drug and selectively delivering the drug to the target part to maximize therapeutic efficacy and effect.

[0148] In one embodiment of the present invention, the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention exist in a dead cell state with lost cellular viability, so that not only is the transportation, storage, and preservation of the cells easy, but also the intracellular physiologically active substances can be stably preserved, so that they can be used as an effective drug delivery system.

[0149] In a specific embodiment of the present invention, when the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention are used as a drug delivery system, the drug may be loaded onto nanoparticles (NPs).

[0150] In one specific example of the present invention, it was confirmed that polyphenol-treated therapeutic cryocells (lyocells) produced after absorbing nanoparticles loaded with therapeutic drugs into mesenchymal stem cells (MSCs) were mostly located in the lungs after administration and exhibited high in vivo persistence.

[0151] That is, it was confirmed that the nanoparticles loaded with the therapeutic drug loaded within the cells exhibited excellent in vivo persistence, particularly, high persistence in lung tissue.

[0152] Therefore, in one embodiment of the present invention, the drug applied to the present invention may be a drug for treating lung diseases, but is not limited thereto.

[0153] The term "pulmonary disease" as used herein means a disease related to the lung, and examples of the lung disease include interstitial lung disease, progressive fibrosing interstitial lung disease, idiopathic interstitial pneumonia, nonspecific interstitial pneumonia, pulmonary fibrosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, alveolitis, pneumonia, emphysema, bronchitis, chronic obstructive pulmonary disease (COPD), combined pulmonary fibrosis and emphysema (CPFE), asthma, and respiratory infectious diseases (e.g., coronavirus disease 2019 (COVID-19)). The main pathogenic mechanisms of the lung disease include lung damage and inflammatory response, and fibrosis.

[0154] Specifically, in one embodiment of the present invention, the lung disease may be a respiratory disease, and specifically, may be at least one selected from the group consisting of pneumonia, asthma, chronic bronchitis, pneumoconiosis, tuberculosis, emphysema, chronic obstructive pulmonary disease, and cystic fibrosis, but is not limited thereto.

[0155] In one specific example of the present invention, free MF-NPs are present in the liver and lungs at the initial stage of administration, but are rapidly eliminated within 3 days, whereas MF-NPs loaded on polyphenol-treated therapeutic frozen cells (lyocells) of the present invention were confirmed to be distributed in the lungs for a long period of time immediately after administration and then migrate to the liver. In addition, MF-NPs loaded on normal cells (norcells) were also confirmed to migrate rapidly to liver tissue, whereas MF-NPs loaded on therapeutic cells (lyocells) of the present invention were confirmed to exhibit long-term persistence in lung tissue.

[0156] Accordingly, the polyphenol-treated therapeutic frozen cells (lyocells) of the present invention exhibit high persistence at the target site after in vivo administration of the loaded therapeutic drug, thereby promising excellent therapeutic effects. In particular, when the cells (lyocells) are used as a drug delivery system for the treatment of lung diseases, excellent therapeutic effects in lung diseases can be expected due to their high persistence rate in the lungs.

[0157] Since the pharmaceutical composition of the present invention includes the polyphenol-treated therapeutic frozen cell and the method for preparing the same, which is one aspect of the present invention described above, redundant contents are cited and the description thereof is omitted in order to avoid excessive complexity of the description herein.

[0158]

[0159] The polyphenol-treated therapeutic freeze-dried cells (lyocells) of the present invention can serve as a bioreservoir that controls the release of various bioactive molecules, including intracellular endogenous substances and therapeutic drugs. In addition, the polyphenol-treated therapeutic freeze-dried cells contain increased and / or concentrated intracellular bioactive substances, such as growth factors and immunomodulators, and thus can be used as a composition for preventing or treating ischemic diseases through angiogenesis-promoting effects, and can be used as a composition for preventing, improving, or treating inflammatory diseases through enhanced immunomodulatory functions. Furthermore, when the therapeutic cells (lyocells) are utilized with an appropriate drug delivery technology prior to freeze-drying, the construction of a simple and effective cell medicine system can be expected.

[0160]

[0161] Figure 1 shows a schematic diagram of a polyphenol-treated freeze-dried cell (lyocell) for treatment of the present invention.

[0162] Figure 2 shows the results of the preparation and characterization of polyphenol-treated freeze-dried cells (lyocells) using mesenchymal stem cells (MSCs): (A) analysis of the viability of MSCs according to the concentration of tannic acid (TA) treatment; (B) evaluation of the morphology, (C) total protein, and (D) total RNA levels of lyocells according to the concentration of TA treatment; (E) analysis of the release pattern of soluble proteins from lyocells; and (F) confirmation of the release of particles similar to extracellular vesicles (EVs) from lyocells.

[0163] Figure 3 shows the results of the biodistribution and retention of mesenchymal stem cells (MSCs) after intravenous (IV) injection into BALB / c mice and intramuscular (IM) injection into C57BL / 6 mice: (A) in vivo biodistribution of cells and (B) cell retention ratio after intravenous (IV) injection, respectively; (C) in vivo biodistribution of cells in different organs and (D) cell retention ratio in the liver versus lung at day 6; and in vivo visualization of (E) cell signaling and (F) cell retention ratio after intramuscular (IM) injection, respectively.

[0164] Figure 4 shows the results of confirming the protective effect of various polyphenols on the cell morphology and maintenance of intracellular physiologically active substances of therapeutic freeze-dried cells (lyocells) of the present invention: (A) Results of evaluating cell morphology using an optical microscope system. Scale bar: 100 μm; and (B, C) Evaluation of total protein levels and total RNA levels of each cell according to polyphenol treatment (n=3). The data are expressed as mean ± SD, analyzed using one-way ANOVA, and corrected for multiple comparisons using the two-step stepwise method of Benjamini, Krieger, and Yekutieli.

[0165] Figure 5 shows the results of preparation of polyphenol-treated freeze-dried cells (lyocells) from various cells and analysis of their characteristics: (A) morphology of lyocells prepared from mouse macrophages (RAW264.7), human fibroblasts (MRC-5), and mouse bone marrow cells; and (B) total protein content.

[0166] Figure 6 shows the results verifying the feasibility of using polyphenol-treated therapeutic freeze-dried cells (lyocells) with enhanced angiogenic effects: (A) western blot bands showing the expression of VEGF-A in MSCs before and after priming with hypoxia-inducing conditions (H; 1% oxygen) and brefeldin A (B; 0.1 μg / ml) at 37°C for 24 h; (B) quantification of VEGF-A in manipulated normal cells (norcells) and lyocell groups; (C) kinetics of VEGF-A release from cells cultured in serum-free medium; (D) quantification of VEGF-A in conditioned medium (CM) extracted from control and manipulated cells; (E) viability of each cell, (F) motility of cells, and (G) tube formation of HUVECs following treatment with conditioned medium (CM) extracted from control and manipulated cells; (H) Body weight changes, (I) ischemic score evaluation, (J) angiogenic and inflammatory gene levels evaluation in mice treated with HB-lyocell.

[0167] Figure 7 shows the results of comparative analysis of polyphenol-treated therapeutic freeze-dried cells (lyocells) of the present invention with cells killed by methods other than polyphenol treatment: (A) live / dead staining analysis using acridine orange (AO) (green, live) and propidium iodide (PI) (red, dead); (B) flow cytometry-based assessment of cell size patterns; (C) relative total protein levels of each cell preparation normalized to cell number (N = 4-5); (D) results of total protein separation on SDS-PAGE gels; (E) relative total RNA levels of each cell preparation normalized to cell number; normal cell (norcell) and lyocell groups (N = 7); HI cells (N = 6); LNT cells (N = 3); (F) Schematic diagram of cell priming under hypoxia (H) and brefeldin A (B), and treatment in the hindlimb ischemic model (HLI); (G) Evaluation of ischemic scores in HLI mice over 14 days; (H) Gene expression profile analysis of mouse thigh muscle tissue collected on day 14.

[0168] Figure 8 shows the results of characterization of polyphenol-treated therapeutic freeze-dried cells (lyocells) with enhanced immunomodulatory factors by IFN-γ treatment: (A) Schematic diagram of the IFN-γ-treated lyocell manufacturing process; (B) Evaluation of IDO levels in IFN-γ-treated lyocells according to TA treatment; (C) and (D) Total bioactive protein (PD-L1 and IDO, respectively) levels in norcell and lyocell groups before and after priming with IFN-γ as measured by specific ELISA kits (norcell group (N = 2), other cell groups (N = 6), the data were analyzed using one-way ANOVA test and suitable for multiple comparison using the two-step stepwise method of Benjamini, Krieger, and Yekutieli); (E) and (F) Retention and release profiles of PD-L1 in various engineered cell groups, respectively.

