KR102224187B1 - Method for tracking stem cells injected in vivo using complexed nanoparticles comprising iron oxide nanoparticles encapsulated by chitosan glycol nanoparticles and method for checking brain stroke lesion - Google Patents

Abstract

The present invention relates to a method for tracking stem cells implanted in the body using composite nanoparticles including iron oxide nanoparticles trapped in chitosan glycol nanoparticles optimized to generate a maximized signal, and a method for identifying stroke lesions using the same.

Description

Method for tracking stem cells injected in vivo using complexed nanoparticles comprising iron oxide nanoparticles encapsulated by chitosan glycol nanoparticles and method for checking brain stroke lesion}

The present invention relates to a method for tracking stem cells implanted in the body using composite nanoparticles including iron oxide nanoparticles trapped in chitosan glycol nanoparticles optimized to generate a maximized signal, and a method for identifying stroke lesions using the same.

Cell labeling and tracking technology is an important technology for studying the therapeutic effects and biological mechanisms of inoculated cells in vivo. Cell labeling and tracking techniques can be used to differentiate host cells from transplanted cells, monitor distribution and migration after transplantation, and evaluate the functional effects of transplanted cells. Indeed, various approaches have been developed to visualize cells of interest in vivo.

For direct labeling and tracking of cells, various reagents such as fluorescent probes, super-paramagnetic iron oxide and radioactive tracers are used. Basically, the cells are labeled with these probes before stem cells are implanted and visualized with an appropriate imaging device. The use of these probes that directly label cells facilitates the visualization of cells, but due to the biological effects by these probes, cell traceability is quite limited. For example, dead cells and released image probes are secondarily phagocytized by host macrophages. Therefore, it is difficult to distinguish whether a signal to be detected originates from a targeted cell or a macrophage. In order to overcome the shortcomings of such direct labeling, genetic modification methods have been used as indirect labeling and tracking methods for the expression of foreign genes. However, genetic modification using non-viral and viral systems has a disadvantage in that there is a possibility of carcinogenesis and it is difficult to regulate gene expression. Therefore, devising a novel cell labeling and tracking method to solve this problem is important to improve biological mechanisms and therapeutic effects in vivo.

Bio-orthogonal non-isotope chemical reaction is a very rapid and effective reaction carried out by an alkyne-azide cyclization reaction as an azide-alkyne cyclization bioconjugation reaction that does not require a copper catalyst having cytotoxicity.

Such bio-orthogonal non-autonomous click-chemical reactions have been used for more powerful applications in combination with metabolic glycoengineering, especially in the field of biology. Metabolic sugar chain manipulation is a method of introducing non-natural glycans into cells using intrinsic metabolism by introducing an appropriate precursor.

Since labeling using metabolic sugar chain manipulation may be suitable for living organisms as a bio-orthogonal chemical reporter for cell imaging, there is a number of evidence that metabolic sugar chain manipulation labeling can be visualized by chemical ligation with a fluorescent probe. In particular, the non-natural analogue of N-acetyl mannoseamine is metabolized by cultured cells and converted to azidosialic acid on the surface of various cells without any significant physiological risk. Azide groups on the cell surface have been treated with dibenzyl cyclooctin (DBCO)-labeled fluorescent probes for cell imaging. However, these studies have not focused on the tracking of specific cells in vivo, and in fact, no studies have shown a response for tracking in specific cells in vivo. When applied to cell labeling and tracking, this method can overcome limitations due to biological effects since the reaction is not normally found in biological systems. In addition, this reaction is rapid, selective, and highly efficient. Therefore, when it is used for detection of a specific cell in vivo, it exhibits a low background signal and high sensitivity without difficulty in controlling the expression of an external gene.

On the other hand, unlike in vitro, cells injected in vivo detect images using a device outside the body, so it is also an important task to discover optimized conditions that can generate strong signals.

Patent Document 1: Korean Registered Publication No. 10-1416290; Patent Document 2: Korean Registered Publication No. 10-1627271.

Non-Patent Document 1: ARTHURS, Owen J. and GALLAGHER, Ferdia A., Pediatric radiology, 2011, 41(2): 185-198; Non-Patent Document 2: Hong, V. et al., Bioconjugate chemistry, 2010, 21(10): 1912-1916; Non-Patent Document 3: Gutierrez-Fernandez, M. et al., Journal of Cellular and Molecular Medicine, 2012, 16(10): 2280-2290; Non-Patent Document 4: Kim, D. E. et al., Stroke, 2004, 35(4): 952-957; Non-Patent Document 5: Kircher, M. F. et al., Nature Reviews Clinical Oncology, 2011, 8: 677-688; Non-Patent Document 6: Lee, S. et al., Biomaterials, 2017, 139: 12-29; Non-Patent Document 7: Yi, P. et al., Biomaterials, 2013, 34(12): 3010-3019; Non-Patent Document 8: Li, L. et al., Theranostics, 2013, 3(8): 595-615; Non-Patent Document 9: Du, J. et al., Glycobiology, 2009, 19(12): 1382-1401; Non-Patent Document 10: Kang, S.-W. et al., Theranostics, 2014, 4(4): 420-431; Non-Patent Document 11: Nam, T. et al., Bioconjugate chemistry, 2010, 21(4); 578-582; Non-Patent Document 12: Han, S.-S. et al., Scientific Reports, 2018, 8(1): 13212; Non-Patent Document 13: Xie, R. et al., Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(19): 5173-5178; Non-Patent Document 14: Kawabori, M. et al., Neurosurgery, 2011, 68(4): 1036-1047; Non-Patent Document 15: Hill, W. D. et al., Journal of Neuropathology & Experimental Neurology, 2004, 63(1): 84-96.

The present inventors discovered a direct labeling method that does not affect the proliferation and/or differentiation of stem cells without cytotoxicity using metabolic sugar chain manipulation, and a tracking method of stem cells transplanted into the body using the same, and an improved signal optimized therefor As a result of intensive research efforts to establish the conditions of the labeling substance to be generated, composite nanoparticles in which iron oxide nanoparticles are captured in chitosan glycol nanoparticles are used, but the chitosan glycol nanoparticles have a cycloalkyne group and a fluorescent molecule on the surface. Molecule-bound particles are used, and as the iron oxide nanoparticles, particles whose surface is modified with oleic acid and whose size is adjusted are used to rapidly bind to stem cells that introduce azide groups to the surface through bio-orthogonal non-moving click chemistry, By directly labeling stem cells capable of double detection through maximized MR imaging and fluorescent signals, it was confirmed that it can be sensitively and efficiently tracked in vivo, and the present invention was completed.

As one aspect for achieving the above object, the present invention is formed by encapsulating two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which is surface-modified with oleic acid, by two or more chitosan glycol nanoparticles. , Provides composite nanoparticles having an average diameter of 200 to 300 nm and an iron oxide content of 13 to 18%.

In another aspect, the present invention is a surface-modified with oleic acid, wherein at least two iron oxide nanoparticles having an average diameter of 15 to 25 nm are encapsulated by at least two chitosan glycol nanoparticles, an average diameter of 200 to A composition for labeling stem cells comprising complex nanoparticles having a size of 300 nm and an iron oxide content of 13 to 18%, wherein the chitosan glycol nanoparticles include cycloalkyne groups bound to the surface, through metabolic sugar chain manipulation. It provides a composition for labeling stem cells, which can be bound to stem cells including the introduced azide group on the surface through bio-orthogonal non-moving click chemistry.

In yet another aspect, the present invention is formed by encapsulating two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which is surface-modified with oleic acid, by two or more chitosan glycol nanoparticles, with an average diameter of 200 to 300. composite nanoparticles having a size of nm and an iron oxide content of 13 to 18%; And isolated stem cells; a composition for detecting stroke lesions, wherein the chitosan glycol nanoparticles contain a cycloalkyne group bound to the surface, and the isolated stem cells contain an azide group introduced through metabolic sugar chain manipulation on the surface. Thus, it provides a composition for detecting stroke lesions that can be bound to each other through bio-orthogonal non-moving click chemistry.

In another aspect, the present invention is a stem cell that can be traced when injected in vivo, wherein two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are added to two or more chitosan glycol nanoparticles. Composite nanoparticles, formed encapsulated by, having an average diameter of 200 to 300 nm and an iron oxide content of 13 to 18%; And isolated stem cells; wherein the chitosan glycol nanoparticles include a cycloalkyne group bonded to the surface, and the isolated stem cells include an azide group introduced through metabolic sugar chain manipulation on the surface, and bio-orthogonal non-moving click chemistry It provides a stem cell that is bound to each other through.

In yet another aspect, the present invention is formed by encapsulating two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which is surface-modified with oleic acid, by two or more chitosan glycol nanoparticles, with an average diameter of 200 to 300. composite nanoparticles having a size of nm and an iron oxide content of 13 to 18%; And isolated stem cells; a composition for detecting stroke lesions, wherein the chitosan glycol nanoparticles contain a cycloalkyne group bound to the surface, and the isolated stem cells contain an azide group introduced through metabolic sugar chain manipulation on the surface. Thus, it provides a composition for detecting stroke lesions that can be bound to each other through bio-orthogonal non-moving click chemistry.

In yet another aspect, the present invention is formed by encapsulating two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which is surface-modified with oleic acid, by two or more chitosan glycol nanoparticles, with an average diameter of 200 to 300. A method for producing stem cells traceable by magnetic resonance imaging, comprising the step of treating and labeling complex nanoparticles having a size of nm and having an iron oxide content of 13 to 18% on stem cells, wherein the chitosan glycol nanoparticles are It provides a method comprising a cycloalkyne group bonded to a surface, and the stem cell includes an azide group introduced through metabolic sugar chain manipulation on the surface, and binds to each other through bio-orthogonal non-click chemistry.

