WO2021086139A1 - Method for promoting stem cell-derived exosome production by means of magnetic nanoparticle cluster - Google Patents

Abstract

The present invention relates to a method for promoting mesenchymal stem cell-derived exosome production by means of a magnetic nanoparticle cluster. Particularly, it is confirmed that stem cell-derived exosome production is promoted by treating a stem cell with a polymer-magnetic nanoparticle cluster and applying a magnetic force to the stem cell. Therefore, a large amount of high-quality exosomes exhibiting a clinical therapeutic efficacy can be provided in vitro and in vivo.

Description

Stem cell-derived exosome generation promotion method using magnetic nanoparticle cluster

The present invention relates to a method for promoting the generation of stem cell-derived exosomes using magnetic nanoparticle clusters, and more particularly, to a method for promoting the generation of stem cell-derived exosomes using a polymer-magnetic nanoparticle cluster.

Stem cells are well known as cells that have the ability to proliferate indefinitely and differentiate into various cells under specific conditions. Stem cell therapeutics are in the spotlight as the only future therapeutics that can treat diseases that are currently incurable or incurable by using these differentiation capabilities. Stem cell therapy can regenerate damaged cells and tissues without immune rejection by differentiating and proliferating the stem cells extracted from the patient into necessary cells in the body, such as the cardiovascular system, nervous system, cartilage system, and skin, and injecting them back into the patient. .

Among stem cells, mesenchymal stem cells (MSCs) have been widely studied for many years for their role in regulating the immune response, immune system activity, and the body's response to inflammation and disease. According to several studies, cultured mesenchymal stem cells have the ability to differentiate into bone and cartilage as well as other cell types and tissues in vitro and in vivo. In addition, mesenchymal stem cells secrete various cytokines and chemokines such as endothelial growth factor, IL-6 (interleukin-6), IL-8 (interleukin-8), etc. Promotes the formation of blood vessels. In addition, mesenchymal stem cells are known to secrete extracellular vesicles (EVs), and extracellular vesicles are known to affect a variety of aspects such as cell fate, function, and differentiation through signal transduction between cells.

On the other hand, extracellular vesicles are a generic term for vesicles having a membrane structure secreted by cells out of the cell, and are classified into microvesicles, exosomes, ectosomes, and apoptotic bodies. Double exosomes are defined as extracellular vesicles having a diameter of about 40 to 150 nm and a density of 1.09 to 1.18 g/ml, and have various kinds of active functions. Exosomes contain cell-specific components that reflect the unique biological function of the cell of origin (donor cell). In addition to phospholipids, messenger RNA (mRNA), microRNA (microRNA, miRNA), various soluble proteins, exogenous proteins, and It includes transmembrane protein components and the like. As marker proteins for exosomes, CD63 and CD81 are well known. These include receptors on the surface of cells such as EGFR, molecules involved in signal transduction, proteins involved in cell adhesion, and heat shock proteins. protein) and Alix related to endoplasmic reticulum formation. These exosomes can be isolated from various types of body fluids, such as saliva, urine, plasma, serum, and amniotic fluid, and can also be isolated from various types of cell culture supernatant.

In addition, purely isolated exosomes have been found to have clinical therapeutic efficacy in vitro and in vivo , so exosomes against various diseases that have been attempted using mesenchymal stem cells. It is emerging as a new alternative. Exosomes also play a role in immune regulation and signal transduction between cells, inducing functional changes in cells to activate cell regeneration programs, and because they are cell-free system, there is no risk of tumor formation and at -20℃ without cryopreservatives It is preserved in a state in which biological activity is maintained for months and is encapsulated, so there is an advantage that substances in exosomes are not decomposed.

However, despite these excellent effects, in order to use exosomes commercially, a large amount of and high quality exosomes are required. However, the amount of exosomes that can be obtained from mesenchymal stem cells is currently only a very small amount, and the development of methods and materials capable of increasing the production of exosomes derived from mesenchymal stem cells is still insufficient. Accordingly, the need for the development of novel methods and materials for promoting the production of mesenchymal stem cells-derived exosomes has been raised.

Accordingly, the present inventors have endeavored to develop a method that can increase the occurrence of exosomes in mesenchymal stem cells, and as a result, the amount of inflow into the mesenchymal stem cells varies depending on the surface modification properties and magnetic stimulation of the magnetic nanoparticle cluster. , It was confirmed that the amount of exosomes was affected by this. Therefore, it was found that the generation and activity of exosomes in mesenchymal stem cells can be maximized using magnetic nanoparticle clusters and magnetic stimulation, and thus the present invention was completed.

