Polyphenol-treated therapeutic freeze-dried cells, method for preparing same, and pharmaceutical composition comprising same

Abstract

The present invention relates to a therapeutic freeze-dried or frozen cell in which the cell surface is treated with a polyphenol, a method for preparing the same, and a pharmaceutical composition comprising the same. The polyphenol-treated lyocell for treatment of the present invention can act as a biological repository for controlling the release of a variety of physiologically active molecules including endogenous substances and therapeutic drugs in the cell. Furthermore, the polyphenol-treated frozen cells for treatment contain an intracellular physiologically active substance increased and / or concentrated, for example, a growth factor, an immunomodulatory factor or the like, and can be used as a composition for preventing or treating ischemic diseases by an angiogenesis promoting effect. The composition can be used as a composition for preventing, ameliorating or treating inflammatory diseases by the improved immunomodulatory function. Meanwhile, when the therapeutic cell (lyocell) is used as an appropriate drug delivery technique before freezing (drying), it is expected to construct a cell drug system that can be simply and efficiently used.

Description

Technical Field

[0001] This invention was achieved with the support of the Ministry of Science and ICT of South Korea through project number 00215511. The research management agency for this project was the Interdepartmental Regenerative Medicine Technology Development Project (Foundation), the project name was "Interdepartmental Regenerative Medicine Technology Development Project", the research topic was "Development of a High-Functional Cell Therapy Agent for Pulmonary Fibrosis Based on Sustained-Release Drug Delivery System-Stem Cell Hybrid Technology", the supervising authority was the Sungkyunkwan University Industry-Academia Collaboration Group, and the research period was from April 1, 2023 to December 31, 2023.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0125918, filed on September 20, 2023, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to polyphenol-treated freeze-dried therapeutic cells, methods for their preparation, and pharmaceutical compositions comprising them. Specifically, this invention relates to freeze-dried or freeze-dried polyphenol-treated therapeutic cells, methods for their preparation, and pharmaceutical compositions comprising them. Background Technology

[0004] According to reports, cell-based therapies, which include mesenchymal stem cells (MSCs), fibroblasts, dendritic cells, and T cells, have shown great potential in the treatment of a variety of intractable diseases, but existing treatments using these cells have limitations due to low efficacy.

[0005] In particular, the commercial application of living-cell-based therapies faces numerous constraints. Specifically, the standardization of isolation, culture, and evaluation of therapeutic cells, including mesenchymal stem cells, presents challenges. These cells also carry the risk of exhibiting efficacy outside the target site, making it difficult to anticipate appropriate therapeutic effects based on each patient's condition and / or disease severity. Furthermore, therapeutic cells exhibit low cell viability in the harsh in vivo environment. Moreover, the storage, transportation, and utilization of cells before their use as therapeutic agents present significant difficulties.

[0006] Lyophilization (freeze drying) is widely known as an important technique for the long-term preservation, delivery, and use of biological products (antibodies, proteins, peptides, etc.) without damaging their biological activity before clinical application (Arto Merivaara et al., 2021). However, in the case of freeze-drying live cells, there is a problem of cell structure destruction during the freeze-drying process, leading to apoptosis. Therefore, various freeze-drying protectants such as dimethyl sulfoxide (DMSO), glycerol, trehalose, and serum albumin have been used to prevent cell damage during freeze-drying and to enable long-term cell preservation. However, there are currently no studies validating the therapeutic potential of freeze-dried therapeutic cells.

[0007] The inventors intend to verify the therapeutic potential of using freeze-dried therapeutic cells based on the hypothesis that only partial treatment (e.g., chimeric antigen receptor (CAR)-T cells) requires maintaining high cell viability, while apoptotic cells or dead cells can also induce specific therapeutic effects. Summary of the Invention

[0008] Technical issues

[0009] To effectively treat various diseases, the inventors have made numerous efforts to develop therapeutic cells containing a variety of physiologically active substances, including endogenous substances and / or therapeutic drugs. As a result, in mesenchymal stem cells that have been cryopreserved (dried) after polyphenol treatment, it was confirmed that although the cells themselves lose their viability due to apoptosis, the intracellular physiologically active substances are stably preserved. This has demonstrated that the polyphenol-treated cryopreserved (dried) mesenchymal stem cells possess angiogenesis-promoting effects, enhanced immunomodulatory functions, and the potential for use as a drug delivery system, thus completing this invention.

[0010] Therefore, the object of the present invention is to provide polyphenol-treated cryo-(dried) cells for therapeutic use.

[0011] Another object of the present invention is to provide a method for preparing polyphenol-treated cryo-(dried) cells for therapeutic use.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising polyphenol-treated cryo-(dried) cells for the prevention or treatment of ischemic diseases.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising polyphenol-treated cryo-(dried) cells for the prevention, improvement or treatment of inflammatory diseases.

[0014] Another object of the present invention is to provide a pharmaceutical composition for drug delivery comprising polyphenol-treated cryo-(dried) cells for therapeutic use.

[0015] Technical solution

[0016] To effectively treat various diseases, the inventors have made numerous efforts to develop therapeutic cells containing a variety of physiologically active substances, including endogenous substances and therapeutic drugs. As a result, in mesenchymal stem cells that have been cryopreserved (dried) after polyphenol treatment, it was confirmed that although the cells themselves lose their biological activity due to apoptosis, the intracellular physiologically active substances are stably preserved. This demonstrates that the polyphenol-treated cryopreserved (dried) mesenchymal stem cells possess angiogenesis-promoting effects, enhanced immunomodulatory functions, and the potential for use as a drug delivery system.

[0017] According to one embodiment of the present invention, the present invention provides cryopreserved cells for therapeutic use treated with polyphenols.

[0018] As used in this specification, the term "therapeutic cell" refers to various types of cells used for the treatment of a disease. For example, the therapeutic cell can be a variety of cells, including mesenchymal stem cells, dendritic cells, T-lymphocytes, natural killer (NK) cells, fibroblasts, and myeloid cells, encompassing both primary and secondary cultured cells.

[0019] The term "mesenchymal stem cells (MSCs)" used in this specification, as a type of adult stem cell, refers to multipotent progenitor cells derived from various tissues, including the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta.

[0020] Mesenchymal stem cells (MSCs) possess the ability to differentiate into various connective tissues such as cartilage, bone, ligaments, and bone marrow matrix. In addition to their structural support functions, they also promote angiogenesis, regulate immunity, and suppress inflammation, making them suitable stem cells for inhibiting inflammatory responses and promoting tissue regeneration. They hold significant potential as cell therapy agents. However, mesenchymal stem cell-based treatments generally face numerous limitations in terms of promotion and commercialization, especially those based on live stem cells, which are difficult to scale up for clinical use and preserve.

[0021] Typically, in order to preserve living cells isolated from various human tissues containing mesenchymal stem cells for a long period of time, freezing (drying) methods must be used. Such freezing (drying) methods are not only useful for the long-term preservation of living cells in emergency situations, but also for the differentiation and transplantation of various cells needed for tissue regeneration and treatment in the future.

[0022] Therefore, in one embodiment of the present invention, the frozen cells can be obtained by freeze-drying.

[0023] The term "freezing" as used in this specification refers to the process of cooling a liquid sample to solidify or crystallize it into ice by rapidly lowering the temperature of the container, or converting a coolable solution sample into ice. Typically, biologically active cells or microorganisms are placed in environments 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 to prepare them for long-term storage.

[0024] The freezing can include all suitable methods commonly used in the technical field to which this invention pertains. In small-scale operations (e.g., in a laboratory setting), it can be carried out by placing the substance in a freeze-drying bottle, rotating the bottle in a bath called a shell freezer, and cooling it with, for example, a freezer, dry ice mixed with alcohols such as ethanol or methanol, or liquid nitrogen. Commercial freeze-drying equipment can also be used.

[0025] The term "freeze drying," as used in this specification, also known as lyophilisation, lyophilization, or cryodesiccation, refers to a drying method. This method involves rapidly lowering the temperature of a container to cool a sample in a liquid state, then evacuating the container to a vacuum, causing the water in the solidified (or crystallized into ice) sample to be removed through sublimation.

[0026] Freezing (drying) is widely used as an effective long-term preservation method that minimizes damage to heat-sensitive substances. It preserves the original state of the sample, prevents contamination, and offers advantages in terms of storage, transportation, and cost-effectiveness. However, the formation of ice particles during freezing (drying) can induce structural or functional damage to the sample. Therefore, to protect the sample during freezing and to restore its function upon rehydration, a process of adding a "freezing (drying) protectant" is necessary.

[0027] The term "freeze-drying protectant" refers to a substance that provides protection during freezing or freeze-drying, commonly used in the technical field to which this invention pertains. This freeze-drying protectant can be a commercially available product, and its type is not particularly limited. Freeze-drying protectants commonly used in the technical field to which this invention pertains can be prepared by including substances such as sugars, proteins, and water-soluble polymers. For example, they can be combined according to the desired composition, such as dimethyl sulfoxide (DMSO), dextran, sucrose, glycerol, mannitol, sorbitol, fructose, trehalose, raffinose, serum albumin, propylene glycol, or polyacrylamide.

[0028] However, while the use of the cryoprotectant to freeze-dry bacteria, viruses, serum, vaccines, etc. has the advantage of long-term preservation, in the case of eukaryotic cells, even with the use of the cryoprotectant, it is difficult to stably preserve cells without damaging cell structures such as cell membranes during the freezing or freeze-drying process, thus having the disadvantage of not being able to maintain the function of physiologically active substances within the cells.

[0029] The therapeutic cryopreservation cells treated with polyphenols of the present invention are characterized in that intracellular physiologically active substances are stably preserved during the freezing (drying) process.

[0030] Specifically, the characteristic is that, although the polyphenol-treated therapeutic frozen cells, as biologically inactive cells (i.e., as apoptotic cells with damaged cell membranes) during freezing or freeze-drying, maintain the same / similar cell morphology as before freezing (drying), and intracellular physiologically active substances are stably preserved.

[0031] As used in this specification, the term "polyphenol" refers to an aromatic alcohol compound found in plants. Typically, the core structure of a polyphenol comprises one or more phenolic units with one or more hydroxyl groups (-OH) bonded to a benzene ring (a ring structure consisting of six carbon atoms and six hydrogen atoms). Polyphenols are formed by the combination of multiple such phenolic units. There are many types and categories of polyphenols, but some of the most well-known include, for example, flavonoids (anthocyanins, flavanols, catechins, etc.), stilbenes (resveratrol), lignans, and tannins.

[0032] In one embodiment of the present invention, the polyphenol may be a water-soluble polyphenol.

[0033] The water-soluble polyphenols refer to polyphenols with two or more hydroxyl groups. Specifically, examples include those selected from 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, and tannic acid. One or more of the following groups are included in the group consisting of, but not limited to, gallotannins, ellagitannins, and their derivatives: any polyphenols included in the group consisting of, but not limited to, water-soluble polyphenols.

[0034] Additionally, in one embodiment of the present invention, the polyphenol may be a polyphenol derivative, including drug-polyphenol conjugates and polymer-polyphenol conjugates, or polyphenol-containing biomaterials.

[0035] More specifically, although the polyphenol-treated cryopreserved cells for therapeutic use are characterized by the fact that the polyphenols are treated before freezing / freeze-drying, as cells that have lost their biological activity, i.e., as apoptotic cells with damaged cell membranes, they maintain the same / similar cell morphology as before freezing (drying), and the physiologically active substances within the cells are stably preserved.

[0036] In an embodiment of the present invention, mesenchymal stem cells (MSCs) were treated with a variety of polyphenol groups before freezing (drying), and the morphology of the cells and the intracellular physiologically active substances (proteins, RNA, etc.) were stably preserved in all polyphenol-treated frozen (dried) cells.

[0037] Furthermore, in embodiments of the present invention, mesenchymal stem cells were treated with polyphenols of various concentrations before freeze-drying. It was confirmed that as the concentration of the polyphenols increased, the cells lost their biological activity, and most cells existed in an apoptotic state. However, although the cells lost their biological activity and existed in an apoptotic state, it was confirmed that the cell structure and morphology were not damaged during the freeze-drying process, and they maintained an intact surface like normal cells.

[0038] Moreover, the cell structure and morphology were not damaged after freezing (drying), but rather maintained an intact surface like normal cells. It was confirmed that various physiologically active substances and genetic materials, such as proteins and RNA, were stably preserved within the cells, and the secretion of physiologically active substances within the cells continued.

[0039] The term "intact" means that after the freeze-drying process, the structure and morphology of the frozen (dried) cells remain the same as or similar to those of normal cells.

[0040] The "normal cells" used as the control group in this invention refer to live cells that have not been treated with polyphenols or frozen (dried).

[0041] Therefore, the present invention is characterized in that although the cells lose their biological activity when treated with polyphenols, the cell structure is not damaged during the freezing (drying) process and the cell has a surface whose structural morphology is completely preserved. Thus, the physiologically active substances in the cells are stably preserved, thereby providing polyphenol-treated cryopreserved cells for therapeutic use.

[0042] In the following embodiments of the present invention, the mesenchymal stem cells whose cell surface has been treated with polyphenols and then frozen (dried) are named "lyocell", and the normal cells used as the control group, i.e., the live cells that have not been treated with polyphenols and have not been frozen (dried), are named "norcell".

