WO2023055129A1 - Pharmaceutical composition comprising magnetic nanoparticle-bearing mesenchymal stem cell for prevention or treatment of nerve disease and use thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising magnetic nanoparticle-bearing mesenchymal stem cells for preventing or treating nerve diseases and the use thereof. When migrated and engrafted to a target site by an externally induced magnetism, the magnetic nanoparticle-bearing mesenchymal stem cells promote the reduction of amyloid plaques. Thus, the composition can be applied as a therapeutic for incurable nervous system diseases such as Alzheimer's disease and can solve the problems of clinical application through in-vivo monitoring of stem cells.

Description

Pharmaceutical composition containing mesenchymal stem cells containing magnetic nanoparticles for preventing or treating neurological diseases and uses thereof

The present invention relates to a pharmaceutical composition containing mesenchymal stem cells containing magnetic nanoparticles for preventing or treating neurological diseases and uses thereof.

Alzheimer's Disease (AD) is a representative neurodegenerative disease related to aging. When Alzheimer's disease develops, the disease progresses, starting in the frontal and temporal lobes and gradually spreading to other parts of the brain. The main pathological features of Alzheimer's disease are amyloid plaques caused by the accumulation of amyloid beta protein and tau protein related to microtubules forming neurofibrillary tangles (NFTs) in nerve cells. is to do The formation of amyloid plaques and neurofibrillary tangles (NFTs) leads to neurodegeneration, synaptic dysfunction and dementia.

Amyloid plaques are produced by amyloid precursor protein (APP). Amyloid precursor protein is a transmembrane protein that penetrates the cell membrane of nerve cells and is very important for the growth, survival and recovery of nerve cells after damage. In the case of Alzheimer's disease, amyloid precursor protein (APP) is degraded and cleaved by γ-secretase and β-secretase enzymes, resulting in amyloid composed of 39 to 43 amino acids. Amyloid beta is produced, which forms an amyloid plaque, a densely accumulated clump on the outside of nerve cells.

Until now, no fundamental treatment method for Alzheimer's disease has been developed, but most of the treatments used in each country are acetylcholinesterase inhibitors, which cannot completely prevent the progression of the disease, but alleviate some pathological symptoms or slow down the progress. There is only effect. Drugs in this class include donepezil, rivastingmine, galantamine, and tacrine.

Stem cells are considered promising treatments for intractable neurological diseases such as Alzheimer's due to their ability to replace damaged or lost cells in the nervous system. In the meantime, it has been reported that embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into various nerve cells and can replace the damaged nervous system. However, clinical trials have not been made because these cells can cause unwanted cancer. Therefore, currently ongoing clinical trials are studying human mesenchymal stem cells (hMSC) among adult stem cells as a cell therapy agent for treating the damaged nervous system due to their high plasticity and low immune rejection response.

However, the biggest problem with existing stem cell therapeutics used in Alzheimer's disease is that it is very difficult to pass through the blood-brain barrier and be delivered to the brain. That is, most of the intravenously injected stem cells do not pass through the capillaries of the lungs, so very few reach the brain, and even if they pass through the lungs, they are hardly delivered to the brain parenchyma due to the blood-brain barrier. Therefore, many studies have been conducted to find various administration routes such as arterial administration, intraventricular administration, subventricular administration, and spinal administration. The administration routes that are predicted to be practically effective are intra-parenchymal and intra-cerebroventricular administration. However, damage to the brain region cannot be avoided depending on the administration location. In the case of intracerebroventricular administration, although the amount of stem cells administered is expected to be higher, brain damage is relatively less and there are advantages in that stem cells can be delivered throughout the brain area. However, when stem cells are directly injected into the 'Cerebral ventricle', the stem cells move to the spinal cord by the flow of cerebrospinal fluid (CSF) before moving to the 'Brain Parenchyma', so the remaining amount in the brain is higher than the dose. The ability may fall, so a way to overcome it is needed.

Accordingly, the present inventors have improved the efficiency of stem cell migration to the target site, improved cellular viability and engraftment efficiency, so as to increase brain targeting efficiency while maximizing the therapeutic efficacy of existing Alzheimer's stem cell therapeutics. Efforts were made to develop ways to increase it. As a result, the present inventors contained magnetic iron oxide nanoparticles in Wharton's jelly-derived mesenchymal stem cells and injected them into the ventricle, and then, using a magnetic field outside the body, prevented the stem cells from flowing into the spinal cord by the cerebrospinal fluid flow and It was confirmed that the Alzheimer's treatment effect was doubled by promoting the penetration into the brain parenchyma where the cells are located. In addition, it was found that Wharton's jelly-derived mesenchymal stem cells containing magnetic iron oxide nanoparticles showed a higher degree of expression of therapeutically effective substances than mesenchymal stem cells that did not contain magnetic iron oxide nanoparticles. In the case of autologous mesenchymal stem cells, due to the nature of patients with Alzheimer's disease, they are often elderly, so their efficacy is not high, and they require a culture and purification process after obtaining cells from patients, so they cannot be treated immediately after a patient visits. Wharton's jelly-derived mesenchymal stem cells of the present invention can be treated immediately, and can also be said to have higher differentiation potential or therapeutic efficacy. In addition, in the case of existing stem cell therapeutics, it was very difficult to track their location in the body after administration to a patient. The present invention was completed by identifying that the amount can be confirmed, stem cell tracking, stem cell survival evaluation, and the effect of stem cell treatment can be accurately analyzed.

Accordingly, one object of the present invention is to provide a pharmaceutical composition for preventing or treating neurological diseases, including mesenchymal stem cells containing magnetic nanoparticles.

In addition, another object of the present invention is to provide a cell therapy agent for treating neurological diseases, including mesenchymal stem cells containing magnetic nanoparticles.

In addition, another object of the present invention is to provide a method for preparing a pharmaceutical composition for preventing or treating neurological diseases.