An object of the present invention is to provide a method for promoting the generation of exosomes derived from stem cells.

In order to achieve the above object, the present invention provides a method for promoting the generation of stem cells-derived exosomes using a polymer-magnetic nanoparticle cluster.

In the present invention, it was confirmed that the generation of stem cell-derived exosomes was promoted when a polymer-magnetic nanoparticle cluster was treated on stem cells and a magnetic force was applied to the stem cells. As a method of promoting production, it has the advantage of providing a large amount of high-quality exosomes with clinical therapeutic efficacy in vitro and in vivo.

1 is a schematic diagram showing a mechanism by which the generation of exosomes in stem cells is promoted by polymer-magnetic nanoparticle clusters and magnetic stimulation.

FIG. 2 is a diagram showing a polymer-superparamagnetic nanoparticle cluster surface-modified with negative charge (FIG. 2, upper) or positive charge (FIG. 2, lower).

3A is a diagram illustrating the size of a polymer-magnetic nanoparticle cluster surface-modified with a positive charge.

3B is a diagram showing a polymer-magnetic nanoparticle cluster observed by zeta potential (ζ(zeta, z)-potential), SEM and TEM of a polymer-magnetic nanoparticle cluster.

4 is a diagram showing the expression level of exosomes according to the surface charge and concentration of the polymer-magnetic nanoparticle cluster.

5 is a diagram showing the size of the expressed exosomes.

6 is a diagram showing the expression levels of CD9, CD63, and CD81, which are exosome-specific markers according to the strength of magnetic force.

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

The present invention

1) treating a macromolecular-magnetic nanoparticle cluster on stem cells; And

2) applying a magnetic force to the stem cells of step 1); provides a method for promoting the generation of stem cell-derived exosomes.

The polymer of step 1) may be a biocompatible polymer, and more specifically, as the biocompatible polymer, poly(beta-hydroxyethyl methacrylate) (Poly(beta-hydroxyethyl Methacrylate), PHEMA); Polyacrylamide (PA); Polyvinyl alcohol (PVA); Polyacrylic acid (PAA), its salt and its derivative; Polymethacrylic acid (Poly (metha acrylic acid), PMAA) or a derivative thereof; Poly(acrylic amide) or a derivative thereof; Poly(undecenoic acid) or a derivative thereof, or a copolymer of the polymer; Dextran or a derivative thereof, polyvinylpyrrolidone (PVP); Polyethylene oxide (PEO); Polyethyleneglycol (PEG) or a derivative thereof; Polypropylene Glycol (PPG) or a derivative thereof; A copolymer of the polyethylene glycol and polypropylene glycol, or a monoesterified derivative thereof; Poly(ethylene oxide-b-propylene oxide, PEO-PPO); Polyl-l-lysine; Polyethylenimine; Polylactic acid lactic acid, PLA); Polyglycolic acid (PGA); Poly(lactic-co-glycolic acid) (PLGA) copolymer; Chitosan; Hyaluronic acid (Hyaluronic acid) acid); And may be one or more selected from the group consisting of a mixture thereof, but is not limited thereto.

In addition, the magnetism of step 1) may be superparamagnetic, but is not limited thereto.

The magnetic nanoparticles of step 1) are ferric oxide (iron(II) oxide: FeO), iron(III) oxide, magnemite; Fe ₂ O ₃ , α-Fe ₂ O ₃ , β- Fe ₂ O ₃ , γ-Fe ₂ O ₃ , ε-Fe ₂ O ₃ ), magnetite (Fe ₃ O ₄ ) and iron(II, Ⅲ) oxides; Fe ₄ O ₅ , Fe ₅ O ₆ , Fe ₅ O ₇ , Fe ₁₃ O ₁₉ , Fe ₂₅ O ₃₂ ) may be one or more selected from the group consisting of, but is not limited thereto.

In addition, the polymer-magnetic nanoparticle cluster in step 1) is preferably a polymer-supermagnetic nanoparticle cluster surface-modified with a positive charge.