In yet another aspect, the present invention is formed by encapsulating two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which is surface-modified with oleic acid, by two or more chitosan glycol nanoparticles, with an average diameter of 200 to 300. treating and labeling the stem cells to be injected with the composite nanoparticles having a size of nm and having an iron oxide content of 13 to 18%; Injecting the stem cells labeled with the composite nanoparticles into the individual; And measuring a magnetic resonance imaging (MRI) signal from an individual injected with the stem cells, comprising the step of measuring a magnetic resonance imaging (MRI) signal, wherein the chitosan glycol nanoparticles Includes a cycloalkyne group bonded to the surface, and the stem cell includes an azide group introduced through metabolic sugar chain manipulation on the surface and binds to each other through bio-orthogonal non-click chemistry.

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

The present invention is a particle that generates magnetic resonance imaging signals in diagnosing stroke by labeling particles that generate magnetic resonance imaging signals through biological orthogonal non-moving click chemistry on stem cells capable of targeting stroke lesions when injected into the body. A composite nanoparticle having a diameter of several hundred nm formed by the nanoparticles surrounded by chitosan glycol nanoparticles is used, but the composite nanoparticles prepared by containing particles having a size adjusted to 15 to 25 nm in diameter as the iron oxide nanoparticles are different. It is characterized by the discovery that it generates a significantly increased magnetic resonance imaging signal compared to that formed from iron oxide nanoparticles of the size.

In the present invention, two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm. Treating and labeling the stem cells to be injected with the iron oxide content of 13 to 18%, the composite nanoparticles; Injecting the stem cells labeled with the composite nanoparticles into the individual; And measuring a magnetic resonance imaging (MRI) signal from an individual injected with the stem cells. At this time, the chitosan glycol nanoparticles include a cycloalkyne group bonded to the surface, and the stem cells include an azide group introduced through metabolic sugar chain manipulation on the surface and bind to each other through bio-orthogonal non-click chemistry.

For example, in the tracking method of the present invention, the chitosan glycol nanoparticles may be additionally labeled with a fluorescent molecule, and accordingly, multiple detection is possible.

For example, the fluorescent molecule may be a material having an absorption peak in a wavelength range of 450 to 800 nm and a fluorescence peak in a wavelength range of 550 to 1000 nm.

Non-limiting examples of the fluorescent molecule include 7-AAD (7-Amionoactinomycin D), Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750 , Allophycocyanin(APC), PharRed(APC-Cy7), BOBO-3/BO-PRO-3, BODIPY TR-X, BODIPY 630/650-X, BODIPY 650/665-X, Calcium Crimson, 5-Carboxynaphthofluoroscein, Carboxy -X-rhodamine(5-ROX), CFC Formazan, Cy5, Cy5.5, Cy7, PE-Cy5(Cychrome, Quantum Red, R670, Tri-color), DiD(DilC18(5)), Dir(DilC18(7) )), Ethidium bromide, Ethidium homodimer-1(EthD-1), Fura Red/Fluo-3, Indodicarbocyanine, Indotricarbocyanine, LDS 751(+DNA), LDS 751(+RNA), Nile Red, PE-Cy7, PE- Texas Red (Red 613), PerCP (Peridinin chlorphyll protein), PerCP-Cy5.5 (TruRed), C-phycocyanin, R-phycocyanin, PI (Propidium Iodide), RH 414, SNAFL-1 (high pH), SNARF- 1 (high pH), SpectrumRed, SYTO 17, Texas Red/Texas Red-X, Thiadicarbocyanine, TOTO-3/TO-PRO-3, TO-PRO-5, WW 781, X-Rhodamine (XRITC), YOYO-3 /YO-PRO-3 may be included.

For example, the stem cells may be treated with one or more compounds selected from the group consisting of compounds represented by the following Formulas 1 to 3 to introduce an azide group to the surface, but is not limited thereto:

[Formula 1]

[Formula 2]

[Formula 3]

.

.

Specifically, the stem cell may be one containing non-natural sialic acid having an azide group generated on the surface of the cell by treatment with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz), but is not limited thereto. Does not.

Further, the chitosan glycol nanoparticles have at least one compound selected from the group consisting of compounds of the following formulas 4 to 16 on the surface to form a bond with the azide group introduced on the surface of the stem cell through bio-orthogonal click chemistry. May be combined:

[Formula 4]

[Formula 5]

[Formula 6]

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

[Formula 15]

[Formula 16]

.

.

However, the present invention is not limited thereto, and the material introduced to the surface of the chitosan glycol nanoparticles is not limited to the above formula, as long as a bond can be formed with the azide group on the surface of the stem cell by bio-orthogonal click chemistry.

For example, the stem cells labeled with the composite nanoparticles injected into the individual according to the present invention may maintain the same level of cellular characteristics as the unlabeled pure stem cells themselves. Maintaining the cellular characteristics of the stem cells themselves, ie growth, proliferation, migration and/or differentiation characteristics, may be an important factor for use in therapeutic purposes. For example, when the label of a composite nanoparticle causes toxicity of the cell, kills the cell, and is ingested into the macrophage, it may provide unclear tracking information by imaging the macrophage that predated it, not the stem cell to be tracked. . Or, if it affects proliferation or differentiation, it may cause cancer or abnormal tissue formation in the body due to its injection.

_{In addition, the r 2} relaxation degree calculated based on the MR phantom image measured from the composite nanoparticles labeled on the stem cells according to the present invention and/or as such may be 200 to 1000 /mM·sec. Since it has an improved r ₂ relaxation degree as described above, its use may be advantageous for obtaining a more clear magnetic resonance image. In a specific embodiment of the present invention, the composite prepared particles have a similar iron oxide content, except that iron oxide nanoparticles having a diameter of 5 nm and 30 nm are used instead of iron oxide nanoparticles having different sizes, for example, 20 nm in diameter. Compared to the prepared composite nanoparticles, it was confirmed that _{the r 2} relaxation was increased by about 4.9 to 5.7 times, respectively.

Furthermore, the composite nanoparticles used in the stem cell tracking method of the present invention may exhibit a small variation in size within 10% up to 14 days in a physiologically active solution condition. This is because the composite nanoparticles, which are the labeling materials used in the stem cell tracking method of the present invention, maintain their original shape without changing their size for a long period of 2 weeks or more, that is, not decomposing or swelling, even if they are injected into the living body. It is suitable for follow-up and can be usefully applied to diagnosis and/or treatment of a disease.

_{For example, the composite nanoparticles used in the stem cell tracking method of the present invention may exhibit a T 2} -weighted signal size that is 10 to 30 times increased compared to using the same amount of iron oxide nanoparticles of the same size. This suggests that sensitive tracking is possible even with a small amount of labeling material.

In another aspect, the present invention is a surface-modified with oleic acid, wherein at least two iron oxide nanoparticles having an average diameter of 15 to 25 nm are encapsulated by at least two chitosan glycol nanoparticles, an average diameter of 200 to Injecting stem cells labeled with complex nanoparticles with a size of 300 nm and an iron oxide content of 13 to 18% into individuals suspected of having a stroke except for humans; And measuring a magnetic resonance imaging (MRI) signal from an individual injected with the stem cells. At this time, the chitosan glycol nanoparticles include a cycloalkyne group bound to the surface, and the stem cells are bio-orthogonal between the azide group introduced through metabolic sugar chain manipulation on the surface thereof and the cycloalkyne group bound to the surface of the chitosan glycol nanoparticles. It may be labeled with the composite nanoparticles through binding by click chemistry.

The term "individual" of the present invention refers to monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits, or guinea pigs, including humans who have or may develop the stroke. It can mean any animal, including.

In a specific embodiment of the present invention, stem cells labeled with the composite nanoparticles were injected into an animal model of a stroke-causing mouse, followed by 14 days at intervals of 1 to 4 days. As a result, the injected stem cells moved to the stroke lesion and selectively It was confirmed that the location was confirmed, and this indicates that the stroke lesion, that is, the specific location where the stroke occurred, can be identified using the method of tracking stem cells labeled with the composite nanoparticles according to the present invention.

As described above, since the stem cells labeled with the composite nanoparticles according to the present invention move to the stroke lesion and can stay there selectively, the multiple functions of confirming the lesion and simultaneously delivering the drug to the treatment by loading a drug therein are performed. It can be performed, but is not limited thereto. As the drug, a drug for treating stroke known in the art may be used, but is not limited thereto. In addition, it can be combined with a treatment method in which a drug is loaded and injected into the stem cells labeled with the composite nanoparticles, and an existing therapeutic agent is administered through another route.

According to the present invention, the composite nanoparticles including magnetic nanoparticles on the surface of stem cells using metabolic sugar chain manipulation and bio-orthogonal non-moving click chemistry that do not show toxicity to stem cells and do not affect proliferation and/or differentiation characteristics In tracking stem cells injected into the body through direct labeling, the maximized signal can be displayed by adjusting the size of the magnetic nanoparticles contained in the composite nanoparticles, and additionally, by introducing a fluorescent molecule into the composite nanoparticles, multiplex It enables more accurate tracking of stem cells by detection.