[0043] Additionally, in embodiments of the present invention, it was confirmed that the polyphenol-treated therapeutic cryocells exhibited high distribution primarily in lung tissue when administered intravenously (IV injection) and high distribution at the administration site when administered intramuscularly (IM injection).

[0044] That is, the polyphenol-treated cryocells of the present invention exhibit high persistence when administered in vivo, and excellent therapeutic effects can be expected in the target body (site) to be treated subsequently.

[0045] In this invention, any cell that can be used for the purpose of treating a disease can be used as the therapeutic cell of this invention.

[0046] In one embodiment of the present invention, the cells are preferably mesenchymal stem cells (MSCs), but are not particularly limited and may be one or more selected from the group consisting of mesenchymal stem cells, macrophages, fibroblasts and bone marrow cells.

[0047] According to research, mesenchymal stem cells contain a variety of physiologically active substances within their cells. In particular, they are known to secrete these intracellular physiologically active substances by sensing changes in the microenvironment through direct / indirect interactions with host cells. When these mesenchymal stem cells are applied to the polyphenol-treated therapeutic cryopreserved cells (lyocells) of the present invention, the various physiologically active substances contained within the mesenchymal stem cells exhibit beneficial therapeutic effects and can be used clinically for pharmaceutical or cosmetic compositions, etc.

[0048] In one embodiment of the invention, the polyphenol-treated cryocells of the present invention can be primed under the following conditions: (i) hypoxia; (ii) protein transport inhibitors; (iii) cytokines; (iv) protein synthesis inhibitors; (v) small molecule compounds; (vi) growth factors; or (iv) combinations thereof. However, the invention is not limited thereto.

[0049] Specifically, in one embodiment of the present invention, the cell may be, but is not limited to, a mesenchymal stem cell, which may be a mesenchymal stem cell in which a variety of physiologically active substances exhibiting beneficial therapeutic effects are increased through the priming.

[0050] As used in this specification, the term "priming" refers to the process of increasing / concentrating the content of physiologically active substances within cells by stimulating and activating them; that is, the process of enhancing the therapeutic effect of the polyphenol-treated cryocells of the present invention by increasing the beneficial physiologically active substances within the cells.

[0051] As used in this specification, the term "biomaterials" refers to specific substances that possess beneficial therapeutic effects and can influence cellular or bodily functions. Specifically, this 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 (EVs), and cyclooxygenase 2 (Cyclooxygenase 2). 2. (The list includes, but is not limited to, 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 (KGF), collagen, fibronectin, and superoxide dismutase (SOD).

[0052] In one embodiment of the invention, after the intracellular physiologically active substances are increased by activation, the polyphenols are processed and then frozen (dried). During the freezing (drying) process, the cells have a surface in which the cell morphology (structure) is completely preserved, thereby allowing the physiologically active substances increased by activation to be stably preserved intracellularly.

[0053] Specifically, in one embodiment of the invention, polyphenol-treated cryocells (lyocells) are able to preserve intracellular physiologically active substances increased by the activation at levels corresponding to those of normal cells (norcells).

[0054] Therefore, when using the polyphenol-treated cryocells of the present invention, a variety of physiologically active substances that exhibit beneficial therapeutic effects can be stably preserved within the cells by activating increased levels, thereby providing the possibility of using them as effective therapeutic agents capable of demonstrating enhanced therapeutic effects.

[0055] Furthermore, in yet another specific embodiment of the invention, the lyocell may contain a therapeutic drug.

[0056] Specifically, the lyocell is characterized in that its morphology (structure) remains intact during the freeze-drying process, and the cell is further characterized by containing a therapeutic drug within it before the freeze-drying process, thereby allowing the therapeutic drug to be stably preserved within the cell and used as an effective therapeutic agent later.

[0057] In one specific embodiment of the invention, the drug may be loaded into nanoparticles.

[0058] The term "nanoparticle" refers to a drug delivery system that can effectively deliver a therapeutic drug to a target site while improving its stability. For example, the nanoparticles of this invention include, but are not limited to, polymer nanoparticles 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 their derivatives.

[0059] On the other hand, in one embodiment of the present invention, the lyocell is characterized by long-term storage stability under low-temperature conditions.

[0060] Specifically, in one embodiment of the present invention, the lyocells retain stability when stored at temperatures ranging from -120°C to 10°C for 5 to 12 months or more. For example, the temperature conditions can be -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℃, -80℃ to 10℃, -80℃ to 0℃, -80℃ to -20℃, -80℃ to -40℃, -80℃ to -60℃, -60℃ to 10℃, -60℃ to 0℃, -60℃ to -20℃, -60℃ to -40℃, -40℃ to 10℃, -40℃ to 0℃, -40℃ to -20℃, -20℃ to 10℃, -20℃ to 0℃ or 0℃ to 10℃, but not limited to these.

[0061] The term "stability" refers to the fact that, under the above conditions, the cell morphology, cell size, level of intracellular physiologically active substances, and therapeutic effects achieved through the cells remain at the same level as before storage.

[0062] According to another embodiment of the present invention, a method for preparing polyphenol-treated cryopreserved cells for therapeutic use is provided, comprising the following steps:

[0063] Step (a) involves treating cells with polyphenols; and

[0064] Step (b): Freeze or freeze-dry the cells.

[0065] In one embodiment of the present invention, the therapeutic cells may be mesenchymal stem cells, dendritic cells, T lymphocytes, NK cells, fibroblasts, or bone marrow cells, etc.

[0066] In one embodiment of the present invention, the therapeutic cells may include therapeutic cells derived from all mammals such as humans, monkeys, pigs, sheep, cattle, horses, dogs, and cats, but are not limited thereto. Specifically, they may be therapeutic cells of human origin.

[0067] In one embodiment of the present invention, in step (a), the polyphenol may be a water-soluble polyphenol.

[0068] In one embodiment of the present invention, the polyphenol is characterized in that it is selected from one or more of the group consisting of gallic acid, caffeic acid, chlorogenic acid, catechin, epicatechin gallate, epicatechin, proanthocyanidins, luteolin, apigenin, scutellarin, sennatin, naringenin, hesperidin, sennaol, 2-methyl-1,4-naphthoquinone, flavonoid quinone, tannic acid, gallantanine, ellagitanninine, and their derivatives.

[0069] In one embodiment of the present invention, the polyphenol may be tannic acid.

[0070] In one embodiment of the present invention, the polyphenol may be a polyphenol derivative, including drug-polyphenol conjugates and polymer-polyphenol conjugates, or a polyphenol-containing biological substance.

[0071] Polyphenols containing tannins aggregate into large molecules and proteins in solution. In particular, when cells are treated with an appropriate concentration of tannins (TA), the cells lose their biological activity and undergo apoptosis, but the structure of the apoptotic cells remains intact, thus allowing them to be used to preserve / concentrate intracellular physiologically active components.

[0072] In this regard, the polyphenol-treated cryocells of the present invention are characterized in that, although they exhibit an apoptotic cell state due to loss of biological activity, the structural morphology of the cells after freezing (drying) still has a surface that is as intact as that of normal cells.

[0073] In a specific example of the present invention, in the case of cells that have been killed by methods other than polyphenol treatment, specifically in the case of heat-inactivated (HI) cells, liquid nitrogen-treated (LNT) cells, and spontaneously apoptotic (SA) cells, similar to the therapeutic cells (lyocells) of the present invention only in that they are apoptotic cells, in the three groups of cells that have been killed by methods other than polyphenol treatment, it was confirmed that the cell structure was not maintained intact and the preservation of intracellular physiologically active substances (e.g., proteins, mRNA, etc.) was not achieved.

[0074] In another specific example of the present invention, therapeutic cryopreserved cells (lyocells) treated with polyphenols were found to have increased effective physiologically active substances in the cells by the cell activation step prior to polyphenol treatment, and were confirmed to show improved therapeutic effects on ischemic diseases. However, in cell groups treated with methods other than the above-mentioned polyphenol treatment, such as HI cells, weak therapeutic effects were found compared with the lyocells of the present invention.

[0075] Therefore, the characteristics of the therapeutic cryocells of the present invention are manifested through polyphenol treatment.

[0076] In one embodiment of the invention, the polyphenol treatment can be carried out at various concentrations.

[0077] Specifically, in one embodiment of the present invention, as the treatment concentration of the polyphenol increases, the number of cells that lose biological activity, i.e., the number of cells that undergo apoptosis due to loss of biological activity, also increases.

[0078] Specifically, when treating tannic acid as the polyphenol of the present invention, the concentration of the tannic acid can be from 0.02% to 10%, more specifically, the concentration of the tannic acid can be from 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%, or 0.1%. The concentrations are 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 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 are not limited thereto. Preferably, in this invention, the tannic acid can be treated at a concentration of 1.0%.

[0079] In this invention, in order to improve the stability of cells during the freeze-drying process in step (b), a freeze-drying protectant may be added, which may include monosaccharides, disaccharides, or polysaccharides.

[0080] Specifically, in one embodiment of the present invention, the freeze-drying can be carried out by adding sugars selected from the group consisting of dextrose, maltose, glucose, lactose, sucrose, trehalose, mannose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, fructose, melezitose, melibiose, sorbitol, and triose.

[0081] In the preparation of the polyphenol-treated cryocells for therapeutic use, step (b) of freezing or freeze-drying the cells can be performed using conventional freeze-drying methods known in the art to which this invention pertains.

[0082] On the other hand, in order to obtain the beneficial therapeutic effects of various physiologically active substances within cells, it may also include a process of enhancing the beneficial physiologically active substances within cells by activating cells.

[0083] Typically, when cells are exposed to harsh environments and undergo apoptosis, the levels of beneficial physiologically active substances within the cells decrease due to insufficient cell initiation. From this perspective, the therapeutic cryocells of the present invention are characterized by utilizing apoptotic cells that have lost their biological activity through polyphenol treatment. Therefore, an additional cell initiation process is required before inducing apoptosis through polyphenol treatment, for example, through tannic acid (TA) treatment.

[0084] Therefore, in one embodiment of the present invention, the method for preparing the therapeutic cryocells may further include a cell initiation step before performing step (a) of treating the cells with polyphenols.

[0085] Specifically, in one embodiment of the present invention, by stimulating and activating cells by including a cell-initiating step before the polyphenol-treated step (a), the content of various physiologically active substances in the cells can be increased and concentrated.

[0086] More specifically, in one embodiment of the invention, the therapeutic lyocell of the invention increases and / or concentrates the content of beneficial physiologically active substances within the cell by activating the cell, thereby demonstrating excellent therapeutic effects based on the increased and / or concentrated physiologically active substances within the cell.

[0087] In one embodiment of the invention, the initiation can be performed under the following conditions: (i) hypoxia; (ii) protein transport inhibitors; (iii) cytokines; (iv) protein synthesis inhibitors; (v) small molecule compounds; (vi) growth factors; or (iv) combinations thereof.

[0088] The term "hypoxia" used in this manual is related to in vitro experiments and refers to a state in which the amount of oxygen supplied to cultured cells is significantly lower than the normal atmospheric oxygen concentration (approximately 20%). It is usually set to an oxygen concentration between 1% and 5%, but can be adjusted according to different experimental requirements.

[0089] Specifically, in one embodiment of the present invention, the hypoxic condition can be an oxygen concentration of 0.5% to 10%. More specifically, the hypoxic condition can be 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 ... The oxygen concentrations are 1% to 8%, 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%. Preferably, the low-oxygen conditions of the present invention can be oxygen concentration conditions of 1% to 5%, and more preferably, the low-oxygen conditions of the present invention can be oxygen concentration conditions of 1%.

[0090] The term "protein transport inhibitor" as used in this specification refers to a substance that can accumulate proteins within cells by blocking the transport process of proteins within the cells.

[0091] Specifically, in one embodiment of the invention, the protein transport inhibitor is characterized as brefeldin A (BFA), monensin, or a combination thereof.

[0092] In one specific example of the present invention, after initiating mesenchymal stem cells under hypoxic conditions and protein transport inhibitors, and then preparing polyphenol-treated therapeutic frozen cells by processing polyphenols, it was confirmed that the initiation process increased / concentrated intracellular physiologically active substances. Although the therapeutic frozen cells (lyocells) were apoptotic cells that had lost their biological activity, they possessed physiologically active substances at levels similar to normal cells (norcells). Furthermore, it was demonstrated that not only was a continuous release of intracellular physiologically active substances formed, but the amount of released physiologically active substances also continuously increased, thus confirming a state distinct from normal cells.

[0093] More specifically, the increased levels of the intracellular angiogenesis factor have been confirmed in mouse models to be effective in the treatment of ischemic diseases through high angiogenesis effects.

[0094] Therefore, in this invention, a cell-initiating step, such as treating the cells to be prepared with polyphenols, can be added before step (a) to increase the concentration of effective physiologically active substances within the cells, specifically increasing and / or increasing the content of growth factors to expect excellent therapeutic effects.

[0095] As used in this specification, the term "cytokines" refers to low-molecular-weight signaling proteins secreted by immune cells, which are the primary communication mechanisms used in the immune system. These include inflammatory cytokines that induce various inflammatory symptoms in vivo and / or anti-inflammatory cytokines that suppress these symptoms. Examples of cytokines may include chemokines (CCLs), tumor necrosis factors (TNFs), interferons (IFNs), interleukins (ILs), colony-stimulating factors (CSFs), growth factors, and monocyte chemoattractant proteins.