Another object of the present invention is to provide a composition for transplantation and differentiation tracking of mesenchymal stem cells containing magnetic nanoparticles.

In addition, another object of the present invention is to provide a method for treating a neurological disease, comprising administering the cell therapy agent to a subject and applying an external magnetic body to position the cell therapy agent in a target tissue.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The terms used herein are used for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

Further, hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable way.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention is a method for preventing neurological diseases, including at least one selected from the group consisting of mesenchymal stem cells containing magnetic nanoparticles, cell-derived exosomes, cell membrane-derived nanoparticles, and cell secretions Or providing a pharmaceutical composition for treatment, wherein the composition is one selected from the group consisting of mesenchymal stem cells containing the magnetic nanoparticles, cell-derived exosomes, cell membrane-derived nanoparticles, and cell secretions by applying an external magnetic body It is characterized by migrating and targeting the abnormality to a treated tissue or organ of the subject.

Mesenchymal stem cells containing magnetic nanoparticles, mesenchymal stem cells containing magnetic nanoparticles, cell-derived exosomes, cell membrane-derived nanoparticles and At least one selected from the group consisting of cell secretions may be used, preferably mesenchymal stem cells containing magnetic nanoparticles, but not limited thereto.

The administration method of the composition of the present application may apply a known inhibitor administration method, and is not particularly limited thereto, and parenteral administration (eg, ventricular, intravenous, subcutaneous, intraperitoneal or local administration) according to the desired method application) or, in particular, administration by intraventricular injection is preferred.

That is, the mesenchymal stem cells containing the magnetic nanoparticles according to the present invention can be administered by an administration route suitable for a subject in need thereof, for example, intraventricular or intravenous administration, preferably containing the magnetic nanoparticles of the present invention. It is administered to the ventricle in consideration of the characteristics of mesenchymal stem cells, and after the administration, appropriate magnetism is applied to the target organ or tissue of the subject to transfer the mesenchymal stem cells containing the administered magnetic nanoparticles to the organ or tissue. It can be positioned (targeted), and also increases the therapeutic efficacy of existing mesenchymal stem cells by containing magnetic nanoparticles.

As used herein, the term "magnetic" refers to the magnetic properties exhibited by a material. All materials interact with magnetic fields to generate attractive or repulsive forces. That is, when a magnetic field is applied to a material, it is magnetized, and the object is classified into a ferromagnetic substance, a paramagnetic substance, a diamagnetic substance, a ferrimagnetc substance, etc. according to the magnetization aspect.

As used herein, the term “magnetic nanoparticles” refers to nanometer-sized structures or materials that are magnetic. The magnetic nanoparticles may be prepared by solution synthesis, co-precipitation, sol-gel method, high-energy milling, hydrothermal synthesis, microemulsion synthesis, synthesis by thermal decomposition, or sonochemical synthesis, but are not limited thereto.

The magnetic nanoparticles may be selected from the group consisting of iron (Fe), cobalt (Co), nickel (Ni), manganese (Mn), gadolinium (Gd), oxides thereof, or alloys thereof. It is not limited.

The magnetic nanoparticle of the present invention refers to a metal nanocomposite having magnetism, and may be made of a metal, a magnetic material, or a magnetic alloy.

Any magnetic nanoparticles known in the art may be used as the magnetic nanoparticles as long as they can achieve the object of the present invention, and materials constituting the magnetic nanoparticles may be included without limitation, , for example iron oxides (FeO, Fe ₂ O ₃ , Fe ₃ O ₄ ), alloys (FePt, CoPt) and ferrites (MnFe ₂ O ₄ , CoFe ₂ O ₄ , NiFe ₂ O ₄ , ZnFe ₂ O ₄ ) It may be one or more selected from the group consisting of, but is not limited thereto.

Preferably, the magnetic nanoparticles may be one or more iron oxide nanoparticles selected from the group consisting of FeO, Fe ₂ O ₃ and Fe ₃ O ₄ , and in one embodiment of the present invention, Fe ₂ O ₃ is used, Not limited to this.

In addition, the iron oxide nanoparticles may be coated with a biocompatible polymer. The biocompatible polymer is dextran, polyethylene glycol, polypropylene glycol, polyoxyethylene, polytrimethylene glycol, polylactic acid, polyacrylic acid, polyamino acid, polyvinyl alcohol, polyurethane, polyphosphazine, poly(L-lysine) , polyalkylene oxide, polysaccharide, polyvinyl pyrrolidone, and may be at least one selected from the group consisting of polyacrylamide, preferably dextran (dextran), but is not limited thereto. As described above, when coated with a biopolymer, problems that can cause toxicity in vivo, such as chemical bonding, viscosity and immunity due to contrast agent aggregation, and interference with enzymes such as lysozyme, which are disadvantages of magnetic nanoparticles, can be solved. It may have biocompatibility such as toxicity, high efficiency, dispersibility in tissue specificity, and good solubility.

In addition, the magnetic nanoparticles of the present invention may be synthesized or formed through any method known in the art, as long as the object of the present invention can be achieved, and additives may be additionally used for this purpose.

The additives include, for example, heparin, protamine, targeting ligand, cell penetrating peptide, albumin, albumin derivative, histone, Cremophor EL, Solutol, cyclodextrin, RGD tripeptide, It may be selected from the group consisting of cholesterol, phospholipids and polyethylene glycol (PEG), but is not limited thereto.

In addition, the iron oxide nanoparticles further include a shell around them, wherein the shell is selected from the group consisting of proteins or peptides, surfactants, lipids, ligands, amino acids, carbohydrates, nucleic acids, small molecules, and biocompatible polymers. components may be included.

In addition, the magnetic nanoparticles of the present invention may be used by modifying the surface of the magnetic nanoparticles as long as the object of the present invention can be achieved. For example, a functional group may be introduced, but is not limited thereto.