1 is a diagram showing the result of characterization of composite nanoparticles (DMCNs) carrying iron oxide nanoparticles. (A) shows the iron oxide content of the composite nanoparticles according to the diameter of the used iron oxide nanoparticles, the diameter of the total particle, and the MR r ₂ relaxation time, (B) the size distribution of DMCNs 20 and the TEM image of the individual particles, ( C) is the diameter change over time in the physiologically active solution of DMCNs 20, (D) is the MRI contrast effect according to the iron oxide concentration in DMCNs 20, (E) is the fluorescence intensity according to the concentration of BCN-CNPs in DMCNs 20 Represents.
FIG. 2 is a diagram showing the results of characteristic analysis of composite nanoparticles (DMNs 5 and DMCNs 30, respectively) carrying iron oxide nanoparticles having an average diameter of 5 nm and 30 nm. (A) shows the size distribution of DMCNs 5 and DMCNs 30 and TEM images of individual particles, (B) shows the MRI T ₂ contrast efficacy according to the iron oxide concentration in DMCNs 5, DMCNs 20, and DMCNs 30, and (C) shows the DMCNs. _{5, MR r 2} relaxation degree according to the iron oxide concentration in DMCNs 20 and DMCNs 30 is shown.
3 is a diagram showing the results of in vitro cytotoxicity and differentiation analysis of metabolic-labeled stem cells (hMSCs). (A) shows the results of the toxicity evaluation analysis for stem cells according to the treatment concentration of DMCNs 20, (B) shows the proliferation trend of DMCNs 20-labeled stem cells and non-labeled stem cells according to the culture period by orthogonal click chemistry. (C) is the differentiation ability of DMCNs 20-labeled stem cells and non-labeled stem cells into chondrocytes, adipocytes, and bone cells by orthogonal click chemistry, and (D) is the stem cells labeled with DMCNs 20 by orthogonal click chemistry. It shows the relative degree of differentiation according to the differentiation of unlabeled stem cells into chondrocytes, adipocytes, and bone cells.
4 is a diagram showing the results of analysis of the cytotoxicity of Ac4ManNAz in vitro and _{the efficacy of the generated azide (N 3 ).} (A) shows the toxicity and fluorescent labeling effect on hMSCs according to the concentration of Ac4ManNAz, (B) shows the Western blot results for confirming the amount of azide expression in stem cells according to the incubation time of Ac4ManNAz (10 μM), ( C) and (D) are the fluorescence labeling effects and FACS analysis results using DBCO-Cy5 according to the incubation time of Ac4ManNAz using a confocal microscope, respectively, and (E) and (F) are respectively using a confocal microscope. The fluorescence labeling effect and FACS analysis results according to the incubation concentration of DBCO-Cy5 are shown.
5 is a diagram showing the labeling efficacy of metabolic-labeled stem cells with DMCNs 20. (A) and (B) represent the degree of metabolic labeling and fluorescence intensity through FACS analysis according to the incubation time with DMCNs 20 in Ac4ManNAz-treated stem cells and untreated stem cells through confocal laser scanning microscope images, respectively, ( C) shows the results of confocal laser scanning microscopy analysis of cells treated with DMCNs after incubation with DMCNs 20 from which BCN, a metabolic marker was removed, and TCEP, a competitive inhibitor of BCN, respectively, in Ac4ManNAz-treated stem cells, (D) Prussian blue staining results of DMCNs 20-labeled hMSCs, (E) shows the results of low-temperature-electron microscopy (Cyro-TEM) analysis of DMCNs 20-labeled hMSCs, and (F) is ICP-MS. _{The decrease in the content of Fe 3} O ₄ in DMCNs 20-labeled hMSCs according to the culture period measured by using, (G) shows the T ₂ -weighted MR signal analysis results of DMCNs 20-labeled hMSCs.
6 is a diagram showing the results of _{T 2} -weighted MR signal analysis according to the cell concentration of DMCNs 20-labeled hMSCs.
7 is a diagram showing in vivo tracking results using optical imaging for DMCNs 20-labeled hMSCs implanted in a photothrombotic stroke (PTS)-induced mouse model. (A) is an animal model using a mouse, (B) is a result of TTC (2,3,5-triphenyltetrazolium chloride) staining in the brains of normal mice and PTS-induced mice, and (C) is metabolic-labeled hMSCs. The results of NIRF signal analysis in stroke mice according to the injection concentration of are shown.
8 is a diagram showing cell behavior and immunostaining analysis results using NIRF after injection of metabolic-labeled hMSCs in a stroke-induced mouse model. (A) is an NIRF image over time after transplantation of metabolic-labeled hMSCs transplanted to stroke-induced and non-induced mice, and (B) is the relative NIRF from the injection site and the lesion site of the metabolic-labeled hMSCs. Changes in signal, (C) is an NIRF image of the excised brain of a stroke-inducing mouse injected with metabolic-labeled hMSCs, and (D) is a fluorescence signal of metabolic-labeled hMSCs injected into the stroke-inducing mouse using a confocal microscope. And human nuclear antibody (HNA-FITC) immunostaining results, (E) shows the fluorescence signals and macrophage (F4/80-FITC) immunostaining results of metabolic-labeled hMSCs injected into stroke-inducing mice using a confocal microscope. Show.
9 is a diagram showing the results of fluorescence and immunostaining analysis of a stroke-induced mouse model. (A) is an NIRF image of the excised brain of a stroke-induced mouse, (B) is a fluorescence signal and human nuclear antibody (HNA-FITC) immunostaining result of a stroke-induced mouse using a confocal microscope, (C) Represents a Prussian blue stained image of the excised brain of a stroke-induced mouse.
10 is a diagram showing MR images over time after injection of metabolic-labeled hMSCs in a stroke-induced mouse model. _{(A) is an MR T 2} image over time of metabolic-labeled hMSCs transplanted into stroke-induced and non-induced mice, and (B) relative MR signals at the injection site and lesion site of metabolic-labeled hMSCs. Changes, (C) and (D) show the results of Prussian blue staining in the brain and #1 stroke lesion, #2 hippocampus, and #3 injection site enlarged image of the mouse transplanted with metabolic-labeled hMSCs after stroke induction, respectively. .

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited by these examples.

<substance>

Oleic acid-coated superparamagnetic iron oxide nanoparticles with diameters of 5, 20 and 30 nm ((OA-Fe ₃ O ₄ ), glycol chitosan (MW=2.5×10 ⁵ Da, degree of deacetylation) of deacetylation)=82.7%), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide; EDC), N-hydroxysuccinimide ( N-hydroxysuccinimide; NHS), 5β-cholanic acid, tetrahydrofuran (THF, purity >99%), methanol (purity >99%), dimethylsulfoxide (DMSO, purity> 99%), alcian blue, alizarin red S and oil red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). NHS ester and tetraacetylated N-azidoacetyl-D-mannosamine (Ac ₄ ManNAz) were purchased from Click Chemistry Tools (Scottsdale, AZ, USA). adipogenesis differentiation kit), chondrogenesis differentiation kit, osteogenesis differentiation kit, fetal bovine serum (FBS), minimum essential media (MEM), streptavidin -Horseradish peroxidase (streptavidin-HRP), subcellular protein fractionation kit on kit) and phosphine -PEG ₃ - biotin (phosphine-PEG ₃ -biotin) was purchased from Thermo Fisher Scientific (Rockford, IL, USA). Bicyclo[6.1.0]nonyne N-hydroxysuccinimide ester II (BCN-PEG ₂ -NHS) was obtained from Berry & Associates (Dexter, MI, USA). I bought it. FITC conjugated anti-F4/80 antibody (FITC-F4/80 antibody; FITC-F4/80) and iron stain kit were purchased from Abcam (Cambridge, MA, USA). Alexa 488 conjugated anti-human nuclei antibody (FITC-HNA) was purchased from Merck Millipore (Darmstadt, Germany). All compounds were purchased for analytical grade and used without further purification.

For cell culture experiments, human adipose tissue-derived mesenchymal stem cells (hMSCs) were obtained from ATCC (American Type Culture Collection, VA, USA). FBS and MEM were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Dulbecco's phosphate buffered saline (DPBS, pH 7.4) and antibiotics-antimycotics (AA) were purchased from WelGENE Inc. (Daegu, Republic of Korea).

Example 1: glycol chitosan nanoparticles ( CNPs ) Of the manufacture

Glycol chitosan nanoparticles (CNPs) were prepared by self-assembly of an amphiphilic glycol chitosan polymer bound to 5β-cholanic acid. First, 5β-cholanic acid (120 mg, 800 μmol) was dissolved in 124 mL methanol mixed with NHS (72 mg, 1.2 mmol). Glycol chitosan (500 mg, 4 μmol) was dissolved in 60 mL deionized water and added to a 60 mL methanol solution containing EDC (120 mg, 1.2 mmol). The prepared 5β-cholanic acid solution was mixed with the glycol chitosan solution, and the mixed solution was stirred at room temperature for 12 hours. Thereafter, the mixed solution was 1:3, 1:2, 1:1 and 1:0 (v/v) of deionized water/methanol using a dialysis membrane (dialysis membrne, MWCO 12-14,000, Spectrum Laboratories, CA, USA). ) Dialysis was performed against a mixed solvent. The purified solution was lyophilized to obtain glycol chitosan nanoparticles (CNPs) as white powder.

Example 2: Cy5 .5-labeled glycol chitosan nanoparticles ( CNP - Cy5 . 5) of Produce

To track metabolically labeled hMSCs based on near infrared fluorescence (NIRF) imaging, CNPs were chemically modified with Cy5.5, a NIR fluorescent dye. Specifically, 2 mg of Cy5.5-NHS ester (1.7 μmol) was dissolved in 1 mL of DMSO, and CNPs (200 mg) prepared according to Example 1 were dispersed in 10 mL of DMSO. The Cy5.5-NHS ester solution was slowly added dropwise to the CNPs solution, and the mixed solution was stirred at room temperature for 12 hours. Thereafter, the mixed solution was dialyzed against deionized water for 2 days using a dialysis membrane (MWCO 12-14,000). The recovered solution was lyophilized to obtain CNP-Cy5.5, which is Cy5.5-labeled CNPs.

Example 3: BCN - Conjugated Cy5 .5-labeled glycol chitosan nanoparticles ( BCN - CNP -Cy5.5) preparation

For metabolic labeling of hMSCs to CNP-Cy5.5 through bioorthogonal click chemistry, CNP-Cy5.5 was chemically modified with _{BCN-PEG 2 -NHS ester.} Specifically, 10 mg of BCN-PEG _2- NHS ester (20 μmol) was dissolved in 1 mL of DMSO, and CNP-Cy5.5 (100 mg) prepared according to Example 2 was dispersed in 10 mL of DMSO. . The BCN-PEG _2- NHS ester solution was added dropwise to the CNP-Cy5.5 solution, and the mixed solution was stirred at room temperature for 12 hours. Thereafter, the mixed solution was dialyzed against deionized water for 2 days using a dialysis membrane (MWCO 12-14,000). The recovered solution was lyophilized to obtain BCN-CNP-Cy5.5, which is BCN-modified CNP-Cy5.5.