[0169] Figure 9 shows the results of confirming the therapeutic effect of polyphenol-treated therapeutic freeze-dried cells (lyocells) with enhanced immunomodulatory factors by IFN-γ treatment: (A) flow cytometry-based evaluation of T cell proliferation in co-cultures of splenocytes and mesenchymal stem cells (MSCs); and (B) body weight changes and (C) colon length in mice with DSS-induced acute colitis model following lyocell treatment.

[0170] Figure 10 shows the results of molecular mechanism analysis on the therapeutic effect of IFN-γ-treated polyphenol-treated therapeutic freeze-dried cells (lyocells) on colitis: (A) Schematic diagram showing the steps of siRNA transfection, priming with IFN-γ, preparation of lyocells, and subsequent colitis treatment; (B) Measurement of colon length in mice (N = 5); (C) MPO assay (N = 5); (D) to (F) Flow cytometry-based evaluation of CD3 T cells, MHC2+ cells, and CD11b+ / 11c+ cells in the colon of mice (N = 3). The data were analyzed using one-way ANOVA test and suitable for multiple comparisons using the two-step method of Benjamini, Krieger, and Yekutieli.

[0171] Figure 11 shows the results of confirming the therapeutic stability of polyphenol-treated therapeutic freeze-dried cells (lyocells) during long-term storage at 4°C: (A) HB (hypoxic condition / protein transport inhibitor treated)-lyocells refrigerated (4°C) storage; (B) Morphological changes of HB-lyocells observed during 12 months of storage; (C) VEGF-A levels of HB-lyocells before and after storage according to ELISA analysis; (D) Ischemic score assessment of HLI mice for 14 days; (E) Gene expression profile assessment of thigh muscle tissues collected on day 14.

[0172] Figure 12 shows the results of the preparation and characterization of polyphenol-treated therapeutic freeze-dried cells (lyocells) for potential drug delivery systems: (A) Schematic diagram of cellular internalization of MF-NPs and subsequent lyocell preparation process; (B) Size distribution of mometasone furoate (MF)-loaded PLGA-NPs measured using dynamic light scattering (DLS) method; (C) Imaging of MF-NP-uptaken MSCs; (D) Absolute content of MF in normocytes and lyocells; (E) Release kinetics of MF in free MF-NPs and MF-NP / lyocell systems; (F) Phagocytosis assay by flow cytometry; (G) and (H) Generation and maturation analysis of dendritic cells (DCs) by flow cytometry; The data in (F) to (H) above were analyzed using a one-way ANOVA test and were suitable for multiple comparisons using the two-step method of Benjamini, Krieger, and Yekutieli.

[0173] Figure 13 shows the results of confirming the distribution of nanoparticles (NPs) in vivo according to polyphenol-treated therapeutic freeze-dried cells (lyocells) used as a drug delivery system: (A) Distribution of nanoparticles in vivo by lyocells; (B) and (C) Biodistribution of cells and particles in MF-NPs / normal cells (MF-NPs / norcells) and MF-NPs / lyocell groups over time as analyzed in the IVIS system.

[0174] Figure 14 shows the results confirming the excellent therapeutic effect of MF-NPs / lyocell on the OVA-induced asthma model: (A) the level of IgE in serum, (B) the level of IL-5 in lung tissue, and (C) the severity of inflammation and the degree of mucus production in lung tissue; (D) a schematic diagram for the induction, treatment, and evaluation of asthma; (E) evaluation of BALF cell counts by flow cytometry; (F) measurement of serum IgE levels using ELISA. The data in (E) and (F) were analyzed using a one-way ANOVA test and were suitable for multiple comparisons using the two-step method of Benjamini, Krieger, and Yekutieli.

[0175]

[0176] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0177]

[0178] Example

[0179] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0180]

[0181] Manufacturing Example 1. Manufacturing of polyphenol-treated freeze-dried cells (Lyocell)

[0182] Manufacturing Example 1.1. Culturing of mesenchymal stem cells (MSCs)

[0183] To enhance the stability of cells during lyophilization, MSCs were cultured in low-glucose DMEM (Dulbecco's Modified Eagle's Medium) containing 10% FBS, 1% antibiotics, and 100 mM trehalose at 37°C and 5% CO2 for 24 hours.

[0184]

[0185] Manufacturing Example 1.2. Pretreatment with polyphenol (tannic acid, TA)

[0186] After detaching the mesenchymal stem cells (MSCs) cultured in the above Preparation Example 1.1 by trypsinization, the MSCs were washed twice with Lyophilization Washing Buffer (LWB) (saline (pH 7.0) buffered with 10 mM HEPES and 10 mM NaHCO3), and then the cells were treated by adding polyphenol, e.g., tannic acid (TA), to the LWB containing 100 mM trehalose. Depending on the concentration of tannic acid (TA) treated, the viability of the MSCs was confirmed through a live / dead assay.

[0187] The results are shown in Figure 2A below.

[0188] As a result of treating mesenchymal stem cells (MSCs) with various concentrations of tannic acid (TA), it was confirmed that the number of dead cells increased as the concentration of tannic acid (TA) increased, and as a result, it was confirmed that the cells were completely dead under conditions of 1.0% concentration of tannic acid (TA) (Fig. 2A).

[0189]

[0190] Manufacturing Example 1.3. Freeze-drying process

[0191] Next, after treating the cells with tannic acid (TA), which corresponds to polyphenol, in the above manufacturing example 1.2, a freeze-drying technique was applied to the obtained cells to manufacture a “ready-to-use” frozen or freeze-dried cell product (hereinafter referred to as “lyocell”).

[0192] First, the cells were washed twice with lyophilization washing buffer (LWB) to remove unreacted materials, and then the cell pellet was mixed with lyophilization washing buffer (LWB) containing 100 mM trehalose and lyophilized at -80°C for 16-24 hours to obtain cell powder.

[0193]

[0194] Example 1. Characterization of polyphenol-treated freeze-dried cells (Lyocell)

[0195] Example 1.1. Confirmation of lyocell cell morphology and content of intracellular physiologically active substances according to tannic acid treatment concentration

[0196] The characteristics of the lyocell obtained in the above manufacturing example 1 were confirmed according to the concentration of tannic acid (TA) treatment.

[0197] First, the morphology of lyocells according to the concentration of pretreated tannic acid (TA) was confirmed through SEM images. As shown in Fig. 2B, compared to normal (living) cells (Norcell), it was confirmed that the cell morphology was maintained more intact as the concentration of pretreated tannic acid (TA) increased.

[0198] Next, the content of cell active components in lyocell was analyzed according to the concentration of tannic acid (TA) treatment. As a result, it was confirmed that as the concentration of pretreated tannic acid (TA) increased, the levels of cellular protein and RNA in lyocell increased together. In particular, lyocell pretreated with 1.0% tannic acid (TA) showed a corresponding content level compared to the normal cell group. Specifically, it was shown that the cellular protein level was maintained at approximately 97±3% and the RNA level was maintained at 107±13% (Fig. 2C and D). In addition, it was confirmed that cell soluble proteins (Fig. 2E) and extracellular vesicle (EV)-like particles (Fig. 2F) were continuously released from lyocell obtained through lyophilization after tannic acid (TA) treatment.

[0199]

[0200] Through this, it was confirmed that mesenchymal stem cells (MSCs) were treated with tannic acid (TA) and their cell activity was the same as that of dead cells, but the cell structure was maintained intact like that of normal cells, and intracellular physiologically active substances were also condensed and preserved.

[0201]

[0202] Example 1.2. Confirmation of body distribution (in vivo persistence) after administration of Lyocell

[0203] Mesenchymal stem cells (MSCs) can reside in the lungs immediately after intravenous injection, making them a common treatment for lung diseases. However, most cells migrate from lung tissue to other tissues (e.g., the liver) or are eliminated within a day, potentially reducing the therapeutic efficacy of MSCs in lung disease treatment. Therefore, the biodistribution characteristics (tissue persistence) of MSCs, which exert therapeutic effects in the target site (site) of treatment, are a key factor in achieving superior therapeutic outcomes.

[0204] The present inventors administered the cells to mice and conducted an in vivo biodistribution study to confirm the in vivo distribution characteristics (tissue persistence) of freeze-dried mesenchymal stem cells.

[0205] First, before cell administration, they were labeled with 1,1-Dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine (DiR) for in vivo observation. Specifically, in the case of normal cells (norcells), they were labeled with DiR before administration, and in the case of lyocells of the present invention, DiR labeling was performed before treatment with tannic acid (TA). In vivo administration of cells was performed through intravenous (IV) administration to BALB / c mice, and intramuscular (IM) administration was also performed to C57BL / 6 mice for comparison by administration route. Each mouse was administered 1 x 10 6 Cell concentrations were administered, and at regular time intervals after administration, mice were imaged at 745 / 780 nm (excitation / emission) using the IVIS® Spectrum In Vivo imaging system.