For example, folic acid, hyaluronic acid, cyclodextrin, imidazole-based compounds, histidine, lysine, arginine, cysteine, thiolalkylamine, spermine, spermidine, polyethyleneimine of various weight average molecular weights, polyhistidine, polylysine, poly One or more functional groups may be further introduced into the nanoparticles by reacting with polymers such as arginine, protamine, heparin, chitosan, and protamine.

The protecting group usable in the present invention may be any protecting group commonly used for protection of each functional group, which can be easily selected and used by anyone with ordinary knowledge in the art to which the present invention belongs.

The magnetic nanoparticle may have a diameter of 1 nm to 1000 nm, preferably 1 nm to 100 nm, more preferably 1 to 50 nm, but is not limited thereto.

According to a preferred embodiment of the present invention, the composition of the present invention is directly administered or transplanted into the brain ventricle of a subject, thereby preventing target stem cells from flowing into the spinal cord by the cerebrospinal fluid flow using a magnetic field such as a magnet outside the body, and nerve cells Since it promotes penetration into the 'brain parenchyma' where there is , it is easy to achieve an effective therapeutic effect and apply to biological research, compared to other routes of administration.

In addition, in order to enhance the engraftment rate of mesenchymal stem cells containing magnetic nanoparticles, which are target cells, various embedding materials, such as sodium alginate, hyaluronic acid, or collagen, are mixed with cells. It can also be formed and used.

Mesenchymal stem cells of the present invention can be mesenchymal stem cells known in the art, regardless of the method of induction, as long as the objects of the present invention can be achieved, and mesenchymal stem cells isolated or isolated from donors Cells or commercially available mesenchymal stem cell lines may be included without limitation.

Although the mesenchymal stem cells exist in very small amounts in bone marrow, etc., the process of isolating and culturing them is well known in the art, and they do not lose their differentiation ability after being separated from hematopoietic stem cells in bone marrow by adhesion characteristics according to a known method. It can be obtained by proliferating in an undeveloped state. Identification of these mesenchymal stem cells can be performed, for example, through flow cytometry. This flow cytometric analysis is performed using specific surface markers of mesenchymal stem cells. For example, mesenchymal stem cells are positive for CD44, CD29 and/or MHC class I. As the medium used in the process, any medium generally used for culturing stem cells may be used. Preferably, it is a medium containing serum (eg, fetal bovine serum, horse serum, and human serum). Mediums that can be used in the present invention are, for example, RPMI series, Eagles' MEM (Eagle's minimum essential medium), α-MEM, Iscove's MEM, 199 medium, CMRL 1066, F12, F10, DMEM (Dulbecco's modifation of Eagle's medium), mixtures of DMEM and F12, Way-mo, h's MB752/1, McCoy's 5A and MCDB series. The medium may contain other components, such as antibiotics or antifungal agents (eg, penicillin, streptomycin, gentamicin) and glutamine.

Preferably, the mesenchymal stem cells of the present invention are derived from one or more selected from the group consisting of Wharton's jelly, umbilical cord, bone marrow, adipose tissue, blood, umbilical cord blood, liver, skin, gastrointestinal tract, placenta and uterus, and more Preferably, it is derived from Wharton's jelly, but is not limited thereto.

In addition, the mesenchymal stem cells of the present invention may be derived from mammals. The mammal may be a human, monkey, chimpanzee, dog, cat, cow, goat, pig, mouse or rat, but is not limited thereto.

The neurological disease may include any neurological disease as long as the composition of the present invention achieves an effect, and preferably, the neurological disease is amyloid beta plaque formation in nerve tissue, phosphorylation of tau protein in nerve cells, neurite It is a disease caused by at least one selected from the group consisting of abnormalities of and decreased expression of neprilysin in nerve cells, more preferably selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis, and mania. more than one species, most preferably Alzheimer's disease.

According to the present invention, when MSC-IONP targeting is maximized by magnetic induction after transplanting (ventricularly administered) mesenchymal stem cells (MSC-IONP) containing magnetic nanoparticles to an Alzheimer's disease model at a target location in vivo, Alzheimer's disease Not only did it exhibit an excellent and significant reduction effect on amyloid plaques, which are the main cause of the disease, but also a behavioral improvement effect was achieved while observing its survival and distribution, so the composition of the present invention can be applied as a treatment for Alzheimer's disease. prove that there is

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, including, but not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil; it is not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity. It can be. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The term "treatment" as used herein, unless stated otherwise, means reversing, alleviating, inhibiting the progression of, or preventing one or more symptoms of a disease or disorder to which the term applies. .

The composition according to the present invention comprises mesenchymal stem cells containing a therapeutically effective amount of magnetic nanoparticles. By therapeutically effective amount is meant the amount necessary to alleviate, ameliorate or beneficially alter one or more symptoms of a neurological disorder.

The composition of the present invention contains the above-mentioned mesenchymal stem cells containing the magnetic nanoparticles according to the present application as an active ingredient, and in addition to that, one or more active ingredients exhibiting the same or similar functions, or the solubility and /or may further contain a compound that maintains/increases absorbency.

In addition, the composition of the present invention can be used alone or in combination with methods using surgery, drug therapy, and biological response modifiers.

In addition, according to another aspect of the present invention, the present invention provides a cell therapy agent for the treatment of neurological diseases, including mesenchymal stem cells containing magnetic nanoparticles, wherein the cell therapy agent applies an external magnetic material to the magnetic nanoparticles. The mesenchymal stem cells containing the particles are migrated and targeted to a treated tissue or organ of a subject.

In another embodiment of the present invention, "cell therapy" refers to living cells used for cell therapy in which live cells are directly injected into a patient, and living autologous cells, allogeneic cells, or xenogeneic cells are used in vitro in the safety regulations of pharmaceuticals, etc. It refers to drugs that are manipulated and manufactured by methods such as culture, propagation, or selection. The cell therapy is divided into somatic cell therapy and stem cell therapy depending on whether it is differentiated. In particular, stem cells are tissue tissue when stem cells show symptoms related to neuron deterioration in neurodegenerative disorders as a result of genetic symptoms or damage. Neuronal damage or neurological disease as well as brain injury, brain disease, spinal cord injury, peripheral nerve injury, peripheral nerve disease, and amyotrophic lateral sclerosis through the basic characteristics of neuroprotection and immunomodulation (amyotrophic lateral sclerosis).