Example 4: double Mode of Magnetic chitosan nanoparticles ( DMCNs ) Of manufacturing and in vitro ( in vitro ) Characteristic analysis

To prepare NIRF/MR dual-modal magnetic chitosan nanoparticles (DMCNs), oleic acid-coated iron oxide nanoparticles (OA-Fe _{3) having a diameter of 5, 20, and 30 nm through dialysis.} O ₄ NPs) were encapsulated with BCN-CNP-Cy5.5. Specifically, BCN-CNP-Cy5.5 (20 mg) prepared according to Example 3 was dispersed in 2 mL of THF: deionized water solution (1:1 v/v). 6 mg of OA-Fe ₃ O ₄ NPs was dispersed in 0.2 mL of THF, and mixed with a BCN-CNP-Cy5.5 solution. The mixed solution was dialyzed against deionized water for 24 hours using a dialysis membrane (MWCO 12-14,000) to remove THF. The recovered solution was filtered through a 0.45 μm syringe filter to remove the residue. Finally, 5 nm OA-Fe ₃ O ₄ NPs-encapsulated BCN-CNP-Cy5.5 (DMCNs 5), 20 nm OA-Fe ₃ O ₄ NPs-encapsulated BCN-CNP-Cy5.5 through lyophilization. (DMCNs 20) and 30 nm OA-Fe ₃ O ₄ NPs-encapsulated BCN-CNP-Cy5.5 (DMCNs 30) were obtained, respectively. _{The amount of OA-Fe 3} O ₄ NPs in DMCNs 5, DMCNs 20 and DMCNs 30 was quantified by inductively coupled plasma mass spectrometry (ICP-MS, Perkin Elmer, MA, USA). In order to analyze the diameter and size distribution of DMCNs 5, DMCNs 20 and DMCNs 30, each nanoparticle (1 mg) was dispersed in 1 mL of PBS (pH 7.4), and a Zeta-sizer (Nano ZS , Malvern Instruments, UK) were used to measure diameter and size distribution. The morphologies of DMCNs 5, DMCNs 20 and DMCNs 30 were observed with transmission electron microscopy (TEM, Tecnai F20, FEI, Netherlands) at an acceleration voltage of 200 keV. To monitor the size stability of DMCNs 20, 1 mg of DMCNs 20 was dispersed in PBS or PBS containing 10% FBS and incubated at 37°C. The size of DMCNs 20 was monitored with a zeta-sizer for 14 days. In order to observe the contrast effect of DMCNs 20 in vitro NIRF, DMCNs 20 and CNs (0, 16, 31, 63, 125, and CNs at 250 μg/mL concentration) were added in PBS (pH 7.4). Was dispersed in, and NIRF images were obtained using IVIS Lumina Series III (Perkin Elmer, MA, USA). The NIRF intensity of DMCNs 20 and CNs was quantified using Living Image ^® software (Perkin Elmer, MA, USA). _{To observe the effect of MR in vitro, DMCNs 20 (containing Fe 3} O _{4 at} 16, 31, 63, 125 and 250 μM concentrations in DMCNs 20), OA-Fe ₃ O ₄ NPs with a diameter of 20 nm (OA- Fe ₃ O ₄ containing 16, 31, 63, 125 and 250 μM concentrations of Fe ₃ O _{4 in} Fe 3 O 4 NPs), CNs and PBS were dispersed in PBS containing 1% (w/v) agarose (pH 7.4), and 4.7 Tesla _{(tesla, T) T 2} -weighted MR phantom images were obtained using an animal MRI system (Axgilent Technologies, CA, USA). Relative MR T ₂ signals were quantified using Image J software (National Institute of Health, Bethesda, USA).

Example 5: in vitro Azide Formation analysis

hMSCs in 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin-amphotericin B (10 units/mL penicillin, 10 mg/mL streptomycin, It was cultured in MEM supplemented with 24 ng/mL amphotericin B) and 5 ng/mL human recombinant fibroblast growth factor (rhFGF). hMSCs were incubated at 37° C. in wet 5% CO _2. For the following experiments, hMSCs of 5 to 7 passage numbers were used.

In order to confirm the formation of azide on the surface of hMSCs after metabolic engineering, azide-containing glycans of hMSCs were visualized by Western blot analysis. Specifically, 5×10 ⁵ hMSCs were dispensed on a plate with a diameter of 100 mm, and _{stabilized in a 37° C. 5% CO 2} incubator for 24 hours. After stabilization, the hMSCs were treated with 10 μM Ac ₄ ManNAz and incubated for 0, 6, 12, 24, 48 and 72 hours. Thereafter, the hMSCs were washed twice with DPBS, and 1 mL of lysis buffer containing a protease inhibitor cocktail (lysis buffer, 1% SDS, 100 mM Tris-HCl, pH 7.4) was used at 4° C. Dissolved in for 30 minutes. The cell lysate was centrifuged at 12,000 rpm for 20 minutes at 4° C., and the supernatant was recovered and protein concentration was determined using a bicinchoninic acid protein assay (BCA, Pierce, USA). 100 μL of each cell lysate (5 mg/mL total protein) was _{mixed with 10 μL of 5 mM phosphine-PEG 3} -biotin (Pierce, USA) and incubated for 12 hours at room temperature. Proteins from each sample were mixed with 1× sodium dodecyl sulfate (SDS) gel-loading dye and boiled for 5 minutes. 20 μg of protein was separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 1 hour at room temperature with a 1×TBST solution (10 mol/L Tris, 100 mol/L NaCl and 0.1% Tween 20, pH 7.4) containing 5% bovine serum albumin (BSA). Finally, the membrane was washed 3 times using 1×TBST, and the protein band was detected with an enhanced chemiluminescence kit (Millipore, Billerica, USA). The signal of the band was obtained using LAS 3000 (Fujifilm, Tokyo, Japan). As a control, total protein was visualized by Coomassie brilliant blue stain.

Example 6: metabolic labeled hMSCs Cytotoxicity assay

In order to confirm the cytotoxicity of metabolic markers for hMSCs, hMSCs _{were incubated with Ac 4} ManNAz or DMCNs 20 for 24 hours, and then the survival rate of hMSCs was measured in a CCK-8 assay kit (Dojindo Molecular Technologies, Inc., Rockvile, USA). Specifically, 5×10 ³ hMSCs were dispensed on a 96-well plate and _{stabilized in a 37° C. 5% CO 2} incubator for 24 hours. After stabilization, hMSCs were _{incubated with Ac 4} ManNAz (0-100 μM) for 48 hours or DMCNs 20 (0-1000 μg/mL) and 37° C. for 24 hours, respectively. Thereafter, the hMSCs were washed twice with DPBS and further incubated in 200 μL of CCK-8 solution (10 μL of CCK-8 and 190 μL medium) at 37° C. for 2 hours. The absorbance at 450 nm for each well was ^{recorded using a microplate reader (VERSAmax™} , Molecular Devices Corp., CA, USA). ③ To monitor the proliferation of metabolic-labeled hMSCs, 1×10 ⁵ hMSCs were dispensed into a cell culture plate having a diameter of 100 mm, and incubated for 48 hours in an incubator _{with 10 μM Ac 4} ManNAz and 37°C 5% CO ₂ Thus, the formation of an azide group (N ₃ -hMSCs) on the surface of hMSCs was induced. In order to cover the N ₃ -hMSCs the DMCNs 20, followed by incubation for 2 hours, the N ₃ -hMSCs at 300 μg / mL of DMCNs 20 and 37 ℃. _{After N 3} -hMSCs labeling with DMCNs 20, metabolically-labeled hMSCs were washed twice with DPBS, and the number of metabolic-labeled hMSCs was collected every 3 days for 12 days. For cell counting, metabolic-labeled hMSCs were suspended in DPBS containing 0.4% (v/v) trypan blue (Sigma-Aldrich, St. Louis, USA), and a cell counter (R1, Olympus, Japan).

Example 7: metabolic labeled hMSCs Differentiation assay in vitro

In order to confirm the differentiation properties of metabolic-labeled hMSCs, chondrogenic, adipogenic, and osteogenic differentiation were performed using metabolic-labeled hMSCs. For metabolic labeling of hMSCs, 2×10 ⁶ hMSCs were dispensed onto a 150 mm diameter cell culture plate and stabilized for 24 hours. After stabilization, hMSCs were _{incubated with 10 μM of Ac 4} ManNAz and 37° C. 5% CO ₂ incubator for 48 hours, and labeled with 300 μg/mL of DMCNs 20 at 37° C. for 2 hours. In order to confirm chondrogenic differentiation, 1×10 ⁷ metabolic-labeled hMSCs were maintained at suspension in a chondrogenic differentiation kit (Thermo Fisher Scientific, Rockford, USA), and the medium was maintained every 3 days for 14 days. It was exchanged every time. After 14 days, the cell pellet was fixed with an OCT compound, frozen at -20°C for 24 hours, and stained with alcian blue (Sigma-Aldrich, St. Louis, USA) solution. To confirm lipogenic differentiation, 5×10 ⁴ metabolic labeled hMSCs were dispensed on a 6-well plate and _{stabilized in a 37°C 5% CO 2} incubator for 48 hours. The metabolic labeled hMSCs were incubated in an adipogenic differentiation kit (Thermo Fisher Scientific, Rockford, USA), and the medium was changed every 3 days for 14 days. After 14 days, metabolic-labeled hMSCs were fixed with a fixation buffer (DPBS containing 10% formaldehyde, 1% glutaraldehyde) at room temperature for 20 minutes, and stained with Oil Red O solution. In order to confirm osteogenic differentiation, 5×10 ⁴ metabolic labeled hMSCs were dispensed on a 6-well plate and _{stabilized in a 37°C 5% CO 2} incubator for 48 hours. The metabolic labeled hMSCs were incubated in an osteogenic differentiation kit (Thermo Fisher Scientific, Rockford, USA), and the medium was changed every 3 days for 14 days. After 14 days, metabolic-labeled hMSCs were fixed with fixation buffer at room temperature for 20 minutes, and stained with Alizarin Red S solution. Chondrogenesis-, adipogenesis- and osteogenic differentiation images of metabolic-labeled hMSCs were obtained using an optical microscope (CK40, Olympus, Japan).