*The positively charged polymer-magnetic nanoparticle cluster may have a cationic transformant attached to the surface of the polymer-magnetic nanoparticle cluster, and more specifically, polyethyleneimine (PEI) as the cationic transformant, Pegyl Pegylated polyethyleneimine, Histidylated polyethyleneimine, Lactosylated polyethyleneimine, Folate-conjugated polyethyleneimine, Melittin-conjugated Polyethyleneimine derivatives such as polyethylinimine; Polylysine, Spermine, Protamine sulfate, Polyamidoamine (PAMAM), Polypropyleneimine, Polybrene, and DEAE-dextran At least one cationic transformant selected from the group consisting of may be attached, but is not limited thereto.

In step 1), the polymer-magnetic nanoparticle cluster may be prepared by the following method, but is not limited thereto:

a) dissolving the polymer;

b) adding magnetic nanoparticles dropwise;

c) mixing the polymer of step a) and the magnetic nanoparticles of step b);

d) ultrasonicating the mixture of step c) and stirring at room temperature.

In addition, in order to prepare a polymer-magnetic nanoparticle cluster surface-modified with a positive charge, the following step a') may be added to the step a):

a′) adding and reacting a cationic transformant and a linker connecting the cationic transformant and the polymer to step a) to obtain a polymer to which the cationic transformant is attached.

The linker may be specifically N,N'-dicyclohexylcarbodiimide (N,N'-Dicyclohexylcarbodiimide, DCC) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS), but is not limited thereto.

In addition, the mixing in step c) may be performed by vortexing for 3 to 7 minutes, but is not limited thereto.

In addition, a fluorescently attached polymer may be added in step c) to prepare a fluorescently-labeled polymer-magnetic nanoparticle cluster.

In addition, the ultrasonic treatment in step d) may be specifically performed for 1 to 5 minutes, more specifically for 2 to 4 minutes. Stirring in step d) may be performed for 1 to 24 hours, specifically for 4 to 8 hours, and more specifically for 5 to 7 hours.

The polymer-magnetic nanoparticle cluster prepared by the above method has a cluster form in which a polymer and magnetic nanoparticles are aggregated.

In addition, the polymer-magnetic nanoparticle cluster may be treated at 1 to 1000 µg/ml in step 1), but is not limited thereto.

The stem cells may be derived from one or more tissues selected from the group consisting of bone marrow, adipose tissue, peripheral blood, liver, muscle, lung, amniotic fluid, placental chorion, and umbilical cord blood, but is not limited thereto.

In addition, the stem cells may be embryonic stem cells or adult stem cells, but are not limited thereto.

The adult stem cells may be one or more adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells, but are not limited thereto. .

In step 2), the magnetic force may be applied in an amount of 0.1, 0.2, 0.3, 0.4, 0.6, or 1.0 T or more, specifically 0.1 to 2 T, and more specifically 0.3 to 1 T, but is not limited thereto. Does not.

In addition, the present invention comprises the steps of: 1) treating a polymer-magnetic nanoparticle cluster on stem cells;

2) applying a magnetic force to the stem cells of step 1); And

3) separating the exosomes from the stem cells of step 2); and a method for preparing an exosome including, and an exosome prepared according to the preparation method.

In addition, the present invention provides a composition for promoting the generation of exosomes derived from stem cells comprising a polymer-magnetic nanoparticle cluster (cluster).

As a medium used for culturing the composition, any general medium used for culturing stem cells may be used.

Preferably, it is a medium containing serum (e.g., fetal bovine serum, horse serum, and human serum), and the medium that can be used in the present invention is, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α -MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and may be KnockOut DMEM, more preferably a minimum essential nutrient medium (minimum essential medium, MEM) It may be, but is not limited thereto.

In addition, the medium may contain other components, such as antibiotics or antifungal agents (eg, penicillin, streptomycin), glutamine, and the like.

In a specific embodiment of the present invention, the present inventors confirmed that the generation of exosomes derived from mesenchymal stem cells is promoted by polymer-magnetic nanoparticle clusters and magnetic stimulation.

More specifically, the present inventors confirmed that when a macromolecular-magnetic nanoparticle cluster was treated on stem cells and a magnetic force was applied to the stem cells, the generation of stem cell-derived exosomes was promoted, so that the generation of the exosomes The method of maximizing the value can be usefully used.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Experimental Examples.