[0206] Additionally, 144 hours after intravenous administration, major organs (heart, lungs, liver, stomach, kidney, spleen, and intestine) were removed from the mice and imaged to observe the distribution of DiR-labeled cells.

[0207]

[0208] Example 1.2.1. In vivo distribution after intravenous administration (IV)

[0209] First, the results of confirming the distribution of cells in vivo at 0, 4, 24, 48 and 144 hours after intravenous administration are shown in Figures A to D of the following Figure 3.

[0210] As shown in Figure 3A, both norcells and lyocells were completely captured in the lungs of BALB / c mice after intravenous administration. However, some of the norcells signal migrated to the liver tissue within 4 hours, whereas most of the lyocells signal remained confined to the lungs even after 24 hours. In particular, when comparing the total cell ratios within the mice in Figure 3B, the ratio of lyocells was observed to be much higher than that of normal cells (norcells) in most cases.

[0211] Additionally, the results of confirming the distribution of cells in major organs 144 hours after intravenous administration are shown in Figures 3C and D.

[0212] As a result, the lyocell signal remained at approximately 60% compared to 0 hours after intravenous administration, while the normal cell (norcell) signal remained at only approximately 40%. It was confirmed that the lung / liver retention ratio of the lyocell group was approximately twice as high as that of the normal cell (norcell) group.

[0213]

[0214] Example 1.2.2. In vivo distribution after intramuscular injection (IM)

[0215] Next, normal cells (norcells) and lyocells were intramuscularly administered into the right (R) and left (L) thighs of the mouse, respectively, and the cell distribution in vivo was confirmed 0, 24, 48, 72, 96, 120, 144, and 168 hours after administration.

[0216] The results are shown in E and F of Figure 3 below.

[0217] First, as shown in E of Fig. 3, the fluorescent signal of lyocell was found to be much stronger than that of normal cells (norcell) after observation for one week, and in particular, while normal cells (norcell) were removed by approximately 50% 48 hours after intramuscular administration, lyocell was found to remain mostly at the injection site.

[0218] In addition, as a result of measuring the in vivo persistence of cells at each time point compared to 0 hour after intramuscular administration in Fig. 3F, it was confirmed that 26 ± 8% of normal cells (norcells) and 33 ± 4% of lyocells remained at the injection site on the 7th day (168 hours).

[0219]

[0220] Manufacturing Example 2. Manufacturing of lyocell using polyphenols other than tannic acid (TA) and analysis of its characteristics.

[0221] In order to confirm the applicability of various polyphenols other than tannic acid (TA) in the production of lyocell of the present invention, lyocell was produced from various cells and its characteristics were confirmed.

[0222]

[0223] Manufacturing Example 2.1. Manufacturing of lyocell using polyphenols other than tannic acid (TA)

[0224] In this manufacturing example, in addition to tannic acid (TA), polyphenols such as baicalein, catechin hydrate, gallic acid hydrate, chlorogenic acid (CGA), and epigallocatechin gallate (EGCG) (TCI Chemical, Japan) were used.

[0225] Specifically, the various polyphenols described above were dissolved in PBS (pH 7.0, buffered with 10 mM HEPES and 10 mM bicarbonate) at an initial concentration of 1%, and then the undissolved material was removed by centrifugation, and the clear supernatant was used for cell treatment. Then, mesenchymal stem cells (MSCs) at passage 4 were detached by trypsinization and washed twice with PBS buffer (pH 7.0). The obtained cells were treated with each of the various polyphenol solutions described above for 1 hour under continuous rotation conditions. The polyphenol-treated cells were washed twice with PBS buffer, dispersed in freeze-drying buffer (PBS, pH 7.0, containing 100 mM trehalose), and frozen at -80°C at a cooling rate of 1°C / min. Then, they were freeze-dried for 16 to 24 hours to obtain polyphenol-treated freeze-dried cell (lyocell) powder.

[0226]

[0227] Manufacturing Example 2.2. Characterization of polyphenol-treated lyocells other than tannic acid (TA)

[0228] The present inventors analyzed the characteristics of lyocells treated with various polyphenols obtained in the above-mentioned Preparation Example 3.1, and confirmed the morphology of the lyocells and the content of intracellular physiologically active substances. Here, normal (living) cells (Norcells) and cells not treated with polyphenols (lyocell-control) were used as controls.

[0229]

[0230] Morphological analysis of lyocells

[0231] First, in order to analyze the morphological characteristics of lyocell according to treatment with polyphenols other than tannic acid (TA), the lyocell powder obtained in the above manufacturing example 3.1 was dissolved in distilled water, reconstituted, and centrifuged to obtain a cell pellet. Then, the pellet was dispersed in PBS, and the cells in the suspension were plated to observe the morphology of lyocell according to treatment with polyphenols other than tannic acid under an optical microscope system.

[0232] The results are shown in Figure 4A.

[0233] As shown in Figure 4A below, in the case of cells not treated with polyphenol (lyocell-control), it was confirmed that the morphology and shape of the cells were significantly damaged and collapsed. On the other hand, it was confirmed that the morphological stability of all polyphenol-treated lyocells was maintained even after freeze-drying, and among them, the EGCG, catechin, and gallic acid treatment groups showed the best maintenance effect.

[0234]

[0235] Analysis of the content of bioactive substances in lyocells

[0236] Next, the total cellular protein and RNA levels of lyocells were measured according to polyphenol treatment other than tannic acid to analyze the content of bioactive substances in lyocells.

[0237]

[0238] 1. Measurement of intracellular protein levels

[0239] First, to measure the intracellular protein level, the pelleted cells were collected in a microtube and then lysed in a 10% trichloroacetate (TCA) solution on ice for 30 min. The precipitated protein was collected by centrifugation at 15,000 rpm for 10 min at 4°C. The precipitate was then dissolved in a bicarbonate buffer (200 mM, pH 8.5, 1% sodium dodecyl sulfate (SDS)) and probe-sonicated on ice for 10 s. The total protein amount was measured by fluorescamine analysis (Thermo Fisher Scientific), and the data were normalized according to the cell number of each group.

[0240] The results are shown in B of Fig. 4.

[0241] As shown in B of the following Figure 4, in the case of cells not treated with polyphenol (lyocell-control), only 60% or less of the protein content within the cells was maintained compared to normal cells (norcell), but it was confirmed that the retention rate of intracellular physiologically active substances, i.e., proteins, was greatly improved in all polyphenol-treated lyocells. In particular, it was confirmed that the proteins within the lyocells were most effectively preserved in the gallic acid and EGCG treatment groups, and specifically, a protein retention rate of more than 75% was shown compared to normal cells (norcell).

[0242]

[0243] 2. Measurement of intracellular RNA levels

[0244] Additionally, to measure intracellular RNA levels, total cellular RNA was extracted from lyocells obtained according to the manufacturer's instructions using the Rneasy Mini Kit (Qiagen). After removing DNA contamination from the column with DNase solution (Qiagen), RNA was eluted with DEPC-treated water and its content was measured using a Nanodrop2000 instrument. Data were normalized to the cell number in each group.

[0245] The results are shown in C of Fig. 4.

[0246] As shown in C of the following Figure 4, in the case of cells not treated with polyphenol (lyocell-control), only less than 30% of the RNA content within the cells was maintained compared to normal cells (norcell), but it was confirmed that the retention rate of RNA content was greatly improved in all polyphenol-treated lyocells. In particular, it was confirmed that the RNA within the lyocells was most effectively preserved in the gallic acid and EGCG treatment groups, and specifically, it showed an RNA preservation rate of more than 75% compared to normal cells (norcell).

[0247]

[0248] In summary, our results suggest that various polyphenols, including tannic acid (TA), play a crucial role in preserving the cell morphology and intracellular bioactive substances of freeze-dried apoptotic cell therapeutic agents before freeze-drying.

[0249]

[0250] Manufacturing Example 3. Manufacturing of lyocell using cells other than mesenchymal stem cells and characterization analysis

[0251] In order to confirm the applicability of various cell groups other than mesenchymal stem cells (MSCs) in the production of lyocell of the present invention, lyocell was produced using various cells and its characteristics were confirmed.

[0252] Specifically, lyocells were prepared using the same method as above using mouse macrophages (RAW264.7), human fibroblasts (MRC-5), and mouse bone marrow cells, and after freeze-drying, the structure of the cells was observed and their total protein content was evaluated.

[0253] The results are shown in Figure 5 below.