[0096] Specifically, in one embodiment of the present invention, the cytokine is characterized in that it is one or more selected from the group consisting of IL-1β, IL-4, IL-10, IL-17, GM-CSF, TGF-β, TNF-α, and IFN-γ.

[0097] According to research, the most prominent feature of mesenchymal stem cells, one of the therapeutic cells applicable to this invention, is their ability to suppress inflammation. This can be induced by soluble factors or direct cell-cell contact. Specifically, it is known that mesenchymal stem cells suppress inflammation by secreting anti-inflammatory factors through activation of inflammatory cytokines expressed at sites of inflammation, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). These factors include prostaglandin E2 (PGE2), interleukin-10 (IL-10), HLA-G, nitric oxide, and indoleamine 2,3-dioxygenase (IDO). Alternatively, they can regulate the immune system through various mechanisms, such as inducing the generation of regulatory T cells (Tregs) or inducing apoptosis of immune cells associated with apoptosis.

[0098] In one specific embodiment of the present invention, it was confirmed that when mesenchymal stem cells were initiated using cytokines (e.g., IFN-γ) and polyphenols were then treated to prepare polyphenol-treated therapeutic cryocells, the levels of beneficial physiologically active substances within the cells, such as immunomodulatory factors like PD-L1 and IDO, increased. Furthermore, it was confirmed that, compared to normal cells (norcells), the increased intracellular immunomodulatory factors were maintained, and their prolonged release pattern was observed.

[0099] More specifically, it was confirmed that the cells can exhibit excellent T-cell immunosuppressive effects by continuously releasing immunomodulatory factors, and that the prolonged release pattern of immunomodulatory factors can be effectively used in the treatment of inflammatory diseases such as colitis.

[0100] Therefore, in this invention, a cell-initiating step, such as a cytokine treatment step, can be added before step (a) of treating the cells to be prepared with polyphenols. This can increase and / or concentrate the effective physiologically active substances within the cells, specifically, increase and / or concentrate the content of immunomodulatory factors to expect excellent therapeutic effects.

[0101] The term "small molecules" as used in this specification refers to pharmacological and chemical drugs such as rapamycin, tetrandrine, kynurenic acid, curcumin, valproic acid, sphingosine-1-phosphate, and lipopolysaccharide (LPS).

[0102] As used in this specification, the term "growth factor" refers to an important protein or peptide that regulates cell growth, division, and survival. For example, the growth factor may be one or more selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), but is not limited thereto.

[0103] In another specific embodiment of the invention, the method for preparing the therapeutic cryocell may further include a step of containing a therapeutic drug within the cell before performing step (a) of treating the cell with polyphenols.

[0104] The polyphenol-treated cryocells of the present invention are characterized by the stable preservation of intracellular physiologically active substances. In cases where the cells also contain therapeutic drugs exhibiting specific therapeutic effects, the therapeutic drugs are stably preserved within the cells, thereby allowing for the expectation of excellent therapeutic efficacy.

[0105] The therapeutic drug may include various glucocorticoid drugs, specifically, for example, one or more drugs selected from the group consisting of dexamethasone, fluticasone, betamethasone, budesonide, and mometasone furoate, but is not limited thereto.

[0106] The drug may have a specific particle size, specifically, it may have a size of about 300 nm to 1000 nm. More specifically, the drug may have a size of about 300 nm to 1000 nm, 500 nm to 1000 nm, 700 nm to 1000 nm, 800 nm to 1000 nm, 900 nm to 1000 nm, 300 nm to 900 nm, 500 nm to 900 nm, 700 nm to 900 nm, 800 nm to 900 nm, 300 nm to 800 nm, 500 nm to 800 nm, 700 nm to 800 nm, 300 nm to 700 nm, 500 nm to 700 nm, or 300 nm to 500 nm, preferably, it may have a size of about 700 nm.

[0107] In another specific embodiment of the invention, the drug may be loaded in nanoparticles (NPs).

[0108] Typically, to achieve sustained and targeted drug release, the use of polymer nanoparticles (NPs) such as PLGA-NPs as drug delivery systems is well-known for therapeutic applications. These polymer nanoparticles are characterized by their ability to effectively deliver drugs to the target site while improving drug stability. Recent research has demonstrated that smaller nanoparticles (below 100 nm) have higher permeability than larger nanoparticles. Several studies have confirmed that surface coatings on nanoparticles can increase permeability to the target site; for example, surface modifications of nanoparticles utilize various substances such as surfactants, lipids, ligands, chitosan, and albumin.

[0109] For specific examples, the polymer nanoparticles used in this invention may be one or more selected from the group consisting of poly(alkyl cyanoacrylate), poly(ε-caprolactone), poly(lactic-co-hydroxyacetic acid copolymer), polylactic acid, poly-L-lysine, polyethylene glycol, chitosan, gelatin, albumin, collagen, hyaluronic acid, and their derivatives, but are not limited thereto.

[0110] However, in most cases, nanoparticles exhibit diverse biodistribution and short retention times in vivo due to rapid phagocytosis and non-specific absorption by other cells. Therefore, in order to use therapeutic drugs more effectively, it is necessary to improve the stability and persistence of the drugs in vivo.

[0111] The polyphenol-treated cryocells of the present invention are characterized by not only the stable preservation of intracellular physiologically active substances, but also the excellent persistence of drug delivery in vivo. When a therapeutic drug loaded in 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 can be expected by the high persistence of the therapeutic drug loaded therein at the target site after drug delivery in vivo.

[0112] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of ischemic diseases is provided, comprising polyphenol-treated cryocells.

[0113] The term "ischemic disease" in this specification refers to diseases in which a portion of the blood supply is insufficient due to various causes such as blood leakage caused by vascular damage, narrowing or constriction of arteries, thrombosis or embolism. In particular, the proper functioning of various parts of the body requires an adequate supply of oxygen and nutrients through blood vessels. However, if the blood supply to the heart and other tissues is blocked due to various causes, preventing the supply of oxygen and the various nutrients contained in the blood, ischemic disease occurs. This can be considered a typical disease requiring 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, lower extremity arterial ischemic disease, peripheral vascular disease, vascular dementia, cerebral insufficiency, vascular insufficiency, and coronary artery insufficiency. All ischemic diseases requiring angiogenesis are included within the scope of this invention.

[0114] The term "angiogenesis" refers to the process of generating new blood vessels, that is, the process by which new blood vessels emerge from cells, tissues, or organs. This includes the formation of new blood vessels (vasculogenesis) and the emergence of new blood vessels from existing blood vessels (angiogenesis).

[0115] That is, the pharmaceutical composition of the present invention provides blood vessels with nutrients, growth factors and oxygen required for metabolism in hypoxic states or ischemic tissues and organs caused by the aforementioned circulatory disorders, thereby expecting to functionally improve ischemia-related tissue damage and conditions.

[0116] In one specific embodiment of the invention, after initiating mesenchymal stem cells under hypoxic conditions and / or with protein transport inhibitors, polyphenol-treated therapeutic cryopreserved cells (lyocells) were prepared by treating the cell surface with polyphenols. It was confirmed that the cells contained increased physiologically active substances, specifically increased angiogenic factors (VEGF), thereby demonstrating an enhanced angiogenesis effect.

[0117] More specifically, treatment of HUVEC cells with the modified (specifically, by initiation to contain increased and / or concentrated angiogenic factors) therapeutic cells (lyocells) confirmed improved cell viability, cell motility, and angiogenesis, confirming results similar to those of EGM-2 (Endothelial growth medium 2).

[0118] Furthermore, more specifically, the modified therapeutic cells (lyocells) were shown to have therapeutic effects on ischemic diseases in mice with induced ischemic symptoms by regulating the expression of angiogenesis-related genes (e.g., vegfa, fgf2, and pdgf genes) and inflammation-related genes (e.g., tnfa gene).

[0119] Therefore, in this invention, the polyphenol-treated cryocells for therapeutic use exhibit excellent angiogenesis effects by having increased and / or concentrated growth factors within the cells, thus making them convenient for use in the prevention or treatment of ischemic diseases.

[0120] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of inflammatory diseases is provided, comprising freeze-dried polyphenol-treated cryocells.

[0121] As used in this specification, the term "inflammatory disease" refers to a disease caused by an excessive response of the body's immune system due to the interaction of environmental and genetic factors, characterized by pain, damage, and dysfunction caused by chronic inflammation resulting from the excessive immune response.

[0122] In a specific example of the present invention, it was confirmed that when mesenchymal stem cells were activated using cytokines (e.g., IFN-γ) and polyphenols were processed to prepare polyphenol-treated therapeutic cryocells, enhanced immunomodulatory function was achieved by containing increased physiologically active substances within the activated cells, specifically by containing increased IDO and PD-L1 (programmed death ligand 1).

[0123] More specifically, treatment of the modified therapeutic cells in a dextran sodium sulfate (DSS)-induced acute colitis model (mice), specifically treatment of lyocells containing increased and / or concentrated immunomodulatory factors (e.g., IDO and PD-L1), confirmed improvements in the weight and colon length of the mice, demonstrating a therapeutic effect against colitis.

[0124] Therefore, in this invention, the polyphenol-treated cryocells for therapeutic use can not only stably preserve intracellular physiologically active substances, such as IDO and PD-L1 and other immunomodulatory factors, but can also be effectively used in the prevention, improvement or treatment of inflammatory diseases such as colitis by increasing and / or concentrating the immunomodulatory function therefrom.

[0125] 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 disease enteritis, infectious enteritis, radiation enteritis, ischemic bowel disease, irritable bowel syndrome, etc., but is not limited thereto, and may include all inflammatory diseases caused by excessive immune response in the body.

[0126] In another specific embodiment of the invention, the inflammatory disease can be a lung-related disease. Specific examples of lung diseases include interstitial lung disease, progressive fibrotic 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., novel coronavirus infection (COVID-19)). The lung disease can be an inflammatory lung disease caused by an inflammatory response.

[0127] As used in this specification, the term "prevention" refers to all actions that inhibit or delay the development, spread, or recurrence of a disease, illness, or symptom by administering the compositions of the present invention.

[0128] As used in this specification, the term "treatment" refers to all actions that inhibit, reduce, or eliminate the development of a disease, ailment, or symptom by administering the compositions of the present invention. Furthermore, it refers to all actions that change a disease, ailment, or symptom into improvement or cure.

[0129] As used in this specification, the term "administration" means providing the prescribed composition of the invention to the subject by any suitable method.

[0130] As used in this specification, the term "subject" refers to an animal in which the composition of the present invention can be administered to alleviate a disease, ailment, or symptom. This can be a vertebrate, preferably a mammal, and for example, can 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 can be a human.

[0131] The term "therapeutic effective amount" as used in this specification means an adequate amount sufficient to provide a therapeutic or preventive effect to a subject to whom the composition is to be administered, including the meaning of "preventive effective amount". This can be determined based on factors including the type and severity of the subject's disease, the activity of the drug, the subject's sensitivity to the drug, the timing of administration, the route of administration and metabolic rate, the duration of treatment, concomitant drugs, and other factors well-known in the medical field.

[0132] The pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a suitable organic or inorganic carrier substance that does not adversely react with the cells, compositions, or components of the present invention, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, physiological saline, phosphate-buffered saline (PBS), or culture medium.

[0133] In addition to the ingredients described above, the pharmaceutical compositions of the present invention may also contain lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0134] The composition of the present invention, as a cell therapy agent, can be administered orally or parenterally. When administered parenterally, it can be achieved through intraperitoneal administration, rectal administration, subcutaneous administration, intravenous administration, intramuscular administration, endometrial administration, intraspinal administration, intraventricular administration, intravascular injection, or intrathoracic injection, and can be directly administered to the disease site.

[0135] Solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc. Such solid dosage forms can be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid dosage forms for oral administration include suspensions, internal solutions, emulsions, syrups, etc. Besides water and liquid paraffin, which are commonly used as simple diluents, they may also contain various excipients, such as humectants, sweeteners, flavorings, preservatives, etc.

[0136] The formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried formulations, suppositories, etc. Non-aqueous solvents and suspensions may use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. The base of suppositories may use Witepsol, polyethylene glycol, Tween 61, cocoa butter, lauryl acetate, glycerin gelatin, etc.

[0137] The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the preparation method, route of administration, patient's age, weight, sex, disease state, diet, administration time, route of administration, metabolic rate, and response sensitivity. The usual dosage of mesenchymal stem cells, the active ingredient in the pharmaceutical composition of the present invention, based on an adult, can be 10 mg / day. 2 -10 10 Cells, but not limited to them.

[0138] The pharmaceutical compositions of the present invention can be prepared in a single-dose form or packaged in a multi-dose container by methods readily practiced by those skilled in the art to which this invention pertains, using pharmaceutically acceptable carriers and / or excipients. In this case, the dosage form can be a solution, suspension, syrup, or emulsion in an oil or aqueous solvent, or an extract, powder, granule, tablet, or capsule, and may also contain a dispersant or stabilizer.

[0139] Furthermore, according to another embodiment of the present invention, a pharmaceutical composition for drug delivery is provided, comprising polyphenol-treated cryocells loaded with a drug.