<Example 1> Cell culture

Mesenchymal stem cells (MSC) were purchased from Cyagen (MUBMX-01001, US). Mesenchymal stem cells were cultured in MEM-α medium (Hyclone, US) containing 10% fetal bovine serum (FBS) (Gibco, US) and antibiotics (Antibiotic-Antimycotic, US). After washing the cultured mesenchymal stem cells with DPBS (Dulbecco's Phosphate-Buffered Saline), the medium was changed every 3 days, and maintained at _{37°C and 5% CO 2.}

<Example 2> Synthesis of negatively charged surface-modified polymerized clustered superparamagnetic iron oxide (PCS) nanoparticles

Polymerized clustered superparamagnetic iron oxide (PCS) nanoparticles were made of iron oxide-based nanoparticles, and the size of the nanoparticles was about 100 nm. In order to determine the PCS nanoparticle treatment conditions, the concentrations of the nanoparticles were prepared at 20, 40 and 60 μg/ml for 24 hours.

To synthesize negatively charged surface-modified PCS nanoparticles, poly(D, L-lactide-co-glycolide) (Poly(D, L-lactide-co-glycolide, PLGA)) (50:50, M _W 38,000 to 54,000) (Sigma-Aldrich, US), superparamagnetic iron oxide (SPIO) nanoparticles, SPIONs) (Sigma-Aldrich, US) were used, and 1-ethyl-3-(3-dimethylaminopropyl) Carbodiimide hydrochloride (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC) (Thermo-fisher Scientigic, US) and N-hydroxysuccinimide (NHS) were used as fluorescent molecules (fluorescence). molecule) between the linker (EDC-NHS). In addition, cyanine 5.5 (Cyanine5.5, Cy5.5) (Cyanine5.5 amine, Lumiprobe Co., US) was used as a fluorescent molecule to detect nanoparticles in cells.

Specifically, 330 mg of PLGA was dissolved in DMSO solution with 60 mg of EDC, 132 mg of NHS and 9.9 mg of Cy5.5 and incubated for 24 hours so that PLGA and Cy5.5 were bound by EDC-NHS. Then, the reaction mixture was dialyzed with deionized water (M _w cutoff = 10 k) to remove excess EDC, NHS and Cy5.5 to obtain a PLGA-Cy5.5 fluorescent polymer (PLGA-Cy5.5). I did.

Then, 0.4 mg of PLGA-Cy5.5 and 0.1 mg of PLGA were dissolved in 0.1 ml of DMSO, and 0.1 ml of SPIONs (5 mg/ml) was added dropwise to 3 ml of deionized water. Thereafter, the vial was vortexed for 5 minutes, sonicated for 3 minutes, and stirred at room temperature for 6 hours to obtain negatively charged surface-modified PCS nanoparticles. The obtained negatively charged surface-modified PCS nanoparticles were freeze-dried to obtain a powder sample (Fig. 2, above).

<Example 3> Synthesis of positively charged surface-modified polymerized clustered superparamagnetic iron oxide (PCS) nanoparticles

Positively charged surface-modified PCS nanoparticles were prepared as follows. In order to determine the PCS nanoparticle treatment conditions, the concentrations of the nanoparticles were prepared at 20, 40 and 60 μg/ml for 24 hours.

To synthesize positively charged surface-modified PCS nanoparticles, poly(D, L-lactide-co-glycolide) (Poly(D, L-lactide-co-glycolide, PLGA))(50:50, M _W 38,000~ 54,000) (Sigma-Aldrich, US) and polyethyleneimine (PEI) (M _W ~25,000 by LS, M _n ~ 10,000 by GPC) (Sigma-Aldrich, US) was used, and N,N'-dicyclo Hexylcarbodiimide (N,N'-Dicyclohexylcarbodiimide, DCC) and N-Hydroxysuccinimide (NHS) were used to prepare a linker between PLGA and PEI.

To synthesize the PLGA-PEI copolymer, 330 mg of PLGA, 113.67 mg of PEI, 18.33 mg of DCC and 11 mg of NHS were dissolved in 100 ml of DMSO solution and incubated for 24 hours, and the reaction mixture was then used in deionized water. Dialysis (M _w cutoff = 10 k) was performed to remove excess DCC, NHS and PEI. The obtained PLGA-PEI copolymer bath liquid was freeze-dried to obtain a powder sample.

The PLGA-Cy5.5 fluorescent polymer was synthesized in the same manner as described in <Example 2>.

