Stem cell-drug delivery system comprising melatonin-containing drug carrier and stem cells, and use thereof

Abstract

The present invention relates to a stem cell-drug delivery system comprising a melatonin-containing drug carrier and stem cells, and, more specifically, to a use of the stem cells for treating intestinal epithelial damage diseases and inflammatory bowel disease. It has been identified that the stem cell-drug delivery system according to the present invention continuously releases PGE2 and has an excellent ability to induce revival stem cells. Therefore, the intestinal epithelial cell regeneration effect thereof is excellent, and thus the stem cell-drug delivery system of the present invention can be variously used in the fields of treating intestinal damage diseases and inflammatory bowel disease.

Description

Stem cell-drug delivery system comprising melatonin-containing drug delivery system and stem cells and use thereof

[0001] The present invention relates to a melatonin-containing drug delivery system and a stem cell-drug delivery system comprising stem cells, and more particularly, to the use of the stem cells for the treatment of intestinal epithelial damage diseases and inflammatory bowel diseases.

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disorder, typified by ulcerative colitis and Crohn's disease. While the pathogenesis of IBD remains unclear, genetic factors, abnormalities in intestinal barrier function, intestinal microbiota imbalances, and environmental factors are believed to be potential causes. Patients with IBD are characterized by severe damage to the intestinal barrier, so restoring barrier integrity by promoting tissue regeneration is a key treatment goal. Current treatments for IBD suffer from refractoriness, resistance, and side effects, and exhibit limitations in restoring intestinal mucosal integrity and tissue regeneration.

[0003] Meanwhile, revival stem cells (revSCs), a newly discovered stem cell population within the intestinal epithelium, have been found to be specialized in the restoration of damaged tissue. Stem cells expressing Lgr5 (Leucine-rich repeat-containing G-protein coupled receptor 5) present in the crypts of the intestinal epithelium replenish intestinal epithelial cells through rapid proliferation and differentiation and are responsible for maintaining homeostasis. However, these stem cells are easily lost by signals that cause epithelial damage, such as dextran sulfate sodium (DSS) and radiation exposure. Instead, regenerative stem cells expressing fetal transcripts, including Clu, Ly6a (Sca-1), and Anxa1, have been found to be induced to regenerate epithelial tissue. It has been reported that regenerative stem cells can be induced by prostaglandin E2 (PGE2), and that PGE2 secreted from fibroblasts within the intestinal mucosa binds to the EP4 receptor on intestinal epithelial cells, activating YAP signaling, thereby inducing regenerative stem cells.

[0004] Accordingly, the present inventors, while researching the treatment of intestinal epithelial damage and inflammatory bowel disease, developed a "stem cell-drug delivery system (heterospheroid)" by mixing melatonin microspheres with three-dimensional mesenchymal stem cells. The heterospheroids of the present invention were confirmed to continuously release melatonin and PGE2 and to have excellent regenerative stem cell induction capabilities, thereby completing the present invention.

[0005] Accordingly, the purpose of the present invention is to provide a stem cell-drug delivery system comprising a drug delivery system containing melatonin; and stem cells.

[0006] Another object of the present invention is to provide a composition comprising the stem cell-drug delivery system.

[0007] Another object of the present invention is to provide a method for producing a cell therapeutic agent for regenerating intestinal epithelial cells, comprising the steps of: (a) mixing and homogenizing melatonin and a polymer to produce a melatonin-containing drug carrier; and (b) mixing the melatonin-containing drug carrier produced in step (a) and stem cells to produce a stem cell-drug carrier.

[0008] Another object of the present invention is to provide a method for treating intestinal epithelial damage disease, comprising a step of administering the stem cell-drug delivery system to a subject in need thereof.

[0009] Another object of the present invention is to provide a method for treating inflammatory bowel disease, comprising administering the stem cell-drug delivery system to a subject in need thereof.

[0010] To achieve the above purpose, the present invention provides a stem cell-drug delivery system comprising a drug delivery system containing melatonin and stem cells.

[0011] The present invention also provides a composition for regenerating intestinal epithelial cells comprising the stem cell-drug delivery system.

[0012] The present invention also provides a composition for inducing regenerative stem cells comprising the stem cell-drug delivery system.

[0013] The present invention also provides a pharmaceutical composition for preventing or treating intestinal epithelial damage disease, comprising the stem cell-drug delivery system.

[0014] The present invention also provides a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising the stem cell-drug delivery system.

[0015] In addition, the present invention provides a method for producing a cell therapeutic agent for intestinal epithelial cell regeneration, comprising the steps of: (a) mixing and homogenizing melatonin and a polymer to produce a melatonin-containing drug carrier; and (b) mixing the melatonin-containing drug carrier produced in step (a) and stem cells to produce a stem cell-drug carrier.

[0016] The present invention also provides a method for treating an intestinal epithelial damage disease, comprising a step of administering the stem cell-drug delivery system to a subject in need thereof.