[0140] The polyphenol-treated cryocells of the present invention are characterized in that, although they exist in an apoptotic state due to loss of cellular biological activity, the physiologically active substances within the cells are stably preserved. That is, the cryocells of the present invention exist in an apoptotic state, thus facilitating transportation, storage, and preservation, and possessing the advantage of stably preserving the substances loaded within the cells.

[0141] Therefore, therapeutic drugs can be loaded into the polyphenol-treated lyocells of the present invention to provide a pharmaceutical composition for drug delivery, which can be regarded as the therapeutic lyocells of the present invention being used as an effective drug delivery system.

[0142] The "Drug Delivery System (DDS)" is used to control the release, absorption, or delivery of drugs to specific sites in the body. It refers to a system that maximizes therapeutic efficacy and effect by reducing drug side effects and selectively delivering drugs to target sites.

[0143] In one embodiment of the present invention, the polyphenol-treated therapeutic cryopreserved cells (lyocells) of the present invention exist in an apoptotic state due to the loss of cellular biological activity. This not only facilitates cell transport, storage and preservation, but also stably preserves intracellular physiologically active substances. Therefore, they can be used as an effective drug delivery system.

[0144] In another specific embodiment of the invention, when the polyphenol-treated therapeutic cryocells of the invention are used as a drug delivery system, the drug can be loaded into nanoparticles (NPs).

[0145] In one specific example of the present invention, it was confirmed that after mesenchymal stem cells absorbed nanoparticles loaded with therapeutic drugs, the prepared polyphenol-treated cryocells were mostly located in the lungs after administration, demonstrating high in vivo persistence.

[0146] That is, it was confirmed that the nanoparticles loaded with therapeutic drugs loaded within the cells exhibited excellent persistence in vivo, especially high persistence in lung tissue.

[0147] Therefore, in one embodiment of the present invention, the drug used in the present invention can be a drug for treating lung diseases, but is not limited thereto.

[0148] As used in this specification, the term "lung disease" refers to diseases related to the lungs. Examples of lung diseases include interstitial lung disease, progressive fibrotic 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, pulmonary fibrosis with emphysema, asthma, and respiratory infectious diseases (e.g., novel coronavirus infection). The main pathogenesis of these lung diseases can be lung injury, inflammatory response, and fibrosis.

[0149] Specifically, in one embodiment of the present invention, the lung disease can be a respiratory disease, specifically, it can be one or more 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.

[0150] In one specific embodiment of the present invention, it was confirmed that free MF-NPs were present in the liver and lungs at the initial stage of administration, but disappeared rapidly within 3 days. In contrast, MF-NPs loaded in the polyphenol-treated therapeutic cryopreserved cells (lyocells) of the present invention were immediately and persistently distributed in the lungs after administration, and then migrated to the liver. Furthermore, it was confirmed that MF-NPs loaded in normal cells (norcells) also migrated directly and rapidly to the liver, while in contrast, MF-NPs loaded in the therapeutic cells (lyocells) of the present invention showed long-term persistence in the lungs.

[0151] Therefore, the polyphenol-treated cryocells of the present invention, when administered in vivo, exhibit high persistence of the loaded therapeutic drug at the target site, thus promising excellent therapeutic effects. In particular, when these lyocells are used as a drug delivery system for treating lung diseases, excellent therapeutic effects in lung diseases can be expected due to the high residual rate in the lungs.

[0152] The pharmaceutical composition of the present invention includes the polyphenol-treated therapeutic frozen cells and their preparation method described in the first embodiment of the present invention. To avoid excessive complexity in this specification, repeated content is cited but omitted.

[0153] The effects of the invention

[0154] The polyphenol-treated therapeutic cryocells of the present invention can serve as a biological reservoir for controlling the release of various physiologically active molecules, including intracellular endogenous substances and therapeutic drugs. Furthermore, the polyphenol-treated therapeutic cryocells contain increased and / or concentrated intracellular physiologically active substances, such as growth factors and immunomodulatory factors, and can be used in compositions for preventing or treating ischemic diseases through angiogenesis promotion effects, or in compositions for preventing, improving, or treating inflammatory diseases through enhanced immunomodulatory function. Simultaneously, when the therapeutic cells are used as a suitable drug delivery technology before freezing (drying), it is expected that a simple and effective cell-based drug system can be constructed. Attached Figure Description

[0155] Figure 1 A simplified diagram is shown regarding the polyphenol-treated cryocells (lyocells) of the present invention.

[0156] Figure 2 The results of the preparation and analysis of polyphenol-treated cryocells (lyocells) for therapeutic use using mesenchymal stem cells are shown: Part A, analysis of the viability of mesenchymal stem cells at different concentrations of tannic acid (TA) treatment; Part B, morphology of lyocells at different concentrations of TA treatment; Part C, total protein; Part D, assessment of total RNA levels; Part E, analysis of the release patterns of soluble proteins in lyocells; and Part F, confirmation of the release of particles similar to extracellular vesicles (EVs) in lyocells.

[0157] Figure 3 The results of intravenous (IV) and intramuscular (IM) injections of mesenchymal stem cells into BALB / c mice and C57BL / 6 mice are shown: in vivo distribution of cells (Part A) and cell retention rate (Part B) after IV injection; in vivo distribution of cells in other organs on day 6 (Part C); cell retention rate in the liver compared to the lung (Part D); and in vivo visualization of cell signaling (Part E) and cell retention rate (Part F) after each IM injection.

[0158] Figure 4The results confirming the protective effects of various polyphenols on the preservation of cell morphology and intracellular physiological activity in the therapeutic cryocells (lyocells) of the present invention are shown: Part A, results of cell morphology assessment using an optical microscopy system. Scale: 100 μm; and Parts B and C, assessment of total protein and total RNA levels (n=3) in each polyphenol-treated cell. The data are expressed as mean ± standard deviation (SD) and analyzed using one-way ANOVA. For multiple comparisons, corrections were made using the Benjamini, Krieger, and Yekutieli two-stage ascending method.

[0159] Figure 5 The results show the preparation of polyphenol-treated cryocells for therapeutic use (lyocells) from various cell types and the analysis of their properties: Part A, morphology of lyocells prepared from mouse macrophages (RAW264.7), human fibroblasts (MRC-5), and mouse bone marrow cells; and Part B, total protein content.

[0160] Figure 6 Results demonstrating the potential of polyphenol-treated cryo- (dried) cells (lyocells) with enhanced angiogenesis effects are shown: Part A, Western blot bands in MSCs showing VEGF-A expression before and after 24-hour induction under hypoxia-induced conditions (H; 1% oxygen) and brefidobacterium A (B; 0.1 μg / ml) at 37°C; B) quantification of VEGF-A in modified normal cells (norcells) and lyocells; Part C, VEGF-A release kinetics in cells cultured in serum-free medium; Part D, quantification of VEGF-A in conditioned medium (CM) extracted from control and modified cells; viability (Part E), cell motility (Part F), and tube formation of HUVECs (Part G) of each cell treated with CM extracted from control and modified cells; and changes in body weight (Part H), ischemia fraction assessment (Part I), and assessment of angiogenesis and inflammatory gene levels (Part J) in mice treated with HB-lyocells.

[0161] Figure 7The results of a comparative analysis of the therapeutic cryocells (lyocells) of the present invention treated with polyphenols and cells killed by methods other than polyphenol treatment are shown: Part A, live / dead staining analysis using acridine orange (AO) (green, viable) and propidium iodide (PI) (red, apoptotic); Part B, cell size pattern assessment based on flow cytometry; Part C, relative total protein levels of each cell preparation normalized to cell number (N=4-5); Part D, results of total protein separation in SDS-PAGE gel; Part E, relative total RNA levels of each cell preparation normalized to cell number; normal cells (norcell) and lyocell groups (N=7); HI cells (cell) (N=6); LNT cells (N=3); Part F, cell initiation under hypoxic conditions (Hypoxia, H) and brefeldin A (B) and related HLI (hindlimb ischemic model). A schematic diagram of the model treatment; Part G, assessment of ischemic scores in HLI mice during 14 days; Part H, analysis of gene expression profiles of mouse thigh muscles collected on day 14.

[0162] Figure 8 The results of the analysis of the properties of polyphenol-treated therapeutic cryocells (lyocells) with enhanced immunomodulatory factors obtained through IFN-γ treatment are shown: Part A, a schematic diagram of the IFN-γ-treated lyocell preparation process; Part B, assessment of IDO levels in IFN-γ-treated lyocells after TA treatment; Parts C and D, total physiologically active protein (PD-L1 and IDO, respectively) levels in normal cells (norcells) and lyocells before and after IFN-γ initiation, measured using specific enzyme-linked immunosorbent assay (ELISA) kits (normal cell group (N=2), other cell groups (N=6). The data were analyzed using one-way ANOVA, suitable for multiple comparisons using the two-stage ascending method of Benjamini, Krieger, and Yekutieli; Parts E and F, retention and release patterns of PD-L1 in various modified cells.

[0163] Figure 9The therapeutic efficacy of polyphenol-treated cryocells (lyocells) with enhanced immunomodulatory factors obtained through IFN-γ treatment is demonstrated in Part A, with T cell proliferation assessed by flow cytometry in a co-culture of splenocytes and mesenchymal stem cells; and in Part B, with confirmation of changes in body weight (part B) and colon length (part C) in a DSS-induced acute colitis model mouse with lyocell treatment.

[0164] Figure 10 The results of molecular mechanism analysis of the therapeutic effect of IFN-γ-treated polyphenol-treated cryocells (lyocells) on colitis are presented: Part A shows a schematic diagram of siRNA transfection, IFN-γ initiation, lyocell preparation, and subsequent colitis treatment steps; Part B shows the measurement of colon length in mice (N=5); Part C shows MPO analysis (N=5); Parts D to F show the evaluation of CD3 T cells, MHC2+ cells, and CD11b+ / 11c cells in the mouse colon based on flow cytometry. + The results for the cells (N=3) were analyzed using one-way ANOVA, with multiple comparisons adapted using the two-stage ascending method of Benjamini, Krieger, and Yekutieli.

[0165] Figure 11 Results confirming the therapeutic stability of polyphenol-treated cryo-(dried) cells (lyocells) during long-term storage at 4°C are presented: Part A, HB (hypoxia / protein transport inhibitor-treated) lyocells stored under refrigeration (4°C); Part B, morphological changes of HB-lyocells observed during 12 months of storage; Part C, VEGF-A levels of HB-lyocells before and after storage, analyzed by ELISA; Part D, assessment of ischemic fraction in HLI mice during 14 days; Part E, assessment of gene expression profiles in thigh muscle tissue collected on day 14.

[0166] Figure 12The results of the preparation and characterization of polyphenol-treated cryocells for therapeutic use in potential drug delivery systems are shown: Part A, a schematic diagram of MF-NP cellular internalization and subsequent lyocell preparation; Part B, size distribution of PLGA-NPs loaded with mometasone furoate (MF) measured by dynamic light scattering (DLS); Part C, imaging of MSCs absorbing MF-NPs; Part D, absolute MF content in normal cells (norcells) and lyocells; Part E, MF release kinetics in MF-free and MF-NP / lyocell systems; Part F, phagocytosis assay by flow cytometry; Parts G and H, dendritic cell (DC) generation and maturation assays by flow cytometry; data from Parts F through H were analyzed using one-way ANOVA, suitable for multiple comparisons using the two-stage ascending method of Benjamini, Krieger, and Yekutieli.

[0167] Figure 13 The results confirm the distribution of in vivo nanoparticles (NPs) in polyphenol-treated therapeutic cryocells (lyocells) used as drug delivery systems: Part A, distribution of in vivo nanoparticles in lyocells; Parts B and C, in vivo distribution of cells and particles in the MF-NPs / normal cell (MF-NPs / norcell) and MF-NPs / lyocell groups over time, analyzed in the IVIS system.

[0168] Figure 14 Results confirming the excellent therapeutic effect of MF-NPs / lyocells on an OVA-induced asthma model are shown in Part A, serum IgE levels; Part B, IL-5 levels in lung tissue; Part C, confirmation of the severity of inflammation and mucus production in lung tissue; Part D, a simplified diagram for asthma induction, treatment, and assessment; Part E, assessment of BALF cell count by flow cytometry; and Part F, measurement of serum IgE levels using ELISA. Data from Parts E and F were analyzed using one-way ANOVA, with multiple comparisons performed using the two-stage ascending method of Benjamini, Krieger, and Yekutieli. Detailed Implementation

[0169] The present invention will now be described in more detail through embodiments. However, it will be apparent to those skilled in the art that these embodiments are merely for illustrating the invention more specifically, and the scope of the invention is not limited to these embodiments according to the spirit of the invention.

[0170] Example

[0171] Unless otherwise specified throughout this specification, the percentage used to indicate the concentration of a particular substance is (weight / )% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0172] Preparation Example 1. Preparation of Lyocells (freeze-dried cells) treated with polyphenols

[0173] Preparation Example 1.1. Culture of Mesenchymal Stem Cells (MSCs)

[0174] To improve stability during freeze-drying, MSCs were cultured for 24 hours in low-glucose DMEM (Dulbecco's Modified Eagle's Medium) containing 10% fetal bovine serum (FBS), 1% antibiotics, and 100 mM trehalose at 37°C and 5% CO2.