Example 8: DMCNs Metabolized as 20 hMSCs

In order to label hMSCs with DMCNs 20 through metabolic engineering and bio-orthogonal click chemistry, 1×10 ⁵ hMSCs were dispensed onto 35 mm cell culture plates and stabilized for 24 hours. After stabilization, hMSCs were _{incubated with 10 μM of Ac 4} ManNAz in a 37° C. 5% CO ₂ incubator for 48 hours and labeled with 300 μg/mL of DMCNs 20 at 37° C. for 2 hours. The labeling efficacy of hMSCs was observed and quantified using a confocal laser scanning microscope (CLSM) and a flow cytometer (FACS), respectively. In addition, metabolic-labeled DMCNs 20-hMSCs and non-metabolic-labeled DMCNs 20-hMSCs were stained with iron through Prussian blue staining and observed with an optical microscope. Specifically, metabolic-labeled hMSCs on a 35 mm cell culture plate were washed twice with DPBS and fixed with 4% formaldehyde at 4° C. for 2 hours. After fixation, DMCNs 20-labeled hMSCs were stained with DAPI and fluorescence signals were observed with CLSM (Leica, Germany) equipped with a 405-diode (405 nm) and HeNe-Red (633 nm) laser. To quantify the fluorescence signal from DMCNs 20-labeled hMSCs, cells were washed twice with DPBS and the fluorescence signals from DMCNs 20-labeled hMSCs were analyzed using FACS (FACS Calibur, BD Biosciences, CA, USA). I did. Flow cytometric data was analyzed using Cell Quest (BD Biosciences, CA, USA). In addition, Prussian blue staining was performed to stain the iron supported on DMCNs in DMCNs 20-labeled hMSCs. A solution of 5% hydrochloric acid and 5% potassium ferrocyanide diluted in the same ratio (1:1) was treated in DMCNs 20-labeled hMSCs for 5 minutes, and then 3 times with distilled water. Washed. After washing, it was treated with a Nuclear-fast Red solution for 5 minutes, washed three times with purified water, and observed using an optical microscope. In order to observe high-magnified images of DMCNs 20-labeled hMSCs, 1×10 ⁴ hMSCs were dispensed onto collagen-coated disks (Wako, Osaka, Japan) in a 24-well cell culture plate. In order to label hMSCs with DMCNs 20, hMSCs were _{incubated with 10 μM of Ac 4} ManNAz and 37° C. 5% CO ₂ incubator for 48 hours and then labeled with 300 μg/mL of DMCNs 20 at 37° C. for 2 hours. After labeling, DMCNs 20-labeled hMSCs were washed twice with DPBS to remove unlabeled DMCNs 20 and fixed at 4° C. for 3 hours with a DPBS solution of 2.5% glutaraldehyde (Sigma-Aldrich, MO, USA). . Thereafter, DMCNs 20-labeled hMSCs were post-fixed at 4° C. for 3 hours with a DPBS solution of 2% osmium tetroxide (Sigma-Aldrich, MO, USA). For negative staining of DMCNs 20-labeled hMSCs, the immobilized cells were subjected to en bloc staining for 12 hours with a 2% uranyl acetate solution dissolved in distilled water. Thereafter, the samples were dehydrated with ethanol diluted in distilled water at a series of concentrations, and embedded in Spurr's resin (Sigma-Aldrich, MO, USA). Finally, the embedded sample was continuously cut to a thickness of 300 nm and collected on formvar coated grids and observed with a cryo-electron microscopy (Cryo-electron microscopy, Tecnai F20, FEI, Netherlands).

Example 9: DMCNs 20-labeled hMSCs undergarment Fe ₃ O ₄ Quantitative analysis of content

_{The amount of Fe 3} O ₄ in DMCNs 20-labeled hMSCs was quantified by inductively coupled plasma mass spectrometry (ICP-MS). Specifically, 1×10 ⁵ hMSCs were dispensed on a 100 mm cell culture plate and stabilized for 24 hours. After stabilization, hMSCs were _{incubated with 10 μM of Ac 4} ManNAz and 37° C. 5% CO ₂ incubator for 48 hours and labeled with 300 μg/mL of DMCNs 20 (45.9 μg of Fe ₃ O _{4) at 37° C. for 2 hours.} . As a control experimental group, hMSCs _{were incubated with 20 nm-sized Fe 3} O ₄ NPs (45.9 μg) for 2 hours or with 300 μg/mL of DMCNs 20 without metabolic manipulation. Thereafter, all cells were washed twice with DPBS and incubated for another 2 days. To quantify Fe ₃ O ₄ , cells were lysed in RIPA buffer (Sigma-Aldrich, MO, USA) and _{the amount of Fe 3} O ₄ was determined by ICP-MS (Perkin Elmer, MA, USA).

Example 10: in the test tube ( In vitro ) Cell level MR phantom Imaging (phantom imaging)

_{To confirm the T 2} -weighted MR signal from DMCNs 20-labeled hMSCs, 1×10 ⁵ hMSCs were dispensed into 100 mm cell culture plates and stabilized for 24 hours. After stabilization, hMSCs were _{incubated with 10 μM of Ac 4} ManNAz and 37° C. 5% CO ₂ incubator for 48 hours and labeled with 300 μg/mL of DMCNs 20 at 37° C. for 2 hours. As a control experimental group, hMSCs were incubated with 300 μg/mL of DMCNs 20 at 37° C. for 2 hours. Thereafter, all cells were washed twice with DPBS and then recovered by trypsin treatment. Transfer 1×10 ⁵ , 3×10 ⁵ , 5×10 ⁵ and 1×10 ⁶ DMCNs 20-labeled hMSCs or DMCNs 20-treated hMSCs to a 0.2 mL PCR tube and add a 4.7T animal MRI system (TR = 10000 ms , 1 mm slice thickness, 7.5 TE in the range from 7.5 ms to 960 ms) was used _{to measure the T 2} -weighted signal.

Example 11: mouse Photothrombotic stroke( photothrombotic stroke; PTS ) Manufacturing of the model

All experiments using live animals were performed in accordance with the relevant laws and regulations within the Korea Institute of Science and Technology (KIST) and institutional guidelines of the Institutional Animal Care and Use Committee (IACUC). Approved by IACUC (approval number 2017-005). In order to prepare a mouse brain stroke model, Balb/c nude mice (10 weeks old, 20 to 25 g, male) were purchased from Orient (Orient Bio, Seoul, Korea). Zoletil 50 (30 mg/kg) was injected intraperitoneally to anesthetize the mouse and fix it on a stereotaxic apparatus (restrained). In order to induce stroke, 100 μ1 of rose bengal (10 mg/mL in saline) solution was injected through the penile vein to the left frontal cortex (bregma) of the brain. The 5 mm anterior and 2.5 mm side from the midline) were exposed to a 561 nm laser for 16 minutes. To confirm stroke induction, brain tissue slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37° C. for 30 minutes.

Example 12: NIRF /MR Dual mode Imaging Used DMCNs 20-labeled hMSCs In vitro tracking ( in vivo tracking)

Prior to tracking of DMCNs 20-labeled hMSCs using NIRF/MR bimodal imaging, the number of hMSCs traceable by NIRF imaging after injection was optimized. Specifically, saline, 5×10 ⁴ , 1×10 ⁵ , 5×10 ⁵ and 1×10 ⁶ DMCNs 20-labeled hMSCs were respectively used in the right frontal lobe contralateral to stroke lesions. the stroke lesion). Thereafter, NIRF signals from DMCNs 20-labeled hMSCs were followed for 14 days using IVIS Lumina Series III (Perkin Elmer, MA, USA). ^{Finally, 1×10 6} DMCNs 20-labeled hMSCs injected in the contralateral part of the right frontal lobe for stroke lesions were followed for 14 days after injection using NIRF/MR dual-mode imaging. _{NIRF signals and T 2} -weighted MR signals from DMCNs 20-labeled hMSCs were transferred to IVIS Lumina System III and 9.4 T animal MRI systems (TR = 4000 ms, TE = 32.5 ms, 1 mm slice thickness, Agilent Technologies, CA, USA).

Example 13: Histological analysis

Brain tissue was fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) at 4° C. for 24 hours and dehydrated using a concentration gradient ethanol (70, 80, 90, 95 and 100%). (dehydrated). The dehydrated sample was immersed in xylene to make it transparent and embedded in a paraffin solution. The embedded sample was cut into 6 μm-thick slices, and Alexa fluor 488-conjugated human nuclei antibody (HNA-488) to observe DMCNs 20-labeled hMSCs in stroke-induced brain tissue. Dyed with. Specifically, brain tissue slides were deparaffinized in xylene and concentration gradient ethanol. For HNA-488 staining, brain tissue slides were washed 3 times with DPBS (pH 7.4), and PBST (1% BSA, 0.1% Tween 20 in PBS) and 30 minutes to block non-specific binding of HNA-488 antibody After incubation during, the brain tissue was incubated with HNA-488 (1:100, 1% BSA in PBST) in a wet chamber for 24 hours at 4°C. Finally, the brain tissue was stained with DAPI and the fluorescence signal was observed with CLSM equipped with a 405-diode laser (405 nm), Ar laser (488 nm) and HeNe-red laser (633 nm). In order to estimate the non-specific signal of DMCNs 20 due to phagocytosis of macrophages in stroke lesions, the brain tissue was stained with fluorescein isothiocyanate (FITC)-conjugated F4/80 antibody (F4/80-FITC). The brain tissue slides were deparaffinized in xylene and gradient ethanol. For F4/80-FITC staining, wash brain tissue slides 3 times with DPBS (pH 7.4) and PBST (1% BSA, 0.1% Tween 20 in PBS) and After incubation for 30 minutes, the brain tissue was incubated with F4/80-FITC (1:200, 1% BSA in PBST) in a wet chamber for 24 hours at 4°C. Finally, the brain tissue was stained with DAPI and the fluorescence signal was observed with CLSM equipped with a 405-diode laser (405 nm), Ar laser (488 nm) and HeNe-red laser (633 nm). To indicate whether the T ₂ -weighted MR signal was derived from DMCNs 20-labeled hMSCs in the stroke-induced brain, the brain tissue slides were stained with a Prussian blue staining kit. Specifically, brain tissue slides were deparaffinized in xylene and concentration gradient ethanol. The brain tissue slides were immersed in a mixture of hydrochloric acid and potassium ferrocyanide solutions (1:1, v/v) for 20 minutes. Thereafter, the brain tissue slides were washed three times with deionized water, counterstained with nuclear fast red solution for 5 minutes, and dehydrated with gradient ethanol and xylene. Finally, the tissue was placed on a cover glass and observed with an optical microscope (CK40, Olympus, Japan).