[0017] The present invention also provides a method for treating inflammatory bowel disease, comprising administering a stem cell-drug delivery system to a subject in need thereof.

[0018] The stem cell-drug delivery system of the present invention was confirmed to continuously release melatonin and PGE2 and to have excellent regenerative stem cell induction properties. This indicates an excellent intestinal epithelial regeneration effect, and thus, the stem cell-drug delivery system of the present invention can be utilized in various fields of treatment for intestinal damage and inflammatory bowel diseases.

[0019] Figure 1a is a diagram showing the manufacturing process of a stem cell-drug delivery system (i.e., heterospheroid) according to the present invention.

[0020] Figure 1b is a diagram showing the results of measuring the diameter of a heterospheroid according to the present invention.

[0021] Figures 1c and d are diagrams showing the results of confirming the cell viability of heterospheroids according to the present invention through LIVE / DEAD staining.

[0022] Figure 2a is a diagram showing the results of observing intestinal epithelial organoids co-cultured with heterospheroids according to the present invention.

[0023] Figure 2b is a diagram showing the results of classifying intestinal epithelial organoids co-cultured with heterospheroids according to the present invention according to cell morphology.

[0024] Figure 2c is a diagram showing the results of analyzing the expression of intestinal epithelial markers in intestinal epithelial organoids co-cultured with heterospheroids according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0025] Figure 2d is a diagram showing the results of counting cells expressing regenerative stem cell markers in intestinal epithelial organoids co-cultured with heterospheroids according to the present invention through flow cytometry analysis (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0026] Figure 3a is a diagram showing the results of body weight measurement of a mouse intestinal inflammation model administered with a heterospheroid according to the present invention.

[0027] Figure 3b is a diagram showing the results of confirming the survival rate of a mouse intestinal inflammation model administered with a heterospheroid according to the present invention.

[0028] Figure 3c is a diagram showing the results of disease activity index evaluation of a mouse intestinal inflammation model administered with heterospheroids according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0029] Figure 3d is a diagram showing the results of measuring the colon length of a mouse intestinal inflammation model administered with a heterospheroid according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0030] Figure 3e is a diagram showing the results of H&E staining of intestinal epithelial tissue of a mouse intestinal inflammation model administered with a heterospheroid according to the present invention.

[0031] Figure 3f is a diagram showing the results of evaluating intestinal epithelial damage in a mouse intestinal inflammation model administered with heterospheroids according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0032] Figures 3g to 3i are diagrams showing the results of analyzing inflammatory indicators (TNF-α, IL-17, and IL-10) in a mouse intestinal inflammation model administered with a heterospheroid according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0033] Figure 3j is a diagram showing the results of analyzing MPO (Myeloperoxidase) activity in the colon of a mouse intestinal inflammation model administered with heterospheroids according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0034] Figure 3k is a diagram showing the results of analyzing the induction of regenerative stem cells in a mouse intestinal inflammation model administered with a heterospheroid according to the present invention (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0035] Figure 4a is a diagram showing the results of analyzing cells expressing the reconstructive stem cell marker Ly6a in intestinal epithelial organoids co-cultured with the stem cell-drug delivery system according to the present invention through flow cytometry (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0036] Figure 4b is a diagram showing the results of analyzing cells expressing the regenerative stem cell marker Ly6a in intestinal epithelial organoids co-cultured with stem cell-drug delivery system (quercetin) using flow cytometry.

[0037] Figure 4c is a diagram showing the results of analyzing the expression of the reconstructive stem cell marker Cldn4 in intestinal epithelial organoids co-cultured with the stem cell-drug delivery system according to the present invention using real-time qPCR (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0038] Figure 4d is a diagram showing the results of analyzing the expression of the reconstructive stem cell marker Cldn4 in intestinal epithelial organoids co-cultured with stem cell-drug delivery system (quercetin) using real-time qPCR (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0039] Figure 4e is a diagram showing the results of analyzing the expression of Cldn4 in colon organoids co-cultured with stem cell-drug delivery system (quercetin) using real-time qPCR (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0040] Hereinafter, the present invention will be described in detail.

[0041] According to an aspect of the present invention, the present invention provides a stem cell-drug delivery system comprising a drug delivery system containing melatonin; and stem cells.

[0042] The drug delivery system containing melatonin of the present invention has melatonin encapsulated within the delivery system. In an embodiment of the present invention, it was confirmed that melatonin induces regenerative stem cells without adversely affecting the cell viability of stem cells. In contrast, quercetin, a clinically used treatment for inflammatory bowel disease, has been proven to be effective in treating inflammatory bowel disease, but when administered together with stem cells, it was confirmed to have no significant effect on the induction of regenerative stem cells. Therefore, the drug delivery system of the present invention is particularly characterized by inducing the regeneration and reconstruction of damaged intestinal epithelial cells.

[0043] The stem cell-drug delivery system of the present invention is characterized by a mixture of a drug delivery system containing melatonin. Due to these technical features, the stem cell-drug delivery system of the present invention not only enables in vivo delivery of stem cells, but also enhances the duration of drug release.