[0254] First, as shown in Fig. 5A, it was confirmed that the cell structure was well maintained after freeze-drying even in lyocells manufactured using cells other than mesenchymal stem cells. In addition, as a result of measuring the total protein content in the manufactured lyocells, it was confirmed that lyocells pretreated with 1.0% tannic acid (TA) in the same manner as in Example 1 showed a total protein content at a corresponding level compared to normal cells (norcells) (Fig. 5B).

[0255] Accordingly, the cells that can be used as lyocells of the present invention are not limited to mesenchymal stem cells (MSCs), but also suggest the possibility of using various cells other than mesenchymal stem cells (e.g., T cells, dendritic cells, stromal vascular fraction cells, etc.) as lyocells.

[0256]

[0257] Example 2. Validation of the availability of lyocells with enhanced angiogenic effects.

[0258] The inventors of the present invention confirmed in Example 1 above that the lyocell of the present invention has a high preservation capacity for intracellular physiologically active substances and excellent in vivo persistence. Therefore, in this Example, mesenchymal stem cells (MSCs) with increased intracellular angiogenic factors were manufactured as lyocell, and the possibility of utilizing lyocell with an enhanced intracellular angiogenic effect was confirmed.

[0259]

[0260] Example 2.1. Preparation of lyocell with enhanced angiogenic effect

[0261] First, to prepare mesenchymal stem cells (MSCs) in which angiogenic factors including VEGF-A are accumulated within the cells, hypoxia induction conditions (indicated as 'H'), brefeldin A (indicated as 'B') treatment, or hypoxia induction conditions and brefeldin A treatment (indicated as 'HB') were performed for 24 hours.

[0262] The results are shown in Figures 6A and B.

[0263] First, as shown in Figure 6A, it was confirmed that treatment with both 1% hypoxic conditions and 0.1 μg / ml of brefeldin A (HB) induced high intracellular VEGF-A content.

[0264]

[0265] Next, HB-lyocell was prepared from mesenchymal stem cells (MSCs) in which angiogenic factors had accumulated using the same method as in Manufacturing Example 1, using 1% tannic acid (TA) treatment and freeze-drying technology.

[0266] As a result, as shown in B of Fig. 6, the VEGF-A content of HB-lyocell was 10 ng / 10 6It was confirmed that the level of VEGF-A was maintained at a level similar to that of HB-normal cells. In addition, as can be seen in Figure 6C, when HB-lyocells were cultured in serum-free medium, it was confirmed that VEGF-A was continuously released for at least 11 days.

[0267]

[0268] Example 2.2. Confirmation of enhanced angiogenic effects by engineered lyocells.

[0269] In order to confirm the effect of lyocell (HB-lyocell) manufactured in Example 2.1 above on angiogenesis, the following experiment was performed.

[0270]

[0271] Example 2.2.1. Evaluation of angiogenic factors in engineered lyocells

[0272] First, to evaluate the angiogenic factors in the manipulated lyocell, normal cells (norcell) or lyocell were cultured in serum-free medium at 37℃ for 2 days, and then conditioned medium (CM) was extracted, and the level of VEGF-A in the extracted CM was measured. Here, HB untreated-normal cell group (Control norcell), HB-normal cell (HB-norcell) group, and HB untreated-lyocell group (Control lyocell) were used as comparative groups for HB lyocell, and serum-free medium (Free medium) was used as a negative control group, and EGM-2 (Endothelial growth medium 2, PromoCell) treatment group was used as a positive control group.

[0273] As a result, as shown in Figure 6D, it was confirmed that the level of VEGF-A release from HB-treated lyocell (HB-lyocell) was increased compared to the EGM-2 treatment group used as a positive control. In addition, it was confirmed that HB-lyocell released significantly increased VEGF-A compared to HB-treated normal cells (HB-norcell), HB-untreated-normal cell group (Control norcell), and HB-untreated-lyocell (Control lyocell).

[0274] This confirmed that the increased VEGF-A level in HB-lyocell was specifically observed in HB-lyocell prepared by performing freeze-drying treatment on mesenchymal stem cells treated with both hypoxic conditions and brefeldin A.

[0275]

[0276] In addition, when the above-mentioned extracted conditioned medium (CM) was treated on HUVEC (Human umbilical vein endothelial cells), it was confirmed that when the CM of HB-lyocell was treated, cell viability, cell mobility, and tube formation were improved compared to the CM extracted from other control groups, which was almost similar to the EGM-2 treatment group (EG in Fig. 6).

[0277]

[0278] Example 2.2.2. Therapeutic effect of engineered lyocell on ischemic disease

[0279] Next, in vitro experiments were performed to confirm the therapeutic effect of the manipulated lyocell on ischemic disease in mice.

[0280] Specifically, C57BL / 6 mice (male, 8 weeks old, Orient Bio, Korea) were anesthetized with 2% isoflurane at a flow rate of 2 L / min. The left limb muscle tissue of the mouse was exposed, and the superior and inferior branches of the femoral artery were ligated using 6-0 silk suture (Ethicon, USA). The ligated artery between the two branches was then resected. Then, the skin of the mice was sutured, disinfected with povidone-iodine, and randomly divided into different groups. For treatment, on the 1st day after induction, PBS, MSCs (1 × 10 6 cells) or cell lysate (1 x 10 6 (Identical to cells) were injected intramuscularly into the central hindlimb muscle of the ischemic vessel-resected area. The severity of the disease was then monitored by measuring the change in body weight of the mice and scoring the limb health status for up to 2 weeks. In addition, the diseased limbs of the mice were harvested and the levels of angiogenic and inflammatory genes were evaluated. The primer information for this is shown in Table 1 below:

[0281] Primer information for assessing angiogenesis and inflammation gene levels Gene Forward primer Reverse primer Vegfa 5'-CTG CTG TAA CGA TGA AGC CCT G-3'5'-GCT GTA GGA AGC TCA TCT CTC C-3'Fgf 2 5'-AAG CGG CTC TAC TGC AAG AAC G-3'5'-CCT TGA TAG ACA CAA CTC CTC TC-3'Pdgfb 5'-AAT GCT GAG CGA CCA CTC CAT C-3'5'-TCG GGT CAT GTT CAA GTC CAG C-3'tnfa 5'-GGT GCC TAT GTC TCA GCC TCT T-3'5'-GCC ATA GAA CTG ATG AGA GGG AG-3'beta-actin 5'-CAT TGC TGA CAG GAT GCA GAA GG-3'5'-TGC TGG AAG GTG GAC AGT GAG G-3'

[0282] In this example, HB-lyocell was used as the experimental group, HB-normal cells (HB-norcell) and HB-lyocell lysate were used as the control group, and PBS, normal cells (norcell), and lyocell were used as the control group, respectively.

[0283] The results are shown in H to J of Figure 6 below.

[0284] First, as shown in H and I of Fig. 6, the results of two weeks of observation confirmed that treatment with HB-lyocell was most effective in controlling weight changes in mice and reducing ischemic scores.

[0285] Next, the levels of angiogenic and inflammatory genes were measured according to each treatment group, and it was confirmed that the HB-lyocell administration group effectively regulated the expression of the above genes by upregulating angiogenic genes (i.e., vegfa, fgf2, and pdgf) and downregulating inflammatory genes (i.e., tnfa) compared to the other groups.

[0286]

[0287] In summary, we confirmed that lyocells, which are made from mesenchymal stem cells (MSCs) with enhanced angiogenic factors through hypoxic conditions and treatment with brefeldin A, have a high angiogenic effect and can be effectively used in the treatment of ischemic diseases.

[0288]

[0289] Comparative Example 1. Cell preparation treated with a method other than polyphenol

[0290] The present inventors prepared a cell preparation killed by a different treatment method other than polyphenol, and performed a comparative analysis with the polyphenol-treated therapeutic lyophilized cells (lyocell) of the present invention, thereby confirming the superior properties of the lyocell prepared by the polyphenol treatment of the present invention.

[0291]

[0292] Comparative Example 1.1. Preparation of cell preparations treated with methods other than polyphenols

[0293] The present inventors prepared cell preparations killed by different methods other than polyphenol treatment, namely heat-inactivated (HI) cells, liquid nitrogen treated (LNT) cells, and spontaneously apoptotic (SA) cells, respectively.

[0294] Specifically, i) heat-inactivated (HI) cells were cultured in PBS (2 x 10 6 MSCs (2 x 10 cells / ml) were prepared by heating at 50°C for 30 min and then cooling on ice. ii) Liquid nitrogen-treated (LNT) cells were prepared by heating MSCs (2 x 10 cells / ml) in a microtube. 6iii) Spontaneously apoptotic (SA) cells were prepared by collecting free-floating cells from adherent MSC cultures. The three types of cells were washed twice with PBS before use.