[0175] Preparation Example 1.2. Pretreatment with Polyphenols (Tanic Acid)

[0176] After detaching the mesenchymal stem cells cultured in Preparation Example 1.1 by trypsinization, the MSCs were washed twice with lyophilization washing buffer (LWB) (physiological saline buffered with 10 mM HEPES and 10 mM sodium bicarbonate (NaHCO3) (pH 7.0)). The cells were then treated with polyphenols, for example, by adding tannic acid (TA) to lyophilization washing buffer (LWB) containing 100 mM trehalose. The survival rate of the MSCs was confirmed by a survival / apoptosis assay based on the concentration of tannic acid (TA) used in the treatment.

[0177] The results are as follows: Figure 2 As shown in Part A.

[0178] Treatment of mesenchymal stem cells with various concentrations of tannic acid (TA) confirmed that the number of apoptotic cells increased with increasing TA concentration. Furthermore, it was confirmed that complete cell death occurred at a 1.0% TA concentration. Figure 2 Part A).

[0179] Preparation Example 1.3. Freeze-drying process

[0180] Then, after treatment with tannic acid (TA), which is equivalent to polyphenols, in Preparation Example 1.2, the obtained cells were freeze-dried to prepare "ready-to-use" frozen or freeze-dried cell products (hereinafter referred to as "lyocell").

[0181] First, the cells were washed twice with freeze-drying wash buffer (LWB) to remove unreacted substances. Then, the cell pellets were mixed with freeze-drying wash buffer (LWB) containing 100 mM trehalose and freeze-dried at -80°C for 16-24 hours to obtain cell powder.

[0182] Example 1. Analysis of the characteristics of polyphenol-treated freeze-dried cells (Lyocells).

[0183] Example 1.1. Confirmation of cell morphology and intracellular physiological activity of lyocells with varying concentrations of tannic acid treatment. Quality content

[0184] For the lyocell obtained in Preparation Example 1, the characteristics of the treatment concentration with tannic acid (TA) were confirmed.

[0185] First, the morphology of lyocells was confirmed using scanning electron microscopy (SEM) images with varying concentrations of pretreated tannic acid (TA). Figure 2 As shown in Part B, it was confirmed that, compared with normal (live) cells (Norcell), the cell morphology was more intact with increasing concentration of tannic acid (TA) in the pretreatment.

[0186] Then, analysis of the cellular bioactive components in lyocells with varying tannic acid (TA) treatment concentrations confirmed that cellular protein and RNA levels in lyocells increased with increasing TA concentration during pretreatment. Specifically, lyocells pretreated with 1.0% TA showed a corresponding increase in levels compared to the normal cell (norcell) group. Specifically, cellular protein levels remained at approximately 97 ± 3%, and RNA levels remained at 107 ± 13%. Figure 2 Parts C and D). Furthermore, it was confirmed that the cell-soluble proteins in lyocells obtained by freeze-drying after tannic acid (TA) treatment ( Figure 2 The E portion) and extracellular vesicle (EV)-like particles ( Figure 2 (F) continues to be released.

[0187] This confirms that, although the activity of mesenchymal stem cells is the same as that of dead cells after treatment with tannic acid (TA), the cell structure is preserved intact, just like that of normal cells, and the physiologically active substances within the cells are also condensed and preserved.

[0188] Example 1.2. Confirmation of in vivo distribution (persistence in vivo) after administration of Lyocell

[0189] Mesenchymal stem cells (MSCs) can be present in the lungs immediately after intravenous injection, and are therefore commonly used in the treatment of lung diseases. However, most of the cells migrate from the lung tissue to other tissues (e.g., the liver) or are cleared within a day, thus reducing the therapeutic efficacy of MSCs in the treatment of lung diseases. Therefore, the in vivo distribution characteristics (tissue persistence) of therapeutically effective MSCs in the target area become one of the important factors for excellent therapeutic effects.

[0190] To confirm the in vivo distribution characteristics (tissue persistence) of freeze-dried mesenchymal stem cells, the inventors administered the cells to mice and conducted an in vivo distribution study.

[0191] First, before administration to the cells, they were labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine (DiR) for in vivo observation. Specifically, in the case of normal cells (norcell), DiR labeling was performed before administration; in the case of the lyocells of this invention, DiR labeling was performed before treatment with tannin (TA). For BALB / c mice, in vivo administration of the cells was performed intravenously (IV), and for comparisons along the route of administration, intramuscular administration (IM) was also performed in C57BL / 6 mice. Each mouse was administered 1 × 10⁻⁶ 6 Mice were administered at cellular concentrations and imaged at 745 / 780 nm (excitation / emission) using the IVIS® Spectrum In Vivo imaging system at specified time intervals after administration.

[0192] Additionally, 144 hours after intravenous administration, major organs (heart, lung, liver, stomach, kidney, spleen, and intestine) were harvested from mice and imaged to observe the distribution of DiR-labeled cells.

[0193] Example 1.2.1. Biodistribution after intravenous injection

[0194] First, the distribution of cells in the organism was confirmed at 0, 4, 24, 48, and 144 hours after intravenous administration, as follows. Figure 3 Parts A through D are shown.

[0195] As follows Figure 3As shown in Part A, it was confirmed that both normal cells (norcells) and lyocells were completely captured in the lungs of BALB / c mice after intravenous administration. However, a portion of the normal cell (norcell) signal was observed to migrate to the liver tissue within 4 hours, while the majority of the lyocell signal remained confined to the lungs after 24 hours. In particular, in… Figure 3 In Part B, when comparing the proportion of total cells in mice, the proportion of lyocells was observed to be significantly higher than that of most normal cells (norcells).

[0196] Furthermore, 144 hours after intravenous administration, the results of confirming cell distribution in major organs were as follows: Figure 3 As shown in parts C and D.

[0197] As a result, it was confirmed that compared with 0 hours after intravenous administration, approximately 60% of the lyocell signal remained, while the signal of normal cells (norcell) was only about 40%. The cumulative signal ratio of liver cells in the lyocell group relative to the lung (lung / liver retention ratio) was about twice that of the normal cell group.

[0198] Example 1.2.2. In vivo distribution after intramuscular injection (IM)

[0199] Next, normal cells (norcell) and lyocell were administered into the thigh muscles of mice on the right (R) and left (L) sides, respectively. Cell distribution in the organisms was confirmed at 0, 24, 48, 72, 96, 120, 144 and 168 hours after administration.

[0200] The results are as follows: Figure 3 Parts E and F are shown.

[0201] First, such as Figure 3 As shown in section E, the results of a week of observation showed that the fluorescence signal of lyocells was significantly stronger than that of normal cells (norcells). In particular, it was confirmed that about 50% of normal cells (norcells) were eliminated after 48 hours of intramuscular administration, while in the case of lyocells, most of them remained at the administration site.

[0202] and, Figure 3 In Part F, the results of in vivo persistence of cells at different time points relative to 0 hours after intramuscular administration were measured. On day 7 (168 hours), 26 ± 8% of normal cells (norcell) and 33 ± 4% of lyocells were found to remain at the administration site.

[0203] Preparation Example 2. Preparation and Analysis of Lyocells Using Polyphenols Other Than Tannic Acid (TA)

[0204] In the lyocell preparation process of the present invention, in order to confirm the application potential of various polyphenols other than tannic acid (TA), lyocells were prepared from various cells and their properties were confirmed.

[0205] Preparation Example 2.1. Preparation of lyocells using polyphenols other than tannic acid (TA).

[0206] In this preparation example, scutellarin, catechin hydrate, gallic acid hydrate, chlorogenic acid (CGA), and epigallocatechin gallate (EGCG) (TCI Chemical Co., Ltd., Japan) were used as polyphenols other than tannic acid (TA).

[0207] Specifically, the aforementioned polyphenols were dissolved in PBS (pH 7.0, buffered with 10 mM HEPES and 10 mM bicarbonate) at an initial concentration of 1%. Undissolved substances were removed by centrifugation, and the clear supernatant was used for cell processing. Then, after trypsinization to detach passage 4 mesenchymal stem cells, they were washed twice with PBS buffer (pH 7.0). Cells were treated with the aforementioned polyphenol solutions under continuous rotation. After washing the polyphenol-treated cells twice with PBS buffer, they were dispersed in freeze-drying buffer (PBS, pH 7.0, containing 100 mM trehalose) and frozen at a cooling rate of 1°C to -80°C. Then, they were freeze-dried for 16–24 hours to obtain polyphenol-treated freeze-dried lyocell powder.

[0208] Preparation Example 2.2. Analysis of the characteristics of lyocells treated with polyphenols other than tannic acid (TA).

[0209] To analyze the characteristics of the various polyphenol-treated lyocells obtained in Preparation Example 3.1, the inventors confirmed the morphology of the lyocells and the content of intracellular physiologically active substances. Normal (live) cells (Norcells) and untreated cells (lyocell-control) were used as control groups.

[0210] Analysis of the morphological characteristics of lyocell

[0211] First, in order to analyze the morphological characteristics of lyocels treated with polyphenols other than tannic acid (TA), the lyocell powder obtained in Preparation Example 3.1 was dissolved in distilled water and reconstituted. After obtaining cell particles by centrifugation, the particles were dispersed in PBS. The cells in the suspension were plated and the morphology of lyocells treated with polyphenols other than tannic acid was observed in a microscope system.

[0212] The results are as follows Figure 4 As shown in Part A.

[0213] As follows Figure 4 As shown in Part A, it was confirmed that in the case of untreated lyocells (lyocell-control), the morphology and shape of the cells were significantly damaged and collapsed. In contrast, in all polyphenol-treated lyocells, it was confirmed that the cells maintained their morphological stability even after freeze-drying, with the EGCG, catechin, and gallic acid treatment groups showing the best retention effect.

[0214] Analyze the content of physiologically active substances in lyocells

[0215] Then, the content of physiologically active substances in lyocells was analyzed by measuring the total cellular protein and RNA levels of lyocells treated with polyphenols other than tannic acid.

[0216] 1. Measuring intracellular protein levels

[0217] First, to measure intracellular protein levels, the granular cells were collected in microtubes and dissolved on ice in a 10% trichloroacetic acid (TCA) solution for 30 minutes. The precipitated proteins were then collected by centrifugation at 15,000 rpm for 10 minutes at 4°C. The precipitate was then dissolved in bicarbonate buffer (200 mM, pH 8.5, 1% sodium dodecyl sulfate (SDS)) and subjected to probe sonication on ice for 10 seconds. Fluoroscamine analysis (Thermo Fisher Scientific) was then performed to measure total protein levels, with data standardized according to cell count for each group.

[0218] The results are as follows Figure 4 As shown in Part B.

[0219] As follows Figure 4As shown in Part B, it was confirmed that in untreated lyocells (lyocell-contro), the intracellular protein content was retained by less than 60% compared to normal cells (norcell), while in all polyphenol-treated lyocells, the retention rate of intracellular physiologically active substances, i.e., proteins, was significantly improved. In particular, the most effective protein preservation was confirmed in the gallic acid and EGCG treatment group, specifically showing a protein retention rate of over 75% compared to normal cells (norcell).

[0220] 2. Measure intracellular RNA levels

[0221] Furthermore, to measure intracellular RNA levels, total cellular RNA was extracted from the obtained lyocells using the Rneasy microkit (Qiagen) according to the manufacturer's instructions. After removing DNA contamination in the column using DNase solution (Qiagen), RNA was eluted with DEPC-treated water and its concentration was measured using a Nanodrop 2000 instrument. Data were standardized according to the cell count of each group.

[0222] The results are as follows Figure 4 As shown in section C.

[0223] As follows Figure 4 As shown in Section C, it was confirmed that in untreated lyocells (lyocell-contro), the intracellular RNA content was retained by less than 30% compared to normal cells (norcell), while in all polyphenol-treated lyocells, the RNA content retention rate was significantly improved. In particular, the most effective RNA preservation was confirmed in the gallic acid and EGCG-treated groups of the lyocells, specifically showing an RNA retention rate of over 75% compared to normal cells (norcell).

[0224] In conclusion, it is believed that various polyphenols, including tannic acid (TA), play a decisive role in preserving the cell morphology and intracellular physiologically active substances of freeze-dried apoptotic cell therapy agents before freeze-drying.

[0225] Preparation Example 3. Preparation and Characterization of Lyocells Using Cells Other Than Mesenchymal Stem Cells

[0226] In the lyocell preparation process of the present invention, in order to confirm the application potential of various cell groups other than mesenchymal stem cells, lyocells were prepared using various cells and their characteristics were confirmed.

[0227] Specifically, lyocells were prepared using mouse macrophages (RAW264.7), human fibroblasts (MRC-5), and mouse bone marrow cells using the same method. The structure of the cells after freeze-drying was observed, and their total protein content was assessed.

[0228] The results are as follows: Figure 5 As shown.

[0229] First, such as Figure 5 As shown in Part A, it was also confirmed that the cell structure was well preserved after freeze-drying in lyocells prepared using cells other than mesenchymal stem cells. Furthermore, measurements of the total protein content in the prepared lyocells, in lyocells pretreated with 1.0% tannic acid (TA) using the same method as in Example 1, showed a corresponding level of total protein content compared to normal cells (norcells). Figure 5 Part B).