[0044] In the present invention, the drug delivery vehicle refers to a particle formed by forming a polymer coating layer centered on a drug, and for convenience, it can be expressed in the form of "type of polymer - drug delivery vehicle." For example, when the type of polymer is poly(lactic-co-glycolic acid), PLGA, it is expressed as a PLGA drug delivery vehicle.

[0045] In a specific embodiment of the present invention, the drug delivery vehicle may be a biodegradable polymer drug delivery vehicle known in the art.

[0046] In a specific embodiment of the present invention, the drug delivery system is preferably made of at least one polymer selected from the group consisting of polylactide-co-glycolide, polylactide-co-glycolide-co-ethylene glycol, polystyrene-co-ethylene glycol, polyethyleneimine-co-ethylene glycol, polyphosphagen-co-ethylene glycol, polylactide-co-ethylene glycol, polycaprolactone-co-ethylene glycol, polyanhydride-co-ethylene glycol, polymalic acid-co-ethylene glycol and derivatives thereof, polyalkylcyanoacrylate-co-ethylene glycol, polyhydroxybutyrate-co-ethylene glycol, polycarbonate-co-ethylene glycol and polyorthoester-co-ethylene glycol, polyethylene glycol, poly-L-lysine-co-ethylene glycol, polyglycolide-co-ethylene glycol, polymethylmethacrylate-co-ethylene glycol, polyvinylpyrrolidone-co-ethylene glycol and copolymers thereof, most preferably polyvinyl alcohol, but the scope of the present invention is not limited thereto. Doesn't.

[0047] The stem cells of the present invention are not limited thereto, but may be of autologous or allogenic origin.

[0048] In a specific embodiment of the present invention, the stem cell may be an embryonic stem cell, a mesenchymal stem cell, or an induced-pluripotent stem cell, and preferably a mesenchymal stem cell.

[0049] In the present invention, embryonic stem cells (ESCs), also commonly abbreviated as ES cells, refer to pluripotent cells derived from the inner cell mass of a blastocyst, an early-stage embryo. For the purposes of the present invention, the term "ESC" is also sometimes used broadly to include embryonic germ cells.

[0050] In the present invention, mesenchymal stem cells (MSCs) refer to pluripotent precursor cells before differentiation into cells of specific organs such as bone, cartilage, fat, tendon, nerve tissue, fibroblasts, and muscle cells.

[0051] In the present invention, induced pluripotent stem cells (iPSCs), also commonly abbreviated as iPS cells, refer to a type of pluripotent stem cell artificially induced from a normally non-pluripotent cell, such as an adult somatic cell, by inducing the “forced” expression of a specific gene.

[0052] In a preferred embodiment of the present invention, the mesenchymal stem cells may be derived from embryonic yolk sac, placenta, umbilical cord, umbilical cord blood, tonsil, skin, peripheral blood, bone marrow, adipose tissue, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, anterior cruciate ligament, articular chondrocytes, deciduous cells, pericytes, periodontal bone, subpatellar fat pad, spleen or thymus, and preferably, mesenchymal stem cells derived from umbilical cord blood or tonsil.

[0053] The stem cells are preferably derived from a human, but may also be derived from a fetus or a non-human mammal. The non-human mammal may more preferably be a canine, feline, simian, cow, sheep, pig, horse, rat, mouse, or guinea pig, and the like, without limitation to their origin.

[0054] In a specific embodiment of the present invention, the stem cells may be in the form of a spheroid.

[0055] In a specific embodiment of the present invention, the spheroid may have an average size of 50 to 300 μm, preferably an average size of 162.09 μm, but the scope of the present invention is not limited thereto.

[0056] Also, the stem cell-drug delivery system is 1×10 1 1×10 5 cells, preferably 1×10 2 1×10 4 may contain cells.

[0057] The drug delivery system containing melatonin according to the present invention has a spherical shape with melatonin encapsulated therein.

[0058] Additionally, the stem cell-drug delivery system of the present invention may include one or more drug delivery systems containing melatonin for each stem cell unit.

[0059] In addition, it is preferable that the stem cell drug delivery system of the present invention be transplantable into a living body.

[0060] In an embodiment of the present invention, a 'stem cell-drug delivery system' was manufactured as shown in Fig. 1a. Specifically, 90 mg of PLGA and 10 mg of melatonin were added to 1 ml of dichloromethane to prepare a first solution, and the first solution was dropped into 5 mL of a 1% PVA (polyvinyl alcohol, Sigma) solution. After that, the mixture was emulsified and evaporated to manufacture a 'drug delivery system containing melatonin'. The manufactured drug delivery system containing melatonin and 1.2 × 10 mesenchymal stem cells were mixed. 6 A 'stem cell-drug delivery system' was created by culturing dog cells for 24 hours.

[0061]

[0062] According to another aspect of the present invention, the present invention provides a composition for regenerating intestinal epithelial cells, or a composition for inducing regenerative stem cells, comprising the cell-drug delivery system.