[0295] Next, to confirm the presence or absence of cell death in all cells, including normal cells (norcells) and lyocells, a live / dead staining assay using acridine orange (AO, green, live) and propidium iodide (PI, red, dead) was performed.

[0296] As a result, as shown in Figure 7A, it was confirmed that all cells were stained red by PI, indicating a dead cell state.

[0297]

[0298] Comparative Example 1.2. Comparative analysis with polyphenol-treated freeze-dried cells (lyocells) of the present invention.

[0299] Next, the three types of dead cells manufactured in Comparative Example 1.1 were compared and analyzed with the lyocell of the present invention.

[0300]

[0301] Comparative Example 1.2.1. Cell Morphology and Size

[0302] First, flow cytometry was performed on all cells to analyze cell patterns, specifically cell morphology and size.

[0303] As a result, as shown in B of Fig. 7, in the case of HI cells, the cell shape and size were partially collapsed, whereas in the case of the lyocell of the present invention, it was confirmed that the cell shape and size were mostly similar to those of normal cells (norcells).

[0304]

[0305] Comparative Example 1.2.2. Intracellular physiologically active ingredients

[0306] Next, we compared and analyzed the levels of all intracellular physiologically active components, such as total protein or RNA.

[0307] Specifically, i) total protein levels within the cells were measured as follows:

[0308] Cells were collected as pellets in microtubes and precipitated in 10% trichloroacetate (TCA) solution on ice for 30 min. Precipitated proteins were collected by centrifugation at 4°C and 15,000 rpm for 10 min, and the pellet was then dissolved in bicarbonate buffer (200 mM, pH 8.5, 1% SDS) under probe sonication for 10 s on ice. Next, Fluorescamine (Thermo Fisher Scientific) analysis was performed to estimate total protein, and a calibration curve was prepared using BSA as a protein standard. Data were normalized to the cell number in each group.

[0309] As a result, as shown in C of the following Figure 7, it was confirmed that the lyocell group of the present invention maintained the total protein content of the normal cell group up to ~90%, whereas in the case of the three types of dead cell groups prepared in Comparative Example 1.1, only ~70% of the protein level was detected.

[0310] Additionally, proteins from each cell group were separated using SDS-PAGE gels, and the gels were made with 12% acrylamide solution and stained with Coomassie Blue solution to visualize protein bands.

[0311] The results are shown in D of Figure 7 below, and compared to the three types of dead cells manufactured in Comparative Example 1.1, it was confirmed that the lyocell of the present invention also exhibited excellent characteristics in protein content storage.

[0312]

[0313] Meanwhile, ii) the total RNA level within the cell was measured by extracting total RNA from each cell using a Trizol-based method and measuring the absorbance.

[0314] As a result, compared to the normal cell group, it was confirmed that ~88% of total RNA was maintained in lyocells, whereas less than 30% of RNA remained in the HI and LNT cell groups (Fig. 7E).

[0315]

[0316] Comparative Example 1.2.3. Therapeutic Effects of Ischemic Disease

[0317] As in Example 2.1 above, all cell groups were manipulated to exhibit angiogenic properties by performing hypoxia-inducing conditions (H) and brefeldin A (B) treatment, and the therapeutic effect on ischemic disease was confirmed by applying this to the HLI (hindlimb ischemic) model (Fig. 7F).

[0318] First, the therapeutic efficacy of HB-lyocell and HB-HI cells was compared.

[0319] The results, as shown in G of Fig. 7 below, confirmed that the HB-lyocell of the present invention had a long-lasting effect of reducing the ischemic score over a 14-day observation period, but the HB-HI cells showed only temporary disease improvement in the early stage (day 3) and did not show any therapeutic effect on ischemic disease thereafter.

[0320] Additionally, to evaluate the gene expression profiles in each cell population treated with HB, thigh muscle tissues from mice were collected on day 14.

[0321] The specific qRT-PCR analysis was as follows: Thigh muscle tissue from mice was collected and lysed using TRIzol reagent. Total RNA was extracted by chloroform layering and precipitated with isopropanol and 75% ethanol. Total RNA levels were measured using a Nanodrop2000 instrument. Next, cDNA was synthesized using the RevertAid First Strand cDNA Synthesis kit, and qRT-PCR analysis was performed using the SYBR Green amplification method.

[0322] Additional primer sequences are listed in the table below:

[0323] Primer information for assessing angiogenesis and inflammation gene levels Gene Forward primer Reverse primer Il1b 5'-TGG ACC TTC CAG GAT GAG GAC A-3'5'-GTT CAT CTC GGA GCC TGT AGT G-3' Cd 31 5'-CCA AAG CCA GTA GCA TCA TGG TC-3'5'-GGA TGG TGA AGT TGG CTA CAG G-3' MyoD 5'-GCA CTA CAG TGG CGA CTC AGA T-3'5'-TAG TAG GCG GTG TCG TAG CCA T-3' MyoG 5'-CCA TCC AGT ACA TTG AGC GCC T-3'5'-CTG TGG GAG TTG CAT TCA CTG G-3'

[0324] As observed in Figure 7H below, compared to PBS-treated mice, when HB-lyocell was treated, angiogenic factors (Vegfa and Cd31) and muscle regeneration / remodeling / homeostasis factors (MyoD and MyoG) were significantly increased, while inflammatory factors (Tnfa and Il1b) were decreased.

[0325]

[0326] Based on the results of this comparative example, it was confirmed that the lyocell, a freeze-dried polyphenol-treated cell of the present invention, exhibits superior characteristics and efficacy compared to cell preparations killed by methods other than polyphenol-treatment.

[0327]

[0328] Example 3. Preparation of lyocell with activated immunomodulatory function and verification of its usability.

[0329] The inventors of the present invention confirmed in Example 1 that the lyocell of the present invention possesses high preservation capacity for intracellular physiologically active substances and excellent in vivo persistence. Furthermore, by producing lyocell with mesenchymal stem cells (MSCs) that activate the expression of intracellular immune regulatory factors, the inventors attempted to confirm the usability of lyocell with activated immune regulatory functions.

[0330]

[0331] Example 3.1. Preparation of lyocells with enhanced immune-regulatory function through IFN-γ priming.

[0332] It is known that cell priming by IFN-γ activates the expression of important immune regulatory factors, including intracellular IDO (indoleamine 2,3-dioxygenase) and PD-L1 (programmed death-ligand 1). Accordingly, mesenchymal stem cells (MSCs) were primed by treating them with IFN-γ (40 ng / ml), and then, in the same manner as in Manufacturing Example 1, tannic acid (TA) was treated and freeze-drying technology was applied to produce lyocells with activated immune regulatory function (Fig. 8A).

[0333]

[0334] Example 3.2. Characterization of lyocells primed with IFN-γ

[0335] Example 3.2.1. Comparison of lyocell characteristics according to tannic acid (TA) treatment concentration

[0336] First, western blotting was performed to measure the level of intracellular IDO (indoleamine 2,3-Dioxygenase) according to the concentration of tannic acid (TA) treatment for lyocells primed with the above IFN-γ.

[0337] As a result, as shown in B of Fig. 8, it was confirmed that the level of IDO in lyocells primed by IFN-γ increased as the concentration of tannic acid (TA) increased, and in particular, it was confirmed that lyocells treated with a 1.0% concentration of tannic acid (TA) showed an IDO level similar to that of normal cells.

[0338] Accordingly, it was confirmed that the intracellular immune regulatory factors increased by priming could be stably preserved even after freeze-drying in lyocells manufactured after tannic acid (TA) treatment, and in particular, when treated with 1.0% concentration of tannic acid (TA), it was confirmed that it had the same level of preservation ability for intracellular substances as compared to normal cells (norcells).

[0339]

[0340] Example 3.2.2. Comparison of lyocell characteristics according to IFN-γ priming

[0341] (1) Quantification of intracellular protein levels

[0342] Next, an Enzyme-Linked Immunosorbent Assay (ELISA) test was performed to quantify the protein levels of MSCs before and after the priming process with IFN-γ.

[0343] As a result, as can be seen in C and D of Fig. 8, the level of PD-L1 was significantly increased by about 10 times or more in all cell groups following priming with IFN-γ, and in particular, it was confirmed that the level of PD-L1 and IDO was maintained at a high level (> 85%) in IFN-γ-lyocells compared to IFN-γ-norcells.

[0344]

[0345] (2) Confirmation of the ability to retain and release intracellular immune regulatory factors

[0346] Next, the inventors performed retention and release tests of intracellular immune regulatory factors primed by IFN-γ.