Preferably, the cells used in the cell therapy agent may be stem cells or cells differentiated from stem cells, more preferably mesenchymal stem cells derived from Wharton's jelly, but are not limited thereto.

In addition, according to another aspect of the present invention, the present invention contains magnetic nanoparticles comprising the step of treating and culturing magnetic nanoparticles in mesenchymal stem cells to contain the magnetic nanoparticles in the mesenchymal stem cells It provides a method for preparing a pharmaceutical composition for preventing or treating neurological diseases, including mesenchymal stem cells.

The culturing process of the present invention may be performed according to media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected cells.

The treatment concentration of the magnetic nanoparticles may be 1 to 1000 μg/ml, preferably 1 to 100 μg/ml, and the incubation time is 24 hours to 72 hours.

According to one embodiment, the production of mesenchymal stem cells containing the magnetic nanoparticles of the present invention is performed by adding 40 μg/ml of ferumoxytol Feraheme®, Fe ₂ O ₃ as iron oxide nanoparticles to Wharton's jelly. It can be obtained by culturing for 72 hours with mesenchymal stem cells derived from.

In addition, according to another aspect of the present invention, the present invention provides a composition for transplantation and tracking of mesenchymal stem cells containing magnetic nanoparticles, wherein the composition applies an external magnetic material to the medium containing the magnetic nanoparticles. Since mesenchymal stem cells can be targeted and tracked using means known in the art that can analyze them, such as MRI, it is possible to provide an in vitro and in vivo platform capable of verifying the effectiveness and reliability of transplanted stem cell therapeutics in vivo. can

In addition, according to another aspect of the present invention, the present invention provides a step of administering the above-described cell therapy agent of the present invention to a human or non-human animal as a subject and applying an external magnetic body to position the cell therapy agent to a target tissue Including, it provides a method for treating neurological diseases. In addition, the present invention is a cell therapy agent transfer method comprising the steps of injecting the cell therapy agent for treating neurological disorders according to the present invention into a subject and applying an external magnetic field to a target location of the subject to position the cell therapy agent to a target tissue provides

The administration is transplantation of a cell therapy agent into a target tissue of a subject, preferably injected into the ventricle of the subject, and subsequent to the administration step, an appropriate magnetic field is applied to the target site of the subject using an external magnetic material to inject the injected cells positioning and engrafting the therapeutic agent into the target tissue.

In addition, the administration is preferably administered 1 to 5 times.

According to one embodiment of the present invention, the neurological disease is Alzheimer's disease, and by the administration, the effect of removing amyloid plaques, the effect of significantly improving mobility and anxiety is achieved.

An appropriate magnetic field is a magnet capable of inducing an administered composition in a sufficient amount to a target organ, and can be easily determined based on the description of the embodiments herein and the knowledge of a person skilled in the art.

The movement to the target location by the magnetic field overcomes the problem that it is difficult to cause cell viability and engraftment due to the poor biological environment in the pathological area, usually transplanted mesenchymal stem cells far from the lesion site. can

The treatment method of the present invention is applied not only to neurological diseases, but also to nerve damage, brain damage, brain disease, spinal cord injury, peripheral nerve damage, peripheral nerve disease, and amyotrophic lateral sclerosis to enable treatment. can

Since the method of the present invention uses the cell therapy agent described above, duplicate descriptions are omitted to avoid excessive complexity of the present specification.

When the mesenchymal stem cells containing the magnetic nanoparticles of the present invention were migrated and engrafted to the target position by externally induced magnetism, the reduction of amyloid plaques was promoted, so it can be applied as an effective therapeutic agent for intractable nervous system diseases such as Alzheimer's. In addition, problems in clinical application can be solved through in vivo monitoring of stem cells.

Figure 1a schematically shows a method for preparing mesenchymal stem cells (MSC-IONP) containing iron oxide nanoparticles of the present invention for Alzheimer's disease. Figures 1b, 1c, 1d, 1e, 1f and 1g show magnetic results of Wharton's Jelly-derived MSC containing iron oxide nanoparticles according to the present invention.

2a and 2b show the cytotoxicity results of Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention.

3a, 3b, 3c, 3d and 3e show the optimal processing conditions of iron oxide nanoparticles.

4a, 4b, 4c, 4d and 4e show the results of analyzing the efficacy of Wharton's jelly-derived MSC (MSC-IONP) containing the iron oxide nanoparticles of the present invention.

5a, 5b, 5c, 5d and 5e show the neuroprotective effect of Wharton's jelly-derived MSC (MSC-IONP) containing the iron oxide nanoparticles of the present invention in an Alzheimer's disease cell model.

FIG. 6 shows the migration and engraftment enhancement effects of Wharton's jelly-derived-MSC containing the iron oxide nanoparticles of the present invention through magnetic induction (MSC-IONP+Mag) in an Alzheimer's disease model.

7a, 7b, and 7c show the therapeutic effect of Wharton's jelly-derived-MSC containing the iron oxide nanoparticles of the present invention through magnetic induction (MSC-IONP+Mag) in Alzheimer's disease models. (i) Wild type, (ii) Vehicle, (iii) MSC, (iv) MSC-IONP, (v) MSC-IONP+Mag.

8a, 8b, 8c, 8d, and 8e show beneficial effects of multiple administrations of Wharton's jelly-derived-MSC (MSC-IONP+Mag) administration in an Alzheimer's disease model. shows (i) Vehicle, (ii) MSC, (iii) MSC-IONP, (iv) MSC-IONP+Mag.