[0063] In a specific embodiment of the present invention, the stem cell-drug delivery system was confirmed to induce regenerative stem cells in intestinal epithelial or colon-derived organoids and in an animal model of intestinal inflammation. Furthermore, the stem cell-drug delivery system of the present invention was confirmed to suppress colon shortening and improve intestinal epithelial structure in an animal model of intestinal inflammation. Therefore, the stem cell-drug delivery system of the present invention can be utilized in various fields of treatment for intestinal damage and inflammatory bowel diseases.

[0064] The above-mentioned revival stem cells (revSCs) are stem cells that induce intestinal epithelial regeneration in place of existing Lgr5-expressing stem cells. These regenerative stem cells can form self-renewing colonies, thereby regenerating damaged epithelial layers and improving intestinal epithelial structure.

[0065] The reagent composition of the present invention may further include one or more known ingredients having an effect of intestinal epithelial regeneration or regenerative stem cell induction.

[0066]

[0067] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating intestinal epithelial damage disease, or a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising the stem cell-drug delivery system.

[0068] In a specific embodiment of the present invention, the intestinal epithelial damage disease may be at least one selected from the group consisting of Crohn's disease, ulcerative colitis, ulcerative duodenitis, hemorrhagic rectal ulcer, leaky gut syndrome, gastritis, gastric ulcer, pouchitis, enteritis, and ischemic colitis.

[0069] In a specific embodiment of the present invention, the inflammatory bowel disease may be any one of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, transitional colitis, and Behcet's syndrome.

[0070] The pharmaceutical composition of the present invention may be a cell therapy composition.

[0071] In the present invention, cell therapy refers to a medicine (as defined by the US FDA) used for the purposes of treatment, diagnosis, and prevention by separating, culturing, and manufacturing cells and tissues from an individual through special manipulation, and by performing a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological characteristics of cells through other methods.

[0072] The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the active ingredient, and the auxiliary agents can include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents.

[0073] The composition according to the present invention can be formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. In addition, target organ-specific antibodies or other ligands can be combined with the carriers to act specifically on target organs. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the relevant technical field or a method disclosed in Remington's literature.

[0074] The pharmaceutical composition of the present invention may be in the form of a liquid, suspension, dispersion, emulsion, gel, injectable liquid, and sustained-release preparation of an active compound, and preferably, may be an injection.

[0075] When the pharmaceutical composition of the present invention is formulated as an injection, in order to ensure product stability according to the distribution of the injection prescription, the pH is adjusted using a buffer solution such as an acid solution or phosphate solution that can be used as an injection, thereby producing an injection that is very stable both physically and chemically.

[0076] More specifically, the injection can be prepared by dissolving the solution in water for injection together with a stabilizer or a solubilizer, and then sterilizing it, in particular, by high-temperature vacuum sterilization or aseptic filtration. The water for injection can be distilled water for injection or an injection buffer solution, for example, a phosphate buffer solution having a pH range of 3.5 to 7.5 or a sodium dihydrogen phosphate (NaH2PO4)-citric acid buffer solution. The phosphate used may be in the form of a sodium salt or potassium salt, or in the form of an anhydrous or hydrated form, and may also be in the form of citric acid, or in the form of an anhydrous or hydrated form.

[0077] In addition, the stabilizer used in the present invention includes sodium pyrosulfite, sodium bisulfite, sodium metabisulfite or ethylenediaminetetraacetic acid, and the dissolution aid includes a base such as sodium hydroxide, sodium bicarbonate, sodium carbonate or potassium hydroxide, or an acid such as hydrochloric acid or acetic acid.

[0078] The injectable composition of the present invention can be formulated as bioabsorbable, biodegradable, and biocompatible. Bioabsorbable means that the injectable composition can disappear from the body upon initial application, without degradation or disintegration of the dispersed injectable composition. Biodegradable means that the injectable composition can be broken down or decomposed within the body by hydrolysis or enzymatic degradation. Biocompatibility means that all components are non-toxic within the body.

[0079] The injection according to the present invention can be manufactured using a diluent such as a conventional filler, weighting agent, binder, wetting agent, surfactant, or excipient.

[0080] The composition or effective ingredient of the present invention may be administered in a conventional manner, depending on the purpose, through intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, subcutaneous, intrauterine, epidural, inhalation, topical, rectal, oral, intraocular, or intradermal routes, and is preferably administered intravenously. The composition or effective ingredient of the present invention may be administered by injection or catheter.

[0081] In the composition of the present invention, the dosage of the active ingredient is 1 x 10 based on an adult weighing 60 kg. 1 1 x 10 50 dogs / kg, preferably 1 x 10 1 1 x 10 30 dog / kg, more preferably 1 x 10 5 1 x 10 20 dogs / kg, most preferably 1 x 10 7 1 x 10 9 It can be adjusted within the range of dog / kg. However, the optimal dosage to be administered can be easily determined by those skilled in the art, and can be adjusted according to various factors including the type of disease, severity of the disease, content of active ingredients and other ingredients contained in the composition, type of formulation, and age, weight, general health, sex, and diet of the patient, time of administration, route of administration, and excretion rate of the composition, treatment period, and concurrently used drugs.