[0347] For IFN-γ-norcells, 1 mL of medium was cultured in each 12-well cell culture plate, specifically, DMEM medium containing 10% FBS and 1% penicillin-streptomycin or free medium. Meanwhile, IFN-γ-lyocells were cultured in a 1 μm transwell system in a 24-well plate format, and the IFN-γ-lyocells were stored in the upper transwell chamber using 0.2 mL of release medium (PBS buffer containing 200 μg / mL 2-phospho-l-ascorbic acid (AA) and 0.3% BSA), and 0.8 mL of medium was stored in the lower well chamber. The samples were stored at 37°C in a CO2 incubator. Afterwards, the release medium and cells were separated by centrifugation at predetermined times, and in a cellular retention test, the cells were lysed in RIPA buffer containing 1x protease inhibitor (Thermo Fisher Scientific). All samples were then stored at -20°C and subjected to ELISA analysis for PD-L1 levels within 2 weeks.

[0348] The results are shown in E and F of Figure 8 below.

[0349] First, as shown in Figure 8E, it was confirmed that the cellular PD-L1 level rapidly decreased in IFN-γ-norcells for 48 hours, whereas it slowly decreased in IFN-γ-lyocells for 168 hours of culture.

[0350] Similarly, in Figure 8F, PD-L1 from the IFN-γ-norcell group was observed to be mostly released into the medium within 48 hours, and the release level was confirmed to be much lower than that under free medium conditions. On the other hand, IFN-γ-lyocells were confirmed to exhibit a prolonged release pattern for PD-L1 for at least 168 hours.

[0351]

[0352] Example 3.3. Verification of therapeutic efficacy by lyocells primed with IFN-γ

[0353] Example 3.3.1. In vitro immunosuppressive effect of lyocells primed with IFN-γ

[0354] First, since it is known that the suppression of T cell proliferation in splenocytes can be regulated by immune regulatory factors secreted from mesenchymal stem cells, the immune suppression effect of lyocells manipulated in Example 3.1 was evaluated in vitro on splenocytes to confirm the immune suppression effect.

[0355] Splenocytes were labeled using CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) and then co-cultured with mesenchymal stem cells (MSCs) in activated RPMI-1640 medium (with PHA and IL-2 added) for 4 days. Then, flow cytometry analysis was performed on T cell proliferation to confirm the T cell proliferation inhibitory effect of lyocell of the present invention. First, as a control group in this example, i) splenocytes were cultured alone, and the co-culture was performed using two culture methods depending on cell-to-cell interaction: ii) direct contact culture or iii) indirect culture (transwell model).

[0356] Specifically, whole splenocytes were isolated from the spleen of C57BL / 6 mice and labeled with 2 μM CFSE in RPMI-1640 medium supplemented with 10% FBS. Next, splenocytes (5 x 10 6 cells) and mesenchymal stem cells (5 x 10 5 Cells) were co-cultured in direct contact and transwell models in 6-well plates, and specifically, RPMI 1640 medium containing 10% FBS, 1% antibiotics, 100 IU / mL IL-2 (BioLegend), 2 μg / mL PHA (Thermo Fisher Scientific), and 50 μM β-mercaptoethanol was used as an activation medium, and cultured for 4 days. Thereafter, the cultured cells were harvested, stained with APC-anti-CD3 antibody, and analyzed using flow cytometry.

[0357] In the control group of this example, spleen cells were cultured alone and used as positive (stimulated) and negative (non-stimulated) controls depending on the presence or absence of stimulation. In the co-culture with the spleen cells, normal cells (norcells) were used as a control group, and in the co-culture of the spleen cells and lyocells, normal cells (norcells) were used as a control group.

[0358] Specifically, as a comparative experimental group for the lyocell (IFN-γ lyocell) manipulated by treating with a stimulating factor (e.g., IFN-γ), which is the experimental group of this example, normal cells treated with IFN-γ (IFN-γ norcell), normal cells not treated with IFN-γ (control norcell), and lyocells not treated with IFN-γ (control lyocell) were used.

[0359] The results are shown in Figure 9A below.

[0360] As shown in Fig. 9A, i) when splenocytes were cultured alone, it was confirmed that the proliferation rate of T cells increased in the stimulated splenocytes. However, ii) when co-cultured with mesenchymal stem cells by the direct contact model, it was confirmed that the proliferation rate of T cells was significantly reduced by IFN-γ-treated normal cells (IFN-γ norcell) and IFN-γ lyocell, respectively, whereas it was confirmed that the other control groups had little effect on the effect of inhibiting T cell proliferation. iii) When co-cultured with mesenchymal stem cells by the transwell model, it was confirmed that T cell proliferation was partially inhibited only in the IFN-γ lyocell-treated group, and it was confirmed that the T cell inhibitory effect by this was very low in the other control groups including the IFN-γ normal cells (IFN-γ norcell), which was the comparative group.

[0361] Consequently, the IFN-γ lyocell of the present invention suggests that it can exhibit an excellent T cell immunosuppressive effect by continuously releasing immune regulatory factors (e.g., indoleamine 2,3-Dioxygenase, IDO) without direct contact with immune cells, compared to the IFN-γ normal cell group.

[0362]

[0363] Example 3.3.2. Verification of the therapeutic effect of IFN-γ-primed lyocells using an in vivo acute colitis mouse model.

[0364] Additionally, the therapeutic effect of IFN-γ-treated lyocells was evaluated in an acute colitis model induced by dextran sulfate sodium (DSS).

[0365] Specifically, acute colitis was induced in 8-week-old male C57BL / 6 mice (Orient Bio, Korea) by administering 2-3% DSS (MP Biomedicals, Canada) via drinking water for 7 days, and then lyocells treated with IFN-γ were administered at 3 Х 10 6 Lyocell was administered to mice via the intraperitoneal (IP) route at a concentration of 100 cells. The therapeutic effect of Lyocell on colitis was then determined by measuring body weight changes and colon length. To determine the severity of colitis, the body weight changes of the mice were measured for 9 days after Lyocell administration, and on the 9th day of the experiment, the colons of the mice were removed and their lengths were measured.

[0366] Here, the DSS-untreated group (Normal) and the DSS-only treated group (DSS only) were used as negative controls, and the normal cell (norcell) administration group was used as a comparison group in this example. Specifically, the IFN-γ-treated normal cell administration group (IFN-γ-treated norcell), IFN-γ-untreated normal cells (control norcell), and IFN-γ-untreated lyocell (control lyocell) were used as comparative experimental groups for the IFN-γ-treated and manipulated lyocell (IFN-γ-treated lyocell), which is the experimental group of this example.

[0367] The results are shown in B and C of Figure 9 below.

[0368] Compared to IFN-γ-treated normal cells, IFN-γ-treated lyocells showed improvements in mouse body weight and colon length, indicating that IFN-γ-treated lyocells are highly effective in recovering colitis symptoms.

[0369]

[0370] Example 3.3.3. Molecular mechanism analysis of the colitis therapeutic effect of IFN-γ-primed lyocells.

[0371] In addition, in order to identify the molecular mechanism for the therapeutic effect on colitis, the inventors silenced immune regulatory factors related to intracellular colitis treatment, such as PD-L1 and IDO, through siRNA transfection before cell priming with IFN-γ, and then produced lyocells.

[0372] The schematic experimental process is as shown in Figure 10A below. Specifically, mesenchymal stem cells (MSCs) at passage 3-4 were seeded in 100 mm culture plates to reach 80% confluency, and then the medium was replaced with TOM (Transfection Optimized Medium, Welgene) medium containing a pre-made complex of Accufect transfection reagent (Bioneer, Korea) and 50 nM siRNA (Bioneer, Korea) according to the manufacturer's instructions. Mouse PD-L1 siRNA (Cat. No. 60533-1) and mouse IDO1 siRNA (Cat. No. 15930-1) were used as target siRNA molecules, and scramble siRNA (AccuTarget™ Negative control siRNA) was used as a control siRNA molecule. After culturing the cells at 37°C for 6 h, the medium was supplemented with 100 mM trehalose (Tokyo chemical industry, Japan) and 40 ng / ml murine IFN-γ (biological activity 1–4 Х 10 6 The medium was replaced with complete MEM-α containing 10 U / mg; Cat. No. 575306; BioLegend) and incubated for an additional 24 h at 37°C. The cells were then collected and prepared into lyocells as described in Preparation Example 1 above, and the transfection efficiency was tested using qRT-PCR.

[0373] Here, the comparative groups that silenced the immune regulatory factors associated with colitis treatment were denoted as si-PD-L1 and si-IDO, respectively, and the control group was denoted as si-Scr, and the colitis treatment effect with the engineered lyocell of the present invention was compared and analyzed.