Example 14: statistical analysis

In the present invention, the difference between the experimental group and the control group was analyzed using a one-way ANOVA, and was determined to be relatively significant (indicated by an asterisk (*) in the figure).

Experimental example One: OA - Fe ₃ O ₄ NPs Encapsulated BCN - Of CNPs Manufacturing and optimization

First, hydrophobic 5β-cholanic acid is directly bonded to glycol chitosan through amide bond formation, and as a delivery carrier of Cy5.5 and SPION (superparamagnetic iron oxide nanoparticles), which are contrast agents, self-assembled Glycol chitosan nanoparticles (CNPs) were formed. Next, for near-infrared fluorescence (NIRF) imaging, a fluorescent dye, Cy5.5, was directly bound to CNPs. Thereafter, Cy5.5-linked CNPs were modified with bicyclo[6.1.0]nonyne (BCN) to form BCN-CNP-Cy5.5 (CNs) for bio-orthogonal click chemistry. Finally, OA-Fe ₃ O ₄ NPs were mounted in CNs to form dual-modal magnetic glycol chitosan nanoparticles (DMCNs). In particular, OA-Fe ₃ O ₄ Optimizing the NPs-mounted conditions gave the desired nanoparticle size and MR contrast effect. _{Specifically, OA-Fe 3} O ₄ of 5, 20 and 30 nm sizes, respectively Disperse NPs in THF and mount in CNs by a simple dialysis method, 5 nm-OA-Fe ₃ O ₄ NPs-loaded CNs (DMCNs 5), 20 nm-OA-Fe ₃ O ₄ NPs-loaded CNs (DMCNs 20) and 30 nm-OA-Fe ₃ O ₄ NPs-loaded CNs (DMCNs 30) were obtained, respectively. _{Subsequently, the Fe 3} O ₄ content, hydrodynamic diameter and r ₂ -relaxivity of the DMCNs 5, DMCNs 20 and DMCNs 30 were respectively determined by inductively coupled plasma mass spectrometry (ICP-MS), dynamic laser scattering. (dynamic laser scattering; DLS) and 4.7 Tesla magnetic resonance (MR) imaging. _{The Fe 3} O ₄ content, hydrodynamic diameter and r ₂ -relaxation degree of DMCNs 5, DMCNs 20 and DMCNs 30 measured as described above are shown in FIG. 1A. _{The OA-Fe 3} O ₄ contents in DMCNs 5, DMCNs 20 and DMCNs 30 were 15.0±1.9, 15.3±1.7 and 13.8±1.1, respectively, as quantified using ICP-MS. The hydrodynamic diameters of DMCNs 5, DMCNs 20 and DMCNs 30 newly dispersed in phosphate buffered saline (PBS) were measured by DLS, and as a result, diameters of 369.9±10.0, 238.9±6.6, and 366.4±8.6 nm were measured. I got it. The DMCNs 5, DMCNs 20, and DMCNs 30 are a plurality of Fe ₃ O _{4 in the inner space of CNs.} By retaining NPs, a spherical nanoparticle shape with a unimodal size distribution was shown (FIGS. 1B and 2A). _{The r 2} relaxation of DMCNs 5, DMCNs 20 and DMCNs 30 was calculated as ^{107.7, 526.1 and 92.5 mM -1} s ^-1 , respectively, based on the MR phantom images of DMCNs at various concentrations (0 to 1000 μM of Fe ₃ O _{4 ).} Became (Fig. 2B). In particular, the r ₂ -relaxation degree of DMCNs 20 was observed to be 4.88 times and 5.69 times higher than that of DMCNs 5 and DMCNs 30, respectively (Fig. 2C). This size of 20 nm Fe ₃ O ₄ NPs are relatively high Fe ₃ O ₄ in the CNs By forming the NPs-density, it is due to increasing the local magnetic dose (LMD) in the hydrophobic core. Accordingly, the present inventors selected and used DMCNs 20 as nanoparticles optimized for metabolic labeling and tracking of hMSCs under in vivo conditions in the following experiments. Next, the size stability of DMCNs 20 in PBS containing 10% FBS was confirmed for 14 days using DLS. The mean diameter of DMCNs 20, when dispersed in PBS and 10% FBS-containing PBS, respectively, did not show any significant change for 14 days (FIG. 1C). This indicates that neutral surface charge properties and hydrophilic ethylene glycol moieties on glycol chitosan can prevent the interaction of CNs with proteins.

The high contrast effect and labeling efficiency of non-toxic DMCNs 20 are important factors for effective tracking of hMSCs in vivo as well as in shipbuilding in vitro. Therefore, in order to confirm the contrast effect in NIRF and MR imaging in vitro, NIRF and MR phantom imaging were performed using DMCNs 20 of various concentrations. As shown in Fig. 1D, it was clearly observed that the NIRF signals from DMCNs 20 and CNs gradually increased in a dose-dependent manner (CNs from 0 to 250 μg/ml). In addition, the NIRF signal of DMCNs 20 did not show any significant difference from that of CNs, indicating that the NIRF signal of DMCNs 20 was not affected by _{Fe 3} O _{4 NPs.} Dose-dependent T _{2 -weighted MR signals for Fe 3} O ₄ of 16, 31, 63, 125 and 250 μM were also observed in DMCNs 20, where the T ₂ MR signals of DMCNs 20 at 250 μM were PBS, CNs and 250 μM of 20 nm-Fe ₃ O ₄ Compared to that of NPs, they were 37 times, 37.5 times, and 18.9 times stronger, respectively (Fig. 1E). The strong MR T ₂ -/NIRF-contrast effect and long-term stability of DMCNs 20 indicate that DMCNs 20 can be used for effective labeling of hMSCs.

Experimental example 2: Effect of metabolic markers on cell viability, proliferation and differentiation

For successful labeling and tracking of hMSCs by metabolic labeling method, the labeling parameters and toxicity of the imaging agent must be optimized along with the labeling method. Prior to optimizing the metabolic labeling of hMSCs using DMCNs 20, in vitro cytotoxicity was evaluated in hMSCs by CCK8 assay after treatment with _{DMCNs 20 or Ac 4 ManNAz for 24 hours, respectively.} As shown in FIG. 3A, when hMSCs were treated with DMCNs 20 (0 to 1000 μg/ml) for 24 hours, no significant change in cell viability was observed. In addition, Ac ₄ ManNAz-treated hMSCs also showed no significant change in cell viability up to _{50 μM of Ac 4 ManNAz (FIG. 4A ).} However, when treated with 100 μM of Ac ₄ ManNAz for 24 hours, the cell viability of hMSCs was significantly reduced. This is because a high concentration of Ac ₄ ManNAz can reduce the glycolytic activity of cells by hyperglycemia-induced damage, and can reduce mitochondrial oxygen consumption in hMSCs. This indicates that it can inhibit the proliferation of hMSCs. Therefore, 10 μM of Ac ₄ ManNAz was selected as an optimized concentration for _{azide production (N 3} -hMSCs) through metabolic manipulation of hMSCs. Next, the azide production effect was confirmed at various incubation times (0, 6, 12, 24, 48 and 72 hours) of _{hMSCs and Ac 4 ManNAz (10 μM).} As shown in FIG. 4B, a _{strong band intensity was observed in Western blot analysis after 6 hours treatment with 10 μM Ac 4} ManNAz, and the intensity of the band gradually increased with incubation time until 24 hours, 48 and 72 hours. Was saturated, indicating that the azide group can successfully bind to the glycans of hMSCs within 24 hours. In order to confirm the labeling efficiency of the N ₃ -hMSCs by DMCNs 20 through the biological orthogonal click chemistry, by 300 μg / ml of DMCNs 20 1, 2, and observing the fluorescence of the labeled N ₃ for 3 and 6 hours -hMSCs Quantified. Confocal microscopy images showed a fluorescent signal gradually increasing up to 2 hours by incubation with DMCNs 20, and a strong fluorescent signal was mainly observed on the surface of metabolic-labeled hMSCs (FIG. 4C). In addition, flow cytometry results indicated that formation signals from metabolic-labeled hMSCs were saturated 2 hours, 3 hours, and 6 hours after treatment with DMCNs 20, which was determined by bio-orthogonal click chemistry within 2 hours of DMCNs 20. It _{indicates binding to N 3} -hMSCs (Fig. 4D). Accordingly, the incubation time with DMCNs 20 was set to 2 hours as a suitable time for labeling _{N 3} -hMSCs through bio-orthogonal click chemistry. Finally, the proliferation of metabolic-labeled hMSCs was confirmed using the CCK8 assay. For metabolic labeling of hMSCs with DMCNs 20, 1×10 ⁵ hMSCs were treated with 10 μM of Ac ₄ ManNAz for 48 hours. Then, the azide group on N ₃ -hMSCs was chemically labeled with 300 μg/ml of DMCNs 20 and bio-orthogonal click chemistry for 2 hours. The number of metabolic-labeled hMSCs did not show a significant difference in proliferation compared to hMSCs treated with _{untreated or 10 μM Ac 4 ManNAz (FIG. 3B ).} This indicates that the metabolic labeling method provides an efficient efficacy method for hMSCs that do not induce toxicity.