[0082]

[0083] According to another aspect of the present invention, the present invention provides a method for producing a cell therapeutic agent for intestinal epithelial cell regeneration, comprising the steps of: (a) mixing and homogenizing melatonin and a polymer to produce a melatonin-containing drug delivery system; and (b) mixing the melatonin-containing drug delivery system produced in step (a) and stem cells to produce a stem cell-drug delivery system.

[0084] The cell therapeutic agent manufactured by the method of the present invention induces regeneration of damaged intestinal epithelial tissue through induction of regenerative stem cells, and can be used for the prevention or treatment of intestinal damage disease or inflammatory bowel disease.

[0085] In a specific embodiment of the present invention, the polymer of step (a) may be a biodegradable polymer as described above.

[0086] In a specific embodiment of the present invention, the step (a) may be mixing and homogenizing an aqueous solution and an oily solution containing melatonin and a polymer.

[0087]

[0088] In another aspect, the present invention provides a method for treating an intestinal epithelial damage disease, comprising administering the stem cell-drug delivery system to a subject in need thereof. The present invention also provides a method for treating an inflammatory bowel disease, comprising administering the stem cell-drug delivery system to a subject in need thereof.

[0089] In a specific embodiment of the present invention, the subject may be, but is not limited to, a subject expected to develop an intestinal epithelial damage disease or an inflammatory bowel disease; a subject that has developed the disease; or a subject that has been judged to be cured.

[0090]

[0091] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0092] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0093]

[0094] Example 1. Production of stem cell-drug delivery systems

[0095] 1-1. Production of heterospheroids

[0096] Heterospheroids (HS) are drug delivery systems containing melatonin; and stem cells; and are stem cell-drug delivery systems, manufactured as shown in Fig. 1a. The heterospheroids are 3D-cultured mesenchymal stem cells that secrete large amounts of PGE2 together with melatonin microspheres, and are intended to be used as therapeutic agents combining PGE2 and melatonin.

[0097] Specifically, to produce the heterospheroids (HS), a solution of 90 mg of PLGA (Sigma) and 10 mg of melatonin dissolved in 1 ml of dichloromethane (Junsei Chemical) was added dropwise to 5 mL of a 1% PVA (polyvinyl alcohol, Sigma) solution. After dropping, the mixture was emulsified at 13,000 rpm for 5 minutes. The emulsion was then transferred to 60 mL of a 1% PVA aqueous solution and stirred at room temperature for 5 hours to evaporate the organic solvent, thereby obtaining 'melatonin microspheres'.

[0098] Umbilical cord blood-derived mesenchymal stem cells were cultured in KSB-3 medium (Kangstem Biotech). The cultured umbilical cord blood-derived mesenchymal stem cells were 1.2 × 10 6 704 μg of dog and melatonin microspheres were mixed in 2 mL of KSB-3 medium. The mixture was then cultured in AggreWell™400 Microwell Culture Plates 24-well plate (STEMCELL Technologies) for 24 h to generate three-dimensional mesenchymal stem cells (3D-MSCs) and heterospheroids (HS). After culture, the cell culture dish was washed with PBS to remove mesenchymal melatonin microspheres. The heterospheroids were cultured at a density of 1 × 10 3 Contains melatonin microspheres with canine cells.

[0099] Two-dimensional mesenchymal stem cells (2D) and three-dimensional mesenchymal stem cells (3D) were used as controls in the experiments described below.

[0100]

[0101] 1-2. Characteristics of heterospheroids

[0102] To analyze the characteristics of the fabricated 3D-MSCs and HS, diameter measurement, LIVE / DEAD staining, and CCK-8 analysis were performed, and the results are shown in Figures 1b to d, respectively.

[0103] As shown in Fig. 1b, the heterospheroids (HS) were confirmed to have an average diameter of 162.09 μm.

[0104] As shown in Figures 1c and d, HS was confirmed to have cell viability similar to that of three-dimensional mesenchymal stem cells (3D).

[0105]

[0106] Example 2. Effect of heterospheroids on regenerative stem cell induction

[0107] 2-1. Coculture of heterospheroids and intestinal epithelial organoids

[0108] The heterospheroids prepared in Example 1 were confirmed to induce regenerative stem cells in the intestinal epithelium. Specifically, intestinal epithelial organoids were seeded on Matrigel and cultured for 24 hours. One heterospheroid was added to each crypt containing intestinal epithelial organoids and co-cultured for 48 hours. The control group was 1 × 10 2D mesenchymal stem cells per crypt. 3 Dog; or 1 3D mesenchymal stem cell (3D) was added.