[0230] Thus, the cells that can be used as lyocells in this invention are not limited to mesenchymal stem cells, and it is believed that there is a possibility of using various cells other than mesenchymal stem cells (e.g., T cells, dendritic cells, stromalvascular fraction cells, etc.) as lyocells.

[0231] Example 2. Examining the potential for use of lyocells with increased angiogenesis effects.

[0232] In Example 1, the inventors confirmed that the lyocell of the present invention has a high retention capacity for intracellular physiologically active substances and exhibits excellent persistence in vivo. Therefore, in this example, mesenchymal stem cells with increased intracellular angiogenesis factors were used to prepare lyocells to confirm the applicability of lyocells with increased intracellular angiogenesis effects.

[0233] Example 2.1. Preparation of lyocells with increased angiogenesis effect

[0234] First, in order to prepare mesenchymal stem cells with intracellular accumulation of angiogenesis factors including VEGF-A, the cells were subjected to hypoxia induction (H) and brefeldin A (B) treatment or hypoxia induction and brefeldin A treatment (HB) for 24 hours.

[0235] The results are as follows Figure 6 Part A and Part B are shown.

[0236] First, such as Figure 6As shown in Part A, it was confirmed that high intracellular VEGF-A levels were induced under both 1% hypoxia and 0.1 μg / ml brefidobacterium A (HB).

[0237] Then, HB-lyocells were prepared from mesenchymal stem cells with accumulated angiogenesis factors using the same method as in Preparation Example 1, with treatment with 1% tannic acid (TA) and freeze-drying.

[0238] As a result, Figure 6 As shown in Part B, the VEGF-A content of HB-lyocell is 10 ng / 10 6 The cells maintained the same high level as HB-normal cells (HB-norcell). Furthermore, in Figure 6 Part C confirms that HB-lyocells cultured in serum-free medium showed continuous VEGF-A release for 11 days.

[0239] Example 2.2. Confirmation of improved angiogenesis through modified lyocells.

[0240] To confirm the effect of the lyocell (HB-lyocell) prepared in Example 2.1 on angiogenesis, the following experiment was conducted.

[0241] Example 2.2.1. Evaluation of angiogenesis factors in modified lyocells

[0242] First, to evaluate the angiogenesis factors within the modified lyocells, normal cells (norcells) or lyocells were cultured in serum-free medium at 37°C for 2 days. Conditioned medium (CM) was then extracted, and the VEGF-A level in the extracted CM was measured. The HB-untreated normal cell group (Control norcell), the HB-normal cell group (HB-norcell), and the HB-untreated lyocell group (Control lyocell) were used as comparative groups for HB lyocells. Serum-free medium was used as a negative control group, and the EGM-2 treated group was used as a positive control group.

[0243] As a result, Figure 6As shown in Part D, increased VEGF-A release levels were confirmed in HB-treated lyocells (HB-lyocells) compared to the EGM-2-treated group used as a positive control. Furthermore, significantly increased VEGF-A release was confirmed in HB-lyocells compared to HB-treated normal cells (norcells) (HB-norcells), untreated HB normal cells (Control norcells), and untreated HB lyocells (Control lyocells).

[0244] Thus, it can be confirmed that the increased VEGF-A level in HB-lyocells is specifically observed in HB-lyocells prepared by freeze-drying mesenchymal stem cells treated with both hypoxia and brevidin A.

[0245] Furthermore, the results of treating human umbilical vein endothelial cells (HUVECs) with the extracted conditioned medium (CM) showed that, in the case of CM treated with HB-lyocells, cell viability, cell motility, and tube formation were improved compared with CM extracted from other control groups, exhibiting levels very similar to those in the EGM-2 treatment group. Figure 6 (Part E - Part G).

[0246] Example 2.2.2. Therapeutic effect of modified lyocell on ischemic diseases

[0247] Then, in vivo experiments were conducted in mice to confirm the therapeutic effect of the modified lyocell on ischemic diseases.

[0248] Specifically, C57BL / 6 mice (male, 8 weeks old, Orient Bio, South Korea) were anesthetized with 2% isoflurane at a flow rate of 2 L / min. The left limb muscle tissue was then exposed. The femoral artery was ligated at both ends (upper and lower) using 6-0 silk suture (Ethicon, USA), and the ligated artery between the two ends was excised. The skin of the mice was then sealed and disinfected with povidone-iodine before randomization to other groups. For treatment, on the first day after induction, PBS and MSCs (1×10⁻⁶) were intramuscularly injected into the central hind leg muscle, which served as the ischemic vessel resection area. 6 Cells or cell lysate (equivalent to 1×10⁻⁶ cells)6 (Cells) Then, the weight change of mice was measured for up to two weeks, and the severity of the disease was observed by fractionating limb healthy status. In addition, the diseased limbs of the mice were recovered to assess the levels of angiogenesis and inflammatory genes. The primer information used for this is shown in Table 1 below.

[0249] Table 1

[0250] Primer information for assessing angiogenesis and inflammatory gene levels

[0251]

[0252] In this embodiment, HB-lyocell was used as the experimental group, HB-norcell and HB-lyocell lysate were used as the comparison group, and PBS, normal cells (norcell) and lyocell were used as the control group.

[0253] The results are as follows: Figure 6 The H section to the J section are shown.

[0254] First, such as Figure 6 As shown in sections H and I, the results observed over two weeks confirm that HB-lyocell treatment is most effective in regulating body weight changes and reducing ischemic fraction in mice.

[0255] Then, the levels of angiogenesis and inflammatory genes in each treatment group were measured. Compared with other groups, the angiogenesis genes (i.e., vegfa, fgf2, and pdgf) and inflammatory genes (i.e., tnfa) were upregulated in the HB-lyocell treatment group, thus confirming the effective regulation of the expression of these genes.

[0256] In conclusion, it has been confirmed that when mesenchymal stem cells with enhanced angiogenesis factors obtained through hypoxia and treatment with brevidin A are prepared as lyocells, they exhibit high angiogenesis efficacy and can be effectively used in the treatment of ischemic diseases.

[0257] Comparative Example 1. Cell preparations treated with methods other than polyphenols

[0258] The inventors prepared apoptosis-inducing cell preparations using different treatment methods than polyphenols and compared them with the polyphenol-treated lyocells of the present invention to confirm the superior properties of the lyocells prepared by polyphenol treatment of the present invention.

[0259] Comparative Example 1.1. Preparation of cell preparations treated with methods other than polyphenols

[0260] The inventors prepared apoptotic cell preparations using different methods other than polyphenol treatment, respectively preparing heat-inactivated (HI) cells, liquid nitrogen-treated (LNT) cells, and spontaneously apoptotic (SA) cells.

[0261] Specifically, i) heat-inactivated (HI) cells were prepared by loading PBS (2×10⁻⁶ cells into a solution of ... 6 MSCs (cells / ml) were prepared by heating at 50°C for 30 minutes and then cooling on ice. ii) Liquid nitrogen treatment (LNT) was performed on MSCs (2 × 10⁶ cells / ml) in microtubes. 6 (i) Cells / tube particles are immersed in liquid nitrogen for 12 hours and then cooled on ice for preparation. (ii) Spontaneously apoptotic (SA) cells are collected from free-floating cells in an adherent MSC culture for preparation. All three cell types are washed twice with PBS before use.

[0262] Then, to confirm whether apoptosis occurred in all cells, including normal cells (norcell) and lyocells, acridine orange (AO, green, live) and propidium iodide (PI, red, apoptosis) staining analysis was performed to determine whether apoptosis occurred.

[0263] As a result, Figure 7 As shown in Part A, all cells were stained red with PI, thus confirming that they all exhibited an apoptotic cell state.

[0264] Comparative Example 1.2. Comparative Analysis with the Polyphenol-treated Freeze-dried Lyocells of the Present Invention

[0265] Then, the three apoptotic cells prepared in Comparative Example 1.1 were compared and analyzed with the lyocell of the present invention.

[0266] Comparative Example 1.2.1. Cell Morphology and Size

[0267] First, flow cytometry was performed on all cells to analyze cell patterns, specifically cell morphology and size.

[0268] As a result, Figure 7 As shown in Part B, it can be confirmed that in HI cells, the cell morphology and size are partially disrupted, whereas in the case of the lyocell of the present invention, the cell morphology and size are mostly similar to those of normal cells (norcell).

[0269] Comparative Example 1.2.2. Intracellular physiologically active components

[0270] Then, a comparative analysis was performed on all intracellular physiologically active components, such as total protein or RNA levels.

[0271] Specifically, i) Intracellular total protein levels were measured as follows: Cells were collected in granular form in microtubes and precipitated on ice in a 10% trichloroacetate (TCA) solution. The precipitated protein was collected by centrifugation at 4°C and 15,000 rpm for 10 minutes. The precipitate was then dissolved on ice in bicarbonate buffer (200 mM, pH 8.5, 1% SDS) under probe sonication for 10 seconds. Fluoroamine (Thermo Fisher Scientific) analysis was then performed to measure total protein, with bovine serum albumin (BSA) used as a protein standard to plot calibration curves. Data were normalized to cell count for each group.

[0272] The result is as follows Figure 7 As shown in section C, it was confirmed that the lyocell group of the present invention maintained the total protein content of the normal cell (norcell) group up to 90%, while in the case of the three apoptotic cell groups prepared in Comparative Example 1.1, only up to 70% protein level was detected.

[0273] Furthermore, proteins from each cell group were separated using SDS-PAGE gels prepared with 12% acrylamide solution and stained with Coomassie Blue solution to visualize the protein bands.

[0274] The results are as follows: Figure 7 As shown in section D, it was confirmed that the lyocell of the present invention also exhibits excellent properties in terms of protein content storage compared to the three types of apoptotic cells prepared in Comparative Example 1.1.

[0275] On the other hand, ii) Intracellular total RNA level, the intracellular total RNA level was determined by absorbance measurement after extracting total RNA from each cell using a Trizol-based method.

[0276] As a result, it was confirmed that compared with the normal cell (norcell) group, lyocells retained up to 88% of total RNA, while the HI and LNT cell groups had less than 30% of RNA remaining. Figure 7 Part E).

[0277] Comparative Examples 1, 2, and 3: Treatment Efficacy of Ischemic Diseases

[0278] Similar to Example 2.1, all cell groups were treated with hypoxia-induced conditions (H) and brefidobacterium A (B) to exert angiogenesis properties, and applied to a hindlimb ischemic (HLI) model to confirm the therapeutic effect on ischemic diseases. Figure 7 (part F).

[0279] First, we compared the therapeutic efficacy of HB-lyocell and HB-HI cells.

[0280] The results are as follows: Figure 7 As shown in section G, the long-term effect of the HB-lyocell of the present invention in reducing ischemic fraction was confirmed during a 14-day observation period, but HB-HI cells only showed temporary disease improvement in the initial stage (day 3) and thereafter showed no therapeutic effect on ischemic disease at all.

[0281] In addition, to assess the gene expression profiles in each cell group treated with HB, thigh muscle tissue from mice was collected on day 14.

[0282] The specific quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed as follows: Mouse thigh muscle tissue was collected and dissolved using TRIzol reagent. Total RNA was extracted using chloroform layering and precipitated using isopropanol and 75% ethanol. Total RNA levels were measured using a Nanodrop 2000 instrument. cDNA was then synthesized using the RevertAid First Strand cDNA Synthesis kit, and qRT-PCR analysis was performed using the SYBR Green amplification method.

[0283] The additional primer sequences are shown in the table below.

[0284] Table 2

[0285] Primer information for assessing angiogenesis and inflammatory gene levels

[0286]

[0287] Observe the following Figure 7 In the H portion, it was confirmed that compared with PBS-treated mice, HB-lyocell treatment significantly increased angiogenesis factors (Vegfa and Cd31) and muscle regeneration / remodeling / homeostasis factors (MyoD and MyoG), while decreasing inflammatory factors (Tnfa and Il1b).

[0288] Based on the results of this comparative example, it is confirmed that, compared with apoptosis-inducing cell preparations using methods other than polyphenol treatment, the lyocell, a freeze-dried polyphenol-treated cell of the present invention, exhibits superior properties and efficacy.

[0289] Example 3. Preparation of lyocells with activated immunomodulatory function and their potential utilization in inflammation.

[0290] In Example 1, the inventors confirmed that the lyocell of the present invention has a high retention capacity for intracellular physiologically active substances and excellent persistence in vivo. Furthermore, mesenchymal stem cells that activate the expression of intracellular immunomodulatory factors were prepared as lyocells to confirm the potential use of lyocells with activated immunomodulatory functions.

[0291] Example 3.1. Preparation of lyocells with enhanced immunomodulatory function via IFN-γ initiation.

[0292] Cell initiation via IFN-γ is known to activate the expression of important intracellular immunomodulatory factors, including IDO and PD-L1. Therefore, after initiating mesenchymal stem cells by treating them with IFN-γ (40 ng / ml), tannin (TA) was treated using the same method as in Preparation Example 1, and lyocells with activated immunomodulatory function were prepared using freeze-drying. Figure 8 Part A).

[0293] Example 3.2. Analysis of the characteristics of lyocells initiated using IFN-γ.

[0294] Example 3.2.1. Comparison of lyocell characteristics with varying concentrations of tannic acid (TA) treatment.