Then, PLGA-PEI copolymer, PLGA-Cy5.5 and SPIONs were used. 0.4 mg of PLGA-Cy5.5 and 0.1 mg of PLGA-PEI were dissolved in 0.1 ml of DMSO, and 0.1 ml of SPIONs (5 mg/ml) was added dropwise to 3 ml of deionized water. Thereafter, the vial was vortexed for 5 minutes, sonicated for 3 minutes, and stirred at room temperature for 6 hours to obtain positively charged surface-modified PCS nanoparticles. The obtained positively charged surface-modified PCS nanoparticles were freeze-dried to obtain a powder sample (Fig. 2, bottom).

<Example 4> Separation of exosomes from mesenchymal stem cells treated with PCS nanoparticles by applying micro-magnetofection

After the PCS nanoparticles prepared in <Examples 2 and 3> were treated to the mesenchymal stem cells of <Example 1> and magnetic force was applied, exosomes were separated.

Specifically, 2×10^6 mesenchymal stem cells of <Example 1> were cultured in a cell culture plate. Then, the PCS nanoparticles prepared in <Examples 2 and 3> were treated with the cultured mesenchymal stem cells for 24 hours, and then magnetic force was applied for 24 hours. Thereafter, the cell culture media was recovered and centrifuged for 30 minutes with a centrifugal force of 2,000 xg, and the supernatant was transferred to a new tube. 0.5 volume of exosome separation reagent (cat.4478359, Thermo-fisher Scientigic, US) was added to the supernatant transferred to a new tube, and mixed by vortexing or pipetting. The mixture was incubated at 6° C. overnight, and then centrifuged at 6° C. for 1 hour with a centrifugal force of 10,000 xg, and the supernatant was discarded and a pellet settled on the bottom of the tube was recovered. Subsequently, the recovered pellet was re-suspended in 1X PBS and stored at -20°C for a long period of time.

<Example 5> Transmission Electron Microscopy (TEM)

The cells were fixed in 2.5% glutaraldehyde for 2 hours at 4°C, and solidified with 2% agar. After washing with 0.1 M cacodylate buffer, it was fixed in _{1% osmium tetroxide (OsO 4 ).} The dehydration step was carried out using 50-100% ethanol and embedded in Epon resin. Ultrathin sections were cut using an ultramicrotome, and stained using uranyl acetate and lead citrate. Then, it was observed using TEM under the condition of 63 kV.

<Example 6> Western blot

After preparing exosomes in <Example 4>, specific markers of exosomes were measured by exosome proteins. Primary antibodies against CD9 (ab92726, Abcam, US), CD63 (ab216130, Abcam, US) and CD81 (ab109201, Abcam, US) were used at 1:1000, goat anti-rabbit immunoglobulin G (Goat anti-rabbit immunoglobulin G, Goat anti-rabbit IgG) was used as a secondary antibody. The expression level of exosome proteins was measured and normalized using SPSS (Statistical Package for the Social Science) (SPSS Inc., US).

<Example 7> Statistical analysis

Statistical analysis was performed using SPSS statistical package version 21.0 (SPSS Inc., US). Descriptive results of continuous variables were expressed as mean ± standard deviation (SD) of normally distributed variables. Means were compared using a two-way analysis of variance, and the statistical significance level was 0.05.

<Experimental Example 1> Confirmation of the properties of PCS nanoparticles

The size and surface charge of the surface-modified PCS nanoparticles prepared according to <Example 3> were analyzed.

Specifically, the size and surface charge of the PCS nanoparticles surface-modified with positive charges prepared according to the <Example 3> were measured by DLS (dynamic light scattering) Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK).

In addition, the PCS nanoparticles were placed on a carbonyl-coated 400 mesh copper grid (CF400-CU-UL, Electron Microscopy Sciences, US) for 15 minutes. Then, 10 µl of 2% uranyl acetate was placed on the grid for 10 minutes, dried and negatively stained. Then, using an electron microscope (JEM-2100F, JEOL Ltd, Japan), a TEM (transmission electron microscopic) image was obtained under the condition of 63 kV to confirm the structure of the nanoparticles. In addition, a scanning electron microscopic (SEM) image was obtained using an electron microscope to confirm the shape of the nanoparticles.

As a result, as shown in FIGS. 3A to 3D, it was confirmed that the PCS nanoparticles surface-modified with a positive charge exhibited a cluster form in which PLGA and iron oxide nanoparticles were aggregated as shown in FIG. 2, and have a positive charge. I did. Accordingly, the PCS nanoparticles were named as polymer-magnetic nanoparticle clusters, and the PCS nanoparticles surface-modified with positive charges were polymer-magnetic nanoparticle clusters surface-modified with positive charges, and the PCS nanoparticles surface-modified with negative charges were negatively charged. It was named as a surface-modified polymer-magnetic nanoparticle cluster.