In order to successfully apply and track stem cell labeling for clinical applications, the labeling method must be non-cytotoxic to cells and must not affect the differentiation properties of stem cells. Therefore, prior to in vivo _{application, the differentiation characteristics of metabolic-labeled hMSCs treated with Ac 4} ManNAz (10 μM, 48 h) and DMCNs 20 (300 μg/ml, 2h) were evaluated for cartilage formation and adipogenesis. And it was confirmed through the evaluation of bone formation. As shown in Figure 3C, compared to untreated hMSCs, almost the same portion (subequal area) of glucosaminoglycan (GAGs) was metabolized in both hMSCs and DMCNs 20-treated hMSCs without metabolism treatment. It was dyed after formation. In addition, when oil-red-O staining was used after adipogenic differentiation, lipid vacuoles of the same frequency were observed in both labeled hMSCs and untreated hMSCs. The osteogenic differentiation of hMSCs was also evaluated after culturing in osteogenic medium for 2 weeks using untreated hMSCs, metabolically labeled hMSCs, and hMSCs treated with DMCNs 20 without metabolic manipulation. In all groups of hMSCs, calcium deposition, a marker of osteogenic differentiation, was observed in almost the same area after Alizarin Rett S staining. The relative differentiation properties of metabolic-labeled hMSCs and DMCNs 20-treated hMSCs without metabolic manipulation showed no significant change in their properties compared to those of untreated hMSCs (Fig. 3D). This differentiation evaluation of metabolic-labeled hMSCs indicates that the metabolic manipulation-based cell labeling method can efficiently label hMSCs without changing the phenotype.

Experimental example 3: DMCNs In vitro to 20 hMSC sign

Based on the optimized metabolic labeling conditions, the labeling efficiency between metabolic labeling and nonspecific DMCNs 20 uptake by hMSCs was compared. As shown in FIG. 5A, the labeling effect of hMSCs using DMCNs 20 was confirmed by a confocal microscope, and the fluorescence intensity from hMSCs treated with DMCNs 20 was remarkably after metabolic manipulation compared to hMSCs treated with untreated or DMCNs 20. It was confirmed that it increased. In addition, flow cytometric data showed that hMSCs metabolically labeled with DMCNs 20 exhibited 3.3 and 2.39 times higher mean fluorescence intensity (MFI), respectively, compared to those of untreated and DMCNs 20 treated hMSCs (Fig. 5B). To confirm that hMSCs metabolically labeled with DMCNs 20 were metabolically labeled, DMCNs 20 from which BCN was removed and TCEP (2-carboxyethyl) phosphine hydrochloride (TCEP), a competitive inhibitor of BCN attached to DMCNs 20, were treated with Ac4ManNAz, respectively. Treated with hMSCs. It was confirmed through confocal microscopy that the labeling of DMCNs 20 was remarkably reduced under metabolic inhibition conditions (FIG. 5C ). Furthermore, the Prussian blue stained image showed a stronger iron oxide intensity in metabolic-labeled hMSCs compared to hMSCs treated with DMCNs 20 without metabolic manipulation (FIG. 5D). In addition, in order to confirm the distribution of DMCNs 20 in hMSCs including the cytoplasm from the cell surface membrane, hMSCs treated with DMCNs 20 and metabolically labeled hMSCs with DMCNs 20 were analyzed using a cryo-electron microscope (Cryo-Electron microscopy). (Fig. 5E). The hMSCs treated with DMCNs 20 showed some DMCNs 20 in the cytoplasm due to non-specific uptake by hMSCs. However, when hMSCs _{were treated with Ac 4} ManNAz, a number of DMCNs 20 were mainly observed in the cell surface membrane. This means that the exogenously provided DMCNs 20 _{can artificially bind to azide groups on the surface of metabolized hMSCs (N 3} -hMSCs) through bioorthogonal click chemistry, resulting in the amount of DMCNs 20 on the surface of hMSCs. It is due to the increase. _{In addition, ICP-MS data of N 3} -hMSCs metabolized by DMCNs 20 also showed _{that 70.7% of Fe 3} O ₄ NPs were maintained in hMSCs 48 hours after labeling (FIG. 5F). However, when hMSCs _{were treated with Fe 3} O ₄ NPs or DMCNs 20 without metabolic manipulation, 38.7% and 42.6% of Fe ₃ O ₄ NPs, respectively, remained in the hMSCs. This is due to the internalization of hMSCs into the cytoplasm through the membrane turnover mechanism of the cell by the DMCNs 20 artificially bound to the azide group on the sialic acid-containing glycan on the cell surface. _{Next, a T 2} -weighted MR signal was measured while changing the number of metabolic-labeled hMSCs, and compared with the values for hMSCs treated with DMCNs 20 without metabolic manipulation. As shown in FIG. 5G, in vitro T ₂ -weighted phantom images are hMSCs concentration-dependent enhancement of _{T 2} -weighted MR signals in metabolic-labeled hMSCs compared to hMSCs treated with DMCNs 20 without metabolic manipulation. Clearly indicated. The relative T ₂ -weighted MR signal of metabolically labeled hMSCs is that the number of metabolic labeled hMSCs is 1×10 ⁵ , 3×10 ⁵ , At 5×10 ⁵ , and 1×10 ⁶ , there was an improvement of 58.4%, 21.3%, 10.6%, and 8.66%, respectively, compared to that of PBS. This is because DMCNs 20 can bind to azide groups on the surface of hMSCs that are exogenously generated through metabolic manipulation, resulting in an increase in the labeling efficiency of hMSCs. _{However, the relative T 2} -weighted MR signal of hMSCs treated with DMCNs 20 without metabolic manipulation decreased to 43.3% at ^{5×10 5} cell concentration, and was saturated (42%) even at ^{1×10 6 cell concentration (Fig. 6).} ).

Experimental example 4: Metabolic Labeled in Mouse Stroke Model hMSCs In vivo optical/MR dual imaging

In order to confirm the in vivo tracking efficiency of metabolic-labeled hMSCs through optical/MR dual imaging, the left cerebral cortical region ( left cortical area), a mouse photothrombotic stroke (PTS) model inducing ischemic damage was used (FIG. 7A). 2,3,5-triphenyltetrazolium chloride (TTC) staining images from the excised brain of the mouse PTS model were compared to normal mouse brain, TTC- The negative region was clearly indicated (Fig. 7B). Prior to tracking hMSCs, after injecting metabolically labeled hMSCs at various concentrations, NIRF signal sensitivity was confirmed and optimized in a mouse PTS model. Each 5×10 ⁴ , 1×10 ⁵ , 5×10 ⁵ , and 1×10 ⁶ metabolic-labeled hMSCs were injected into the right hippocampus of the mouse PTS model, and NIRF signals were monitored for 14 days (Fig. 7C). Compared to the saline-treated mouse PTS model, when the mice were treated with 5×10 ⁴ or 1×10 ⁵ metabolic labeled hMSCs, respectively, a strong NIRF signal was monitored in the head for 3 days. Interestingly, the ^{NIRF signal in the head of the mouse PTS model treated with 5×10 4} or 1×10 ⁵ metabolic-labeled hMSCs, respectively, showed a strong NIRF signal extending up to 14 days after injection. Furthermore, migration of NIRF signals to stroke lesions was observed from day 7 after injection ^{of 5×10 4} or 1×10 ^{5 metabolic-labeled hMSCs.}

^{Based on the NIRF sensitivity result, after injecting 1×10 6} cells into the right hippocampus of the mouse PTS model and the normal mouse model, NIRF signal tracking from metabolic-labeled hMSCs was performed. On day 1 after injection into the mouse PTS model, a strong NIRF signal from metabolically labeled hMSCs was observed at the injection site. The NIRF signal gradually migrated to the stroke lesion on the 3rd day after injection, and a strong NIRF signal appeared in the stroke lesion 7, 10, and 14 days after the injection of metabolically-labeled hMSCs. This indicates that stromal cells derived factor-1α (SDF-1α, CXCL 12) protein that is overexpressed in stroke lesions can induce hMSC migration to stroke lesions. However, when metabolic-labeled hMSCs were injected into a normal mouse model, the NIRF signal gradually decreased at the injection site without any migration (Fig. 8A). As shown in FIG. 8B, in the mouse PTS model, the relative NIRF signal gradually increased at the stroke lesion, while gradually decreased at the injection site. For further observation of metabolic-labeled hMSCs in the brain of the mouse PTS model, all brains were excised on day 14 after injection of metabolic-labeled hMSCs. In normal brain, the NIRF signal from metabolically labeled hMSCs was concentrated at the injected site. In addition, the NIRF signal from stroke-induced brain tissue that was not treated with metabolic-labeled hMSCs showed only the background signal of the brain tissue (FIG. 9A). However, when metabolic-labeled hMSCs were injected into the stroke-induced brain, a strong NIRF signal was observed at the injection site as well as at the stroke lesion (FIG. 8C).

In order to confirm whether the NIRF signal is derived from metabolic-labeled hMSCs in the stroke-induced brain, the stroke-induced brain tissue was subjected to fluorescein isothiocyanate conjugated-human nucleic antibody (FITC). -HNA). As shown in FIG. 8D, the confocal microscope image showed that the fluorescence signal of FITC-HNA was observed both at the stroke lesion and at the injection site, as well as co-localized with the NIRF signal of DMCNs 20. These results indicate that metabolic-labeled hMSCs in brain tissue survive 14 days after injection and can migrate to stroke lesions. However, no fluorescence signals from DMCNs 20 and FITC-HNA were observed in stroke-induced brain tissue without metabolically labeled hMSCs (FIG. 9B ). Thereafter, after injection of metabolic-labeled hMSCs, the distortion of the NIRF signal at both the injection site and the stroke lesion of the stroke-induced brain tissue was confirmed through direct staining of macrophages using FITC-conjugated F4/80 antibody. (Fig. 8E). The injection site of metabolic labeled hMSCs in stroke-induced brain tissue was revealed by the NIRF signal of DMCNs 20 from metabolic labeled hMSCs partially co-localized with fluorescence from macrophages. However, the NIRF signal of DMCNs 20 from the metabolic-labeled hMSCs in stroke lesions was hardly co-localized with the fluorescence of FITC-F4/80, which means that macrophages metabolized the phagocytosis of metabolically-labeled hMSCs in stroke lesions. This indicates that a false signal by is not displayed at all.