[0109]

[0110] 2-2. Morphological analysis

[0111] After co-culturing heterospheroids and intestinal epithelial organoids as described in Example 2-1, the cell morphology was analyzed using a microscope. The results of the microscopic observation are shown in Fig. 2a, and the results of classifying cell morphology based on the microscopic observation results are shown in Fig. 2b.

[0112] As shown in Figures 2a and b, when heterospheroids and intestinal epithelial organoids were co-cultured, it was confirmed that the organoids co-cultured with heterospheroids showed a more prominent change to a cystic form than the control group (2D and 3D).

[0113]

[0114] 2-3. Analysis of intestinal epithelial marker expression

[0115] After co-culturing heterospheroids and intestinal epithelial organoids as in Example 2-1, the expression of reconstructive stem cell markers (Ly6a, Clu), stem cell markers (Lgr5, Olfm4), and intestinal epithelial markers (Lyz, Muc2, Tff3, Alpi, ChgA) was analyzed using real-time qPCR. The results of analyzing the expression of the markers are shown in Fig. 2c.

[0116] As shown in Fig. 2c, the heterospheroid treatment group (HS) and the 3D mesenchymal stem cell treatment group (3D) showed significantly higher expression of reconstructive stem cell markers (Ly6a, Clu) than the negative control group (cont.) and the 2D mesenchymal stem cell (2D) treatment group. In particular, the heterospheroid treatment group showed 1.92- and 2.09-fold higher expression of reconstructive stem cell markers Ly6a and Clu, respectively, than the 3D mesenchymal stem cell treatment group (3D).

[0117]

[0118] 2-4. Flow cytometry

[0119] After co-culturing heterospheroids and intestinal epithelial organoids as in Example 2-1, cells expressing Ly6a, a reconstructive stem cell marker (i.e., reconstructive stem cells) were analyzed using flow cytometry. The results of the flow cytometry analysis are shown in Fig. 2d.

[0120] As shown in Fig. 2d, the heterospheroid treatment group (HS) was confirmed to have the highest number of regenerative stem cells within the intestinal epithelial organoids. In particular, the heterospheroid treatment group (HS) had approximately 1.8 times more regenerative stem cells than the 3D mesenchymal stem cell treatment group (3D), confirming that the heterospheroid treatment group (HS) can induce more regenerative stem cells than the 3D mesenchymal stem cell treatment group.

[0121]

[0122] Example 3. Confirmation of the protective effect of heterospheroid administration in a mouse intestinal inflammation model.

[0123] 3-1. Mouse intestinal inflammation model and heterospheroid production

[0124] To create a mouse intestinal inflammation model, intestinal inflammation was induced in 9-week-old C57BL / 6 mice by supplying drinking water containing 2.5% DSS for 5 days.

[0125] Additionally, heterospheroids were produced using umbilical cord blood (UCB) and tonsil (T)-derived mesenchymal stem cells as in Example 1-1. The produced heterospheroids were named HS#UCB and HS#T, respectively.

[0126] To evaluate the in vivo efficacy of the manufactured heterospheroids, 2,000 heterospheroids (1,000 cells per spheroid) were injected intraperitoneally per mouse on the fifth day of feeding with DSS-containing drinking water. To evaluate the regenerative effect on the already damaged intestinal epithelium due to inflammatory induction, the cell administration time was set to day 5 rather than the initial induction period.

[0127] Positive controls were injected with 3D mesenchymal stem cells (3D) derived from umbilical cord blood (UCB) and tonsil (T) into a mouse intestinal inflammation model.

[0128]

[0129] 3-2. Analysis of body weight and survival rate in a mouse intestinal inflammation model

[0130] The body weight and survival rate of the mouse intestinal inflammation model administered with heterospheroids as described in Example 3-1 above were determined. The body weight measurement results are shown in Figure 3a, and the survival rate results are shown in Figure 3b.

[0131] As shown in Figures 3a and b, the HS#UCB and HS#T groups showed less weight loss than the PBS group (DSS+PBS). Furthermore, the HS#UCB group, in particular, showed a significantly lower weight loss rate and the highest survival rate.

[0132]

[0133] 3-3. Evaluation of colitis severity in a mouse intestinal inflammation model

[0134] The severity of colitis in the mouse intestinal inflammation model was assessed on the 10th day of DSS administration using the disease activity index (DAI). The results of the DAI evaluation of colitis are shown in Figure 3c.

[0135] As shown in Fig. 3c, the disease activity index of the HS#UCB and HS#T administration groups was statistically significantly lower than that of the PBS control group. In particular, HS#UCB showed a significantly lower disease activity index compared to the 3D#UCB administration group. The above results indicate that the severity of colitis was alleviated when HS#UCB and HS#T were administered.

[0136]

[0137] The anti-inflammatory effects of HS#UCB and HS#T administration were evaluated by measuring colon length in a mouse model of intestinal inflammation and improving intestinal epithelial damage using H&E staining. The results of colon length measurement are shown in Fig. 3d, and the results of intestinal epithelial damage evaluation are shown in Figs. 3e and f.