[0374] The results are shown in B to F of Figure 10 below.

[0375] As a result, all lyocell preparations were confirmed to effectively reduce the severity of DSS-induced colitis (Fig. 10B). However, si-PD-L1 or si-IDO significantly inhibited the therapeutic effect of IFN-γ-treated lyocell (IFN-γ-lyocell) on the infiltration of immune cells into the colon, as evidenced by increased MPO activity (Fig. 10C) and flow cytometry-based T cell (Fig. 10D) and MHC2+ cell counts (Figs. 10E and F). This confirmed that the therapeutic effect of IFN-γ-lyocell on colitis was largely dependent on PD-L1 and IDO.

[0376] In addition, in this example, the lysate of si-Scr / IFN-γ-lyocell was used as a control through probe sonication in PBS on ice, and the effect of the engineered lyocell in a mouse model of colitis was evaluated by administering the lysate at an equivalent cell dose. As can be seen in E and F of Figs. 10 below, the administration of the lysate showed a specific effect on DSS-induced colitis, but significantly reduced the function of intact IFN-γ-lyocell.

[0377] Therefore, these results suggest that prolonged release of bioactive molecules from intact IFN-γ-lyocells is essential for higher therapeutic efficacy.

[0378]

[0379] Example 4. Evaluation of the therapeutic stability of Lyocell during long-term storage

[0380] Next, in order to verify the possibility of using the lyocell of the present invention as an “off-the-shelf product,” HB-lyocell was stored at 4°C for a certain period of time to confirm its therapeutic stability (Fig. 11A).

[0381] First, as described above, when HB-lyocell was stored at 4℃ for 12 months, it was confirmed through ELISA that the HB-lyocell still maintained an intact cell shape and size (B in Fig. 11), and the level of VEGF-A within the cells was also maintained at a constant level (C in Fig. 11).

[0382] In addition, when the therapeutic effects of freshly prepared HB-lyocell and HB-lyocell stored for 6 months were compared in the HLI (ischemic limb injury) model, it was confirmed that both HB-lyocell groups showed the same effect in reducing the ischemic score during the 14-day observation period (Fig. 11D). In addition, in all mouse models treated with HB-lyocell, the expression of angiogenic factors (Vegfa and Cd31) and muscle regeneration / remodeling / homeostasis factors (MyoD and MyoG) was significantly increased, and inflammatory factors (Tnfa and Il1b) were decreased, confirming that thigh muscle tissue damage was effectively recovered by all HB-lyocells regardless of the storage period (Fig. 11E).

[0383] From this, it was confirmed that the lyocell preparation of the present invention exhibits excellent therapeutic stability even when stored for a long period of time.

[0384]

[0385] Example 5. Validation of the feasibility of using lyocell as a drug delivery system.

[0386] Next, we attempted to confirm the possibility of using the lyocell of the present invention as a potential cell-based drug delivery system (DDS).

[0387]

[0388] Example 5.1. Preparation of lyocell for drug delivery system

[0389] Typically, therapeutic agents are loaded into polymeric nanoparticles (NPs), such as poly(lactide-co-glycolide) (PLGA)-NPs, to achieve sustained and targeted drug release.

[0390] In this example, drug-loaded PLGA-NPs were absorbed by mesenchymal stem cells (MSCs), and lyocells were manufactured using tannic acid (TA) treatment and freeze-drying technology in the same manner as in Manufacturing Example 1.

[0391] Specifically, in this example, mometasone furoate (MF), a potent glucocorticoid, was loaded onto PLGA NPs (average size of about 700 nm, B in Fig. 12) and used as a model drug, and these MF-NPs were loaded at about 4.5 μg MF / 10 6 After being absorbed into the cells by MSC, it was manufactured into lyocell (denoted as MF-NP / lyocell below).

[0392] Figure 12A below illustrates the cell internalization of MF-NPs and the subsequent lyocell manufacturing process.

[0393]

[0394] Example 5.2. Characterization of lyocells for drug delivery systems

[0395] As a result of observing the MF-NP / lyocell manufactured in Example 5.1 using a confocal laser scanning microscope (CLSM), it was confirmed that MF-NP was present in the lyocell (Fig. 12C), and as a result of measuring the content of MF, it was confirmed that MF was well preserved in the lyocell, which is a freeze-dried mesenchymal stem cell, as in the MF-NP / normal cell (MF-NP / norcell) (Fig. 12D). Meanwhile, in terms of drug release within the cells, in the case of MF-NP / lyocell, the drug release within the cells continued for at least about 10 days, whereas in the case of Free MF-NPs that were not absorbed into the lyocell, the drug release showed a pattern of continuously increasing for about 40 days (Fig. 12E).

[0396] Accordingly, the drug delivery system using the lyocell of the present invention can stably store the drug to be delivered within the cell and enable continuous release of the stored drug.

[0397]

[0398] Meanwhile, in this example, a significant increase in the phagocytosis rate was observed in a co-culture including heat-inactivated (HI) cells and liquid nitrogen-treated (LNT) cells (p< 0.0001 vs. norcell or lyocell group). Interestingly, as shown in F of Fig. 12 below, it was confirmed that the MF-NPs / lyocell group significantly reduced the phagocytic activity of macrophages compared to the lyocell group (p = 0.0005).

[0399]

[0400] Next, the in vitro effects of MSCs on the generation and maturation of dendritic cells (DCs) were evaluated. Specifically, bone marrow cells (BMCs) were isolated and stimulated with GM-CSF and IL-4 to generate DCs. BMCs without cytokine stimulation served as a negative control. BMCs were co-cultured with MSCs from different groups from day 0 and harvested on day 5 of stimulation. The cell ratio of BMCs to MSCs was maintained at a constant 10:1.

[0401] As a result of flow cytometry analysis shown in G of Figure 12 below, both the normal cell group and the lyocell group showed CD11c in culture. + We confirmed that DC production was significantly reduced, and incorporating MF-NPs into MSCs resulted in CD11c + It was confirmed that the DC value decreased further.

[0402] Additionally, maturation of DCs was induced by LPS in the presence or absence of MSCs, leading to immature CD11c + was evaluated by activating DC for 2 days. Similarly, mature CD86 was detected in all MSC groups, especially in the MF-NPs / normal cells (MF-NPs / norcell) and MF-NPs / lyocell groups. + CD11c + A significant decrease in the DC ratio was observed (Fig. 12H). Interestingly, the lyocell group was found to exhibit a slightly higher DC inhibitory effect than the norcell group.

[0403]

[0404] Example 5.3. Confirmation of the distribution of nanoparticles (NPs) in vivo

[0405] The present inventors confirmed the in vivo distribution of nanoparticles (NPs) to evaluate the drug delivery effect by lyocell.

[0406]

[0407] Example 5.3.1. Confirmation of the in vivo distribution of nanoparticles (NPs) applied to lyocells

[0408] First, to confirm the in vivo distribution of nanoparticles (NPs) by lyocell, MF-NPs were labeled with Cy5.5, and labeled MF-NPs / lyocell (1 × 10) were added to 200 μl PBS. 6 BALB / c mice were intravenously administered with MF-NPs (equivalent to ~4.5 μg MF) or free MF-NPs (same dose) via the tail vein. Then, various internal organs including lungs, liver, heart, spleen, and kidneys were collected from the mice 6 days after administration, and the signals of MF-NPs were evaluated in the samples using an IVIS imaging system (675 nm / 710 nm excitation / emission).

[0409] The results are shown in Figure 13A below.

[0410] Specifically, in the early stage immediately after administration, free MF-NPs were distributed to both the lungs and the liver, but these MF-NPs were rapidly eliminated from the lungs within 1 day and from the liver within 3 days. In contrast, MF-NPs loaded on lyocell (MF-NP / lyocell) were located mostly in the lungs from the time of administration, and the signal was still detected in the lungs even 6 days after administration. This suggests the potential of lyocell as a drug delivery vehicle for the treatment of lung diseases.

[0411]

[0412] Example 5.3.2. Comparison of the in vivo distribution of nanoparticles (NPs) in lyocells and normal cells (norcells).

[0413] In addition, the inventors compared the in vivo distribution of cells and particles for MF-NPs loaded on normal cells (norcells) and lyocells, respectively.

[0414] Specifically, cells containing MF-NPs were administered via the tail vein of BALB / c mice, similar to Example 5.2.1. Cells and MF-NPs were labeled with DiR and Cy5.5, respectively, and organs were collected at predetermined times, and fluorescence signals were detected using an IVIS system. The relative fluorescence signal of each organ was then analyzed by dividing the total signal of all organs collected each time.

[0415] The results are shown in B and C of Figure 13 below.