FIG. 9 shows changes in gene expression levels after multiple administration of Wharton's jelly-derived MSC containing iron oxide nanoparticles in an Alzheimer's disease model by magnet induction (MSC-IONP+Mag).

Hereinafter, the examples are only for explaining the present invention in more detail, and those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention It will be self-evident for

Preparation Example 1. Preparation of Wharton's Jelly-derived MSC Incorporated with Iron Oxide Nanoparticles

The present inventors prepared Wharton's jelly-derived mesenchymal stem cells containing magnetic iron oxide nanoparticles.

Briefly, it is as follows: First, Wharton's jelly-derived mesenchymal stem cells produced in GMP grade at Samsung Seoul Hospital were cultured in MEM alpha (L-glutamine, phenol red, sodium pyruvate, low glucose, HEPES-non-added) medium. 10% Fetal Bovine Serum (FBS) and 50 μg/ml gentamicin were mixed and cultured. When treating iron oxide nanoparticles (Ferumoxytol, Feraheme®, Fe ₂ O ₃ ), mix them at concentrations of 80 μg/ml of Ferumoxytol, 1.6 U/ml of heparin, and 32 μg/ml of Protamine Sulfate in an FBS-free culture medium and treat the cells. , After 4 hours, a culture solution containing the same amount of 20% FBS was added to maintain the final concentration of iron oxide nanoparticles at 40 μg/ml. After 24 hours, all the iron oxide nanoparticles were washed, replaced with a culture medium containing 10% FBS, and further cultured for 48 hours to complete the stem cell treatment.

Example 1. Magnetic confirmation of Wharton's jelly-derived MSC containing iron oxide nanoparticles

The present inventors measured the iron content of Wharton's jelly-derived mesenchymal stem cells (MSC-IONP) containing the iron oxide nanoparticles of the present invention prepared in Preparation Example 1 by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) did

As a result, the iron content of MSC-IONP was 272.6 ± 38.7 mg/kg (Fig. 1b), and it was confirmed through scanning electron microscopy (Fig. 1c) and Prussian blue staining (Fig. 1d) that iron oxide nanoparticles were contained in the cells. did

In addition, the inventors attached (positioned) a magnet from the outside and observed that the cells were collected by the magnet at different times (FIG. 1e).

In addition, in order to confirm that there is an effect of increasing the residual by the magnet even when there is a flow of cerebrospinal fluid, a peristaltic pump was used to flow the liquid containing the cells, and the degree of capture by the magnet was confirmed through cell counting (FIG. 1f and FIG. 1g).

The above results show that a movement effect to a target position by magnetism can be achieved by using a magnet in a non-surgical way.

Example 2. Confirmation of Cytotoxicity of Wharton's Jelly-derived MSC Containing Iron Oxide Nanoparticles

In order to confirm the cytotoxicity of iron oxide nanoparticles to mesenchymal stem cells, the present inventors performed Live/dead imaging assay by treating iron oxide nanoparticles at a concentration of 0 to 100 μg/ml for 48 hours.

As a result, as shown in FIG. 2a, it was confirmed that the iron oxide nanoparticles had little cytotoxicity to mesenchymal stem cells.

In addition, CD73 (Ecto-5'-nucleotidase) and CD90 (Thy1) known as mesenchymal stem cell markers were quantified by flow cytometry to confirm the effect of iron oxide nanoparticle treatment on the properties of mesenchymal stem cells.

As a result, as shown in FIG. 2B, it was confirmed that the properties of mesenchymal stem cells did not change even when treated with iron oxide nanoparticles.

Example 3. Confirmation of Optimal Processing Conditions for Iron Oxide Nanoparticles

The present inventors, in order to determine the treatment concentration of iron oxide nanoparticles at which the increase in therapeutic efficacy is maximized, treated mesenchymal stem cells with iron oxide nanoparticles at a concentration of 0 to 100 μg / ml, and then BDNF, a representative nerve growth factor or neurotrophic factor , NGF, NT3 and TGF-β1 were quantitatively compared by qRT-PCR.

As a result, as shown in FIG. 3A, it was confirmed that the optimal processing concentration of the iron oxide nanoparticles was 40 μg/ml.

In addition, the present inventors, in order to determine the appropriate incubation time at the optimal concentration, treated mesenchymal stem cells with 40 μg/ml iron oxide nanoparticles for 24 hours, and then further cultured them for 24 hours (IONP 48h) or 48 hours. (IONP 72h) was cultured and quantitatively analyzed for nerve growth factor (NGF), neurotrophic factor (BDNF, NT3), and anti-inflammatory factor (TGF-β1) through qRT-PCR.

As a result, as shown in Figure 3b, it was confirmed that the mRNA expression level of the corresponding factors increased when additionally cultured for 48 hours (IONP 72h).

In addition, the present inventors treated mesenchymal stem cells with 40 μg/ml iron oxide nanoparticles for 24 hours and cultured them for an additional 48 hours in order to compare the increase in various therapeutically effective substances at the optimal concentration and incubation time. Quantitative analysis was performed on known Alzheimer's disease therapeutic substances (NGF, BDNF, NT-3, NT-4, TGF-β1, AgRP, FGF2, GDF15, Activin A, Ang-1 and Galectin3) through qRT-PCR.

As a result, as shown in Figure 3c, NGF, BDNF, NT-3, NT-4, TGF-β1, AgRP, FGF2, GDF15, Activin A, Ang-1 in the MSC-IONP group according to the optimal conditions of the present invention , it was confirmed that the mRNA expression level of Galectin3 was highly increased.

In addition, the present inventors performed Western blotting to confirm the mechanism of increasing the therapeutic efficacy of mesenchymal stem cells through iron oxide nanoparticles.

As a result, as shown in FIG. 3D, it was confirmed that the p-JNK/JNK ratio and the p-c-Jun/c-Jun ratio increased when the iron oxide nanoparticles were treated with the mesenchymal stem cells under the optimal conditions. Through this, it can be seen that the iron oxide nanoparticles are ionized in the mesenchymal stem cells that have uptaken the iron oxide nanoparticles to stimulate the corresponding pathway.