[0138] As shown in Figure 3d, it was confirmed that the HS#UCB administration group and the HS#T administration group had protection against shortening of the colon length (i.e., relief of colitis symptoms).

[0139] As shown in Figures 3e and f, the HS#UCB administration group and the HS#T administration group showed improvements in intestinal epithelial structure and inflammatory cell infiltration.

[0140]

[0141] 3-5. Analysis of inflammatory markers in a mouse intestinal inflammation model

[0142] As in Example 3-1 above, the inflammatory indicators (TNF-α, IL-17, and IL-10) of the mouse intestinal inflammation model administered with heterospheroids were analyzed, and the results are shown in Figures 3g to i.

[0143] As shown in Figures 3g to i, the HS#UCB and HS#T administration groups showed a significant decrease in TNF-α and IL-17 levels, while the IL-10 level increased. This suggests that HS#UCB and HS#T administration regulates the inflammatory response in a mouse intestinal inflammation model.

[0144]

[0145] 3-6. Analysis of MPO (Myeloperoxidase) activity in colon tissue of a mouse intestinal inflammation model

[0146] MPO is an enzyme found in neutrophil granules and is used as an indicator of neutrophil infiltration and inflammatory activity. In colitis, MPO activity is closely correlated with intestinal damage scores. As described in Example 3-1, MPO activity was analyzed in the colon of a mouse model of intestinal inflammation administered heterospheroids, and the results are shown in Figure 3j.

[0147] As shown in Fig. 3j, the HS#UCB and HS#T administration groups showed significantly reduced MPO activity compared to the PBS group. This suggests that HS reduces inflammation and intestinal damage.

[0148]

[0149] 3-7. Evaluation of regenerative stem cell induction in a mouse intestinal inflammation model

[0150] Ly6a-expressing cells were analyzed in a mouse intestinal inflammation model administered with heterospheroids as described in Example 3-1 using flow cytometry. The results of the flow cytometry analysis are shown in Fig. 3k.

[0151] As shown in Fig. 3k, the HS#UCB and HS#T administration groups showed a significant increase in Ly6a-expressing cells compared to the PBS group. These results suggest that HS contributes to intestinal epithelial regeneration by inducing regenerative stem cells in intestinal epithelium already damaged by inflammation.

[0152]

[0153] Example 4. Comparative experiment on the regenerative stem cell induction ability of heterospheroids according to drug type.

[0154] 4-1. Manufacturing of heterospheroids according to drug type

[0155] In the art, quercetin is known as a therapeutic agent for inflammatory bowel disease. Therefore, in this example, the regenerative stem cell induction ability of heterospheroids comprising melatonin microspheres and three-dimensional mesenchymal stem cells prepared in Example 1-1 (hereinafter referred to as 'Mel-HS') and heterospheroids comprising quercetin microspheres and three-dimensional mesenchymal stem cells (hereinafter referred to as 'Q-HS') was compared. The Q-HS was prepared in the same manner as in Example 1-1, but using quercetin instead of melatonin.

[0156]

[0157] 4-2. Comparison of regenerative stem cell induction abilities

[0158] After treating small intestinal epithelial organoids with Mel-HS, they were co-cultured for 48 hours. After culture, cells expressing the regenerative stem cell marker Ly6a in the small intestinal epithelial organoids were analyzed by flow cytometry. The Q-HS treatment group was also performed in the same manner as above. The control group in this experiment was treated with two-dimensional (2D) or three-dimensional (3D) mesenchymal stem cells. The flow cytometry results of the Mel-HS treatment group are shown in Figure 4a, and the flow cytometry results of the Q-HS treatment group are shown in Figure 4b.

[0159] As shown in Figures 4a and b, the Mel-HS treatment group showed a significant increase in Ly6a-expressing cells within organoids compared to the control group (2D, 3D) (Figure 4a). However, the Q-HS treatment group showed a significant decrease in Ly6a-expressing cells within organoids compared to the control group (2D, 3D) (Figure 4b). These results indicate that quercetin does not affect regenerative stem cell induction, and that the regenerative stem cell induction effect by Mel-HS is a specific effect.

[0160]

[0161] 4-3. Analysis of regenerative stem cell marker expression in organoids

[0162] After treating small intestinal epithelial organoids with Mel-HS, they were co-cultured for 48 hours. After culture, the expression of Claudin-4 (Cldn4), a marker of regenerative stem cells, was measured via real-time qPCR. The Q-HS treatment group was also performed in the same manner as above. The control group in this experiment was treated with two-dimensional mesenchymal stem cells (2D) or three-dimensional mesenchymal stem cells (3D). The real-time qPCR results of the Mel-HS treatment group are shown in Figure 4c, and the real-time qPCR results of the Q-HS treatment group are shown in Figure 4d.

[0163] As shown in Figures 4c and d, the Mel-HS treatment group showed a significant increase in Cldn4 expression compared to the control group (2D, 3D) (Figure 4c). However, the Q-HS treatment group showed higher Cldn4 expression than the control group (2D), but lower than the control group (3D). This indicates that quercetin does not enhance the regenerative stem cell induction effect of stem cells.