[0416] Similar to the comparative analysis results with Free MF-NPs in Example 5.2.1 above, both the MF-NPs / normal cell (MF-NPs / norcell) group and the MF-NPs / lyocell group exhibited major signals in the lung tissue immediately after injection. However, the cell and particle signals of the MF-NPs / normal cell (MF-NPs / norcell) group rapidly migrated to the liver tissue within 24 hours, whereas most of the signals of the MF-NPs / lyocell group remained in the lung tissue for 144 hours of observation.

[0417] Taken together, these in vivo distribution data suggest the advantage of using the lyocell formulation of the present invention to deliver effective drugs to lung tissue.

[0418]

[0419] Example 5.4. Verification of the therapeutic efficacy of MF-NPs / lyocell in an in vivo OVA-induced asthma mouse model.

[0420] Accordingly, the present inventors evaluated the therapeutic efficacy of various MSC preparations using an OVA-induced asthma mouse model in BALB / c mice.

[0421] Specifically, as shown in Figure 14D below, asthma was induced in 8- to 12-week-old female BALB / c mice (Orient Bio, Korea) using ovalbumin (OVA-based model), by intraperitoneally sensitizing the mice with 200 μl of PBS containing 50 μg of OVA (grade V, Sigma-Aldrich) and 2 mg of aluminum hydroxide (Sigma-Aldrich) on days 0, 7, and 14. On day 20, 1 × 10 6 Mice were administered 1 × 10 cells / 200 μl of PBS (+ 150 μg enoxaparin) as a negative control via the tail vein, and each experimental group (MF-NPs / lyocell, control lyocell, Free NPs) (1 × 10 6 Cells and ~4.5 μg MF equivalent) were also administered. Then, PBS containing 20 μg of OVA was administered intranasally to the mice on days 21, 23, and 25. On day 26, blood and lung tissues were collected from the mice, and IL-5 levels in lung tissues and IgE levels in serum were measured using specific ELISA kits. In addition, lung tissues were sectioned and stained with H&E and PAS reagents to evaluate local inflammation and mucus-producing cells in the airways, respectively.

[0422] The results are shown in Figures A to C of Figure 14 below.

[0423] As shown in Figures 14A to C, the intranasal challenge of OVA to sensitized mice significantly increased the levels of serum IgE, IL-5 in lung tissue, and the severity of inflammation and mucus production in lung tissue compared to the results of the negative control group (OVA+PBS). In addition, treatment with (control) lyocell was found to have a minimal effect on reducing asthma symptoms, whereas treatment with free MF-NPs was found to have almost no effect. However, treatment with MF-NP / lyocell was found to be effective in significantly reducing asthma compared to other control groups.

[0424]

[0425] In addition, the inventors of the present invention compared and analyzed MF-NPs / lyocell and MF-NPs / normal cells (MF-NPs / norcell) to more clearly confirm the therapeutic efficacy of the lyocell of the present invention in an OVA-induced asthma mouse model through the excellent drug delivery effect.

[0426] The experiment for this purpose was conducted in the same manner as described above, and the experimental and control groups used here are as shown in Table 3 below:

[0427] Classification Name Dosage Experimental Group MF-NPs / norcell 1 x 10 6 Cells / ~4.5 μg MF / 200 μlMF-NPs / lyocell Control groupPBS (+ 150 μg enoxaparin) 200 μlNormal cells (norcell) 1 x 10 6 Cells / 200 μllyocell1 x 10 6 Cells / 200 μlfree MF-NPs~4.5 μg MF / 200 μl

[0428] On day 26, blood and lung tissue were collected from the mice, and the severity of asthma was assessed by analyzing indicators including bronchoalveolar lavage fluid (BALF) cell counts and serum IgE levels. The results are shown in Figures E and F of Figure 14 below.

[0429] As shown in Figures 14E and F, the norcell, lyocell, free MF-NPs, and MF-NPs / normal cell (MF-NPs / norcell) groups were found to have moderate effects in reducing asthma-related indicators. However, the MF-NPs / lyocell group was found to significantly reduce asthma severity (p = 0.0002 compared to the OVA / PBS group in both BALF cell count and serum IgE level).

[0430]

[0431] Therefore, the above results suggest the possibility of using lyocell as a drug carrier targeting lung tissue for the treatment of lung diseases such as asthma, and excellent therapeutic effects can be expected through this.

Claims

1. Polyphenol-treated frozen cells for therapeutic purposes.

2. In paragraph 1, The above frozen cells are freeze-dried, polyphenol-treated frozen cells for treatment.

3. In paragraph 1, The polyphenol-treated cryotherapy, wherein the polyphenol is at least one selected from the group consisting of gallic acid, caffeic acid, chlorogenic acid (CGA), catechin, epicatechin gallate (EGCG), epicatechin, proanthocyanidins, luteolin, apigenin, baicalein, chrysoeriol, naringenin, hesperidin, eriodictyol, 2-methyl-1,4-naphthoquinone, flavinoquinones, tannic acid, gallotannins, ellagitannins, and derivatives thereof. Cell.

4. In paragraph 1, A polyphenol-treated frozen cell for treatment, wherein the cells are primed with (i) hypoxia; (ii) a protein transport inhibitor; (iii) cytokines; (iv) a protein synthesis inhibitor; (v) a small molecule compound; (vi) growth factors; or (iv) a combination thereof.

5. In paragraph 1, The above cells are polyphenol-treated therapeutic frozen cells containing a therapeutic drug.

6. In paragraph 5, The above drug is a polyphenol-treated frozen cell for therapeutic use, which is loaded into nanoparticles.

7. A method for producing polyphenol-treated frozen cells for therapeutic purposes, comprising the following steps: (a) a step of treating cells with polyphenol; and (b) A step of freezing or lyophilizing the cells.

8. In paragraph 7, A method for producing polyphenol-treated therapeutic frozen cells, wherein the freezing or freeze-drying is performed by adding a sugar selected from the group consisting of dextrose, maltose, glucose, lactose, sucrose, trehalose, mannose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, fructose, melezitose, melibiose, sorbitol, and triose.

9. In paragraph 7, In the above step (a), the polyphenol is at least one selected from the group consisting of gallic acid, caffeic acid, chlorogenic acid (CGA), catechin, epicatechin gallate (EGCG), epicatechin, proanthocyanidins, luteolin, apigenin, baicalein, chrysoeriol, naringenin, hesperidin, eriodictyol, 2-methyl-1,4-naphthoquinone, flavinoquinones, tannic acid, gallotannins, ellagitannins, and derivatives thereof. Method for preparing frozen cells for polyphenol-treated therapy.

10. In paragraph 9, A method for producing polyphenol-treated frozen cells for treatment, wherein the tannic acid is treated at a concentration of 0.02 to 10%.

11. In paragraph 7, A method for producing polyphenol-treated frozen cells for treatment, further comprising a step of priming the cells prior to performing step (a).

12. In paragraph 7, A method for producing polyphenol-treated therapeutic frozen cells, wherein the priming is performed by (i) hypoxia; (ii) a protein transport inhibitor; (iii) cytokines; (iv) a protein synthesis inhibitor; (v) a small molecule compound; (vi) a growth factor; or (iv) a combination thereof.

13. In paragraph 12, A method for producing polyphenol-treated frozen cells for treatment, wherein the hypoxic conditions are comprised of an oxygen concentration of 0.5 to 10%.

14. In paragraph 12, A method for producing polyphenol-treated frozen cells for treatment, wherein the protein transport inhibitor is monensin, brefeldin A (BFA), or a combination thereof.

15. In paragraph 12, A method for producing polyphenol-treated therapeutic frozen cells, wherein the cytokine is at least one selected from the group consisting of IL-1β, IL-4, IL-10, IL-17, GM-CSF, TGF-β, TNF-α, and IFN-γ.

16. In paragraph 7, A method for producing polyphenol-treated therapeutic frozen cells, further comprising a step of incorporating a therapeutic drug into the cells prior to performing the above step (a).

17. A pharmaceutical composition for the prevention or treatment of ischemic disease, comprising polyphenol-treated frozen cells, A pharmaceutical composition for the prevention or treatment of ischemic disease, wherein the polyphenol-treated frozen cells are freeze-dried.

18. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising polyphenol-treated frozen cells, A pharmaceutical composition for the prevention or treatment of an inflammatory disease, wherein the polyphenol-treated frozen cells are freeze-dried.

19. A pharmaceutical composition for drug delivery comprising a polyphenol-treated therapeutic cell carrying a drug, A pharmaceutical composition for drug delivery, wherein the polyphenol-treated frozen cells are freeze-dried.

20. A pharmaceutical composition for drug delivery, wherein the drug in claim 19 is a drug for treating lung disease.

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