Based on the T ₂ -weighted MR signal from DMCNs 20, spatiotemporal tracking of metabolic labeled hMSCs using 4-Tesla MR imaging at 1, 3, 7, 10 and 14 days post-injection. ) Was performed. _{As shown in Fig. 10A, strong T 2} -weighted MR signals were observed from metabolic-labeled hMSCs at the injection site on day 1 of injection in both the mouse PTS model and the normal mouse model. _{Afterwards, the T 2} -weighted MR signal gradually migrated to the stroke lesion on the 3rd day of injection of metabolically-labeled hMSCs in the mouse PTS model _{, and strong T 2} -weighted MR in the stroke lesion on the 7th, 10th and 14th days after injection. The signal appeared. However, in the normal mouse model, the T ₂ -weighted MR signal from metabolically labeled hMSCs gradually decreased at the injection site without any migration. In the mouse PTS model, the relative T ₂ -weighted MR signal and the T ₂ -weighted MR signal gradually increased in stroke lesions, and were mainly localized to the stroke lesions 7, 10 and 14 days after injection (Fig. 8B). . _{Overall, the T 2} -weighted MR signal from the injection site of the mouse PTS model gradually decreased at the injection site on days 7, 10 and 14 after injection. This is highly correlated with the NIRF imaging results, and supports that metabolic-labeled hMSCs can be successfully tracked by in vivo NIRF imaging as well as MR imaging.

Finally, stroke-induced brain tissue was stained with a Prussian blue staining kit _{to confirm whether the T 2} -weighted MR signal originated from metabolic-labeled hMSCs in the stroke-induced brain. Compared with stroke-induced brain tissue without metabolism-labeled hMSCs (Fig. 9C), Prussian blue staining images of stroke-induced brain treated with metabolic-labeled hMSCs were Fe ₃ O _{4 in brain tissue.} It indicated the presence of NPs (Figures 10C and 10D). In addition, the enlarged Prussian blue staining image of the brain tissue is Fe ₃ O ₄ It was clearly shown that NPs were distributed in stroke lesions (#1 in FIGS. 10C and 10D), hippocampus (#2 in FIGS. 10C and 10D) and injection sites (#3 in FIGS. 10C and 10D). The successful non-invasive and accurate tracking of hMSCs based on metabolic labeling methods is non-toxic, high sensitivity, and high spatial resolution in combination with _{in vivo NIRF imaging and T 2 -weighted MR imaging.} resolution), thus showing promising potential.

<Conclusion>

In the present invention, the metabolic labeling strategy of hMSCs allowed to accurately monitor the migration and distribution of hMSCs based on _{NIRF and T 2 -weighted MR dual mode imaging after injection into the brain of a mouse PTS model.} At the optimal Ac ₄ ManNAz concentration, hMSCs _{were metabolized by Ac 4} ManNAz treatment to form azide groups on the cell surface. Thereafter, _{these azide groups formed exogenously by Ac 4} ManNAz treatment were chemically labeled with DMCNs 20 through bio-orthogonal click chemistry. The metabolic labeling strategy was able to control the labeling efficiency of hMSCs in a concentration dependent manner of _{Ac 4 ManNAz and DMCNs 20 in cell culture conditions.} Furthermore, hMSCs metabolized with DMCNs 20 showed a prolonged labeling effect by DMCNs 20 taken through a membrane conversion mechanism without any significant change in stem cell properties such as cell viability, proliferation, and differentiation. Finally, the injected metabolic-labeled hMSCs in the brain of the mouse PTS model showed a high contrast at the injection site, which allows non-invasive tracking of hMSCs for 14 days by _{dual NIRF and T 2 -weighted MR imaging.} Allowed. Furthermore, dual NIRF and T ₂ -weighted MR imaging could be used to track the migration of metabolic labeled hMSCs from the injected site in the brain to the stroke lesion for 14 days. This indicates that the metabolic labeling strategy of hMSCs can provide valuable information for optimizing next-generation therapies for various neurodegenerative diseases including Parkinson's disease as well as stroke based on stem cell therapy.

Claims (23)

Two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm, and the iron oxide content is 13% to 18% by weight,
_{Complex nanoparticles with an r 2} relaxation degree of 200 to 1000 /mM·sec calculated based on MR phantom images when labeled on stem cells.
The method of claim 1,
Composite nanoparticles that exhibit a size change within 10% of deviation up to 14 days in a physiologically active solution condition.
The method of claim 1,
To show an increased magnetic resonance imaging (MRI) signal compared to the composite nanoparticles containing iron oxide nanoparticles having an average diameter of less than 15 nm or more than 25 nm in an equivalent or ±10% by weight content, Composite nanoparticles.
The method of claim 1,
Compared to the use of the same amount of iron oxide nanoparticles of the same size, 10 to 30 times increased T ₂ -weighted signal size to be displayed, the composite nanoparticles.
Two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm, and the iron oxide content is As a composition for labeling stem cells containing 13% to 18% by weight, composite nanoparticles,
The chitosan glycol nanoparticles, including a cycloalkyne group bound to the surface, are capable of binding to stem cells including an azide group introduced through metabolic sugar chain manipulation on the surface through bio-orthogonal non-moving click chemistry, for stem cell labeling Composition.
The method of claim 5,
The composition of the chitosan glycol nanoparticles is one or more compounds selected from the group consisting of compounds of the following formula (4) are bound to the surface:
[Formula 4]

Two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm, and the iron oxide content is 13% to 18% by weight of composite nanoparticles; And
As a composition for detecting a stroke lesion comprising; isolated stem cells,
The chitosan glycol nanoparticles include a cycloalkyne group bound to the surface, and the isolated stem cells include an azide group introduced through metabolic sugar chain manipulation on the surface, and are capable of binding to each other through bio-orthogonal non-click chemistry,
When the composite nanoparticles are labeled on the stem cells, the r ₂ relaxation degree calculated based on the MR phantom image is 200 to 1000 /mM·sec, a composition for detecting stroke lesions.
The method of claim 7,
The chitosan glycol nanoparticles are further labeled with a fluorescent molecule to enable multiple detection.
The method of claim 8,
The composition, wherein the fluorescent molecule has an absorption peak in a wavelength range of 450 to 800 nm, and a fluorescence peak in a wavelength range of 550 to 1000 nm.
The method of claim 7,
The stem cells are treated with one or more compounds selected from the group consisting of compounds of the following formulas 1 to 3, and an azide group is introduced into the surface thereof:
[Formula 1]

[Formula 2]

[Formula 3]

.
The method of claim 7,
The stem cells are treated with tetraacetylated N-azidoacetyl-D-mannoseamine (Ac4ManNAz) to contain non-natural sialic acid having an azide group generated on the surface of the cell.
As stem cells that can be traced when injected in vivo,
Two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm, and the iron oxide content is 13% to 18% by weight of composite nanoparticles; And isolated stem cells; including,
The chitosan glycol nanoparticles contain a cycloalkyne group bonded to the surface, and the isolated stem cells include an azide group introduced through metabolic sugar chain manipulation on the surface, and are bonded to each other through bio-orthogonal non-click chemistry,
When the composite nanoparticles are labeled on the stem cells, the r ₂ relaxation degree calculated based on the MR phantom image is 200 to 1000 /mM·sec.
The method of claim 12,
Stem cells that specifically target a stroke lesion upon in vivo injection.
The method of claim 12,
Stem cells that maintain the same level of cellular characteristics as stem cells that are not combined with the composite nanoparticles.
delete Two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm, and the iron oxide content is As a method for producing stem cells traceable by magnetic resonance imaging, comprising the step of treating and labeling the stem cells of 13% to 18% by weight of the composite nanoparticles,
The chitosan glycol nanoparticles contain a cycloalkyne group bonded to the surface, and the stem cells include an azide group introduced through metabolic sugar chain manipulation on the surface and bind to each other through bio-orthogonal non-moving click chemistry,
When the composite nanoparticles are labeled on the stem cells, the r ₂ relaxation degree calculated based on the MR phantom image is 200 to 1000 /mM·sec.
The method of claim 16,
The chitosan glycol nanoparticles are further labeled with a fluorescent molecule to enable multiple detection.
The method of claim 17,
The method of claim 1, wherein the fluorescent molecule has an absorption peak in a wavelength range of 450 to 800 nm, and a fluorescence peak in a wavelength range of 550 to 1000 nm.
The method of claim 16,
The stem cells are treated with one or more compounds selected from the group consisting of compounds of the following formulas 1 to 3, and an azide group is introduced into the surface thereof:
[Formula 1]

[Formula 2]

[Formula 3]

.
The method of claim 16,
The method of claim 1, wherein the stem cells are treated with tetraacetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) to contain non-natural sialic acid having an azide group generated on the surface of the cell.
The method of claim 16,
The chitosan glycol nanoparticles are one or more compounds selected from the group consisting of compounds of the following formula 4 is bound to the surface, the method:
[Formula 4]

delete Two or more iron oxide nanoparticles having an average diameter of 15 to 25 nm, which are surface-modified with oleic acid, are encapsulated by two or more chitosan glycol nanoparticles, and have an average diameter of 200 to 300 nm, and the iron oxide content is 13% to 18% by weight, treating and labeling the stem cells to be injected with the composite nanoparticles;
Injecting the stem cells labeled with the composite nanoparticles into the individual; And
A method of tracking stem cells injected into a living body of an individual other than humans, comprising the step of measuring a magnetic resonance imaging (MRI) signal from an individual injected with the stem cells,
The chitosan glycol nanoparticles contain a cycloalkyne group bonded to the surface, and the stem cells include an azide group introduced through metabolic sugar chain manipulation on the surface and bind to each other through bio-orthogonal non-click chemistry,
When the composite nanoparticles are labeled on the stem cells, the r ₂ relaxation degree calculated based on the MR phantom image is 200 to 1000 /mM·sec.

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