[0164]

[0165] Furthermore, Q-HS was administered and co-cultured with mouse colon organoids using the same method described above, and Cldn4 expression was analyzed using real-time qPCR. The control group in this experiment, conditioned media (CM), was treated with stem cell culture medium. The real-time qPCR results are shown in Figure 4e.

[0166] As shown in Fig. 4e, Q-HS confirmed that the expression of Cldn4 in mouse colon organoids was lower than that in the control 3D.

[0167]

[0168] These results indicate that Mel-HS melatonin specifically enhances the regenerative stem cell induction efficiency in conjunction with 3D mesenchymal stem cells. Conversely, Q-HS, unlike Mel-HS, does not exhibit a synergistic effect with 3D mesenchymal stem cells.

[0169]

[0170] In summary, the present inventors have created a "stem cell-drug delivery system (i.e., heterospheroid)" by mixing melatonin microspheres and 3D mesenchymal stem cells. The heterospheroids of the present invention were confirmed to continuously release PGE2 and exhibit excellent regenerative stem cell induction. This suggests that the heterospheroids of the present invention have an excellent intestinal epithelial regeneration effect, and thus, the heterospheroids of the present invention can be utilized in various fields of treatment for intestinal damage and inflammatory bowel diseases.

[0171]

[0172] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A drug carrier containing melatonin; and a stem cell; comprising a stem cell-drug carrier.

2. In the first paragraph, the drug delivery system containing melatonin is a stem cell drug delivery system made of at least one polymer selected from the group consisting of polylactide-co-glycolide, polylactide-co-glycolide-co-ethylene glycol, polystyrene-co-ethylene glycol, polyethyleneimine-co-ethylene glycol, polyphosphagen-co-ethylene glycol, polylactide-co-ethylene glycol, polycaprolactone-co-ethylene glycol, polyanhydride-co-ethylene glycol, polymalic acid-co-ethylene glycol and derivatives thereof, polyalkylcyanoacrylate-co-ethylene glycol, polyhydroxybutyrate-co-ethylene glycol, polycarbonate-co-ethylene glycol and polyorthoester-co-ethylene glycol, polyethylene glycol, poly-L-lysine-co-ethylene glycol, polyglycolide-co-ethylene glycol, polymethylmethacrylate-co-ethylene glycol, polyvinylpyrrolidone-co-ethylene glycol, and copolymers thereof.

3. A stem cell-drug delivery system according to claim 1, wherein the stem cell is an embryonic stem cell, a mesenchymal stem cell, or an induced pluripotent stem cell.

4. In the third paragraph, the mesenchymal stem cells are derived from embryonic yolk sac, placenta, umbilical cord, umbilical cord blood, tonsil, skin, peripheral blood, bone marrow, adipose tissue, muscle, liver, nerve tissue, periosteum, fetal membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, anterior cruciate ligament, articular chondrocytes, deciduous cells, pericytes, periodontal bone, subpatellar fat pad, spleen, or thymus.

5. A stem cell-drug delivery system in the first paragraph, wherein the stem cells are in the form of spheroids.

6. A composition for regenerating intestinal epithelial cells comprising a stem cell-drug delivery system according to Article 1.

7. A composition for inducing regenerative stem cells comprising a stem cell-drug delivery system according to Article 1.

8. A composition for inducing reconstructive stem cells, wherein the reconstructive stem cells in paragraph 7 are Lgr5 (Leucine-rich repeat-containing G-protein coupled receptor 5) positive epithelial stem cells.

9. A pharmaceutical composition for preventing or treating intestinal epithelial damage disease, comprising a stem cell-drug delivery system according to Article 1.

10. A pharmaceutical composition for preventing or treating an intestinal epithelial damage disease according to claim 9, wherein the intestinal epithelial damage disease is at least one selected from the group consisting of Crohn's disease, ulcerative colitis, ulcerative duodenitis, hemorrhagic rectal ulcer, leaky gut syndrome, gastritis, gastric ulcer, pouchitis, enteritis, and ischemic colitis.

11. A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a stem cell-drug delivery system according to Article 1.

12. A pharmaceutical composition for preventing or treating inflammatory bowel disease, wherein the inflammatory bowel disease is any one of ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, conversion colitis, and Behcet's syndrome. 13.(a) a step of preparing a melatonin-containing drug delivery system by mixing and homogenizing melatonin and a polymer; and (b) A method for producing a cell therapeutic agent for intestinal epithelial cell regeneration, comprising the step of producing a stem cell-drug carrier by mixing the melatonin-containing drug carrier produced in step (a) and stem cells.

14. A method for treating an intestinal epithelial damage disease, comprising administering a stem cell-drug delivery system according to paragraph 1 to a subject in need thereof.

15. A method for treating inflammatory bowel disease, comprising administering a stem cell-drug delivery system according to paragraph 1 to a subject in need thereof.

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