[0295] First, Western blotting was performed on the lyocells initiated by IFN-γ to measure the level of intracellular IDO with varying concentrations of tannic acid (TA) treatment.

[0296] As a result, Figure 8 As shown in Part B, it was confirmed that the level of IDO in lyocells initiated by IFN-γ increased with increasing tannic acid (TA) concentration. In particular, lyocells treated with 1.0% tannic acid (TA) showed IDO levels similar to those in normal cells (norcells).

[0297] Thus, in lyocells prepared after tannic acid (TA) treatment, it was confirmed that the increased intracellular immunomodulatory factors were stably preserved after freeze-drying. In particular, when treated with 1.0% tannic acid (TA), it was confirmed that the same level of preservation ability of intracellular substances was achieved compared with normal cells (norcell).

[0298] Example 3.2.2. Comparison of IFN-γ-activated lyocell characteristics

[0299] (1) Quantification of intracellular protein levels

[0300] Then, in order to quantify the protein levels of MSCs before and after the initiation process of the IFN-γ, an enzyme-linked immunosorbent assay (ELISA) was performed.

[0301] As a result, through Figure 8 Parts C and D confirm that, with the activation of IFN-γ, the level of PD-L1 in all cell groups increased significantly by more than 10-fold, especially in IFN-γ-lyocells, which maintained higher levels of PD-L1 and IDO (>85%) compared to IFN-γ-norcells.

[0302] (2) Confirm the ability to retain and release intracellular immunomodulatory factors.

[0303] Then, the inventors conducted tests on the retention and release of intracellular immune regulatory factors initiated by IFN-γ.

[0304] In the case of IFN-γ-normal cells (norcells), 1 mL of culture medium was used in each of the 12-well cell culture plates, specifically in DMEM medium containing 10% FBS and 1% penicillin-streptomycin or in free medium without them. On the other hand, IFN-γ-lyocells were cultured in 1 μm Transwell systems in the form of 24-well plates. The IFN-γ-lyocells were placed in the upper Transwell chamber with 0.2 mL of release medium (containing 200 μg / mL 2-phospho-1-ascorbic acid (AA) and 0.3% BSA in PBS buffer) and 0.8 mL of medium in the lower Transwell chamber. Samples were incubated in a CO2 incubator at 37°C. Then, the release medium and cells were separated by centrifugation at pre-set time intervals. In the cellular retention test, cells were dissolved in RIPA buffer containing 1x protease inhibitor (Thermo Fisher Scientific). All samples were then stored at -20°C and analyzed by ELISA for PD-L1 levels within 2 weeks.

[0305] The results are as follows: Figure 8 Parts E and F are shown.

[0306] First, such as Figure 8 As shown in section E, it was confirmed that cellular PD-L1 levels decreased sharply within 48 hours in IFN-γ-normal cells (norcell), while in contrast, they decreased slowly after 168 hours of culture in IFN-γ-lyocells.

[0307] Similarly, Figure 8 In the F segment, it was observed that PD-L1 in the IFN-γ-normal cell (norcell) group was largely released into the culture medium within 48 hours, confirming that its release level remained significantly low under free culture conditions. On the other hand, it was confirmed that IFN-γ-lyocells exhibited a prolonged release pattern of PD-L1 for at least 168 hours.

[0308] Example 3.3. Verification of the therapeutic efficacy of IFN-γ-activated lyocells

[0309] Example 3.3.1. In vitro immunosuppressive effect of IFN-γ-activated lyocells

[0310] First, it is known that the inhibition of T cell proliferation in splenocytes can be regulated by immunomodulatory factors secreted by mesenchymal stem cells. Therefore, in order to confirm the immunosuppressive effect of the modified lyocell in Example 3.1, the immunosuppressive effect of lyocell in splenocytes was evaluated in vitro.

[0311] Splenocytes were labeled with 5,6-carboxyfluoresceindiacetate succinimidyl ester (CFSE) and then co-cultured with mesenchymal stem cells for 4 days in activated RPMI-1640 medium (supplemented with PHA and IL-2). The cell proliferation inhibition effect of the lyocell of this invention was then confirmed by flow cytometry analysis. First, the following methods were used as a control group for this embodiment: i) individual culture of splenocytes; the co-culture was performed using two methods based on cell-cell interactions: ii) direct contact culture; or iii) indirect culture (transwell module).

[0312] Specifically, all spleen cells were isolated from the spleen of C57BL / 6 mice and labeled with 2 μM CFSE in RPMI-1640 medium supplemented with 10% FBS. Then, spleen cells (5 × 10⁶ cells / year) were... 6 Cells) and mesenchymal stem cells (5×10 5Cells were co-cultured in 6-well plates using direct contact and transwell modules. Specifically, RPMI 1640, containing 10% FBS, 1% antibiotic, 100 IU / mL IL-2 (BioLegend), 2 μg / mL PHA (Thermo Fisher Scientific), and 50 μM β-mercaptoethanol, was used as the activation medium for 4 days. The cultured cells were then collected, stained with APC-anti-CD3 antibody, and analyzed by flow cytometry.

[0313] In the separate culture of spleen cells, which serves as the control group in this embodiment, positive (activated) and negative (non-activated) control groups are used according to the presence or absence of stimulation. In the co-culture with spleen cells, normal cells (norcell) are used as the control group. In the co-culture of spleen cells with lyocells, normal cells (norcell) are used as the control group.

[0314] Specifically, 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 as comparative experimental groups to the lyocells modified by treating the stimulating factor (e.g., IFN-γ) used as the experimental group in this embodiment (IFN-γ lyocell).

[0315] The results are as follows: Figure 9 As shown in Part A.

[0316] like Figure 9 As shown in Part A, it was confirmed that in i) culturing spleen cells alone, the proliferation rate of T cells in stimulated spleen cells increased. However, in ii) co-culturing with mesenchymal stem cells via a direct contact model, the T cell proliferation rate was significantly slowed by treating IFN-γ normal cells (IFN-γ norcells) and IFN-γ lyocells, respectively; conversely, in other control groups, there was almost no effect on the inhibition of T cell proliferation. In iii) co-culturing with mesenchymal stem cells via a transwell module, it was confirmed that T cell proliferation was partially inhibited only in the IFN-γ lyocell treatment group; in other control groups, including IFN-γ normal cells (IFN-γ norcells) as a comparison group, the T cell inhibition effect was very low.

[0317] As a result, compared with the IFN-γ normal cell (IFN-γ norcell) group, the IFN-γ lyocell of the present invention continuously releases immunomodulatory factors (e.g., 2,3-dioxygenase (IDO)) even without direct contact with immune cells, thus demonstrating excellent T-cell immunosuppressive effects.

[0318] Example 3.3.2. Validating the in vivo activity of IFN-γ-activated lyocells using an acute colitis mouse model. (in vivo) Treatment efficacy

[0319] Furthermore, the therapeutic effect of treating IFN-γ-lyocells was evaluated in an acute colitis model induced by dextran sulfate sodium (DSS).

[0320] Specifically, acute colitis was induced in 8-week-old male C57BL / 6 mice (Orient Bio, South Korea) by administering 2%-3% DSS (MP Biomedicals, Canada) via drinking water for 7 days. Then, on days 3 and 6, 3 × 10⁻⁶ DSS was administered. 6 The concentration of lyocells was administered to mice via intraperitoneal (IP) administration to treat IFN-γ. The body weight and colon length of each mouse were then measured to confirm the therapeutic effect of lyocells on colitis. To determine the severity of colitis, body weight was measured for 9 days after lyocell administration, and the colon length was measured on day 9 of the experiment.

[0321] In this embodiment, the DSS-untreated group (Normal) and the DSS-only group (DSS only) were used as negative control groups. In this embodiment, the normal cell (norcell) administration group was used as the control group. Specifically, the IFN-γ-treated normal cell administration group, the untreated IFN-γ normal cell (control norcell), and the untreated IFN-γ lyocell (control lyocell) were used as comparative experimental groups to the IFN-γ-treated lyocells used as the experimental groups in this embodiment.

[0322] The results are as follows: Figure 9 As shown in Parts B and C.

[0323] It was confirmed that mouse body weight and colon length were improved by IFN-γ-treated lyocells compared to IFN-γ-treated normal cells, thus confirming that IFN-γ-treated lyocells exhibit high efficacy in the recovery of colitis symptoms.

[0324] Example 3.3.3. Analysis of the molecular mechanism of the therapeutic effect of IFN-γ-activated lyocells on colitis.

[0325] Meanwhile, in order to confirm the molecular mechanism of the therapeutic effect on colitis, the inventors prepared lyocells by silencing intracellular immunomodulatory factors related to colitis treatment, such as PD-L1 and IDO, through small interfering RNA transfection before initiating cells with IFN-γ.

[0326] The simplified experimental procedure is as follows: Figure 10 As shown in Part A. Specifically, mesenchymal stem cells at passage 3-4 were seeded into 100mM culture plates until 80% confluence was achieved. The medium was then replaced with a pre-prepared complex of Accufect transfection reagent (Bioneer, Korea) and 50nm siRNA (Bioneer, Korea) in TOM (Transfecion Optimized Medium, Welgene), 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 scrambled siRNA (AccuTarget™ negative control siRNA) was used as control siRNA molecules. After culturing cells at 37°C for 6 hours, the culture medium was replaced with one containing 100 mM trehalose (Tokyo Chemical Industry Co., Ltd., Japan) and 40 ng / ml murine IFN-γ (biological activity 1–4 × 10⁻⁶). 6 Complete MEM-α (U / mg; Cat. No. 575306; BioLegend) was cultured at 37°C for 24 hours. Cells were then collected and prepared as lyocells according to the method described in Preparation Example 1, and transfection efficiency was checked using qRT-PCR.

[0327] The comparative groups that silenced immunomodulatory factors related to the treatment of colitis were denoted as si-PD-L1 and si-IDO, respectively, while the control group was denoted as si-Scr. The therapeutic effects of colitis were compared with those of the modified lyocell of the present invention.

[0328] The results are as follows: Figure 10 Parts B through F are shown.

[0329] The results confirmed that all lyocell preparations effectively reduced the severity of colitis caused by DSS. Figure 10 Part B). However, significant inhibition of the therapeutic effect of IFN-γ-treated lyocells (IFN-γ-lyocells) on infiltration of immune cells in the colon was confirmed in si-PD-L1 or si-IDO, which was achieved through increased MPO activity ( Figure 10 (part C) and T cells based on flow cytometry analysis ( Figure 10 (part D) and MHC2+ cell count ( Figure 10 The efficacy of IFN-γ-lyocell therapy for colitis is verified using E and F. This confirms that the therapeutic effect of IFN-γ-lyocell therapy for colitis is highly dependent on PD-L1 and IDO.

[0330] Furthermore, in this embodiment, lysates of si-Scr / IFN-γ-lyocells treated with probe sonication on ice in PBS were used as a control group, and the effects of the modified lyocells were evaluated in a mouse model of colitis by administering an equivalent cell dose of the lysates. The results are as follows: Figure 10 As confirmed in parts E and F, administration of the lysate also showed a specific effect in DSS-induced colitis, but significantly reduced the function of undamaged IFN-γ-lyocells.

[0331] Therefore, such results are considered essential for the long-term release of physiologically active molecules from intact IFN-γ-lyocells to achieve greater therapeutic efficacy.

[0332] Example 4. Evaluation of therapeutic stability during long-term storage of Lyocell

[0333] Then, for the lyocell of the present invention, in order to verify its possibility of being used as an "off-the-shlef" product, its therapeutic stability was confirmed after storing HB-lyocell at 4°C for a fixed period of time. Figure 11 Part A).

[0334] First, as mentioned above, after storing HB-lyocells at 4°C for 12 months, the HB-lyocells remained undamaged and maintained their cell morphology and size. Figure 11 Part B), and confirmed by ELISA that the intracellular VEGF-A level also remained at the prescribed level ( Figure 11 Part C).

[0335] Furthermore, in an HLI (ischemic ileus) model, comparing the therapeutic effects of newly prepared HB-lyocell with HB-lyocell stored for 6 months, the results confirmed that both HB-lyocell groups showed the same effect in reducing ischemic fraction during a 14-day observation period. Figure 11 (Part D). Furthermore, in all mouse models treated with HB-lyocell, the expression of angiogenesis factors (Vegfa and Cd31) and muscle regeneration / remodeling / homeostasis factors (MyoD and MyoG) was significantly increased, while the expression of inflammatory factors (Tnfa and Il1b) was decreased. This confirms that, regardless of storage time, HB-lyocell effectively restored thigh muscle tissue damage. Figure 11 E).

[0336] Therefore, it can be confirmed that the lyocell formulation of the present invention also exhibits excellent therapeutic stability during long-term storage.

[0337] Example 5. Testing the feasibility of using lyocell as a drug delivery system

[0338] Then, for the lyocell of the present invention, its potential as a drug delivery system (DDS) for cells was confirmed.

[0339] Example 5.1. Preparation of lyocells for drug delivery systems

[0340] Typically, sustained and targeted drug release is achieved by loading therapeutic formulations into polymer nanoparticles (NPs) such as PLGA-NP.

[0341] In this embodiment, drug-loaded PLGA-NP was absorbed into mesenchymal stem cells and prepared as lyocells using the same method as in Preparation Example 1, employing tannic acid (TA) treatment and freeze-drying techniques.