In addition, the present inventors confirmed the up-regulation of therapeutic factors in mesenchymal stem cells (MSC-IONP) containing the iron oxide nanoparticles of the present invention compared to MSC through Western blotting (FIG. 3e).

Example 4. Efficacy Analysis of Wharton's Jelly-derived MSC (MSC-IONP) Containing Iron Oxide Nanoparticles

The present inventors co-cultured Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention with HUVEC (vascular endothelial cells), astrocytes (astrocytes), neurons, and microglia (macrophage), respectively, to induce angiogenesis Effects on promotion, inhibition of inflammation, inhibition of cell death, and nerve regeneration were quantitatively analyzed.

First, vascular endothelial cells were seeded in a 24-well plate coated with matrigel, and PBS (shown as (i) in FIG. 4), iron oxide nanoparticles (IONP) (shown as (ii) in FIG. 4), iron oxide Mesenchymal stem cells (MSC) not treated with nanoparticles (shown in (iii) in FIG. 4), Wharton's jelly-derived MSC (MSC-IONP) containing iron oxide nanoparticles of the present invention (in (iv) in FIG. 4) ) was co-cultured with HUVECs and quantitatively compared the level of vascular tube production of HUVECs after 18 hours. As shown in FIG. 4a, Wharton's jelly-derived MSC (MSC -IONP) was confirmed to be able to effectively increase the production of vascular tubes.

In addition, as a result of analyzing the proliferation of HUVECs co-cultured with PBS, IONP, MSC, and MSC-IONP of the present invention with a CCK-8 assay, as shown in FIG. 4b, Wharton's jelly containing iron oxide nanoparticles of the present invention It was confirmed that derived-MSC (MSC-IONP) had the best proliferation effect of HUVEC.

In addition, each test substance was co-cultured in LPS-inflammation-induced Raw 264.7 cells, and after 48 hours, the secretion and level of M1 cytokine and M2 cytokine were quantitatively analyzed through qRT-PCR. As shown in FIG. 4c, M1 The expression of cytokine decreased and M2 cytokine increased. Through this, it was confirmed that the MSC-IONP of the present invention can polarize microglia cells into cells having an anti-inflammatory phenotype in the brain.

In addition, the expression of pro-inflammatory factors (IL-1β, iNOS), neurotrophic factors (BDNF), anti-apoptotic factors (FGF2) and angiogenic factors (VEGF) 48 hours after co-culture of the stem cells of the experimental and control groups in astrocytes. As a result of analyzing the level through qRT-PCR, as shown in Fig. 4d, pro-inflammatory factors decreased and the expression levels of neurotrophic, anti-apoptotic, and angiogenic factors increased. Through this, it was confirmed that MSC-IONP can alleviate inflammation by lowering the activity of astrocytes in the brain.

In addition, after co-cultivating neurons (Neuro-2a, PC12) and each test substance, inducing LPS-inflammation and culturing for 24 hours under hypoxic (2% O ₂ ) conditions, the Bcl-2/BAX ratio of neurons As a result of quantification through qRT-PCR, as shown in Figure 4e, it was confirmed that MSC-IONP has the effect of inhibiting apoptosis of neurons.

Example 5. Neuroprotective effect of Wharton's jelly-derived MSC (MSC-IONP) containing iron oxide nanoparticles in Alzheimer's disease cell model

After the present inventors treated Neuro-2a cells with 25 μM of Aβ _25-35 , PBS (shown as (i) in FIG. 5), iron oxide nanoparticles (IONP) (shown as (ii) in FIG. 5), and iron oxide nanoparticles Mesenchymal stem cells (MSC) not treated with particles (shown in (iii) in FIG. 5), Wharton's jelly-derived MSC (MSC-IONP) containing iron oxide nanoparticles of the present invention (shown in (iv) in FIG. 5) mark) were co-cultured for 24 hours, respectively, and the changes were confirmed by methods such as CCK-8 Assay, LDH Assay, Annexin V Assay, ROS scavenging assay, and qRT-PCR.

As a result, as shown in FIG. 5a, when the mesenchymal stem cells treated with iron oxide nanoparticles quantitatively evaluated how much the cytotoxicity by Aβ _25-35 was inhibited through the CCK-8 Assay, iron oxide nanoparticles were not treated. While untreated mesenchymal stem cells did not inhibit cytotoxicity, Wharton's jelly-derived MSC containing iron oxide nanoparticles of the present invention significantly inhibited cytotoxicity compared to the Aβ group.

In addition, as a result of analyzing the cytotoxicity inhibitory effect by Aβ _25-35 through the LDH assay conducted under the same conditions, as shown in FIG. 5B, the iron oxide nanoparticles of the present invention were more It was confirmed that Wharton's jelly-derived-MSC containing cytotoxicity was further suppressed (*p < 0.05 vs (i), †p < 0.05 vs (ii), ¶p < 0.05 vs (iii)).

In addition, as a result of quantitatively evaluating the degree of cell apoptosis through the Annexin V assay, as shown in FIG. 5c, in the group co-cultured with Wharton's jelly-derived-MSC containing the iron oxide nanoparticles of the present invention, cell apoptosis by Aβ was effectively inhibited. inhibition was confirmed.

ROS secreted by neurons by Aβ is known to be a major mechanism that exacerbates Alzheimer's disease by inducing neuroinflammation, and scavenging ROS can play a significant role in treating Alzheimer's disease. Accordingly, the present inventors verified the therapeutic efficacy by quantitatively evaluating the ROS inhibition of Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention through a fluorescence microscope and a flow cytometer.

As a result, as shown in FIG. 5d, it was confirmed that Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention inhibited ROS more significantly than mesenchymal stem cells without it.

In addition, as a result of extracting mRNA from Neuro-2a co-cultured with Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention and analyzing gene expression through qRT-PCR, as shown in FIG. 5e, Aβ It was confirmed that the increased expression level of apoptosis-related genes in nerve cells was decreased by Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention.