<Experimental Example 2> Confirmation of the degree of generation of exosomes according to the surface charge and concentration of the polymer-magnetic nanoparticle cluster

In order to confirm the degree of generation of exosomes according to the surface charge and concentration of the polymer-magnetic nanoparticle cluster, the concentration of the polymer-magnetic nanoparticle cluster surface-modified with positive/negative charges prepared in <Examples 2 and 3> above. In different ways, the mesenchymal stem cells were treated and micromagnetic was applied, and then the amount of exosomes produced in the mesenchymal stem cells was analyzed.

Specifically, the polymer-magnetic nanoparticle cluster surface-modified with a positive charge prepared in <Example 3> was treated on the mesenchymal stem cells at a concentration of 40 μg/ml in the same manner as in the method described in <Example 4>. , 0.3 T, 0.4 T, 0.6 T and 1.0 T magnetic force was applied. Then, the amount of exosomes produced from mesenchymal stem cells was analyzed through TEM observation in the same manner as described in <Example 5>.

In addition, in the same method as described above, the polymer-magnetic nanoparticle clusters surface-modified with positive charges prepared in <Example 3> were treated at concentrations of 20, 40, and 60 ㎍/mL, and after applying magnetic force, mesenchymal stem The amount of exosomes produced from cells was analyzed.

As a control, mesenchymal stem cells without treatment of polymer-magnetic nanoparticle clusters were used.

As a result, as shown in FIGS. 4 and 5, when the polymer-magnetic nanoparticle clusters surface-modified with negative charges were treated, exosomes were not generated, but when the polymer-magnetic nanoparticle clusters surface-modified with positive charges were treated. The generation of exosomes was induced, and as the concentration of the polymer-magnetic nanoparticle clusters surface-modified with positive charges increased, the amount was also observed to increase (FIG. 4). In particular, when the polymer-magnetic nanoparticle clusters surface-modified with positive charges are treated, a large amount of nanoparticle clusters are introduced in a short time by magnetic force, and the nanoparticle clusters introduced by endocytosis pass through exosomes. It was confirmed that it migrates to the lysosome. In addition, the nanoparticle cluster in the lysosome remains as it is, only the amount of exosomes produced increases, and the resulting exosomes were found to have a size of 91 to 169 nm (FIG. 5).

<Experimental Example 3> Confirmation of the degree of generation of exosomes according to magnetic force strength

In order to confirm the degree of generation of exosomes according to the strength of magnetic force, the polymer-magnetic nanoparticle cluster surface-modified with the positive charge prepared in <Example 3> was treated to the mesenchymal stem cells, and micromagnets were applied by varying the strength. Afterwards, the amount of exosomes produced in mesenchymal stem cells was analyzed.

Specifically, in the same method as described in <Experimental Example 3>, the polymer-magnetic nanoparticle clusters surface-modified with positive charges prepared in <Example 3> were treated at concentrations of 20, 40 and 60 µg/ml, and 0.3 After applying magnetic force of T, 0.4 T, 0.6 T, and 1.0 T intensity, Western blotting was performed in the same manner as described in <Example 5> to measure the expression level of exosome proteins.

As a result, as shown in FIG. 6, it can be observed that the expression of all of the exosome-specific markers CD9, CD63, and CD81 during exposure to magnetic force increased at increased levels, and the expression also increased as the strength of the applied magnetic force increased. There was (Fig. 6).

Through the above results, the surface modification of the nanoparticle cluster causes a difference due to the zeta-potential, and the more the nanoparticle cluster has a positive property rather than a negative property, the greater the amount of the nanoparticle cluster flows into the stem cells. As a result, it can be seen that the amount of exosomes produced increases when magnetic force is applied.

The present invention relates to a method for promoting generation of exosomes derived from mesenchymal stem cells using magnetic nanoparticle clusters, and specifically, when a polymer-magnetic nanoparticle cluster is treated on stem cells and magnetic force is applied to the stem cells, Since it promotes the generation of stem cell-derived exosomes, it can be usefully used to provide a large amount of high-quality exosomes with clinical therapeutic efficacy both in vitro and in vivo.