[0342] Specifically, in this embodiment, mometasone furoate (MF), a potent glucocorticoid, is loaded onto a PLGA NP (with an average size of approximately 700 nm). Figure 12 The B portion was used as a model drug, and such MF-NP was absorbed to approximately 4.5 μg MF / 10 6After MSC treatment, the cells are prepared as lyocells (hereinafter referred to as MF-NP / lyocell).

[0343] The following Figure 12 Part A illustrates the cellular internalization of MF-NP and the subsequent lyocell preparation process.

[0344] Example 5.2. Analysis of the characteristics of lyocells used in drug delivery systems

[0345] The results of observing the MF-NP / lyocell prepared in Example 5.1 using a confocal laser scanning microscope (CLSM) confirmed the presence of MF-NP ( ) within the lyocell. Figure 12 The results of measuring the MF content in part C of the lyocell confirmed that, similar to MF-NP / norcell cells, MF was well preserved within the lyocells, which are freeze-dried mesenchymal stem cells. Figure 12 Part D). On the other hand, regarding intracellular drug release, it was confirmed that in the case of MF-NP / lyocell, intracellular drug release lasted for at least about 10 days; conversely, in the case of free MF-NPs that were not absorbed by lyocells, drug release showed a continuous increase over about 40 days. Figure 12 Part E).

[0346] Thus, the drug delivery system using the lyocell of the present invention can stably store the drug to be delivered within the cell and can continuously release the stored drug.

[0347] On the other hand, in this embodiment, a significantly increased rate of phagosytosis was observed in the co-culture of heat-inactivated (HI) cells and liquid nitrogen-treated (LNT) cells (p < 0.0001 vs. normal cell (norcell) group or lyocell group). Interestingly, as follows... Figure 12 As shown in section F, it was confirmed that the phagocytic activity of macrophages in the MF-NPs / lyocell group was significantly reduced compared with that in the lyocell group (p=0.0005).

[0348] Next, the in vitro effects of MSCs on the generation and maturation of dendritic cells (DCs) were evaluated. Specifically, to generate DCs, bone marrow cells (BMCs) were isolated and stimulated with GM-CSF and IL-4, with BMCs without cytokine stimulation used as a negative control. BMCs were co-cultured with MSCs from other groups starting from day 0 and collected on day 5 of stimulation. The cell ratio of BMCs to MSCs was maintained at a prescribed 10:1.

[0349] The following Figure 12 The flow cytometry results shown in section G confirm the presence of CD11c in all cultures of both the normal cell (norcell) and lyocell groups. + DC generation was significantly reduced, and when MF-NP was integrated into MSC, CD11c was confirmed. + The DC value decreased further.

[0350] Additionally, DC maturation utilizes LPS to activate immature CD11c in the presence or absence of MSCs. + DC 2 days were used for evaluation. Similarly, mature CD86 was observed in all MSC groups, especially in the MF-NPs / norcell and MF-NPs / lyocell groups. + CD11c + The proportion of DC decreased significantly ( Figure 12 (H part). Interestingly, it was found that the lyocell group showed a slightly higher DC inhibition effect compared with the normal cell (norcell) group.

[0351] Example 5.3. Confirmation of the distribution of nanoparticles (NPs) in biological organisms.

[0352] To evaluate the drug delivery efficacy via lyocell, the inventors confirmed the in vivo distribution of nanoparticles (NPs).

[0353] Example 5.3.1. Confirmation of the in vivo distribution of nanoparticles (NPs) applied to lyocells

[0354] First, to confirm the in vivo distribution of lyocell nanoparticles (NPs), MF-NPs were labeled with Cy5.5 and the labeled MF-NPs / lyocell (1×10⁻⁶) were added to 200 μl of PBS. 6 MF-NPs (one cell and approximately ~4.5 μg of MF) or free MF-NPs (at the same dose) were administered intravenously to BALB / c mice via the tail vein. Then, within 6 days of administration, various visceral organs, including lungs, liver, kidneys, spleen, and heart, were collected from the mice, and the signal of MF-NPs in the samples was evaluated using an IVIS imaging system (675 nm / 710 nm excitation / emission).

[0355] The results are as follows: Figure 13 As shown in Part A.

[0356] Specifically, in the initial period after administration, free MF-NPs were observed to be distributed in both the lungs and liver, but these MF-NPs rapidly disappeared from the lungs within 1 day and from the liver within 3 days. Conversely, MF-NPs loaded in lyocells (MF-NP / lyocell) were confirmed to be predominantly located in the lungs from the start of administration, and signals were still detected in the lungs on day 6 post-administration. This suggests that lyocells have at least the potential to serve as a drug delivery system for the treatment of lung diseases.

[0357] Example 5.3.2. Comparison of in vivo bioactivity of lyocell and normal cell (norcell) nanoparticles (NPs). cloth

[0358] Furthermore, for MF-NPs loaded in normal cells (norcell) and lyocells respectively, the inventors compared the in vivo distribution of nuclear particles.

[0359] Specifically, cells containing MF-NPs were administered via the tail vein of BALB / c mice using the same method as described in Example 5.2.1. Cells and MF-NPs were labeled with DiR and Cy5.5, respectively, and fluorescence signals were detected in an IVIS system after organs were collected at predetermined times. The relative fluorescence signal of each organ was then analyzed by dividing the total signal of all organs collected each time.

[0360] The results are as follows: Figure 13 As shown in Parts B and C.

[0361] Similar to the comparative analysis results with free MF-NPs in Example 5.2.1, both the MF-NPs / normal cell (MF-NPs / norcell) group and the MF-NPs / lyocell group showed major signals in lung tissue immediately after injection. However, the cellular and particle signals in the MF-NPs / normal cell (MF-NPs / norcell) group rapidly migrated to liver tissue within 24 hours, while in contrast, most of the signals in the MF-NPs / lyocell group remained in lung tissue during the 144-hour observation period.

[0362] In summary, such in vivo distribution data suggest that the lyocell formulation of this invention has the advantage of delivering effective drugs to lung tissue.

[0363] Example 5.4. Examining in vivo therapeutic effects of MF-NPs / lyocells in an OVA-induced asthma mouse model. effect

[0364] Therefore, the inventors used an OVA-induced asthma mouse model in BALB / c mice to evaluate the therapeutic efficacy of various MSC preparations.

[0365] Specifically, as follows Figure 14 As shown in section D, asthma was induced in 8–12 week old male BALB / c mice (Orient Bio, South Korea) using ovalbumin (an OVA-based model). This was achieved by intraperitoneal sensitization on days 0, 7, and 14 using 200 μl of PBS containing 50 μg of OVA (grade V, Sigma-Aldrich) and 2 mg of aluminum hydroxide (Sigma-Aldrich). On day 20, 1 × 10⁻⁶ OVA was used. 6 Mice were administered 1 × 10⁻⁶ cells / 200 μl PBS (+150 μg enoxaparin) as a negative control. The drugs were also administered to mice via tail vein. Additionally, each experimental group (MF-NPs / lyocell, control lyocell, Free NPs) was administered (1 × 10⁻⁶ cells / lyocell, ... 6 Cells and equivalent to ~4.5 μg MF were collected. Then, on days 21, 23, and 25, mice were administered PBS containing 20 μg OVA intranasally. On day 26, blood and lung tissue were collected from the mice, and IL-5 levels in lung tissue and serum IgE levels were measured using an ELISA kit. Furthermore, after dissecting the lung tissue, H&E and PAS staining was performed to assess local inflammation and mucin-producing cells in each airway.

[0366] The results are as follows: Figure 14 Parts A through C are shown.

[0367] like Figure 14 As shown in Parts A through C, the results from the negative control group (OVA + PBS) confirmed that intranasal challenge with OVA in sensitized mice significantly increased serum IgE levels, lung tissue IL-5 levels, lung tissue inflammation severity, and mucus production. Furthermore, treatment with the control lyocell showed negligible effect on asthma symptoms, while treatment with free MF-NP had almost no effect. However, MF-NP / lyocell treatment was confirmed to be significantly effective in reducing asthma compared to other control groups.

[0368] Meanwhile, the inventors intend to more clearly confirm the therapeutic efficacy of the excellent drug delivery effect of the lyocell of the present invention in an OVA-induced asthma mouse model by comparing and analyzing MF-NPs / lyocell with MF-NPs / norcell.

[0369] The experiment was conducted using the same methods as described above, with the experimental and control groups shown in Table 3 below.

[0370] Table 3

[0371]

[0372] On day 26, blood and lung tissue were collected from mice. The severity of asthma was assessed by analyzing indicators including bronchoalveolar lavage fluid (BALF) cell count and serum IgE. The results are as follows. Figure 14 Parts E and F are shown.

[0373] like Figure 14 As shown in sections E and F, normal cells (norcell), lyocells, free MF-NPs, and the MF-NPs / normal cells (MF-NPs / norcell) groups were confirmed to have moderate effects in reducing asthma-related indicators. However, the MF-NPs / lyocell group was confirmed to significantly reduce asthma severity (BALF cell count and serum IgE levels were both compared with the OVA / PBS group, p=0.0002).

[0374] Therefore, based on the above results, lyocell is considered to have the potential to be used as a drug delivery system targeting lung tissue for the treatment of lung diseases such as asthma, and excellent therapeutic effects can be expected through this approach.

Claims

1. A type of cryopreserved cells for therapeutic use treated with polyphenols.

2. The polyphenol-treated cryopreserved cells for therapeutic use according to claim 1, characterized in that, The frozen cells were obtained by freeze-drying.

3. The polyphenol-treated cryopreserved cells for therapeutic use according to claim 1, characterized in that, The polyphenols are selected from one or more of the following groups: gallic acid, caffeic acid, chlorogenic acid, catechin, epicatechin gallate, epicatechin, proanthocyanidins, luteolin, apigenin, scutellarin, naringenin, hesperidin, sennaol, 2-methyl-1,4-naphthoquinone, flavonoid quinone, tannic acid, gallantanine, ellagitanninine, and their derivatives.

4. The polyphenol-treated cryopreserved cells for therapeutic use according to claim 1, characterized in that, The cell is activated under the following conditions: (i) Hypoxic conditions; (ii) Protein transport inhibitors; (iii) Cytokines; (iv) Protein synthesis inhibitors; (v) Small molecule compounds; (vi) growth factors; or (iv) Their combinations.

5. The polyphenol-treated cryopreserved cells for therapeutic use according to claim 1, characterized in that, The cells contain therapeutic drugs.

6. The polyphenol-treated cryopreserved cells for therapeutic use according to claim 5, characterized in that, The drug is loaded in nanoparticles.

7. A method for preparing therapeutic frozen cells treated with polyphenols, characterized in that, include: Step (a), treat cells with polyphenols; as well as Step (b): Freeze or freeze-dry the cells.

8. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 7, characterized in that, The freezing or freeze-drying is carried out by adding sugars selected from the group consisting of dextrose, maltose, glucose, lactose, sucrose, trehalose, mannose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, fructose, melitriose, melbiose, sorbitol, and triose.

9. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 7, characterized in that, The polyphenols in step (a) are selected from one or more of the following groups: gallic acid, caffeic acid, chlorogenic acid, catechin, epicatechin gallate, epicatechin, proanthocyanidins, luteolin, apigenin, scutellarin, naringenin, hesperidin, sennaol, 2-methyl-1,4-naphthoquinone, flavonoid quinone, tannic acid, gallantanine, ellagitanninine, and their derivatives.

10. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 9, characterized in that, The concentration of tannic acid used in the treatment is from 0.02% to 10%.

11. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 7, characterized in that, Prior to step (a), the process also includes a step of initiating the cell.

12. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 7, characterized in that, The startup is performed under the following conditions: (i) Hypoxic conditions; (ii) Protein transport inhibitors; (iii) Cytokines; (iv) Protein synthesis inhibitors; (v) Small molecule compounds; (vi) growth factors; or (iv) Their combinations.

13. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 12, characterized in that, The hypoxic conditions are formed with an oxygen concentration of 0.5% to 10%.

14. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 12, characterized in that, The protein transport inhibitor is monensin, brefidobacterium A, or a combination thereof.

15. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 12, characterized in that, The cytokines are selected from one or more of the group consisting of IL-1β, IL-4, IL-10, IL-17, GM-CSF, TGF-β, TNF-α, and IFN-γ.

16. The method for preparing polyphenol-treated cryopreserved cells for therapeutic use according to claim 7, characterized in that, Prior to step (a), the procedure also includes the step of containing the therapeutic drug within the cell.

17. A pharmaceutical composition for the prevention or treatment of ischemic diseases, comprising polyphenol-treated cryopreserved therapeutic cells, characterized in that, The therapeutic frozen cells treated with polyphenols were obtained by freeze-drying.

18. A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising therapeutically frozen cells treated with polyphenols, characterized in that, The therapeutic frozen cells treated with polyphenols were obtained by freeze-drying.

19. A pharmaceutical composition for drug delivery comprising polyphenol-treated therapeutic cells loaded with a drug, characterized in that, The therapeutic frozen cells treated with polyphenols were obtained by freeze-drying.

20. The pharmaceutical composition for drug delivery according to claim 19, characterized in that, The drug is used to treat lung diseases.

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