Example 6. Confirmation of increased migration and engraftment of Wharton's jelly-derived MSC containing iron oxide nanoparticles through magnetic induction (MSC-IONP+Mag) in Alzheimer's disease model

The inventors of the present invention, in order to confirm the effect of increasing the targeting efficiency to the target site by magnetism by using a magnet in a non-surgical manner on Wharton's jelly-derived MSC containing iron oxide nanoparticles of the present invention, an Alzheimer's disease transgenic mouse model In (5xFAD), a magnet-induced group (MSC-IONP+Mag) in which a magnet was attached (positioned) to the skull after administration of Wharton's jelly-derived MSC containing iron oxide nanoparticles of the present invention to the right ventricle (MSC-IONP + Mag) and a group without magnet attached ( MSC-IONP) were compared.

At this time, as another comparative control group, Wharton's jelly-derived MSC-administered group (MSC) without iron oxide nanoparticles, Alzheimer's disease transgenic mouse group (TG) not administered with MSC, and Alzheimer's disease transgenic mice were injected with stem cells into the ventricle. The experimental group (Positive) sacrificed immediately after treatment was used.

Briefly, Wharton's jelly-derived MSCs or Wharton's jelly-derived MSCs containing the iron oxide nanoparticles of the present invention were administered in a single dose at a dose of 1x10 ⁵ cells/ul to the right ventricle of an Alzheimer's disease transgenic mouse model (5xFAD). . A planned autopsy was performed one week after administration, and real-time PCR was performed using an Alu primer to analyze the remaining level of human Wharton's jelly-derived MSCs in the brain.

In order to evaluate the distribution in the body after administration of stem cell therapy products, it should be evaluated with a sensitive method that can be detected. Since human stem cells are administered to animals, biodistribution can be evaluated by detecting and quantifying human-derived specific DNA sequences in animal tissues. Accordingly, a primer capable of detecting Alu was used as a human-specific DNA sequence.

As a result, as shown in FIG. 6, compared to other control groups, the most remarkable and A significant number of stem cells were detected.

Through this, when Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention is administered to the ventricle of an Alzheimer's disease model and a magnet is placed on the skull, the remaining amount in the brain, which is the target location of mesenchymal stem cells, is the target cell increase can be seen.

Therefore, it was evaluated that the migration and engraftment to the target site by external magnetism are increased and thus effective in an in vivo system.

Example 7. Therapeutic effect through magnetic induction (MSC-IONP+Mag) of Wharton's jelly-derived MSC containing iron oxide nanoparticles in Alzheimer's disease model

The present inventors performed the experiment of FIG. 7a to confirm the treatment effect of Alzheimer's disease according to magnetic induction after administration of MSC derived from Wharton's jelly containing iron oxide nanoparticles of the present invention to the brain, and Amyloid, which is well known as the main cause of Alzheimer's disease Thioflavin S staining, which stains plaques, was performed on histopathology slides.

As a result, as shown in FIG. 7B, amyloid beta plaque (Aβ), which was not observed in the normal group (WT), was observed in the TG experimental group, and Wharton's jelly-derived MSC containing iron oxide nanoparticles of the present invention After administration, it was confirmed that the most significant decrease was observed in the magnet-induced group (MSC-IONP+Mag) in which a magnet was attached to the skull.

In addition, the number of amlyoid plaques and the area occupied by the amyloid plaques (Area fraction) in the whole brain region were analyzed using the fluorescence microscope images.

As a result, as shown in FIG. 7c, the mesenchymal stem cell administration group (MSC) without iron oxide nanoparticles and the experimental group in which magnets were attached to mesenchymal stem cells containing iron oxide nanoparticles of the present invention (MSC-IONP+ The number of plaques in Mag) and the area occupied by amyloid plaques in the entire brain area were significantly reduced.

Based on the above results, when Wharton's jelly-derived MSC containing iron oxide nanoparticles of the present invention was administered to the ventricle of an Alzheimer's disease model and then a magnet was placed on the skull, the effect of removing amyloid plaques, which is the cause of Alzheimer's disease, was high. confirmed to appear.

Example 8. Beneficial effects of multiple administrations of magnet-induced (MSC-IONP+Mag) MSC derived from Wharton's jelly containing iron oxide nanoparticles in Alzheimer's disease model

In an Alzheimer's disease model, the present inventors investigated the beneficial effect achieved by repeating magnetic induction (MSC-IONP+Mag) after ventricular administration of Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention, 5xFAD mouse brain H&E staining, thioflavin S staining, and behavioral changes (activity and anxiety) were observed.

At this time, the stem cells were administered to Alzheimer's disease mice a total of three times at intervals of one week, and sacrificed one week after each administration (FIG. 8a).

As a result, as shown in FIG. 8B, when H&E staining of 5xFAD mouse brains repeated three times with MSC-IONP+Mag, in the MSC-IONP+Mag group of the present invention, Wharton's jelly-derived-MSCs that had been injected aggregated in the lateral ventricle. It was found that most of them remained.

In addition, as shown in FIG. 8c, when thioflavin S staining of 5xFAD mouse brains repeated three times with MSC-IONP+Mag, the amyloid plaque removal effect, which is the cause of Alzheimer's disease, was the highest in the MSC-IONP+Mag group of the present invention. It was confirmed that it appeared high. In addition, when the number of amlyoid plaques and the area occupied by amyloid plaques (Area fraction) in the whole brain area were analyzed using the fluorescence microscopy images taken, as shown in FIG. 8d, the MSC-IONP+Mag of the present invention In the group, the number of plaques and the area occupied by amyloid plaques in the whole brain area were significantly reduced.

In addition, as a result of open field behavioral evaluation that can confirm the activity and anxiety of the experimental animals, behavioral changes began to appear from the second administration, as shown in FIG. 8e. Immediately after the second administration of mesenchymal stem cells containing iron oxide nanoparticles, the time to stay in the center of the open field, the number of entries, and the distance moved increased.