Claims (17)

1) treating a macromolecular-magnetic nanoparticle cluster on stem cells; And

2) The step of applying a magnetic force to the stem cells of step 1); Stem cell-derived exosome production promotion method comprising a.

The method of claim 1,

The method, characterized in that the polymer of step 1) is a biocompatible polymer.

The method of claim 2,

The biocompatible polymer,

Poly(beta-hydroxyethyl methacrylate) (PHEMA); Polyacrylamide (PA); Polyvinyl alcohol (PVA); Polyacrylic acid (PAA), its salt and its derivative; Polymethacrylic acid (Poly (metha acrylic acid), PMAA) or a derivative thereof; Poly(acrylic amide) or a derivative thereof; Poly(undecenoic acid) or a derivative thereof, or a copolymer of the polymer; Dextran or a derivative thereof, polyvinylpyrrolidone (PVP); Polyethylene oxide (PEO); Polyethyleneglycol (PEG) or a derivative thereof; Polypropylene Glycol (PPG) or a derivative thereof; A copolymer of the polyethylene glycol and polypropylene glycol, or a monoesterified derivative thereof; Poly(ethylene oxide-b-propylene oxide, PEO-PPO); Polyl-l-lysine; Polyethylenimine; Polylactic acid lactic acid, PLA); Polyglycolic acid (PGA); Poly(lactic-co-glycolic acid) (PLGA) copolymer; Chitosan; Hyaluronic acid (Hyaluronic acid) acid); And a mixture thereof, characterized in that at least one selected from the group consisting of.

The method of claim 1,

The method, characterized in that the magnetism of step 1) is superparamagnetic.

The method of claim 1,

The magnetic nanoparticles of step 1),

Ferric oxide (iron(Ⅱ) oxide: FeO), ferric oxide (iron(Ⅲ) oxide, magnemite; Fe ₂ O ₃ , α-Fe ₂ O ₃ , β-Fe ₂ O ₃ , γ-Fe ₂ O ₃ , ε-Fe ₂ O ₃ ), magnetite (Fe ₃ O ₄ ) and iron(II, Ⅲ) oxides; Fe ₄ O ₅ , Fe ₅ O ₆ , Fe ₅ O ₇ , Fe ₁₃ O ₁₉ , Fe ₂₅ O ₃₂ ), characterized in that at least one selected from the group consisting of.

The method of claim 1,

The polymer-magnetic nanoparticle cluster of step 1) is a polymer-magnetic nanoparticle cluster surface-modified with a positive charge.

The method of claim 6,

The positively charged polymer-magnetic nanoparticle cluster is characterized in that the cationic transformant is attached to the polymer-magnetic nanoparticle cluster surface.

The method of claim 7,

The cationic transformant,

Polyethyleneimine (PEI), Pegylated polyethyleneimine, Histidylated polyethyleneimine, Lactosylated polyethyleneimine, Folate-conjugated polyethyleneimine, Mellie Polyethyleneimine derivatives, such as a tin covalent bond type (Melittin-conjugated) polyethylinimine; Polylysine, Spermine, Protamine sulfate, Polyamidoamine (PAMAM), Polypropyleneimine, Polybrene, and DEAE-dextran Characterized in that at least one selected from the group consisting of, the method.

The method of claim 1,

In the step 1), the polymer-magnetic nanoparticle cluster is treated with 1 to 1000 μg/ml.

The method of claim 1,

The stem cell is characterized in that derived from one or more tissues selected from the group consisting of bone marrow, adipose tissue, peripheral blood, liver, muscle, lung, amniotic fluid, placental chorionic membrane and umbilical cord blood.

The method of claim 8,

The method, characterized in that the stem cells are embryonic stem cells or adult stem cells.

The method of claim 9,

The adult stem cells are at least one adult stem cell selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells.

The method of claim 1,

The method, characterized in that applying a magnetic force of 0.1 to 2.0 T in step 2).

1) treating a macromolecular-magnetic nanoparticle cluster on stem cells;

2) applying a magnetic force to the stem cells of step 1); And

3) separating the exosomes from the stem cells of step 2); containing, exosomes manufacturing method.

An exosome prepared according to the manufacturing method of claim 14.

A composition for promoting generation of exosomes derived from stem cells comprising a polymer-magnetic nanoparticle cluster.

The method of claim 16,

The composition is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM/F- 12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM The composition, characterized in that cultivated in one or more selected medium.

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