Based on this result, after administering Wharton's jelly-derived MSCs containing iron oxide nanoparticles of the present invention to the ventricle of an Alzheimer's disease model, engraftment occurs when the process of placing magnets on the skull is performed three or more times. It was confirmed that iron oxide nanoparticles continued to remain in Wharton's jelly-derived MSC, and that activity, which had been reduced due to Alzheimer's disease, increased and anxiety was improved.

Example 9. Confirmation of change in gene expression level after multiple administration of magnetic induction (MSC-IONP+Mag) of Wharton's jelly-derived MSC containing iron oxide nanoparticles in Alzheimer's disease model

In the Alzheimer's disease model, the present inventors confirmed changes in gene expression levels according to repetition of magnetic induction (MSC-IONP+Mag) after intraventricular administration of Wharton's jelly-derived MSC containing the iron oxide nanoparticles of the present invention.

As a result, as shown in Fig. 9, Euclidean clustering between the experimental groups in the Alzheimer's disease panel (n = 3) (Fig. 9A); Euclidean clustering between experimental groups in the neuritis panel (n=3) (FIG. 9B); Number of genes increased or decreased (underlined) in the Alzheimer's disease panel after stem cell treatment compared to TG (FIG. 9C); Number of genes increased or decreased (underlined) in the neuritis panel after stem cell treatment compared to TG (FIG. 9D); Heat map showing progressive increase and decrease of Alzheimer's disease-related gene expression (TG, MSC, MSC-IONP, MSC-IONP+Mag) in 5xFAD mouse brain (FIG. 9E); And a heat map (FIG. 9F) showing sequential increases and decreases in neuroinflammation-related gene expression (TG, MSC, MSC-IONP, MSC-IONP+Mag) in the 5xFAD mouse brain.

The above results suggest that the expression of Alzheimer's disease and neuroinflammation-related genes is normally restored in the brain of 5xFAD mice according to Wharton's jelly-derived MSC administration. In addition, this tendency is more pronounced when Wharton's jelly-derived MSC containing iron oxide nanoparticles is administered, and in particular, when magnetic induction is performed after administration of Wharton's jelly-derived MSC containing iron oxide nanoparticles, the change is the greatest. observed to appear. Through this, when Wharton's jelly-derived MSC or Wharton's jelly-derived MSC containing iron oxide nanoparticles are administered to the ventricles of 5xFAD transgenic mice, it is possible to select target genes that affect it, and follow-up studies are needed to identify specific treatment mechanisms. suggests

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (16)

1. A pharmaceutical composition for preventing or treating neurological diseases, comprising at least one selected from the group consisting of mesenchymal stem cells containing magnetic nanoparticles, cell-derived exosomes, cell membrane-derived nanoparticles, and cell secretions,

   The composition applies an external magnetic substance to move at least one selected from the group consisting of mesenchymal stem cells containing the magnetic nanoparticles, cell-derived exosomes, cell membrane-derived nanoparticles, and cell secretions to a tissue or organ to be treated To target by, the composition.
2. According to claim 1,

   Wherein the magnetic nanoparticles are at least one selected from the group consisting of FeO, Fe ₂ O ₃ , Fe ₃ O ₄ , FePt, CoPt, MnFe ₂ O4, CoFe ₂ O4, NiFe ₂ O ₄ and ZnFe ₂ O ₄ , a composition .
3. According to claim 2,

   The magnetic nanoparticles are FeO, Fe ₂ O ₃ And Fe ₃ O ₄ Of one or more types of iron oxide selected from the group consisting of, the composition.
4. According to claim 1,

   Wherein the composition is for ventricular administration or intravenous administration.
5. According to claim 1,

   The mesenchymal stem cells are derived from one or more selected from the group consisting of Wharton's jelly, umbilical cord, bone marrow, adipose tissue, blood, cord blood, liver, skin, gastrointestinal tract, placenta and uterus, composition.
6. According to claim 5,

   The composition, wherein the mesenchymal stem cells are derived from Wharton's jelly.
7. According to claim 1,

   The neurological disease is a disease caused by at least one selected from the group consisting of amyloid beta plaque formation in nerve tissue, phosphorylation of tau protein in nerve cells, abnormality of neurites, decreased expression of neprilysin in nerve cells, and combinations thereof. which is, the composition.
8. According to claim 7,

   The neurological disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania, the composition.
9. As a cell therapy agent for the treatment of neurological diseases, including mesenchymal stem cells containing magnetic nanoparticles,

   The cell therapy agent is a cell therapy agent that targets mesenchymal stem cells containing the magnetic nanoparticles by applying an external magnetic material to a target tissue or organ to be treated.
10. Prevention or prevention of neurological diseases, including mesenchymal stem cells containing magnetic nanoparticles, comprising treating and culturing magnetic nanoparticles in mesenchymal stem cells to contain the magnetic nanoparticles in the mesenchymal stem cells; A method for preparing a therapeutic pharmaceutical composition.
11. According to claim 10,

    The treatment concentration of the magnetic nanoparticles is 1 to 1000 μg / ml, the method.
12. According to claim 10,

    The culture time is 24 hours to 72 hours, the method.
13. A composition for transplantation and differentiation tracking of mesenchymal stem cells containing magnetic nanoparticles,

    Wherein the composition targets mesenchymal stem cells containing the magnetic nanoparticles by applying an external magnetic material.
14. A method for treating a neurological disorder, comprising administering the cell therapy agent of claim 9 to a subject and applying an external magnetic body to position the cell therapy agent to a target tissue.
15. According to claim 14,

    The treatment method, wherein the administration is administered 1 to 5 times.
16. According to claim 14,

    The neurological disease is Alzheimer's disease, and the amyloid plaque removal effect, mobility and anxiety are improved by the administration, the treatment method.

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